Coronavirus immunogenic compositions and their uses

Abstract

The present disclosure provides compositions and methods comprising circular polyribonucleotides comprising sequences encoding one or more coronavirus immunogens, as well as compositions and methods comprising linear polyribonucleotides comprising sequences encoding coronavirus immunogens. Related compositions and methods are provided, for example, for generating polyclonal antibodies using the disclosed circular polyribonucleotides or the disclosed linear polyribonucleotides.

Description

[Background technology]

[0001] COVID-19, a human respiratory disease caused by infection with SARS-CoV-2, originated in Wuhan, China, and spread globally, resulting in millions of deaths worldwide, leading the World Health Organization to declare it a pandemic on March 11, 2020. Therefore, there is an urgent need for effective vaccines and therapeutics against coronaviruses and their use. Summary of the Invention [Means for solving the problem]

[0002] The present disclosure generally relates to cyclic polyribonucleotides comprising sequences encoding coronavirus immunogens and immunogenic compositions comprising cyclic polyribonucleotides. The disclosure further relates to methods of using the cyclic polyribonucleotides and immunogenic compositions comprising sequences encoding coronavirus immunogens. In certain embodiments, the cyclic polyribonucleotides and immunogenic compositions of the present disclosure are used in methods of generating polyclonal antibodies. The generated polyclonal antibodies can be used in methods of prevention in subjects (e.g., human subjects) or methods of treatment of subjects (e.g., human subjects) with a coronavirus infection. The generated polyclonal antibodies can be administered to subjects at high risk of exposure to a coronavirus infection.

[0003] In a first aspect, the present disclosure provides a circular polyribonucleotide comprising an open reading frame encoding a coronavirus immunogen, wherein the coronavirus immunogen comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In certain embodiments, the coronavirus immunogen comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the coronavirus immunogen comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291.

[0004] In certain embodiments, the coronavirus immunogen is an RBD immunogen that has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the amino acid sequence of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In some embodiments, the coronavirus immunogen is an RBD immunogen that has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the amino acid sequence of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In some embodiments, the coronavirus immunogen is an RBD immunogen having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In some embodiments, the coronavirus immunogen is an RBD immunogen having the amino acid sequence of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111.

[0005] In certain embodiments, the coronavirus immunogen is a spike immunogen having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In some embodiments, the coronavirus immunogen is a spike immunogen having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In some embodiments, the coronavirus immunogen is a spike immunogen having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In some embodiments, the coronavirus immunogen is a spike immunogen having the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286.

[0006] In certain embodiments, the coronavirus immunogen is a nonstructural protein (nsp) having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the amino acid sequence of any one of SEQ ID NOs: 291-295. In some embodiments, the coronavirus immunogen is an nsp immunogen having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the amino acid sequence of any one of SEQ ID NOs: 291-295. In some embodiments, the coronavirus immunogen is an nsp immunogen having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 291-295. In some embodiments, the coronavirus immunogen is an nsp immunogen having the amino acid sequence of any one of SEQ ID NOs: 291-295.

[0007] In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 112 to 174 and 292 to 300. In some embodiments, the open reading frame comprises the nucleic acid sequence of any one of SEQ ID NOs: 112 to 174 and 292 to 300.

[0008] In some embodiments, the coronavirus immunogen is an RBD immunogen that has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In some embodiments, the coronavirus immunogen is an RBD immunogen that has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In some embodiments, the coronavirus immunogen is an RBD immunogen having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In some embodiments, the coronavirus immunogen is an RBD immunogen having the nucleic acid sequence of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174.

[0009] In one embodiment, the coronavirus immunogen is a spike immunogen having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In some embodiments, the coronavirus immunogen is a spike immunogen that has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In some embodiments, the coronavirus immunogen is a spike immunogen that has at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In one embodiment, the coronavirus immunogen is a spike immunogen having the nucleic acid sequence of any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291.

[0010] In one embodiment, the coronavirus immunogen is an nsp immunogen having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 296-300, or an nsp immunogen having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) identity to the nucleic acid sequence of any one of SEQ ID NOs: 296-300. In some embodiments, the coronavirus immunogen is an nsp immunogen having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 296-300. In some embodiments, the coronavirus immunogen is an nsp immunogen having the nucleic acid sequence of any one of SEQ ID NOs: 296-300.

[0011] In some embodiments, the open reading frame encoding the coronavirus immunogen is operably linked to an IRES. In some embodiments, the open reading frame encoding the coronavirus immunogen encodes a second polypeptide. In some embodiments, the coronavirus immunogen and the second polypeptide are separated by a polypeptide linker, a 2A self-cleaving peptide, a protease cleavage site, or a 2A self-cleaving peptide together with a protease cleavage site. In some embodiments, the protease cleavage site is a furin cleavage site.

[0012] In some embodiments, the circular polyribonucleotide further comprises a second open reading frame encoding a second polypeptide operably linked to a second IRES. In some embodiments, the second polypeptide is a polypeptide immunogen. In some embodiments, the second polypeptide is a viral immunogen. In some embodiments, the second polypeptide is a coronavirus immunogen. In some embodiments, the second coronavirus immunogen comprises an amino acid sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 1-10, 53, 55, 57, 63-111, and 283-291. In some embodiments, the second coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 1-10, 53, 55, 57, 63-111, and 283-291. In some embodiments, the second polypeptide is an influenza immunogen.

[0013] In some embodiments, the second polypeptide is a polypeptide adjuvant. In some embodiments, the adjuvant is a cytokine, a chemokine, a costimulatory molecule, an innate immune stimulator, a signal transduction molecule, a transcriptional activator, a cytokine receptor, a bacterial component, or a component of the innate immune system. In some embodiments, the cyclic polyribonucleotide further comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator. In some embodiments, the innate immune system stimulator is selected from a GU-rich motif, an AU-rich motif, a structured region comprising dsRNA, or an aptamer.

[0014] In another aspect, the present disclosure provides a cyclic polyribonucleotide comprising a first sequence encoding a coronavirus immunogen and a second sequence encoding a polypeptide adjuvant. In one embodiment, the sequence encoding the coronavirus immunogen is operably linked to a first IRES, and the sequence encoding the polypeptide adjuvant is operably linked to a second IRES. In one embodiment, the coronavirus immunogen and the polypeptide adjuvant are encoded by a single open reading frame operably linked to the IRES. In one embodiment, the coronavirus immunogen and the polypeptide adjuvant are separated by a polypeptide linker, a 2A self-cleaving peptide, a protease cleavage site, or a 2A self-cleaving peptide with a protease cleavage site.

[0015] In some embodiments, the polypeptide adjuvant is a cytokine, chemokine, costimulatory molecule, innate immune stimulator, signaling molecule, transcriptional activator, cytokine receptor, bacterial component, or component of the innate immune system. In some embodiments, the second coronavirus immunogen comprises an amino acid sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 1-10, 53, 55, 57, 63-111, and 283-291. In some embodiments, the second coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 1-10, 53, 55, 57, 63-111, and 283-291.

[0016] In another aspect, the present disclosure provides a circular polyribonucleotide comprising an open reading frame encoding a coronavirus immunogen and a non-coding ribonucleic acid sequence that is an innate immune system stimulator. In some embodiments, the innate immune system stimulator is selected from a GU-rich motif, an AU-rich motif, a structured region comprising dsRNA, or an aptamer. In some embodiments, the second coronavirus immunogen comprises an amino acid sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 1-10, 53, 55, 57, 63-111, and 283-291. In some embodiments, the second coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 1-10, 53, 55, 57, 63-111, and 283-291.

[0017] In some embodiments, the open reading frame encodes a concatemeric coronavirus immunogen. In some embodiments, the open reading frame comprises 2 to 100 coronavirus immunogens, either directly linked to each other or interspersed with linkers. In other embodiments, the immunogen is a concatemeric peptide immunogen comprising multiple peptide epitopes. In some embodiments, the cyclic polyribonucleotide encodes 2 to 10 coronavirus immunogens. In some embodiments, the cyclic polyribonucleotide encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 coronavirus immunogens. In some embodiments, the coronavirus immunogens are separated by a polypeptide linker, a 2A self-cleaving peptide, a protease cleavage site, or a 2A self-cleaving peptide together with a protease cleavage site. In some embodiments, the concatemeric coronavirus immunogen comprises an amino acid sequence having at least 85% identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the concatemeric coronavirus immunogen comprises an amino acid sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the concatemeric coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In one embodiment, the concatemeric coronavirus immunogen comprises a nucleic acid sequence having at least 85% identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 112-174 and 292-300.In some embodiments, the concatemeric coronavirus immunogen comprises a nucleic acid sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the concatemeric coronavirus immunogen comprises the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300.

[0018] In another aspect, the present disclosure provides a circular polyribonucleotide comprising a first sequence encoding a coronavirus immunogen and a second sequence encoding a multimerization domain. In some embodiments, the multimerization domain comprises a T4-foldon domain. In some embodiments, the multimerization domain comprises a ferritin domain. In some embodiments, the multimerization domain comprises a β-annulus peptide. In some embodiments, the multimerization domain is at the N-terminus of the coronavirus immunogen. In some embodiments, the multimerization domain is at the C-terminus of the coronavirus immunogen.

[0019] In another aspect, the present disclosure provides an immunogenic composition comprising any one of the cyclic polyribonucleotides described herein, a pharmaceutically acceptable excipient, and no carrier. In another aspect, the present disclosure provides an immunogenic composition comprising any one of the cyclic polyribonucleotides described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises a second cyclic polyribonucleotide. In some embodiments, the second cyclic polyribonucleotide comprises an open reading frame encoding a second polypeptide immunogen. In some embodiments, the second cyclic polyribonucleotide comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator.

[0020] In another aspect, the disclosure provides a linear polyribonucleotide comprising an open reading frame encoding a coronavirus immunogen, wherein the coronavirus immunogen comprises an amino acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In certain embodiments, the coronavirus immunogen comprises an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the coronavirus immunogen comprises an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the coronavirus immunogen comprises an amino acid sequence having the amino acid sequence of any one of SEQ ID NOs: 63-111 and 283-291.

[0021] In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 112 to 174 and 292 to 300. In some embodiments, the open reading frame comprises a nucleic acid sequence having the nucleic acid sequence of any one of SEQ ID NOs: 112 to 174 and 292 to 300.

[0022] In some embodiments, the open reading frame encoding the coronavirus immunogen is operably linked to an IRES. In some embodiments, the open reading frame encoding the coronavirus immunogen encodes a second polypeptide. In some embodiments, the coronavirus immunogen and the second polypeptide are separated by a polypeptide linker, a 2A self-cleaving peptide, a protease cleavage site, or a 2A self-cleaving peptide together with a protease cleavage site. In some embodiments, the protease cleavage site is a furin cleavage site.

[0023] In some embodiments, the circular polyribonucleotide further comprises a second open reading frame encoding a second polypeptide operably linked to a second IRES. In some embodiments, the second polypeptide is a polypeptide immunogen. In some embodiments, the second polypeptide is a coronavirus immunogen. In some embodiments, the second polypeptide is a polypeptide adjuvant. In some embodiments, the adjuvant is a cytokine, a chemokine, a costimulatory molecule, an innate immune stimulator, a signaling molecule, a transcriptional activator, a cytokine receptor, a bacterial component, or a component of the innate immune system. In some embodiments, the linear polyribonucleotide further comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator. In some embodiments, the innate immune system stimulator is selected from a GU-rich motif, an AU-rich motif, a structured region comprising dsRNA, or an aptamer.

[0024] In another aspect, the present disclosure provides a linear polyribonucleotide comprising a first sequence encoding a coronavirus immunogen and a second sequence encoding a multimerization domain. In some embodiments, the multimerization domain comprises a T4-foldon domain. In some embodiments, the multimerization domain comprises a ferritin domain. In some embodiments, the multimerization domain comprises a β-annulus peptide. In some embodiments, the multimerization domain is at the N-terminus of the coronavirus immunogen. In some embodiments, the multimerization domain is at the C-terminus of the coronavirus immunogen.

[0025] In another aspect, the present disclosure provides an immunogenic composition comprising any one of the linear polyribonucleotides described herein and a pharmaceutically acceptable excipient, without any carrier. In another aspect, the present disclosure provides an immunogenic composition comprising any one of the linear polyribonucleotides described herein and a pharmaceutically acceptable carrier and excipient. In some embodiments, the composition further comprises a second linear polyribonucleotide. In some embodiments, the second linear polyribonucleotide comprises an open reading frame encoding a second polypeptide immunogen. In some embodiments, the second linear polyribonucleotide comprises an open reading frame encoding a polypeptide adjuvant. In some embodiments, the second linear polyribonucleotide comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator.

[0026] In another aspect, the disclosure provides a method of inducing an immune response to a coronavirus immunogen in a non-human animal or human subject by a) administering any one of the immunogenic compositions described herein to the non-human animal or human subject, and b) harvesting antibodies to the coronavirus immunogen from the non-human animal or human subject, which in certain embodiments further comprises administering an adjuvant to the non-human animal or human subject.

[0027] In another aspect, the present disclosure provides a method of treating a subject having or susceptible to SARS-CoV-2 infection, comprising administering to the subject any one of the cyclic polyribonucleotides or immunogenic compositions described herein.

[0028] In another aspect, the present disclosure provides a method for preventing SARS-CoV-2 infection in a subject, the method comprising administering to the subject any one of the cyclic polyribonucleotides or immunogenic compositions described herein. In some embodiments, the human subject is at risk of SARS-CoV-2 infection. In some embodiments, the human subject is over 50 years of age, an immunocompromised human, a human with a chronic health condition, or a healthcare worker. In some embodiments, administering the cyclic polyribonucleotide or immunogenic composition reduces the frequency or severity of symptoms associated with SARS-CoV-2 infection. In some embodiments, the subject is a human subject. In some embodiments, the method further comprises administering an adjuvant to the subject.

[0029] definition The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Terms used hereinafter generally should be understood in their ordinary sense unless otherwise indicated.

[0030] As used herein, the term "adaptive immune response" refers to either a humoral or a cell-mediated immune response. For the purposes of this disclosure, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T lymphocytes and / or other white blood cells.

[0031] As used herein, the term "adjuvant" refers to a composition (e.g., a compound, polypeptide, nucleic acid, or lipid) that increases an immune response, e.g., increases the specific immune response to an immunogen. Increasing an immune response includes increasing or broadening the specificity of either or both the antibody and cellular immune response.

[0032] As used herein, the terms "circRNA," "circular polyribonucleotide," "circular RNA," and "circular polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having a structure with no free ends (i.e., no free 3' and / or 5' ends), e.g., a polyribonucleotide molecule that forms a circular or endless structure via covalent (e.g., covalently closed) or non-covalent bonds. A cyclic polyribonucleotide may be a covalently closed polyribonucleotide.

[0033] As used herein, the term "circularization efficiency" is a measure of the resulting circular polyribonucleotide compared to its non-circular starting material.

[0034] The term "diluent" refers to a vehicle containing an inert solvent in which a composition described herein (e.g., a composition containing a cyclic polyribonucleotide) can be diluted or dissolved. The diluent can be an RNA solubilizing agent, a buffer, an isotonicity agent, or a mixture thereof. The diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, or powdered sugar.

[0035] As used herein, the term "epitope" refers to the portion or entirety of an immunogen that is recognized, targeted, or bound by an antibody or T-cell receptor. An epitope can be a linear epitope, e.g., a contiguous sequence of nucleic acids or amino acids. An epitope can be a conformational epitope, e.g., an epitope containing amino acids that form the epitope in the folded conformation of a protein. A conformational epitope can contain non-contiguous amino acids from the primary amino acid sequence. As another example, a conformational epitope includes a nucleic acid that forms an epitope in the folded conformation of an immunogenic sequence based on its secondary or tertiary structure.

[0036] As used herein, the term "expressed sequence" is a nucleic acid sequence that encodes a product, such as a polypeptide (e.g., an immunogen), or a regulatory nucleic acid. An exemplary expressed sequence that encodes a polypeptide can include multiple nucleotide triplets, each of which can encode an amino acid, and are referred to as a "codon."

[0037] As used herein, the term "fragment" in reference to a polypeptide or nucleic acid sequence, e.g., a polypeptide immunogen or a nucleic acid sequence encoding a polypeptide immunogen, refers to a contiguous, less than all, portion of the polypeptide or nucleic acid sequence. A fragment of a polypeptide immunogen or a nucleic acid sequence encoding a polypeptide immunogen refers to a contiguous, less than all, portion (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the total length) of a sequence, such as, for example, a sequence disclosed herein. It is understood that all of the present disclosure contemplates fragments (e.g., immunogenic fragments) of all immunogens disclosed herein.

[0038] As used herein, the term "GC content" refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence. The formula for calculating GC content is (G+C) / (A+G+C+U) x 100% (for RNA) or (G+C) / (A+G+C+T) x 100% (for DNA). Similarly, the term "uridine content" refers to the percentage of uridine (U) in a nucleic acid sequence. The formula for calculating uridine content is U / (A+G+C+U) x 100%. Similarly, the term "thymidine content" refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula for calculating thymidine content is T / (A+G+C+T) x 100%.

[0039] As used herein, the term "innate immune system stimulator" refers to a substance that induces an innate immune response, in part, by inducing the expression of one or more genes involved in innate immunity, including, but not limited to, type I interferons (e.g., IFNα, INFβ, and / or IFNγ), inflammatory cytokines (e.g., IL-1, IL-12, IL-18, TNF-α, and / or GM-CSF), retinoic acid-inducible gene-I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, also known as IFIH1), 2'-5' oligoadenylate synthase 1 (OAS 1), OAS-like protein (OASL), and / or protein kinase R (PKR). Innate immune system stimulators can act as adjuvants, for example, when administered in combination with or formulated with ribonucleotides encoding immunogens. The innate immune system stimulator can be a separate molecular entity (e.g., not encoded by or incorporated as a sequence in a polyribonucleotide), such as STING (e.g., caSTING), TLR3, TLR4, TLR9, TLR7, TLR8, TLR7, RIG-I / DDX58, and MDA-5 / IFIH1, or a constitutively active mutant thereof. The innate immune system stimulator can be encoded by (e.g., expressed from) a polyribonucleotide. The polyribonucleotide can alternatively or additionally include a ribonucleotide sequence that acts as an innate immune system stimulator (e.g., a structured region including a GU-rich motif, an AU-rich motif, dsRNA, or an aptamer).

[0040] As used herein, the terms "human antibody," "human immunoglobulin," and "human polyclonal antibody" are used interchangeably to refer to one or more antibodies produced in a non-human animal that are inherently indistinguishable from antibodies produced in a human inoculated with the same circular RNA formulation. This is in contrast to "humanized antibodies," which have been modified to have human characteristics, such as by creating chimeras, but retain the attributes of the host animal in which they are produced. Because human antibodies produced according to the methods disclosed herein are composed of fully human IgG, no enzymatic treatment is required to eliminate the risks of anaphylaxis and serum sickness associated with xenogenic IgG.

[0041] As used herein, the term "immunogen" refers to any molecule or molecular structure that contains one or more epitopes that are recognized, targeted, or bound by an antibody or T cell receptor. In particular, an immunogen induces an immune response in a subject (e.g., is immunogenic as defined herein). An immunogen is capable of inducing an immune response in a subject, where an immune response refers to a series of molecular, cellular, and biological events that are induced when an immunogen is encountered by the immune system. An immune response can be a humoral and / or cellular immune response. These can include antibody production and B cell and T cell proliferation. To determine whether an immune response has occurred and track its progress, immunized subjects can be monitored for the appearance of immune reactants against a specific immunogen. Immune responses against most immunogens induce the production of both specific antibodies and specific effector T cells. In certain embodiments, an immunogen is foreign to the host. In certain embodiments, an immunogen is not foreign to the host. An immunogen can include all or part of a polypeptide, polysaccharide, polynucleotide, or lipid. The immunogen may also be a mixture of polypeptides, polysaccharides, polynucleotides, and / or lipids. For example, the immunogen may be a translationally modified polypeptide. A "polypeptide immunogen" refers to an immunogen that comprises a polypeptide. The polypeptide immunogen may also contain one or more post-translational modifications, and / or may form a complex with one or more additional molecules, and / or may adopt a tertiary or quaternary structure, each of which may determine or influence the immunogenicity of the polypeptide.

[0042] As used herein, the term "immunogenicity" refers to the ability to induce a response to a substance that exceeds a predetermined threshold in a particular immune response assay. The assay can be, for example, the expression of a particular inflammatory marker, the production of antibodies, or an assay for immunogenicity as described herein. In certain embodiments, an immune response can be induced when an organism's immune system or a particular type of immune cell is exposed to an immunogen.

[0043] Immunogenic responses can be assessed by evaluating antibodies in a subject's plasma or serum using total antibody assays, confirmatory tests, antibody titration and isotyping, and neutralizing antibody assessment. Total antibody assays measure all antibodies produced as part of an immune response in the serum or plasma of a subject administered an immunogen. The most commonly used test for detecting antibodies is ELISA (enzyme-linked immunosorbent assay), which detects antibodies in the tested serum that bind to the antibody of interest, including IgM, IgD, IgG, IgA, and IgE. Immunogenic responses can be further evaluated by confirmatory assays. After total antibody assessment, confirmatory assays can be used to confirm the results of the total antibody assay. Competitive assays can be used to confirm that antibodies specifically bind to the target and that positive findings in screening assays are not the result of nonspecific interactions between the test serum or detection reagent and other substances in the assay.

[0044] The immunogenic response can be assessed by isotyping and titration. Isotyping assays can be used to assess only the relevant antibody isotype. For example, the expected isotypes can be IgM and IgG, which can be specifically detected and quantified by isotyping and titration, and then compared to the total antibodies present.

[0045] Immunogenic responses can be assessed by neutralizing antibody assays (nAbs). Neutralizing antibody assays (nAbs) can be used to determine whether antibodies produced in response to an immunogen neutralize the immunogen, thereby inhibiting the immunogen from affecting the target and causing aberrant pharmacokinetic behavior. nAb assays are often cell-based assays in which target cells are incubated with an antibody. Various cell-based nAb assays can be used, including but not limited to, cell proliferation, viability, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), cytopathic effect inhibition (CPE), apoptosis, ligand-stimulated cell signaling, enzyme activity, reporter gene assays, protein secretion, metabolic activity, stress, and mitochondrial function. Detection readouts include absorbance, fluorescence, luminescence, chemiluminescence, or flow cytometry. Ligand binding assays can also be used to measure the binding affinity of immunogens and antibodies in vitro to assess neutralizing efficacy.

[0046] Furthermore, the induction of a cellular immune response can be assessed by measuring T cell activation in a subject using cellular markers on T cells obtained from the subject. A blood sample, lymph node biopsy, or tissue sample can be collected from the subject, and T cells from the sample can be assessed for one or more (e.g., two, three, four, or more) activation markers: CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, CD134, CD69, CD62L, or CD44. T cell activation can also be assessed using the same method in an in vivo animal model. This assay can also be performed by adding an immunogen to T cells in vitro (e.g., T cells obtained from a subject, an animal model, a repository, or a commercial source) and measuring the above-mentioned markers to assess T cell activation. Similar techniques can be used to assess the effect on activation of other immune cells, such as eosinophils (markers: CD35, CD11b, CD66, CD69, and CD81), dendritic cells (markers: IL-8, MHC class II, CD40, CD80, CD83, and CD86), basophils (CD63, CD13, CD4, and CD203c), and neutrophils (CD11b, CD35, CD66b, and CD63). These markers can be assessed using flow cytometry, immunohistochemistry, in situ hybridization, and other assays that allow for the measurement of other cell markers. Comparison of results before and after administration of the immunogen can be used to determine efficacy.

[0047] As used herein, the term "impurity" refers to an undesired substance present in a composition, e.g., a pharmaceutical composition described herein. In some embodiments, the impurity is a process-related impurity. In some embodiments, the impurity is a product-related substance other than the desired product in the final composition, e.g., other than the active drug ingredient, e.g., the cyclic or linear polyribonucleotides described herein. As used herein, the term "process-related impurity" refers to a substance used, present, or produced in the manufacture of a composition, preparation, or product, other than the linear polyribonucleotides described herein, that is undesired in the final composition, preparation, or product. In some embodiments, the process-related impurity is an enzyme used in the synthesis or cyclization of polyribonucleotides. As used herein, the term "product-related substance" refers to a substance or by-product produced during the synthesis of a composition, preparation, or product, or any intermediate. In some embodiments, the product-related substance is a deoxyribonucleotide fragment. In some embodiments, the product-related substance is a deoxyribonucleotide monomer. In certain embodiments, the product-related substance is a derivative or fragment of a polyribonucleotide described herein, for example, one or more of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acid, monoribonucleic acid, diribonucleic acid, or triribonucleic acid fragments.

[0048] As used herein, the term "inducing an immune response" refers to initiating, amplifying, or sustaining an immune response by a subject. Inducing an immune response may refer to an adaptive immune response or an innate immune response. Induction of an immune response may be measured as described above.

[0049] As used herein, the terms "linear RNA," "linear polyribonucleotide," and "linear polyribonucleotide molecule" are used interchangeably and refer to a monoribonucleotide molecule or a polyribonucleotide molecule having 5' and 3' ends. One or both of the 5' and 3' ends can be free or can be attached to another moiety. In certain embodiments, the linear RNA has a 5' or 3' end that is modified or protected from degradation (e.g., by a 5' or 3' end blocking agent). In certain embodiments, the linear RNA has a non-covalently linked 5' or 3' end. The linear RNA can be used as starting material for circularization, for example, by splint ligation or chemical, enzymatic, ribozyme-, or splicing-catalyzed circularization methods.

[0050] As used herein, the term "linear equivalent" refers to a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage of sequence similarity therebetween) and has two free ends (i.e., the non-circularized form (and fragments thereof) of a circularized polyribonucleotide). In certain embodiments, a linear equivalent (e.g., pre-circularized form) is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage of sequence similarity therebetween) and the same or similar nucleic acid modifications and has two free ends (i.e., the non-circularized form (and fragments thereof) of a circularized polyribonucleotide). In certain embodiments, a linear equivalent is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence similarity therebetween) and different or no nucleic acid modifications, and has two free ends (i.e., the non-circularized form (and fragments thereof) of a circularized polyribonucleotide). In certain embodiments, a fragment of a polyribonucleotide molecule that is a linear equivalent is any portion of the linear equivalent polyribonucleotide molecule that is shorter than the linear equivalent polyribonucleotide molecule. In certain embodiments, the linear equivalent further comprises a 5' cap. In certain embodiments, the linear equivalent further comprises a polyadenosine tail. In certain embodiments, the linear equivalent further comprises a 3' UTR. In certain embodiments, the linear equivalent further comprises a 5' UTR.

[0051] As used herein, the term "modified ribonucleotide" is a nucleotide having at least one modification to the sugar, nucleobase, or internucleoside linkage.

[0052] As used herein, the term "multimerization domain" refers to a polypeptide domain that self-assembles to form a multimer (e.g., a dimer, trimer, tetramer, or oligomer). In certain embodiments, a multimerization domain can be fused to a polypeptide (e.g., a polypeptide immunogen). In such cases, fusion to a multimerization domain results in the formation of a multimeric immunogen complex having two or more immunogens upon expression of the polypeptide comprising the immunogen covalently linked to the multimerization domain.

[0053] As used herein, the terms "multimeric immunogen complex," "naked delivery," and cognates refer to a formulation for delivery to a cell that is carrier-free and has no covalent modifications to moieties that aid in delivery to the cell. Naked delivery formulations do not include any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of a cyclic polyribonucleotide is a formulation that includes a cyclic polyribonucleotide that has no covalent modifications and does not include a carrier. Naked delivery formulations may include non-carrier pharmaceutical excipients or diluents.

[0054] As used herein, the term "naked delivery" refers to a formulation for delivery to a cell that does not use a carrier and does not have covalent modifications to moieties that aid in delivery to the cell. A naked delivery formulation does not include any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of a cyclic polyribonucleotide is a formulation that includes a cyclic polyribonucleotide that does not have covalent modifications and does not include a carrier.

[0055] As used herein, the terms "nicked RNA," "nicked linear polyribonucleotide," and "nicked linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having 5' and 3' ends resulting from the nicking or degradation of a circular RNA.

[0056] As used herein, the term "non-circular RNA" refers to all nicked RNA and linear RNA.

[0057] The term "pharmaceutical composition" is also intended to disclose that the cyclic polyribonucleotide contained within the pharmaceutical composition can be used for the treatment of the human or animal body by therapy, and is therefore intended to correspond to "cyclic polyribonucleotide for use in therapy."

[0058] As used herein, the term "polynucleotide" refers to a molecule containing one or more nucleic acid subunits, or nucleotides, and may be used interchangeably with "nucleic acid" or "oligonucleotide." A polynucleotide may contain one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide may contain a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO) groups. A nucleotide may contain a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. Polyribonucleotide, or ribonucleic acid, or RNA, may refer to a polymer containing multiple ribonucleotides polymerized via phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0059] "Polydeoxyribonucleotide," "deoxyribonucleic acid," and "DNA" refer to a polymer comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide refers to a deoxyribonucleoside polyphosphate, such as a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, including a detectable tag, such as a luminescent tag or marker (e.g., a fluorophore). A nucleotide can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be specific for A, C, G, T, or U, or one or more complementary A, C, G, T, or U, or any other subunit complementary to a purine (i.e., A or G, or variants thereof) or pyrimidine (i.e., C, T, or U, or variants thereof). In some examples, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a derivative or variant thereof. In some cases, the polynucleotide is a small interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), pre-mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some cases, the polynucleotide molecule is circular. The polynucleotide may have a variety of lengths. The nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. Polynucleotides can be isolated from cells or tissues.As realized herein, polynucleotide sequences can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0060] Polynucleotides, such as polyribonucleotides or polydeoxyribonucleotides, can contain one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylamino Examples of suitable nucleotides include methyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wybutosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 3-(3-amino-3-carboxypropyl)uridine 2,6-diaminopurine, etc. In some cases, nucleotides may contain modifications of their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications of the thiol moiety (e.g., α-thiotriphosphate and β-thiotriphosphate).Nucleic acid molecules can also be modified at the base moiety (e.g., typically at one or more atoms available to form a hydrogen bond with a complementary nucleotide, and / or typically at one or more atoms incapable of forming a hydrogen bond with a complementary nucleotide), sugar moiety, or phosphate backbone. Nucleic acid molecules can also include amine-modified groups such as aminoallyl-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP) to enable covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS). Substitutions for standard DNA or RNA base pairs in the oligonucleotides of the present disclosure can provide higher bit density per cubic mm, greater safety (resistance to accidental or intentional synthesis of natural toxins), easier identification in photoprogrammed polymerases, or lower secondary structure. Such alternative base pairs compatible with native and mutant polymerases for de novo and / or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. NAT. CHEM. BIOL. 2012 Jul;8(7):612-4, incorporated herein by reference for all purposes.

[0061] As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) linked together, most often by peptide bonds. This term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, natural polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs thereof. Polypeptides can be single molecules or multimolecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides, such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0062] As used herein, the term "prevent" means reducing the likelihood of developing a disease, disorder, or condition, or reducing the severity or frequency of symptoms of a disease or disorder that subsequently occur. A therapeutic agent may be administered to a subject who is at increased risk of developing a disease or disorder compared to members of the general population to prevent the development of, or reduce the severity of, the disease or condition. A therapeutic agent may be administered prophylactically, for example, prior to the onset of symptoms or signs of a disease or disorder.

[0063] As used interchangeably herein, the terms "polyA" and "polyA sequence" refer to an untranslated, contiguous region of a nucleic acid molecule that is at least 5 nucleotides in length and consists of adenosine residues. In certain embodiments, the polyA sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In certain embodiments, the polyA sequence is located 3' (e.g., downstream) of an open reading frame (e.g., an open reading frame encoding a polypeptide), and the polyA sequence is 3' to a termination element (e.g., a stop codon) such that the polyA is not translated. In certain embodiments, the polyA sequence is located 3' to the termination element and the 3' untranslated region.

[0064] As used herein, the term "regulatory element" refers to a portion of a nucleic acid sequence or the like that regulates the expression of an expression sequence within a circular polyribonucleotide.

[0065] As used herein, the term "replication elements" are sequences and / or motifs that are useful for replication or initiating transcription of a circular polyribonucleotide.

[0066] As used herein, the term "RNA equivalent" refers to an RNA sequence that is the RNA equivalent of a DNA sequence. Thus, an RNA equivalent of a DNA sequence refers to a DNA sequence in which each thymidine (T) residue is replaced with a uridine (U) residue. For example, the present disclosure provides the DNA sequence of a ribozyme identified by bioinformatics methods. The present disclosure specifically contemplates that any of these DNA sequences can be converted into a corresponding RNA sequence and included in the RNA molecules described herein.

[0067] As used herein, the term "sequence identity" is determined by aligning two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences can then be called "substantially identical" or "essentially similar" when they share at least a certain minimum percentage of sequence identity (e.g., when optimally aligned using the programs GAP or BESTFIT with default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, GAP default parameters are used: gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is the nwsgapdna.cmp scoring matrix, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and score for sequence identity percentage can be determined using computer programs such as GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program "needle"). Alternatively or additionally, percent identity can be determined by searching against a database using algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

[0068] A "signal sequence" refers to a polypeptide sequence, eg, 10-45 amino acids in length, present at the N-terminus of a polypeptide sequence of a nascent protein that targets the polypeptide sequence to the secretory pathway.

[0069] As used herein, the terms "treat" and "treating" refer to the therapeutic treatment of a disease or disorder (e.g., an infection, cancer, toxicity, or allergic reaction) in a subject. The effect of treatment can include ameliorating, alleviating, reducing the severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of, stabilizing the state of (i.e., not worsening) the disease or disorder, and / or preventing the spread of the disease or disorder, compared to the state and / or pathology of the disease or disorder in the absence of therapeutic treatment.

[0070] As used herein, the term "termination element" refers to a portion, such as a nucleic acid sequence, that stops translation of an expressed sequence in a circular polyribonucleotide.

[0071] As used herein, the term "total ribonucleotide molecules" refers to the total amount of any ribonucleotide molecules, including linear polyribonucleotide molecules, cyclic polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, fragments thereof, and modified forms thereof, as measured by the total amount of ribonucleotide molecules.

[0072] As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In certain embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per given amount of transcript encoding the protein or peptide, e.g., in a given translation system, e.g., in an in vitro translation system such as a rabbit reticulocyte lysate, or in an in vivo translation system such as a eukaryotic or prokaryotic cell, for a given period of time.

[0073] As used herein, the term "translation initiation sequence" is a nucleic acid sequence that initiates translation of an expression sequence in a circular polyribonucleotide.

[0074] As used herein, a "variant" refers to a polypeptide that contains at least one modification, e.g., a substitution, insertion, deletion, and / or fusion, at one or more residue positions compared to a parent or wild-type polypeptide. A variant can contain 1 to 10, 10 to 20, 20 to 50, 50 to 100, or more modifications. [Brief explanation of the drawings]

[0075] [Figure 1] 1 shows an exemplary circular polyribonucleotide comprising a sequence encoding a coronavirus immunogen (e.g., spike protein, receptor binding domain (RBD) protein of the spike protein). [Figure 2] 1 shows an exemplary polyribonucleotide construct encoding a coronavirus immunogen and one or more multimerization domains. [Figure 3] 1 is a schematic diagram of an exemplary circular RNA comprising two expression sequences, each expression sequence operably linked to an IRES, where at least one expression sequence is a coronavirus immunogen. [Figure 4] Schematic diagram of an exemplary circular RNA comprising two expressed sequences separated by a cleavage domain (e.g., 2A, furin site, or furin-2A), where at least one expressed sequence is a coronavirus immunogen, and all are operably linked to an IRES. [Figure 5] Schematic diagram of multiple circular RNAs, where a first circular RNA contains an ORF encoding a coronavirus immunogen and a second circular RNA contains an ORF encoding either a second immunogen or a polypeptide adjuvant. [Figure 6] (Figure 6A) Multiple immunogen expression from cyclic polyribonucleotides. RBD immunogen expression was detected from the SARS-CoV-2 RBD immunogen and circular RNA encoding GLuc. (Figure 6B) Multiple immunogen expression from cyclic polyribonucleotides. GLuc activity was detected from the SARS-CoV-2 RBD immunogen and circular RNA encoding GLuc. [Figure 7](Figure 7A) Immunogenicity of multiple immunogens from circular RNA in a mouse model. Mice were inoculated with a first circular RNA encoding a SARS-CoV-2 RBD immunogen and a second circular RNA encoding GLuc. Anti-RBD antibodies were obtained 17 days after injection. (Figure 7B) Immunogenicity of multiple immunogens from circular RNA in a mouse model. Mice were inoculated with a first circular RNA encoding a SARS-CoV-2 RBD immunogen and a second circular RNA encoding GLuc. GLuc activity was detected 2 days after injection. [Figure 8] (Figure 8A) Immunogenicity of multiple immunogens from circular RNA in a mouse model. Mice were inoculated with a first circular RNA encoding a SARS-CoV-2 RBD immunogen and a second circular RNA encoding an influenza hemagglutinin (HA) immunogen. Anti-RBD antibodies were obtained 17 days after injection. (Figure 8B) Immunogenicity of multiple immunogens from circular RNA in a mouse model. Mice were inoculated with a first circular RNA encoding a SARS-CoV-2 RBD immunogen and a second circular RNA encoding an influenza hemagglutinin (HA) immunogen. Anti-HA antibodies were obtained 17 days after injection. [Figure 9] (Figure 9A) Immunogenicity of multiple immunogens from circular RNA in a mouse model. Mice were inoculated with a first circular RNA encoding a SARS-CoV-2 spike immunogen and a second circular RNA encoding an influenza hemagglutinin (HA) immunogen. Anti-RBD (spike domain) antibodies were obtained 17 days after injection. (Figure 9B) Immunogenicity of multiple immunogens from circular RNA in a mouse model. Mice were inoculated with a first circular RNA encoding a SARS-CoV-2 spike immunogen and a second circular RNA encoding an influenza hemagglutinin (HA) immunogen. Anti-HA antibodies were obtained 17 days after injection. [Figure 10]This demonstrates anti-HA antibody responses in mice administered circular RNA encoding multiple immunogens. Mice were administered circular RNA encoding a SARS-CoV-2 RBD immunogen, a SARS-CoV-2 spike immunogen, an influenza HA immunogen, a SARS-CoV-2 RBD immunogen and an influenza HA immunogen, a SARS-CoV-2 RBD immunogen and a GLuc protein, or a SARS-CoV-2 RBD immunogen and a SARS-CoV-2 spike immunogen. Anti-influenza HA antibodies were measured using a hemagglutination inhibition assay (HAI). Figure 10 shows HAI titers in samples administered circular RNA formulations encoding influenza HA immunogens when administered alone or in combination with a SARS-CoV-2 immunogen, such as RBD or spike. [Figure 11] IL-12, as measured using an IL-12-specific ELISA, was expressed from circular RNA in mammalian cells. Circular RNA encoding the SARS-CoV-2 RBD immunogen was included as a negative control. [Figure 12] (Figure 12A) IL-12 expression was detected in serum in a mouse model 2 days after injection of a circular RNA formulation containing a first circular RNA encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen. Injection of PBS or a formulation containing only a circular RNA encoding a SARS-CoV-2 RBD immunogen was included as controls. (Figure 12B) IL-12 expression was detected in serum in a mouse model 2 days after injection of a circular RNA formulation containing a first circular RNA encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen (immediately downstream of IL12 signaling). Injection of PBS or a formulation containing only a circular RNA encoding a SARS-CoV-2 RBD immunogen was included as controls. [Figure 13A]Figure 1 shows that administration of a circular RNA formulation containing a first circular RNA encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen increased the number of SARS-CoV-2 RBD-specific CD4 T cells. Administration of PBS or a formulation containing only the circular RNA encoding the SARS-CoV-2 RBD immunogen was included as a control. Asterisks represent statistical significance as determined by two-way RM ANOVA with protected Tukey's post-hoc test. [Figure 13B] This shows that administration of a circular RNA formulation containing a first circular RNA encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen did not result in a change in the number of RBD-specific CD8 T cells. Administration of PBS or a formulation containing only a circular RNA encoding a SARS-CoV-2 RBD immunogen was included as a control. [Figure 13CD] (Figure 13C) Administration of a circular RNA formulation containing a first circular RNA encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen increased the amount of IFN-γ production by CD4 T cells. Administration of PBS or a formulation containing only the circular RNA encoding the SARS-CoV-2 RBD immunogen was included as a control. Asterisks represent statistical significance as determined by an unpaired t-test. (Figure 13D) Administration of a circular RNA formulation containing a first circular RNA encoding IL-12 and a second circular RNA encoding the SARS-CoV-2 RBD immunogen increased the amount of IFN-γ production by CD8 T cells. Administration of PBS or a formulation containing only the circular RNA encoding the SARS-CoV-2 RBD immunogen was included as a control. Asterisks represent statistical significance as determined by an unpaired t-test. [Figure 14] Figure 1 shows the expression of SARS-CoV-2 spike immunogen in serum of cynomolgus macaques after receiving a 100 μg dose of lipid nanoparticle (LNP)-formulated circular RNA by intramuscular injection on day 0 (prime) and day 28 (boost). [Figure 15]Figure 1 shows the expression of SARS-CoV-2 RBD immunogen fused to the T4 foldon multimerization domain in the serum of cynomolgus monkeys after receiving a 100 μg dose of LNP-formulated circular RNA or a 1000 μg dose of adjuvanted circular RNA by intramuscular injection. [Figure 16A] Figure 1 shows that spike-specific binding antibodies were primed in cynomolgus monkeys 42 days after administration of the first dose of LNP-formulated or adjuvanted circular RNA encoding either a SARS-CoV-2 spike immunogen or a SARS-CoV-2 RBD immunogen fused to a T4 foldon multimerization domain. [Figure 16B] We show that RBD-specific binding antibodies were primed in cynomolgus monkeys 42 days after administration of a first dose of LNP-formulated or adjuvanted cyclic polyribonucleotide encoding either a SARS-CoV-2 spike immunogen or a SARS-CoV-2 RBD immunogen fused to a T4 foldon multimerization domain. [Figure 17A] Figure 1 shows that SARS-CoV-2 neutralizing antibodies were primed in cynomolgus monkeys 42 days after administration of an initial 30 μg or 100 μg dose of LNP-formulated circular RNA encoding a SARS-CoV-2 spike immunogen. [Figure 17B] We show that SARS-CoV-2 neutralizing antibodies were primed in cynomolgus monkeys 42 days after administration of a first dose of either LNP-formulated cyclic polyribonucleotide encoding a SARS-CoV-2 RBD immunogen fused to a T4-foldon multimerization domain or adjuvanted cyclic polyribonucleotide encoding a SARS-CoV-2 RBD immunogen fused to a T4-foldon multimerization domain. DETAILED DESCRIPTION OF THE INVENTION

[0076] The present disclosure provides compositions, pharmaceutical formulations, and methods related to polyribonucleotides (e.g., cyclic polyribonucleotides or linear polyribonucleotides) encoding one or more immunogens and / or epitopes from a coronavirus. The present disclosure also provides methods of using cyclic polyribonucleotides encoding one or more immunogens and / or epitopes from a coronavirus. The cyclic polyribonucleotide compositions and pharmaceutical formulations described herein can induce an immune response in a subject upon administration. The cyclic polyribonucleotide compositions and pharmaceutical formulations described herein can be used to treat or prevent a disease, disorder, or condition (e.g., SARS-CoV, e.g., SARS-CoV-1 or SARS-CoV-2) in a subject.

[0077] cyclic polyribonucleotide The cyclic polyribonucleotides disclosed herein contain one or more expressible sequences encoding one or more immunogens and / or epitopes from a coronavirus. The cyclic polyribonucleotides express the sequences encoding one or more immunogens and / or epitopes from a coronavirus in a subject. In some embodiments, the cyclic polyribonucleotides containing one or more coronavirus immunogens and / or epitopes are used to generate an immune response in a subject. In some embodiments, the cyclic polyribonucleotides containing one or more coronavirus immunogens and / or epitopes are used to generate polyclonal antibodies described herein.

[0078] Coronavirus immunogens and epitopes The cyclic polyribonucleotides described herein include at least one expression sequence encoding a coronavirus immunogen and / or epitope. The cyclic polyribonucleotides described herein can include multiple expression sequences, where at least one expression sequence encodes a coronavirus immunogen and / or epitope. The cyclic polyribonucleotides described herein can include two or more (two, three, four, five, six, or more) expression sequences, where each expression sequence encodes a coronavirus immunogen and / or epitope. The cyclic polyribonucleotides described herein can include a first expression sequence encoding a coronavirus immunogen and / or epitope and a second expression sequence encoding an adjuvant. The cyclic polyribonucleotides described herein can include an expression sequence encoding a coronavirus immunogen and / or epitope and a non-coding sequence that stimulates the innate immune system.

[0079] In some embodiments, the coronavirus is a pathogenic coronavirus. In some embodiments, the coronavirus is a respiratory pathogen. In some embodiments, the coronavirus is a blood-borne pathogen. In some embodiments, the coronavirus is an enteric pathogen.

[0080] Non-limiting examples of coronaviruses of the present disclosure include severe acute respiratory syndrome-associated coronaviruses (SARS-CoV, e.g., SARS-CoV-1, SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), bat coronaviruses, zoonotic coronaviruses that can infect humans or other animals, novel or newly discovered coronaviruses, and other coronaviruses.

[0081] In some embodiments, the cyclic polyribonucleotide comprises a severe acute respiratory syndrome-associated coronavirus (SARS-CoV) immunogen and / or epitope. In some embodiments, the cyclic polyribonucleotide comprises a SARS-CoV-1 immunogen and / or epitope. In some embodiments, the cyclic polyribonucleotide comprises a SARS-CoV-2 immunogen and / or epitope. In some embodiments, the cyclic polyribonucleotide comprises a Middle East respiratory syndrome coronavirus (MERS-CoV) immunogen and / or epitope. In some embodiments, the cyclic polyribonucleotide comprises a zoonotic coronavirus immunogen and / or epitope that can infect humans or other animals. In some embodiments, the cyclic polyribonucleotide comprises an immunogen and / or epitope from a novel coronavirus.

[0082] In certain embodiments, the cyclic polyribonucleotide comprises a Coronaviridae immunogen and / or epitope.

[0083] In some embodiments, the cyclic polyribonucleotide comprises an immunogen and / or epitope from the genus or subgenus Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus, subgenus Merbecovirus, or genus Sarbecovirus. In some embodiments, the cyclic polyribonucleotide comprises a Betacoronavirus immunogen and / or epitope. In some embodiments, the cyclic polyribonucleotide comprises a Sarbecovirus immunogen and / or epitope. In some embodiments, the cyclic polyribonucleotide comprises a Merbecovirus immunogen and / or epitope.

[0084] In some embodiments, the cyclic polyribonucleotide comprises an immunogen and / or epitope from the genus or subgenus of the Omicron coronavirus variant (B.1.1.529). In some embodiments, the Omicron coronavirus variant is selected from the group consisting of BA.2, BA.2.75, BA.4.1, BA.4.1.8, BA.4.6.1, BA.4.6.4, BA.5, BA.5.1, BA.5.1.12, BA.5.1.25, BA.5.10.1, BA.5.2, BA.5.2.1, BA.5.2.6, BA.5.3, BA.5.3.1, BA.5.3.5, BA.5.5, BA. It may be of a sublineage of BA.5.6, BA.5.6.1, BA.5.7, BE.1.1, BF.10, BF.16, BF.31, BF.31.1, BF.7, BQ.1, BQ.1.1, BQ.1.8, XBB, or XBB.1.

[0085] In some embodiments, the cyclic polyribonucleotide comprises a sequence for an immunogen from a coronavirus that is a Biosafety Level 2 (BSL-2) pathogen. In some embodiments, the cyclic polyribonucleotide comprises a sequence from a coronavirus that is a Biosafety Level 3 (BSL-3) pathogen. In some embodiments, the coronavirus is a Biosafety Level 4 pathogen (BSL-4). In some embodiments, there are no approved drugs (e.g., antivirals or antibiotics) available to treat infection with the coronavirus from which the immunogen expressed by the cyclic polyribonucleotide is derived. In some embodiments, there are no approved vaccines available to prevent or reduce the risk of infection with the coronavirus from which the immunogen expressed by the cyclic polyribonucleotide is derived.

[0086] The immunogen and / or epitope can be derived from a coronavirus surface protein, a coronavirus membrane protein, a coronavirus envelope protein, a coronavirus capsid protein, a coronavirus nucleocapsid protein, a coronavirus spike protein, a coronavirus receptor binding domain (RBD) of a spike protein, a coronavirus entry protein, a coronavirus membrane fusion protein, a coronavirus structural protein, a coronavirus nonstructural protein, a coronavirus regulatory protein, a coronavirus accessory protein, a secreted coronavirus protein, a coronavirus polymerase protein, a coronavirus RNA polymerase, a coronavirus protease, a coronavirus glycoprotein, a coronavirus fusion protein, a coronavirus helical capsid protein, a coronavirus icosahedral capsid protein, a coronavirus matrix protein, a coronavirus replicase, a coronavirus transcription factor, or a coronavirus enzyme.

[0087] Immunogens and / or epitopes from different coronaviruses are expressed by the cyclic polyribonucleotide. In some cases, the immunogens and / or epitopes are associated with or expressed by one coronavirus disclosed herein. In some embodiments, the immunogens and / or epitopes are associated with or expressed by more than one coronavirus disclosed herein.

[0088] In some cases, the two or more coronaviruses are phenotypically related. For example, the compositions and methods of the present disclosure may use immunogens and / or epitopes from two or more coronaviruses that are respiratory pathogens, two or more coronaviruses associated with severe disease, two or more coronaviruses associated with adverse outcomes in immunocompromised subjects (e.g., subjects for immunization), two or more coronaviruses associated with acute respiratory distress syndrome (ARDS), two or more coronaviruses associated with severe acute respiratory syndrome (SARS), two or more coronaviruses associated with Middle East respiratory syndrome (MERS), or combinations thereof.

[0089] A cyclic polyribonucleotide can comprise or encode, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more antigens and / or epitopes from coronaviruses. In certain embodiments, a cyclic polyribonucleotide comprises or encodes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more immunogens and / or epitopes from targets other than coronaviruses (e.g., viruses other than coronaviruses, such as influenza viruses).

[0090] In certain embodiments, the cyclic polyribonucleotide comprises or encodes no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, or no more immunogens and / or epitopes from a coronavirus. In certain embodiments, the cyclic polyribonucleotide comprises or encodes no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, or no more immunogens and / or epitopes from targets other than coronaviruses (e.g., viruses other than coronaviruses, such as influenza viruses).

[0091] In some embodiments, the cyclic polyribonucleotide comprises or encodes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 immunogens and / or epitopes from a coronavirus. In some embodiments, the cyclic polyribonucleotide comprises or encodes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 immunogens and / or epitopes from targets other than coronaviruses (e.g., viruses other than coronaviruses, such as influenza viruses).

[0092] In certain embodiments, the immunogens and / or epitopes are derived from a coronavirus, e.g., severe acute respiratory syndrome-associated coronavirus (SARS-CoV, e.g., SARS-CoV-1, SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), or another coronavirus. In certain embodiments, the immunogens and / or epitopes of the present disclosure are derived from predicted open reading frames from a coronavirus genome.

[0093] A novel SARS isolate can be identified by a percent homology of 99%, 98%, 97%, 95%, 92%, 90%, 85%, or 80% of the polynucleotide sequence of a specific genomic region of the novel virus to the polynucleotide sequence of a specific genomic region of a known SARS virus.Furthermore, a novel SARS isolate can be identified by a percent homology of 99%, 98%, 97%, 95%, 92%, 90%, 85%, or 80% of the polypeptide sequence encoded by the polynucleotide of a specific genomic region of the novel SARS virus to the polypeptide sequence encoded by the polynucleotide of a specific region of a known SARS virus. These genomic regions can include regions that are typically common among many coronaviruses (e.g., gene products or ORFs), as well as group-specific regions (e.g., immunogenic groups), such as any one of the following genomic regions that can be readily identified by one skilled in the art of virology: 5' untranslated region (UTR), leader sequence, ORF1a, ORF1b, nonstructural protein 2 (NS2), hemagglutinin-esterase glycoprotein (HE) (also called E3), spike glycoprotein (S) (also called E2), and the like. (SEQ ID NO: 1), ORF3a, ORF3b, nonstructural protein 4 (NS4), envelope (small membrane) protein (E) (also called sM), membrane glycoprotein (M) (also called E1), ORF5a, ORF5b, nucleocapsid phosphoprotein (N), ORF6, ORF7a, ORF7b, ORF8, ORF8a, ORF8b, ORF9a, ORF9b, ORF10, intergenic sequence, receptor-binding domain (RBD) of spike protein, 3'UTR, or RNA-dependent RNA polymerase (pol). SARS virus may have identifiable genomic regions that include one or more of the above-identified genomic regions. SARS virus immunogens include proteins encoded by any one of these genomic regions. SARS virus immunogens may be proteins or fragments thereof that are highly conserved with coronaviruses. SARS virus immunogens may be proteins or fragments thereof that are specific to SARS virus (compared to known coronaviruses).

[0094] In certain embodiments, the immunogens and / or epitopes of the present disclosure are derived from predicted transcripts from the SARS-CoV genome. In certain embodiments, the immunogens and / or epitopes of the present disclosure are derived from proteins encoded by open reading frames from the SARS-CoV genome. Non-limiting examples of open reading frames in the SARS-CoV genome can include ORF1a, ORF1b, spike (S), ORF3a, ORF3b, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF8a, ORF8b, ORF9a, ORF9b, nucleocapsid (N), and ORF10.

[0095] ORF1a and ORF1b encode 16 nonstructural proteins (nsp), e.g., nsp1, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11, nsp12, nsp13, nsp14, nsp15, and nsp16. Nonstructural proteins contribute, for example, to viral replication, viral assembly, immune response regulation, or a combination thereof. In some embodiments, the immunogen is a nonstructural protein or an immunogenic sequence encoding a nonstructural protein. In some embodiments, the epitope is derived from a coronavirus nonstructural protein.

[0096] Spike (S) encodes a spike protein, which, in some embodiments, contributes to binding to a host cell receptor, viral fusion with the host cell membrane, viral entry into the host cell, or a combination thereof. The spike protein can be an immunogen. In some embodiments, the epitope of the present disclosure is derived from the spike protein. In some embodiments, the epitope of the present disclosure comprises the receptor-binding domain of the spike protein. In some embodiments, the epitope of the present disclosure comprises the ACE2-binding domain of the spike protein.

[0097] Envelope (E) encodes an envelope protein, which in some embodiments contributes to viral assembly and morphogenesis. Envelope proteins can be immunogens. In some embodiments, epitopes of the present disclosure are derived from coronavirus envelope proteins.

[0098] Membrane (M) encodes a membrane protein, which in some embodiments contributes to viral assembly. Membrane proteins can be immunogens. In some embodiments, the epitopes of the present disclosure are derived from coronavirus membrane proteins.

[0099] Nucleocapsid (N) encodes the nucleocapsid protein, which, in certain embodiments, may form a complex with genomic RNA, contribute to viral assembly, and / or interact with the M protein. The nucleocapsid protein may be immunogenic. In certain embodiments, epitopes of the present disclosure are derived from coronavirus nucleocapsid proteins.

[0100] ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF8a, ORF8b, ORF9a, ORF9b, and ORF10 encode accessory proteins. In certain embodiments, the accessory proteins may modulate host cell signaling, modulate host cell immune responses, be incorporated into mature virions as minor structural proteins, or a combination thereof. The accessory proteins may be immunogens. In certain embodiments, the epitopes of the present disclosure are derived from coronavirus accessory proteins.

[0101] The compositions and methods of the present disclosure may use immunogens and / or epitopes encoded by or derived from one or more open reading frames of the SARS-CoV genome. For example, the immunogens and / or epitopes may be encoded by or derived from ORF1a, ORF1b, spike (S), ORF3a, ORF3b, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF8a, ORF8b, ORF9a, ORF9b, nucleocapsid (N), ORF10, or any combination thereof.

[0102] In some embodiments, the epitope of the present disclosure is derived from the spike protein. In some embodiments, the epitope of the present disclosure is derived from the Omicron coronavirus spike protein. The Omicron coronavirus spike protein has the amino acid sequence of SEQ ID NO: 283. In some embodiments, the epitope of the present disclosure comprises the receptor binding domain (RBD) of the spike protein. In some embodiments, the epitope of the present disclosure comprises the ACE2 binding domain of the spike protein. In some embodiments, the epitope of the present disclosure comprises the S1 subunit spike protein, the S2 subunit spike protein, or a combination thereof. In some embodiments, the epitope of the present disclosure comprises the extracellular domain of the spike protein. In some embodiments, the epitope of the present disclosure comprises Gln498, Thr500, Asn501, or a combination thereof from the coronavirus spike protein. In some embodiments, the epitope of the present disclosure comprises Lys417, Tyr453, or a combination thereof from the coronavirus spike protein. In some embodiments, the epitope of the present disclosure includes Gln474, Phe486, or a combination thereof from a coronavirus spike protein. In some embodiments, the epitope of the present disclosure includes Gln498, Thr500, Asn501, Lys417, Tyr453, Gln474, Phe486 from a coronavirus spike protein, one or more equivalent amino acids from a spike protein variant or derivative, or a combination thereof. In some embodiments, the spike protein of the present disclosure includes a D614G mutation, i.e., having the amino acid glycine (G) at position 614 instead of aspartic acid (D). In some embodiments, the epitope of the present disclosure includes Gly614 from a spike protein variant or derivative from a coronavirus spike protein, or a combination thereof. In some cases, the D614G mutation may result in reduced S1 shedding, increasing coronavirus infectivity.In some embodiments, the spike protein of the disclosure has any of the following mutations compared to a wild-type spike protein: T19I, L24del, P25del, P26del, A27S, H69del, V70del, V213G, G229D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, The mutations may include one or more of N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, Y144del, P251L, and S256L. In certain embodiments, the spike protein of the disclosure has one or more of the following mutations compared to the wild-type spike protein: T19I, L24del, P25del, P26del, A27S, H69del, V70del, V213G, G229D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, These may include K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, Y144del, P251L, and S256L. In some embodiments, the spike proteins of the disclosure have any of the following mutations compared to a wild-type spike protein: T19I, L24del, P25del, P26del, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, K356T, S371F, S373P, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S446A, S447B, S447C, S447D, S447E, S447F, S447H ... 77N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D574V, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, D1199N, M177T, N185D, N211del, L212I, K444T, N450D, L452R, F486P, F486I, S494P, and H1101Y mutations.In some embodiments, the spike proteins of the disclosure have any of the following mutations compared to the wild-type spike protein: T19I, L24del, P25del, P26del, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, K356T, S371F, S373P, T376A, D405N, R408S, K417N, N440K, G446S, N447K, N448K, N449K, N450K, N451K, N452K, N453K, N454K, N455K, N456K, N457K, N458K, N459K, N460K, N461K, N462K, N463K, N464K, N465K, N466K, N467K, N468K, N469K, N470K, N471K, N472K, N473K, N474K, N475K, N476K, N477K, N478K, N479K, N480K, N481K, N482K, N483K, N484K, N485K, N486K, N487K, N488K, N489K, N490K, N491K, N492K, N493K, N494K, N495K, N496K, N497K, N498K, N499K, N499K, N499K, N499K, N499K, N499K, N499 60K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D574V, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, D1199N, M177T, N185D, N211del, L212I, K444T, N450D, L452R, F486P, F486I, S494P, and H1101Y.

[0103] In some embodiments, the spike protein of the disclosure has any of the following mutations compared to a wild-type spike protein: T19I, L24del, P25del, P26del, A27S, H69del, V70del, G142D, Y144del, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R4 The fragment may include one or more of the following mutations: 08S, K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, and P251H. In some embodiments, spike proteins of the disclosure may include the following mutations relative to the wild-type spike protein: T19I, L24del, P25del, P26del, A27S, H69del, V70del, G142D, Y144del, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

[0104] In some embodiments, the spike proteins of the disclosure have any of the following mutations compared to a wild-type spike protein: T19I, L24del, P25del, P26del, A27S, V83A, G142D, Y144del, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, The mutations may include one or more of D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and H146K. In some embodiments, the spike proteins of the disclosure have any of the following mutations compared to the wild-type spike protein: T19I, L24del, P25del, P26del, A27S, V83A, G142D, Y144del, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F , T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and H146K.

[0105] In some embodiments, the immunogen and / or epitope is encoded by or derived from ORF1a. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF1b. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV spike. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF3a. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF3b. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV envelope (E). In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV membrane (M). In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF6. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF7a. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF7b. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF8. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF8a. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF9a. In some embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF9b. In certain embodiments, the immunogen and / or epitope is encoded by or derived from the SARS-CoV nucleocapsid (N). In certain embodiments, the immunogen and / or epitope is encoded by or derived from SARS-CoV ORF10.In certain embodiments, the immunogens and / or epitopes are encoded by or derived from the SARS-CoV spike (S), envelope (E), membrane (M), and nucleocapsid (N).

[0106] In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF1a. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF1b. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV spike. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF3a. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF3b. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV envelope (E). In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV membrane (M). In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF6. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF7a. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF7b. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF8. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF8a. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF9a. In some embodiments, the immunogen and / or epitope is not encoded by or derived from SARS-CoV ORF9b. In certain embodiments, the immunogen and / or epitope is not encoded by or derived from the SARS-CoV nucleocapsid (N).In certain embodiments, the immunogens and / or epitopes are not encoded by or derived from SARS-CoV ORF 10. In certain embodiments, the immunogens and / or epitopes are not encoded by or derived from SARS-CoV spike (S), envelope (E), membrane (M), and nucleocapsid (N).

[0107] The immunogen and / or epitope may be encoded by or derived from SARS-CoV2.

[0108] A non-limiting example of a SARS-CoV-2 genome is provided in DB Source accession MN908947.3, the complete genome sequence of a SARS-CoV2 isolate, the entire contents of which are incorporated herein by reference. DB Source accession MN908947.3:21563-25384 corresponds to the S protein, the entire contents of which are incorporated herein by reference. A non-limiting example of a SARS-CoV-2 spike protein is provided in GenBank sequence: QHD43416.1, the sequence of the spike protein of a SARS-CoV2 isolate, the entire contents of which are incorporated herein by reference.

[0109] A non-limiting example of a SARS-CoV-2 genome is provided in sequence NCBI Reference Sequence Accession No. NC_045512, version NC_045512.2, the complete genome sequence of SARS-CoV2 isolate Wuhan-Hu-1, the contents of which are incorporated herein by reference in their entirety.

[0110] A non-limiting example of a SARS-CoV-2 genome is provided in sequence NCBI Reference Sequence Accession No. MW450666, the complete genome sequence of a SARS-CoV2 isolate, the contents of which are incorporated herein by reference in their entirety.

[0111] A non-limiting example of a SARS-CoV-2 genome is provided in sequence NCBI Reference Sequence Accession No. MW487270, the complete genome sequence of the SARS-CoV2 lineage B.1.1.7 virus, the contents of which are incorporated herein by reference in their entirety.

[0112] A non-limiting example of SARS-CoV-2 is provided in the sequence GISAID reference sequence accession numbers EPI_-SL_10894052-EPI_ISL_10894090, the complete genome sequence of severe acute respiratory syndrome coronavirus 2, the contents of which are incorporated herein by reference in their entirety.

[0113] A non-limiting example of a SARS-CoV-2 genome is provided in sequence GISAID reference sequence accession number EPI_ISL_792683, the complete genome sequence of SARS-CoV2 lineage P.1 virus, the contents of which are incorporated herein by reference in their entirety.

[0114] A non-limiting example of a SARS-CoV-2 genome is provided in sequence GISAID reference sequence accession number EPI_ISL_678615, the complete genome sequence of the SARS-CoV2 lineage B.1.351 virus, the contents of which are incorporated herein by reference in their entirety.

[0115] Non-limiting examples of SARS-CoV-2 genomes are provided in sequences NCBI Reference Sequence Accession Nos. MW972466-MW974550, the complete genome sequences of SARS-CoV2 lineages B.1.427 and B.1.429 viruses, the contents of which are incorporated herein by reference in their entirety.

[0116] Non-limiting examples of the SARS-CoV-2 genome are provided in sequence NCBI Reference Sequence Accession Nos. MZ156756-MZ226428, the complete genome sequence of the SARS-CoV2 virus, the contents of which are incorporated herein by reference in their entirety.

[0117] In one embodiment, the SARS-CoV-2 genome is provided in the GISAID database at www.gisaid.org. In one embodiment, the SARS-CoV-2 genome is provided in the International Sequence Database Collaboration (INSDC) at www.insdc.org.

[0118] In some embodiments, the immunogens and / or epitopes of the present disclosure are derived from predicted transcripts from the SARS-CoV-2 genome. In some embodiments, the immunogens and / or epitopes of the present disclosure are derived from proteins, or derivatives thereof, encoded by open reading frames from the SARS-CoV-2 genome. Non-limiting examples of open reading frames in the SARS-CoV-2 genome include ORF1a, ORF1b, spike (S), ORF3a, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8, nucleocapsid (N), and ORF10. In some embodiments, the SARS-CoV-2 genome encodes ORF3b, ORF9a, ORF9b, or a combination thereof. In some embodiments, the SARS-CoV-2 genome does not encode ORF3b, ORF9a, ORF9b, or any combination thereof.

[0119] Non-limiting examples of amino acid sequences are shown in Table 1. In certain embodiments, the immunogen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from Table 1.

[0120] [Table 1-1]

[0121] [Table 1-2]

[0122] Further non-limiting examples of proteins encoded by the SARS-CoV-2 genome include those listed under NCBI accession numbers MT334522, MT334523, MT334524, MT334525, MT334526, MT334527, MT334528, MT334529, MT334530, MT334531, MT334532, MT334533, MT334534, MT334535, MT334536, MT334537, MT334538, MT334539, MT334540, MT334541, MT334542, and MT334543. , MT334544, MT334545, MT334546, MT334555, MT334547, MT334548, MT334549, MT334550, MT334551, MT334552, MT334553, MT334554, MT334556, MT3345 57, MT334558, MT334559, MT334560, MT334561, MT334562, MT334563, MT334564, MT334565, MT334566, MT334567, MT334568, MT334569, MT334570, MT334 571, MT334572, MT334573, MT326097, MT326106, MT326107, MT326116, MT326117, MT326124, MT326125, MT326126, MT326127, MT326134, MT326135, MT3 26136, MT326137, MT326138, MT326139, MT326140, MT326141, MT326142, MT326143, MT326144, MT326145, MT326146, MT326148, MT326149, MT326150, MT 326151,MT326152,MT326158,MT326159,MT326160,MT326161,MT326162,MT326168,MT326169,MT326170,MT326171,MT326172,MT326178,MT326179,M T326180, MT326181, MT326182, MT326183, MT326188, MT326189, MT326190, MT326191, MT326129, MT326121, MT326120, MT326119, MT326118, MT326111,MT326023、MT326025、MT326033、MT326035、MT326036、MT326040、MT326043、MT326045、MT326053、MT326055、MT326056、MT326063、MT326066、MT32607 0、MT326071、MT326072、MT326075、MT326076、MT326078、MT326079、MT326089、MT325563、MT325565、MT325566、MT326155、MT326163、MT326177、MT3261 30、MT326128、MT326110、MT326109、MT326108、MT326101、MT326100、MT326099、MT326098、MT326094、MT326093、MT326092、MT325568、MT325569、MT325 590、MT325640、MT325606、MT325607、MT325608、MT325609、MT325610、MT325611、MT325616、MT325618、MT325619、MT325620、MT325622、MT325623、MT32 5624、MT325599、MT325600、MT325601、MT325602、MT325612、MT325613、MT325615、MT325617、MT325625、MT324062、MT324684、MT325573、MT325574、MT3 25577、MT325579、MT325586、MT325592、MT325593、MT325594、MT325598、MT 325605、MT325626、MT325627、MT325633、MT325634、MT326028、MT326031、MT 326091、MT326090、MT326085、MT326084、MT326083、MT326082、MT326081、M T326080、MT326077、MT326067、MT326057、MT326024、MT326026、MT326027、M T326032、MT326034、MT326037、MT326039、MT326041、MT326042、MT326044、 MT326046、MT326047、MT326049、MT326050、MT326051、MT326052、MT326054、MT326059、MT326060、MT326061、MT326062、MT326064、MT326065、MT326068、MT326069、MT326073、MT326074、MT326088、MT327745、MT324679、MT32556 1、MT325571、MT325572、MT325575、MT325583、MT325587、MT325588、MT325589、MT325596、MT325597、MT325603、MT325604、MT325614、MT325621、MT3256 29、MT325630、MT325631、MT325632、MT325635、MT325636、MT325637、MT325638、MT325639、MT326086、MT326096、MT326102、MT326104、MT326105、MT326 112、MT326113、MT326114、MT326115、MT326122、MT328034、MT325564、MT325567、MT326164、MT326165、MT326173、MT326174、MT326184、MT326185、MT32 6186、MT326187、MT325584、MT325585、MT326087、MT326095、MT326103、MT326123、MT326131、MT326132、MT326133、MT328033、MT325562、MT326147、MT3 26153、MT326154、MT326156、MT326157、MT326166、MT326167、MT326175、MT 326176、MT324680、MT325570、MT325576、MT325578、MT325580、MT325581、MT 325582、MT325591、MT325595、MT325628、MT326029、MT326030、MT326038、M T326048、MT326058、MT324681、MT324682、MT324683、MT328032、MT328035、M T322404、MT039874、MT322398、MT322409、MT322421、MT322423、MT322408、MT322413、MT322417、MT322394、MT322407、MT322418、MT322424、MT322411、MT077125、MT322395、MT322396、MT322397、MT322399、MT322400、MT322401、MT322402、MT322403、MT322405、MT322406、MT322414、MT322416、MT32241 9、MT322420、MT322410、MT322412、MT322415、MT322422、MT320538、MT320891、MT308692、MT308693、MT308695、MT308696、MT308698、MT308699、MT3087 01、MT308703、MT308704、MT308694、MT308697、MT308700、MT308702、MT293547、MT304476、MT304474、MT304475、MT304477、MT304478、MT304479、MT304 481、MT304482、MT304484、MT304485、MT304486、MT304487、MT304488、MT304491、MT304480、MT304483、MT304489、MT304490、MT300186、MT292571、MT29 2576、MT292578、MT293186、MT292570、MT292573、MT293173、MT292575、MT293179、MT293180、MT293184、MT293189、MT293192、MT293193、MT293194、MT2 93201、MT293202、MT292572、MT292577、MT293185、MT293187、MT293188、MT291826、MT291832、MT291833、MT291835、MT291836、MT291831、MT293170、MT 292574、MT293178、MT293181、MT293183、MT293195、MT293196、MT293197、MT293203、MT293204、MT293223、MT293212、MT293214、MT293215、MT293216、M T293219、MT293224、MT293225、MT293206、MT293208、MT293209、MT293221、MT295464、MT293160、MT293166、MT293171、MT293190、MT293161、MT293167、MT293168、MT293174、MT293175、MT293182、MT293191、MT293158、MT293162、MT293163、MT293164、MT293156、MT293157、MT293159、MT291834、MT29182 9、MT291827、MT291830、MT291828、MT293169、MT293200、MT293210、MT293211、MT293217、MT293218、MT295465、MT293198、MT293205、MT293207、MT2932 13、MT293220、MT293222、MT292581、MT292569、MT293172、MT293177、MT293176、MT293199、MT292580、MT292582、MT293165、MT292579、MT273658、MT281 577、MT281530、MT276597、MT276598、MT276323、MT276328、MT276331、MT276329、MT276330、MT276324、MT276325、MT276327、MT276326、MT263388、MT26 3392、MT262900、MT262902、MT262906、MT262908、MT262912、MT262913、MT262914、MT262993、MT263074、MT263381、MT263391、MT262901、MT262903、MT2 62907、MT262909、MT262911、MT262899、MT262904、MT262915、MT262916、MT 262897、MT262898、MT262905、MT262910、MT263400、MT263382、MT263383、MT 263384、MT263385、MT262896、MT263407、MT263415、MT263406、MT263408、M T263422、MT263469、MT263439、MT263457、MT263459、MT263432、MT263450、M T263458、MT263467、MT263401、MT263411、MT263413、MT263426、MT263421、MT263443、MT263412、MT263416、MT263417、MT263423、MT263431、MT263461、MT263410、MT263424、MT263425、MT263427、MT263442、MT263402、MT263405、MT263409、MT263418、MT263419、MT263398、MT263399、MT263403、MT263404、M、 T263414、MT263430、MT263390、MT263434、MT263436、MT263446、MT263448、MT263452、MT263453、MT263456、MT263462、MT263463、MT263386、MT263387 MT263389, MT263428, MT263429, MT263433, MT263435, MT263437, MT263438, MT263440, MT263447, MT263449, MT263455, MT263444, MT263445, MT26345 1、MT263466、MT263420、MT263441、MT263454、MT263464、MT263465、MT263468、MT263460、MT263393、MT263394、MT263395、MT263396、MT263397、MT2592 26、MT259275、MT259276、MT259279、MT259247、MT258377、MT258378、MT258379、MT259231、MT259228、MT259238、MT259248、MT256917、MT259227、MT259 236、MT256918、MT258380、MT259235、MT259237、MT259239、MT259281、MT259282、MT259283、MT259240、MT259243、MT259249、MT259250、MT259251、MT2 59256、MT259258、MT259266、MT259267、MT259274、MT259286、MT259287、MT259241、MT259242、MT258381、MT259257、MT259261、MT259262、MT259263、MT 259264、MT259268、MT259269、MT259270、MT259271、MT259272、MT259273、MT259277、MT259278、MT259280、MT258383、MT258382、MT259246、MT256924、M T259244、MT259245、MT259252、MT259253、MT259254、MT259255、MT259259、MT259284、MT259229、MT259230、MT259265、MT259260、MT259285、LC534419、LC534418、MT253710、MT253709、MT253705、MT253708、MT253701、MT253702、MT253703、MT253704、MT253706、MT253707、MT251972、MT251974、MT25197 5、MT251973、MT251976、MT251979、MT253697、MT253699、MT253696、MT253698、MT253700、MT251977、MT251978、MT251980、MT246451、MT246461、MT2464 71、MT246472、MT246474、MT246483、MT246450、MT246453、MT246454、MT246 462、MT246463、MT246464、MT246470、MT246473、MT246480、MT246484、MT24 6449、MT246455、MT246456、MT246478、MT246485、MT246488、MT246452、MT2 46460、MT246465、MT246481、MT246482、MT246490、MT246459、MT246468、MT2 46475、MT246477、MT246479、MT246457、MT246458、MT246466、MT246467、MT 246469、MT246476、MT246486、MT246487、MT246489、MT233526、MT246667、M T240479、MT232870、MT232871、MT233523、MT232869、MT232872、MT233519、MT233521、MT233522、MT233520、MT226610、MT198653、MT198651、MT198652、 MT192773、MT192758、MT192772、MT192765、MT192759、MT188341、MT188340、MT188339、MT186676、MT186681、MT186677、MT186678、MT187977、MT186680 、MT186682、MT186679、MT184909、MT184911、MT184912、MT184913、MT18491 0、MT184907、MT184908、CADDYA000000000、MT163718、MT163719、MT163720、MT163714、MT163715、MT163721、MT163717、MT163737、MT163738、MT163712、MT163716、MT159706、MT159716、MT159719、MT159707、MT159717、MT159709、MT159715、MT159718、MT159722、MT159708、MT161607、MT159705、MT159710、MT159711、MT159712、MT159713、MT159714、MT159720、MT159721、MT121215、MT159778、MT066156、LC529905、MT050493、MT012098、MT152900、MT152824、MT135044、MT135042、MT135041、MT135043、MT126808、MT127113、MT127114、MT127116、MT127115、LC528232、LC528233、MT123293、MT123291、MT123290、MT123292、MT118835、MT111896、MT111895、MT106052、MT106053、MT106054、MT093571、MT093631、MT081061、MT081063、MT081066、MT081062、MT081064、MT081065、MT081067、MT081059、MT081060、MT081068、MT072667、MT072668、MT072688、MT066157、MT066176、MT066159、MT066175、MT066158、LC523809、LC523807、LC523808、MT044258、MT044257、MT050416、MT050417、MT042773、MT042774、MT042775、MT042776、MT049951、MT050414、MT050415、MT042777、MT042778、MT039887、MT039888、MT039890、MT039873、LC522350、MT027062、MT027063、MT027064、MT020881、MT019530、MT019531、MT019533、MT020880、MT019532、MT019529、MT020781、LR757995、LR757998、LR757996、LR757997, MT007544, MT008022, MT008023, MN996531, MN996530, MN996527, MN996528, MN996529, MN9974 09, MN988668, MN988669, MN994467, MN994468, MN988713, MN938384, MN975262, MN985325, MN938386, MN93 8388, MN938385, MN938387, MN938390, MN938389, MN975263, MN975267, MN975268, MN975265, MN975264, MN975266, MN970004, MN970003, MN908947, and OL672836.1, the contents of which are each incorporated herein by reference in their entirety.

[0123] In certain embodiments, the cyclic polyribonucleotide comprises a SARS-CoV-2 immunogen set forth in Table 2. In certain embodiments, the immunogen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from Table 2.

[0124] [Table 2-1]

[0125] [Table 2-2]

[0126] [Table 2-3]

[0127] [Table 2-4]

[0128] [Table 2-5]

[0129] In Table 2, "Proline Substitution" indicates proline substitutions at residues 986 and 987 and a "GSAS" substitution at the furin cleavage site (residues 682-685). In "Cloning Optimization," a single base substitution was made at coordinate 2541 to destroy a BsaI site that aids in Golden Gate Cloning construction of the plasmid DNA template. In "Circularization Optimization," four single nucleotides at positions 2307, 2790, 159, and 315 were substituted to destroy sites that could potentially bind to the circularization element of the splint nucleic acid sequence, thereby potentially inhibiting efficient ligation. In constructs from which the type II terminator was removed (e.g., p33, p35, p36, p39, p41, p44, and p45), two single nucleotides at 1047 and 1049 were substituted to destroy type II terminator sites. In constructs with GC optimization (e.g., p39 and p41), GC optimization was performed so that the GC content was approximately 50%. All single base pair substitutions were designed to be translationally silent. Furthermore, in Table 2, the IRES is either EMCV (SEQ ID NO: 31) or CVB3 (SEQ ID NO: 45).

[0130] In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the SARS-CoV-2 immunogen is an immunogenic fragment comprising a contiguous stretch of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 amino acids of any one of SEQ ID NOs: 63-111 and 283-291. In some embodiments, the SARS-CoV-2 immunogen is an immunogenic fragment comprising a contiguous stretch of at least 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids of any one of the sequences of SEQ ID NOs: 63-111 and 283-291.

[0131] In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-174 and 292-300. In certain embodiments, the circular polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 112-174 and 292-300. In certain embodiments, the polyribonucleotide sequence encoding a SARS-CoV-2 immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 112-174 and 292-300. In one embodiment, the polyribonucleotide sequence encoding the SARS-CoV-2 immunogen is a fragment comprising at least a 50%, 60%, 70%, 80%, 90%, or 95% contiguous stretch of the amino acids of any one of SEQ ID NOs: 112-174 and 292-300.

[0132] In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 219-281. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 219-281. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 219-281. In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 219-281. In some embodiments, the polyribonucleotide sequence encoding the SARS-CoV-2 immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 219 to 281. In some embodiments, the polyribonucleotide sequence encoding the SARS-CoV-2 immunogen is a fragment comprising a contiguous stretch of at least 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids of any one of SEQ ID NOs: 219 to 281.

[0133] In certain embodiments, the cyclic polyribonucleotide comprises a SARS-CoV-2 RBD immunogen set forth in Table 3. In some embodiments, the immunogen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from Table 3. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 RBD immunogen having an amino acid sequence that is at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 RBD immunogen having an amino acid sequence that is at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 RBD immunogen having an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In one embodiment, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111.In some embodiments, the SARS-CoV-2 RBD immunogen is an immunogenic fragment comprising a contiguous stretch of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 amino acids of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111. In some embodiments, the SARS-CoV-2 RBD immunogen is an immunogenic fragment comprising a contiguous stretch of at least 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, and 98-111.

[0134] In certain embodiments, the circular polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 RBD immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 RBD immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 RBD immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In certain embodiments, the circular polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 RBD immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174. In certain embodiments, the polyribonucleotide sequence encoding the SARS-CoV-2 RBD immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174.In one embodiment, the polyribonucleotide sequence encoding the SARS-CoV-2 RBD immunogen is a fragment comprising at least a 50%, 60%, 70%, 80%, 90%, or 95% contiguous stretch of amino acids of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174.

[0135] In certain embodiments, the cyclic polyribonucleotide comprises two or more SARS-CoV-2 RBDs listed in Table 5. In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame listed in Table 5.

[0136] In certain embodiments, the cyclic polyribonucleotide comprises a SARS-CoV-2 spike immunogen set forth in Table 4. In some embodiments, the immunogen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a sequence from Table 4. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 spike immunogen having an amino acid sequence that is at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 spike immunogen having an amino acid sequence that is at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 spike immunogen having an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In one embodiment, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286.In one embodiment, the SARS-CoV-2 spike immunogen is an immunogenic fragment comprising a contiguous stretch of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 amino acids of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286. In one embodiment, the SARS-CoV-2 spike immunogen is an immunogenic fragment comprising at least a 50%, 60%, 70%, 80%, 90%, or 95% contiguous stretch of amino acids from any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, and 283-286.

[0137] In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 spike immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 spike immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 spike immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 spike immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291. In one embodiment, the polyribonucleotide sequence encoding the SARS-CoV-2 spike immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291.In one embodiment, the polyribonucleotide sequence encoding the SARS-CoV-2 spike immunogen is a fragment comprising at least a 50%, 60%, 70%, 80%, 90%, or 95% contiguous stretch of the amino acids of any one of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 287-291.

[0138] In certain embodiments, the cyclic polyribonucleotide comprises a SARS-CoV-2 nonstructural protein (nsp) immunogen. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 nsp immunogen having an amino acid sequence at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 291-295. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 nsp immunogen having an amino acid sequence at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 291-295. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 nsp immunogen having an amino acid sequence at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 291-295. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is any one of SEQ ID NOs: 291-295. In some embodiments, the SARS-CoV-2 nsp immunogen is an immunogenic fragment comprising a contiguous stretch of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 amino acids of any one of SEQ ID NOs: 291-295. In some embodiments, the SARS-CoV-2 nsp immunogen is an immunogenic fragment comprising a contiguous stretch of at least 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids of any one of the sequences of SEQ ID NOs: 291-295.

[0139] In certain embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 nsp immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 296-300. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 nsp immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 296-300. In some embodiments, the cyclic polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 nsp immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 296-300. In certain embodiments, the circular polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 nsp immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 296-300, and 287-291. In certain embodiments, the polyribonucleotide sequence encoding the SARS-CoV-2 nsp immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 296-300. In one embodiment, the polyribonucleotide sequence encoding the SARS-CoV-2 nsp immunogen is a fragment comprising at least a 50%, 60%, 70%, 80%, 90%, or 95% contiguous stretch of the amino acids of any one of SEQ ID NOs: 296-300.

[0140] The present disclosure specifically contemplates that any of the DNA sequences described herein can be converted into a corresponding RNA sequence and included in an RNA molecule described herein.

[0141] [Table 3-1]

[0142] [Table 3-2]

[0143] [Table 3-3]

[0144] [Table 4-1]

[0145] [Table 4-2]

[0146] [Table 5]

[0147] In some embodiments, the GC content of a nucleic acid sequence encoding a SARS-CoV-2 immunogen is at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%). In some embodiments, the GC content of a nucleic acid sequence encoding a SARS-CoV-2 immunogen is 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59%, or 60% or less. In some embodiments, the GC content of a nucleic acid sequence encoding a SARS-CoV-2 immunogen is 51% to 60%, 52% to 60%, 53% to 60%, 54% to 60%, 55% to 60%, 52% to 58%, or 53% to 58%.

[0148] In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the SARS-CoV-2 immunogen is greater than 10% (e.g., greater than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the SARS-CoV-2 immunogen is 30% or less (e.g., 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20% or less). In certain embodiments, the uridine content (in the case of RNA) or thymidine content (in the case of DNA) of the nucleic acid sequence encoding the SARS-CoV-2 immunogen is 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%.

[0149] The GC content of an expressed sequence encoding a SARS-CoV-2 immunogen refers to the GC content of the expressed sequence encoding only the SARS-CoV-2 immunogen, without any other coding sequence encoding peptides other than the SARS-CoV-2 immunogen. Similarly, the uridine content or thymidine content of an expressed sequence encoding a SARS-CoV-2 immunogen refers to the uridine content of the expressed sequence encoding only the SARS-CoV-2 immunogen, without any other coding sequence encoding peptides other than the SARS-CoV-2 immunogen. In one embodiment, the calculation of the GC content or uridine (or thymidine) content of an expressed sequence encoding a SARS-CoV-2 immunogen considers only the contiguous nucleic acid sequence, in the 5'-3' direction, starting from the first nucleoside of the start codon of the open reading frame encoding the SARS-CoV-2 immunogen to the last nucleoside of the stop codon of the same open reading frame. In other embodiments, the calculation of the GC content or uridine (or thymidine) content of an expressed sequence encoding a SARS-CoV-2 immunogen considers only the contiguous nucleic acid sequence in the 5'-3' direction, starting from the first nucleoside of the codon encoding the N-terminal amino acid residue of the SARS-CoV-2 immunogen to the last nucleoside of the codon encoding the C-terminal amino acid residue of the SARS-CoV-2 immunogen.

[0150] In certain embodiments, the immunogen or epitope is derived from cells of the host subject (e.g., the subject being immunized). For example, antibodies that block coronavirus entry can be generated by using immunogens or epitopes from components of host cells that the virus uses as an entry factor.

[0151] In certain embodiments, a coronavirus epitope comprises or contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids, or more. In certain embodiments, a coronavirus epitope comprises or contains 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, or 30 or less amino acids, or fewer. In some embodiments, a coronavirus epitope comprises or contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, a coronavirus epitope contains 5 amino acids. In some embodiments, a coronavirus epitope contains 6 amino acids. In some embodiments, an epitope contains 7 amino acids. In some embodiments, a coronavirus epitope contains 8 amino acids. In some embodiments, an epitope can be about 8 to about 11 amino acids. In some embodiments, an epitope can be about 9 to about 22 amino acids.

[0152] The coronavirus immunogen may comprise an immunogen recognized by B cells, an immunogen recognized by T cells, or a combination thereof. In certain embodiments, the immunogen comprises an immunogen recognized by B cells. In certain embodiments, the coronavirus immunogen is an immunogen recognized by B cells. In certain embodiments, the coronavirus immunogen comprises an immunogen recognized by T cells. In certain embodiments, the immunogen is an immunogen recognized by T cells.

[0153] Coronavirus epitopes include those recognized by B cells, immunogens recognized by T cells, or a combination thereof. In some embodiments, coronavirus epitopes include epitopes recognized by B cells. In some embodiments, the epitopes are epitopes recognized by B cells. In some embodiments, coronavirus epitopes include epitopes recognized by T cells. In some embodiments, coronavirus epitopes are epitopes recognized by T cells.

[0154] Techniques for identifying immunogens and epitopes in silico have been described, for example, in Sanchez-Trincado, et al. (2017), Fundamentals and methods for T- and B-cell epitope prediction., Journal of immunology research; Grifoni, Alba, et al., A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2. Cell HOST & MICROBE (2020); Russi et al., and In silico prediction of T- and B-cell epitopes in PmpD: First step towards the design of a Chlamydia trachomatis vaccine. BIOMEDICAL JOURNAL 41.2(2018):109-17; Baruah, et al., Immunoinformatics-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV. Journal of and Medical Virology (2020); each of which is incorporated herein by reference in its entirety.

[0155] A cyclic polyribonucleotide of the present disclosure can comprise the sequence of any number of coronavirus immunogens and / or epitopes, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more coronavirus immunogens or epitopes (e.g., selected from any of the coronavirus immunogens and / or epitopes described herein).

[0156] In certain embodiments, the cyclic polyribonucleotide comprises the sequence of, for example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or fewer coronavirus immunogens or epitopes.

[0157] In some embodiments, the cyclic polyribonucleotide comprises the sequence of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus immunogens or epitopes.

[0158] The cyclic polyribonucleotide may contain sequences for one or more coronavirus epitopes from a coronavirus immunogen. For example, a coronavirus immunogen may contain an amino acid sequence that can contain multiple coronavirus epitopes (e.g., epitopes recognized by B cells and / or T cells) therein, and the cyclic polyribonucleotide may contain or encode one or more of those coronavirus epitopes. In certain embodiments, the cyclic polyribonucleotide may contain one or more sequences encoding a coronavirus immunogen and one or more sequences encoding an immunogen that represents the coronavirus immunogen. For example, the cyclic polyribonucleotide may contain one or more sequences encoding a coronavirus immunogen and one or more sequences encoding an immunogen from another virus (e.g., an influenza virus immunogen).

[0159] The cyclic polyribonucleotide may, for example, comprise the sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more epitopes from a single coronavirus immunogen.

[0160] In certain embodiments, the cyclic polyribonucleotide comprises a sequence of no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 120, no more than 140, no more than 160, no more than 180, no more than 200, no more than 250, no more than 300, no more than 350, no more than 400, no more than 450, or no more than 500 coronavirus epitopes, e.g., from one coronavirus immunogen.

[0161] In one embodiment, the cyclic polyribonucleotide comprises a sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus epitopes, e.g., from one coronavirus immunogen.

[0162] The cyclic polyribonucleotide can encode a variant of a coronavirus immunogen or epitope. The variant can be a naturally occurring variant (e.g., a variant identified in sequence data from different coronavirus genera, species, isolates, or quasispecies) or can be a derivative sequence disclosed herein that has been generated in silico (e.g., an immunogen or epitope having one or more amino acid insertions, deletions, substitutions, or combinations thereof compared to the wild-type immunogen or epitope).

[0163] The cyclic polyribonucleotide may, for example, comprise the sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more variants of a coronavirus immunogen or epitope.

[0164] In certain embodiments, the cyclic polyribonucleotide comprises a sequence of, for example, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or fewer variants of a coronavirus immunogen or epitope.

[0165] In some embodiments, the cyclic polyribonucleotide comprises the sequence of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 variants of a coronavirus immunogen or epitope.

[0166] The coronavirus immunogen and / or epitope sequences of the cyclic polyribonucleotide may also be referred to as coronavirus expression sequences. In certain embodiments, the cyclic polyribonucleotide comprises one or more coronavirus expression sequences, each of which may encode a coronavirus polypeptide. The coronavirus polypeptides may be produced in significant quantities. The coronavirus polypeptides may be secreted from the cell or localized to the cytoplasm, nucleus, or membrane compartment of the cell. Some coronavirus polypeptides include, but are not limited to, at least a portion of an immunogen disclosed herein, an epitope disclosed herein, a coronavirus protein (e.g., a viral envelope protein, a viral matrix protein, a viral spike protein, a viral receptor-binding domain (RBD) of a viral spike protein, a viral membrane protein, a viral nucleocapsid protein, a viral accessory protein, a fragment thereof, or a combination thereof). In certain embodiments, the coronavirus polypeptide encoded by the cyclic polyribonucleotide of the present disclosure comprises a fragment of a coronavirus immunogen disclosed herein. In certain embodiments, the coronavirus polypeptide encoded by the cyclic polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus immunogens disclosed herein, or a fragment thereof. In some embodiments, the coronavirus polypeptides encoded by the cyclic polyribonucleotides of the present disclosure comprise coronavirus epitopes. In some embodiments, the polypeptides encoded by the cyclic polyribonucleotides of the present disclosure comprise fusion proteins comprising two or more coronavirus epitopes disclosed herein, e.g., artificial peptide sequences comprising multiple predicted epitopes from one or more coronaviruses of the present disclosure.

[0167] In certain embodiments, exemplary coronavirus proteins expressed from the circular polyribonucleotides disclosed herein include secreted proteins, e.g., proteins (e.g., immunogens and / or epitopes) that naturally include a signal peptide, or those that do not normally encode a signal peptide but have been modified to include one.

[0168] In some cases, the cyclic polyribonucleotide expresses a secreted coronavirus protein with a short half-life in the blood, or is a protein with an intracellular localization signal or a protein with a secretory signal peptide. In some cases, the cyclic polyribonucleotide expresses a transmembrane domain with a short half-life in the blood, or is a protein with an intracellular localization signal or a protein with a secretory peptide.

[0169] In some embodiments, the cyclic polyribonucleotide comprises one or more coronavirus expression sequences and is configured for sustained expression in the cells of a subject (e.g., a subject for immunization) in vivo. In some embodiments, the cyclic polyribonucleotide is configured such that expression of the one or more coronavirus expression sequences in the cells at later time points is equal to or higher than at earlier time points. In such embodiments, expression of the one or more coronavirus expression sequences can be maintained at a relatively stable level or can increase over time. In some embodiments, expression of the coronavirus expression sequences is relatively stable over an extended period of time.

[0170] In certain embodiments, the cyclic polyribonucleotide expresses one or more coronavirus immunogens and / or epitopes in a subject (e.g., a subject for immunization), e.g., transiently or long-term. In certain embodiments, expression of the coronavirus expression sequences persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer, or any time in between. In certain embodiments, expression of the coronavirus immunogen and / or epitope occurs within about 30 minutes to about 7 days or less, or within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or less. 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, 45, 60, 75, 90 or less days, or any time in between.

[0171] In some embodiments, the coronavirus expressed sequence has a length of less than 5000 bps (e.g., less than about 5000 bps, 4000 bps, 3000 bps, 2000 bps, 1000 bps, 900 bps, 800 bps, 700 bps, 600 bps, 500 bps, 400 bps, 300 bps, 200 bps, 100 bps, 50 bps, 40 bps, 30 bps, 20 bps, 10 bps, or less). In some embodiments, the coronavirus expressed sequences may, independently or in addition, be greater than 10 bps (e.g., at least about 10 bps, 20 bps, 30 bps, 40 bps, 50 bps, 60 bps, 70 bps, 80 bps, 90 bps, 100 bps, 200 bps, 300 bps, 400 bps, 500 bps, 600 bps, 700 bps, 800 bps, 900 bps, 1000 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5 kb or more in length).

[0172] In some embodiments, the cyclic polyribonucleotide encodes multiple immunogens (e.g., one or more, two or more, three or more, four or more, or five or more immunogens), and the multiple immunogens share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the multiple immunogens also have less than 100% sequence identity. This may indicate immunogens that are related to each other due to genetic drift; thus, a single cyclic polyribonucleotide composition or immunogenic composition may be capable of inducing an immune response against targets that exist in various mutational states in a population, or may induce an immune response against multiple targets with the same immunogen that are related by genetic drift. For example, the immunogens may be related to each other due to genetic drift of the target virus (e.g., a coronavirus such as SARS-CoV-2).

[0173] Derivatives and Fragments The immunogens or epitopes of the present disclosure can include wild-type sequences. When referring to an immunogen or epitope, the term "wild-type" refers to a sequence (e.g., an amino acid sequence) that is naturally occurring and encoded by a genome (e.g., a coronavirus genome). A coronavirus can have one wild-type sequence, or more than one wild-type sequence (e.g., having one standard wild-type sequence present in a reference coronavirus genome and additional variant wild-type sequences present that have arisen from mutations).

[0174] When referring to an immunogen or epitope, the terms "derivative" and "derived from" refer to a sequence (e.g., an amino acid sequence) that differs from the wild-type sequence in one or more amino acids, e.g., contains one or more amino acid insertions, deletions, and / or substitutions relative to the wild-type sequence.

[0175] An immunogenic or epitope derivative sequence is a sequence that has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type sequence, e.g., the wild-type protein, immunogenic, or epitope sequence.

[0176] In some embodiments, the immunogen or epitope contains one or more amino acid insertions, deletions, substitutions, or a combination thereof that affect the structure of the encoded protein. In some embodiments, the immunogen or epitope contains one or more amino acid insertions, deletions, substitutions, or a combination thereof that affect the function of the encoded protein. In some embodiments, the immunogen or epitope contains one or more amino acid insertions, deletions, substitutions, or a combination thereof that affect the expression or processing of the encoded protein by a cell.

[0177] The amino acid insertion, deletion, substitution, or combination thereof may introduce a site for post-translational modification (e.g., introduce a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or a sequence targeted for cleavage). In certain embodiments, the amino acid insertion, deletion, substitution, or combination thereof removes a site for post-translational modification (e.g., removes a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or a sequence targeted for cleavage). In certain embodiments, the amino acid insertion, deletion, substitution, or combination thereof modifies a site for post-translational modification (e.g., a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or modifies a site to alter the efficiency or characteristics of cleavage).

[0178] Amino acid substitutions can be conservative or non-conservative. A conservative amino acid substitution can be the substitution of one amino acid for another amino acid with similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution can be the substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R). A conservative amino acid change can be an amino acid change from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids may include Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A conservative amino acid change may be from one acidic amino acid to another. Acidic amino acids may include Glu (E) and Asp (D). A conservative amino acid change may be from one basic amino acid to another. Basic amino acids may include His (H), Arg (R), and Lys (K). A conservative amino acid change may be from one polar amino acid to another. Polar amino acids may include Asn (N), Gln (Q), Ser (S), and Thr (T). A conservative amino acid change may be from one nonpolar amino acid to another nonpolar amino acid. Nonpolar amino acids may include Leu (L), Val (V), Ile (I), Met (M), Gly (G), and Ala (A). A conservative amino acid change may be from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids may include Phe (F), Tyr (Y), and Trp (W). A conservative amino acid change may be from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids may include Ala (A), Val (V), Leu (L), and Ile (I).In one embodiment, a conservative amino acid substitution is an amino acid change from one amino acid to another within one of the following groups: Group I: ala, pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ile, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0179] In certain embodiments, the immunogenic or epitope derivatives of the present disclosure comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid deletions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0180] In certain embodiments, the immunogenic or epitope derivatives of the present disclosure comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions relative to a sequence disclosed herein (e.g., a wild-type sequence).

[0181] In certain embodiments, an immunogenic or epitope derivative of the disclosure comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0182] In some embodiments, the immunogenic or epitope derivatives of the present disclosure have 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 30, 1 to 40, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 ...16, 1 to 17, 1 to 18, 1 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 31, 2 to 32, 2 to 33, 2 to 34, 2 to 35, 2 to 36, 2 to 37, 2 to 38, 2 to 39, 2 to 40, 2 to 41, 2 to 42, 2 to 43, 2 to 44, 2 to Contains 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, or 20 to 25 amino acid substitutions.

[0183] In certain embodiments, the immunogenic or epitope derivatives of the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0184] The one or more amino acid substitutions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions may be consecutive, non-consecutive, or a combination thereof.

[0185] In certain embodiments, an immunogenic or epitope derivative of the disclosure comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 120, no more than 140, no more than 160, no more than 180, or no more than 200 amino acid deletions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0186] In some embodiments, the immunogenic or epitope derivatives of the present disclosure have 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 30, 1 to 40, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 15, 2 to 20, The deletions include 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, 20 to 25, 20 to 30, 30 to 50, 50 to 100, or 100 to 200 amino acids.

[0187] In certain embodiments, the immunogenic or epitope derivatives of the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0188] The one or more amino acid deletions can be at the N-terminus, C-terminus, or a combination thereof within the amino acid sequence. The amino acid deletions can be contiguous, non-contiguous, or a combination thereof.

[0189] In certain embodiments, the immunogenic or epitope derivatives of the present disclosure comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid insertions relative to a sequence disclosed herein (e.g., a wild-type sequence).

[0190] In certain embodiments, an immunogenic or epitope derivative of the disclosure comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid insertions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0191] In some embodiments, the immunogenic or epitope derivatives of the present disclosure have 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 30, 1 to 40, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 ...16, 1 to 17, 1 to 18, 1 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 2 to 27, 2 to 28, 2 to 29, 2 to 31, 2 to 32, 2 to 33, 2 to 34, 2 to 35, 2 to 36, 2 to 37, 2 to 38, 2 to 39, 2 to 40, 2 to 41, 2 to 42, 2 to 43, 2 to 44, 2 to and 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, or 20 to 25 amino acid insertions.

[0192] In certain embodiments, the immunogenic or epitope derivatives of the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid insertions compared to the sequences disclosed herein (e.g., the wild-type sequences).

[0193] The one or more amino acid insertions may be at the N-terminus, C-terminus, or a combination thereof within the amino acid sequence. The amino acid insertions may be contiguous, non-contiguous, or a combination thereof.

[0194] Circular polyribonucleotide element The cyclic polyribonucleotide comprises the elements described below as well as the coronavirus immunogens or epitopes described herein. In some embodiments, the cyclic polyribonucleotide comprises any feature, or any combination of features, disclosed in WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0195] In certain embodiments, the cyclic polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0196] In some embodiments, the cyclic polyribonucleotide is 500 to 20,000 nucleotides, 1,000 to 20,000 nucleotides, 2,000 to 20,000 nucleotides, or 5,000 to 20,000 nucleotides. In some embodiments, the cyclic polyribonucleotide is 500 to 10,000 nucleotides, 1,000 to 10,000 nucleotides, 2,000 to 10,000 nucleotides, or 5,000 to 10,000 nucleotides.

[0197] internal ribosome entry site In certain embodiments, a circular or linear polyribonucleotide described herein comprises one or more internal ribosome entry site (IRES) elements. In certain embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences, where each expression sequence optionally encodes an immunogen, such as a coronavirus immunogen). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence (e.g., the coding sequence encoding a coronavirus immunogen).

[0198] Suitable IRES elements to be included in the polyribonucleotide comprise an RNA sequence capable of engaging a eukaryotic ribosome, hi certain embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0199] In certain embodiments, the IRES element is derived from the DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA can be derived from picornavirus complementary DNA (cDNA), including, but not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the Antennapedia gene from Drosophila melanogaster.

[0200] In certain embodiments, the IRES sequence is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall enteric virus, Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Small kite P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamo virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus (AEV), acute bee paralysis virus, hibiscus chlorotic mottle virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-l, mouse Rbm3, Drosophila reaper, dog scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, salivirus, cosavirus, parechovirus, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae TFIIDcerevisiae YAP1, human c-src, human FGF-1, simian picornavirus, turnip crinkle virus, Aichi virus, Kurohi virus, echovirus 11, an aptamer against eIF4G, coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1 / 2) IRES sequence. In still other embodiments, the IRES is a coxsackievirus B3 (CVB3) IRES sequence. In a further embodiment, the IRES is an encephalomyocarditis virus IRES sequence. In a further embodiment, the IRES is a Theiler's encephalomyelitis virus IRES sequence.

[0201] The IRES sequence may have a modified sequence compared to the wild-type IRES sequence.In some embodiments, if the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified to be a cytosine residue.For example, the IRES sequence may be a CVB3 IRES sequence in which the terminal adenosine residue is modified to a cytosine residue.In some embodiments, the modified CVB3 IRES is [ka] The nucleic acid sequence may be:

[0202] In one embodiment, the IRES sequence is an enterovirus 71 (EV17) IRES. In one embodiment, the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue. In one embodiment, the modified EV71 IRES is [ka] The nucleic acid sequence may be:

[0203] In some embodiments, the polyribonucleotide comprises at least one IRES flanking at least one (e.g., 2, 3, 4, 5, or more) expressed sequence. In some embodiments, an IRES flanks each side of at least one (e.g., 2, 3, 4, 5, or more) expressed sequence. In some embodiments, the polyribonucleotide comprises one or more IRES sequences on one or both sides of each expressed sequence, resulting in separation of the resulting peptides and / or polypeptides. For example, the polyribonucleotides described herein can comprise a first IRES operably linked to a first expressed sequence (e.g., encoding a first immunogen, such as a first coronavirus immunogen) and a second IRES operably linked to a second expressed sequence (e.g., encoding a second immunogen, such as a second coronavirus immunogen).

[0204] In certain embodiments, a polyribonucleotide described herein comprises an IRES (eg, an IRES operably linked to a coding region). For example, polyribonucleotides can be prepared using the methods described in Chen et al. Mol. Cell 81(20):4300-4318, 2021; Jopling et al. Oncogene 20:2664-2670, 2001; Baranick et al. PNAS 105(12):4733-4738, 2008; Lang et al. Molecular Biology of the Cell 13(5):1792-1801, 2002; Dorokhov et al. PNAS 99(8):5301-5306, 2002; Wang et al. Nucleic Acids Research 33(7):2248-2258, 2005; Petz et al. Nucleic Acids Research 35(8):2473-2482, 2007, Chen et al., each of which is incorporated by reference in its entirety. al. SCIENCE 268:415-417, 1995; Fan et al. NATURE COMMUNICATION 13(1):3751-3765, 2022, and International Publication No. WO 2021 / 263124.

[0205] signal sequence In certain embodiments, the immunogen expressed from a circular or linear polyribonucleotide disclosed herein comprises a secreted protein, e.g., a protein that naturally includes a signal sequence, or one that does not normally encode a signal sequence but has been modified to include one. In certain embodiments, the immunogen includes a secretory signal. For example, the secretory signal can be a naturally encoded secretory signal for a secreted protein. In another example, the secretory signal can be a modified secretory signal for a secreted protein. In other embodiments, the immunogen does not include a secretory signal.

[0206] In some embodiments, the polyribonucleotide encodes multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the same immunogen. In some embodiments, at least one copy of the immunogen includes a signal sequence and at least one copy of the immunogen does not include a signal sequence. In some embodiments, the circular polyribonucleotide encodes multiple immunogens, where at least one of the multiple immunogens includes a signal sequence and at least one copy of the multiple immunogens does not include a signal sequence.

[0207] In some embodiments, the signal sequence is a wild-type signal sequence present on the N-terminus of the corresponding wild-type immunogen, e.g., when endogenously expressed. In some embodiments, the signal sequence is heterologous to the immunogen, e.g., not present when the wild-type immunogen is endogenously expressed. The polyribonucleotide sequence encoding the immunogen can be modified to remove nucleotide sequences encoding the wild-type signal sequence and / or to add sequences encoding a heterologous signal sequence.

[0208] The cyclic polyribonucleotide may further comprise one or more adjuvants, each with or without a signal sequence. In some embodiments, the cyclic polyribonucleotide encodes at least one adjuvant and at least one immunogen. In some embodiments, at least one encoded adjuvant comprises a signal sequence and at least one encoded immunogen does not comprise a signal sequence. In some embodiments, at least one encoded adjuvant comprises a signal sequence and at least one encoded immunogen comprises a signal sequence. In some embodiments, at least one encoded adjuvant does not comprise a signal sequence and at least one encoded immunogen comprises a signal sequence. In some embodiments, neither the encoded adjuvant nor the encoded immunogen comprises a signal sequence.

[0209] In some embodiments, the signal sequence is a wild-type signal sequence present on the N-terminus of the corresponding wild-type adjuvant, e.g., when endogenously expressed. In some embodiments, the signal sequence is heterologous to the adjuvant, e.g., not present when the wild-type adjuvant is endogenously expressed. The polyribonucleotide sequence encoding the adjuvant can be modified to remove nucleotide sequences encoding the wild-type signal sequence and / or to add sequences encoding a heterologous signal sequence.

[0210] A polypeptide encoded by a polyribonucleotide (e.g., an immunogen or adjuvant encoded by a polyribonucleotide) can contain a signal sequence that directs the immunogen or adjuvant into the secretory pathway. In certain embodiments, the signal sequence can direct the immunogen or adjuvant to reside in a specific organelle (e.g., the endoplasmic reticulum, the Golgi apparatus, or an endosome). In certain embodiments, the signal sequence directs the immunogen or adjuvant to be secreted from the cell. In the case of a secreted protein, the signal sequence can be cleaved after secretion to yield the mature protein. In other embodiments, the signal sequence can be embedded in the membrane of a cell or a specific organelle to generate a transmembrane region that anchors the protein to the membrane of the cell, the endoplasmic reticulum, or the Golgi apparatus. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N-terminus of the polypeptide. In other embodiments, the first transmembrane domain acts as a first signal sequence, which targets the protein to the membrane.

[0211] In some embodiments, the secretory signal is a human interleukin-2 (IL-2) secretory signal. In some embodiments, the IL-2 secretory signal has an amino acid sequence that is at least 90% identical to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 199). In some embodiments, the IL2 secretory signal has an amino acid sequence that is at least 95% identical to SEQ ID NO: 199. In some embodiments, the IL-2 secretory signal has an amino acid sequence that is at least 99% identical to SEQ ID NO: 199. In some embodiments, the IL-2 secretory signal has an amino acid sequence that is 100% identical to SEQ ID NO: 199.

[0212] In some embodiments, the secretion signal is a Gaussia luciferase secretion signal. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with at least 90% sequence identity to MGVKVLFALICIAVAEAK (SEQ ID NO: 198). In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 198. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 198. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 198.

[0213] In some embodiments, the secretory signal is an EPO (e.g., human EPO) secretory signal. In some embodiments, the EPO secretory signal has an amino acid sequence with at least 90% sequence identity to MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 197). In some embodiments, the EPO secretory signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 197. In some embodiments, the EPO secretory signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 197. In some embodiments, the EPO secretory signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 197.

[0214] In some embodiments, the secretory signal is a wild-type SARS-CoV-2 secretory signal. In some embodiments, the wild-type SARS-CoV-2 secretory signal has an amino acid sequence with at least 90% sequence identity to MFVFLVLLPLVSS (SEQ ID NO: 200). In some embodiments, the wild-type SARS-CoV-2 secretory signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 200. In some embodiments, the wild-type SARS-CoV-2 secretory signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 200. In some embodiments, the wild-type SARS-CoV-2 secretory signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 200.

[0215] In some embodiments, the adjuvant encoded by the polyribonucleotide comprises a secretory signal sequence. In some embodiments, the immunogen encoded by the polyribonucleotide comprises a secretory signal sequence, a transmembrane insertion signal sequence, or no signal sequence.

[0216] Adjustment element A regulatory element may comprise a sequence located adjacent to an expression sequence encoding an expression product. The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of expressed product compared to the amount of product expressed in the absence of the regulatory element. A regulatory element may be used to increase the expression of one or more immunogens and / or adjuvants encoded by a polyribonucleotide. Similarly, a regulatory element may be used to decrease the expression of one or more immunogens and / or adjuvants encoded by a polyribonucleotide. In certain embodiments, a regulatory element may be used to increase the expression of an immunogen and / or adjuvant, and another regulatory element may be used to decrease the expression of another immunogen and / or adjuvant in the same polyribonucleotide. Furthermore, a single regulatory element may increase the amount of a product (e.g., an immunogen or adjuvant) expressed for multiple expression sequences linked in tandem. Thus, a single regulatory element can promote the expression of one or more expression sequences (e.g., an immunogen or adjuvant). Multiple regulatory elements may also be used, for example, to differentially regulate expression of different expression sequences.

[0217] In certain embodiments, the regulatory elements provided herein may comprise a selective translation sequence. As used herein, the term "selective translation sequence" refers to a nucleic acid sequence that selectively initiates or activates translation of an expression sequence in a polyribonucleotide, for example, a specific riboswitch aptazyme. Regulatory elements may also comprise a selective degradation sequence. As used herein, the term "selective degradation sequence" refers to a nucleic acid sequence that initiates degradation of a polyribonucleotide or an expression product of a polyribonucleotide. In certain embodiments, the regulatory element is a translation modulator. A translation modulator may regulate the translation of an expression sequence in a polyribonucleotide. A translation modulator may be a translation enhancer or suppressor. In certain embodiments, a translation initiation sequence may function as a regulatory element.

[0218] In some embodiments, polyribonucleotides produce expression products in stoichiometric ratios. Rolling circle translation produces expression products sequentially in substantially equal ratios. In some embodiments, polyribonucleotides have stoichiometric translation efficiencies such that expression products are produced in substantially equal ratios. In some embodiments, polyribonucleotides have stoichiometric translation efficiencies for multiple expression products, e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression sequences. In some embodiments, polyribonucleotides produce expression products in substantially different ratios. For example, the translation efficiencies of the multiple expression products can be 1:10,000; 1:7000, 1:5000, 1:1000, 1:700, 1:500, 1:100, 1:50, 1:10, 1:5, 1:4, 1:3, or 1:2. In some embodiments, the ratio of the multiple expression products can be regulated using regulatory elements.

[0219] Further examples of regulatory elements are described in paragraphs

[0154] to

[0161] of WO 2019 / 118919, the entirety of which is incorporated herein by reference.

[0220] Cleavage domain A circular or linear polyribonucleotide of the present disclosure may comprise a cleavage domain (e.g., a stagger element or cleavage sequence).

[0221] As used herein, the term "stagger element" refers to a portion of a nucleotide sequence or the like that induces ribosome pausing during translation. In some embodiments, the stagger element is a non-conserved sequence of amino acids with a strong alpha-helical propensity, followed by the consensus sequence -D(V / I)ExNPG P (SEQ ID NO: 52), where x = any amino acid. In some embodiments, the stagger element can include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof. In some embodiments, a cyclic or linear polyribonucleotide includes at least one stagger element flanking an expressed sequence, such as a sequence encoding a coronavirus immunogen. In some embodiments, a cyclic or linear polyribonucleotide includes a stagger element flanking each expressed sequence. In some embodiments, a stagger element is present on one or both sides of each expressed sequence, resulting in separation of the expression products, e.g., immunogen and / or adjuvant. In some embodiments, a stagger element is part of one or more expressed sequences. In some embodiments, the circular or linear polyribonucleotide comprises one or more expression sequences (e.g., immunogens and / or adjuvants), each of which is separated from a subsequent expression sequence (e.g., immunogens and / or adjuvants) by a stagger element on the circular or linear polyribonucleotide. In some embodiments, the stagger element prevents (a) two translations of a single expression sequence, or (b) the generation of a single polypeptide from one or more translations of two or more expression sequences. In some embodiments, the stagger element is a sequence separated from one or more expression sequences. In some embodiments, the stagger element comprises a portion of an expression sequence of one or more expression sequences.

[0222] Examples of stagger elements are described in paragraphs

[0172] to

[0175] of WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0223] In some embodiments, multiple immunogens and / or adjuvants encoded by the cyclic ribonucleotides can be separated by an IRES between each immunogen (e.g., each immunogen is operably linked to a separate IRES). For example, the cyclic polyribonucleotide can include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence. The IRES can be the same among all immunogens. The IRES can be different among different immunogens.

[0224] In some embodiments, multiple immunogens and / or adjuvants can be separated by a 2A self-cleaving peptide. For example, a cyclic polyribonucleotide can encode an IRES operably linked to an open reading frame encoding a first immunogen, 2A, and a second immunogen. In some embodiments, 2A can have the sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 202).

[0225] In some embodiments, multiple immunogens and / or adjuvants may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first immunogen, a protease cleavage site (e.g., a furin cleavage site), and a second immunogen. In some embodiments, the furin cleavage site may have the sequence of GRLRR (SEQ ID NO: 201). In some embodiments, the furin cleavage site may have the sequence of GRLRR (SEQ ID NO: 203).

[0226] In certain embodiments, multiple immunogens and / or adjuvants may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first immunogen, 2A, a protease cleavage site (e.g., a furin cleavage site), and a second immunogen. A circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first immunogen, a protease cleavage site (e.g., a furin cleavage site), 2A, and a second immunogen. The tandem 2A and furin cleavage site may be referred to as furin-2A (including furin-2A or 2A-furin arranged in either orientation).

[0227] Furthermore, multiple immunogens and / or adjuvants encoded by cyclic ribonucleotides can be separated by both an IRES and a 2A sequence. For example, an IRES can be between one immunogen and / or adjuvant and a second immunogen and / or adjuvant, while a 2A peptide can be between a second immunogen and / or adjuvant and a third immunogen and / or adjuvant. Selection of a particular IRES or 2A self-cleaving peptide can be used to control the expression level of the immunogen and / or adjuvant under the control of the IRES or 2A sequence. For example, depending on the IRES and / or 2A peptide selected, expression in the polypeptide can be higher or lower.

[0228] To avoid the production of continuous expression products, e.g., immunogens and / or adjuvants, while maintaining rolling circle translation, a stagger element can be included to induce pausing of ribosomes during translation. In one embodiment, the stagger element is at the 3' end of at least one of the one or more expressed sequences. The stagger element can be configured to pause ribosomes during rolling circle translation of circular or linear polyribonucleotides. The stagger element can include, but is not limited to, a 2A-like or CHYSEL (SEQ ID NO: 175) (cis-acting hydrolase element) sequence. In one embodiment, the stagger element encodes a sequence containing the C-terminal consensus sequence X1X2X3EX5NPGP, where X1 is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid (SEQ ID NO: 176). Some non-limiting examples of stagger elements include GDVESNPGP (SEQ ID NO: 177), GDIEENPGP (SEQ ID NO: 178), VEPNPGP (SEQ ID NO: 179), IETNPGP (SEQ ID NO: 180), GDIESNPGP (SEQ ID NO: 181), GDVELNPGP (SEQ ID NO: 182), GDIETNPGP (SEQ ID NO: 183), GDVENPGP (SEQ ID NO: 184), GDVEENPGP (SEQ ID NO: 185), GDVEQNPGP (SEQ ID NO: 186), IESNPGP (SEQ ID NO: 187), GDIELNPGP (SEQ ID NO: 188), HDIETPNPGP (SEQ ID NO: 189), HDVETNPGP (SEQ ID NO: 190), HDVEMNPGP (SEQ ID NO: 191), GDMESNPGP (SEQ ID NO: 192), GDVETNPGP (SEQ ID NO: 193), GDIEQNPGP (SEQ ID NO: 194), and DSEFNPGP (SEQ ID NO: 195).

[0229] In some embodiments, the stagger elements described herein cleave the expression product, such as between the G and P of the consensus sequences described herein. As one non-limiting example, a circular or linear polyribonucleotide comprises at least one stagger element for cleaving the expression product. In some embodiments, the circular or linear polyribonucleotide comprises a stagger element adjacent to at least one expressed sequence. In some embodiments, the circular or linear polyribonucleotide comprises a stagger element after each expressed sequence. In some embodiments, the circular or linear polyribonucleotide comprises a stagger element, and the stagger element is present on one or both sides of each expressed sequence to provide for translation of individual peptides and / or polypeptides from each expressed sequence.

[0230] In certain embodiments, the stagger element comprises one or more modified or non-natural nucleotides that induce pausing of ribosomes during translation. Non-natural nucleotides can include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Examples such as these are distinguished from natural DNA or RNA by changes to the backbone of the molecule. Exemplary modifications can include any modification to the sugar, nucleobase, internucleoside linkage (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone), and any combination thereof that can induce pausing of ribosomes during translation. Some of the exemplary modifications provided herein are described elsewhere herein.

[0231] In some embodiments, the stagger element is present in a circular or linear polyribonucleotide in another form. For example, in some exemplary circular or linear polyribonucleotides, the stagger element comprises a termination element of a first expression sequence in the circular or linear polyribonucleotide and a nucleotide spacer sequence separating the termination element from a first translation initiation sequence of expression following the first expression sequence. In some examples, the first stagger element of the first expression sequence is upstream (5'-terminal) of the first translation initiation sequence of expression following the first expression sequence in the circular or linear polyribonucleotide. In some cases, the first expression sequence and the expression sequence following the first expression sequence are two separate expression sequences in the circular or linear polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence may allow for continuous translation of the first expression sequence and the subsequent expression sequence. In some embodiments, the first stagger element comprises a termination element, separating the expression product of the first expression sequence from the expression product of the subsequent expression sequence, thereby generating separate expression products. In some cases, a circular or linear polyribonucleotide containing a first stagger element upstream of a first translation initiation sequence of a subsequent sequence in the circular or linear polyribonucleotide is translated continuously, while a corresponding circular or linear polyribonucleotide containing a stagger element of a second expression sequence upstream of a second translation initiation sequence of an expression sequence following a second expression sequence is not translated continuously. In some cases, there is only one expression sequence in the circular or linear polyribonucleotide, and the first expression sequence and its subsequent expression sequence are the same expression sequence. In some exemplary circular or linear polyribonucleotides, the stagger element includes a first termination element of a first expression sequence in the circular or linear polyribonucleotide and a nucleotide spacer sequence that separates the termination element from the downstream translation initiation sequence. In some such examples, the first stagger element is upstream (5' end) of the first translation initiation sequence of a first expression sequence in the circular or linear polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and any subsequent expressed sequences.In some embodiments, the first stagger element separates the first expression product of a first expression sequence from the next expression product of the first expression sequence, thereby generating distinct expression products. In some cases, a circular or linear polyribonucleotide comprising a first stagger element upstream of a first translation initiation sequence of a first expression sequence in a circular or linear polyribonucleotide is translated continuously, while a corresponding circular or linear polyribonucleotide comprising a stagger element upstream of a second translation initiation sequence of a second expression sequence in a corresponding circular or linear polyribonucleotide is not translated continuously. In some cases, the distance between the second stagger element and the second translation initiation sequence in the corresponding circular or linear polyribonucleotide is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the distance between the first stagger element and the first translation initiation sequence in the corresponding circular or linear polyribonucleotide. In some cases, the distance between the first stagger element and the first translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or more. In some embodiments, the distance between the second staggered element and the second translation start is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or more, greater than the distance between the first staggered element and the first translation start. In some embodiments, the circular or linear polyribonucleotide comprises two or more expressed sequences.

[0232] In some embodiments, the circular or linear polyribonucleotide comprises at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, the cleavage sequence is contained within the expression sequence. In some embodiments, the circular or linear polyribonucleotide comprises 2 to 10 cleavage sequences. In some embodiments, the circular or linear polyribonucleotide comprises 2 to 5 cleavage sequences. In some embodiments, multiple cleavage sequences are between multiple expression sequences; for example, the circular or linear polyribonucleotide may comprise three expression sequences and two cleavage sequences, such that there is a cleavage sequence between each expression sequence. In some embodiments, the circular or linear polyribonucleotide comprises a cleavage sequence, such as in a sacrificial circular RNA, a cleavable circular RNA, or a self-cleaving circular RNA. In some embodiments, the circular or linear polyribonucleotide comprises two or more cleavage sequences, resulting in separation of the circular or linear polyribonucleotide into multiple products, such as miRNA, linear RNA, smaller circular or linear polyribonucleotides, etc.

[0233] In some embodiments, the cleavage sequence comprises a ribozyme RNA sequence. Ribozymes (derived from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. While many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds or the hydrolysis of bonds in other RNAs, they have also been shown to catalyze the aminotransferase activity of ribosomes. Catalytic RNAs can be "evolved" by in vitro methods. Similar to the riboswitch activity described above, ribozymes and their reaction products can regulate gene expression. In some embodiments, catalytic RNAs or ribozymes can be placed within larger non-coding RNAs so that the ribozyme is present in many copies within the cell for chemical conversion of molecules from bulk volume. In some embodiments, both an aptamer and a ribozyme can be encoded within the same non-coding RNA.

[0234] In certain embodiments, the cleavage sequence may encode a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more immunogens, for example, when two or more immunogens are encoded by a single open reading frame (ORF). For example, two or more immunogens may be encoded by a single open reading frame, the expression of which is controlled by an IRES. In certain embodiments, the ORF further encodes a polypeptide linker, such that the expression product of the ORF encodes two or more immunogens, each separated by a sequence encoding a polypeptide linker (e.g., a linker of 5 to 200, 5 to 100, 5 to 50, 5 to 20, 50 to 100, or 50 to 200 amino acids). The polypeptide linker may include a cleavage site, for example, that is recognized and cleaved by a protease (e.g., an endogenous protease in a subject after administration of the polyribonucleotide to the subject). In such embodiments, a single expression product comprising the amino acid sequences of two or more immunogens is cleaved during expression, allowing the two or more immunogens to be separated after expression. Exemplary protease cleavage sites, e.g., amino acid sequences that act as protease cleavage sites recognized by metalloproteinases (e.g., matrix metalloproteinases (MMPs) such as any one or more of MMPs 1-28), disintegrin and metalloproteinases (ADAMs such as any one or more of ADAMs 2, 7-12, 15, 17-23, 28-30, and 33), serine proteases, urokinase-type plasminogen activator, matriptase, cysteine ​​proteases, aspartic acid proteases, or cathepsin proteases, are known to those of skill in the art. In certain embodiments, the protease is MMP9 or MMP2. In certain embodiments, the protease is matriptase.

[0235] In some embodiments, the cyclic or linear polyribonucleotide described herein is a sacrificial cyclic or linear polyribonucleotide, a cleavable cyclic or linear polyribonucleotide, or a self-cleaving cyclic or linear polyribonucleotide. The cyclic or linear polyribonucleotide can deliver cellular components, including, for example, RNA, lncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, the cyclic or linear polyribonucleotide comprises an miRNA separated by (i) a self-cleaving element; (ii) a cleavage recruitment site; (iii) a degradable linker; (iv) a chemical linker; and / or (v) a spacer sequence. In some embodiments, the cyclic RNA comprises an siRNA separated by (i) a self-cleaving element; (ii) a cleavage recruitment site (e.g., ADAR); (iii) a degradable linker (e.g., glycerol); (iv) a chemical linker; and / or (v) a spacer sequence. Non-limiting examples of self-cleaving elements include hammerhead, splicing elements, hairpins, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozyme.

[0236] In some embodiments, the cyclic polyribonucleotide comprises at least one stagger element flanking the expression sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element flanking each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, resulting in separation of the expression products, e.g., peptides and / or polypeptides. In some embodiments, the stagger element is part of one or more expression sequences. In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, each of which is separated from the subsequent expression sequence by a stagger element on the cyclic polyribonucleotide. In some embodiments, the stagger element prevents the production of a single polypeptide from (a) two translations of a single expression sequence, or (b) one or more translations of two or more expression sequences. In some embodiments, the stagger element is a sequence separated from one or more expression sequences. In some embodiments, the stagger element comprises a portion of an expression sequence of one or more expression sequences.

[0237] Examples of stagger elements are described in paragraphs

[0172] to

[0175] of WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0238] Translation initiation sequence In some embodiments, the circular or linear polyribonucleotide encodes an immunogen and includes a translation initiation sequence, e.g., a start codon. In some embodiments, the circular polyribonucleotide encodes an immunogen for generating a desired human polyclonal antibody and includes a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the translation initiation sequence includes a Kozak sequence. In some embodiments, the circular or linear polyribonucleotide includes a translation initiation sequence, e.g., a Kozak sequence, adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, a translation initiation sequence, e.g., a Kozak sequence, is present on one or both sides of each expression sequence, resulting in separation of the expression products. In some embodiments, the circular or linear polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the circular or linear polyribonucleotide. In some embodiments, the translation initiation sequence is within the substantially single-stranded region of the circular or linear polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] to

[0165] of WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0239] A circular or linear polyribonucleotide may contain two or more start codons, for example, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may begin at the first start codon or may begin downstream of the first start codon.

[0240] In some embodiments, a circular or linear polyribonucleotide can initiate at a first start codon, e.g., a codon that is not AUG. Translation of a circular or linear polyribonucleotide can initiate at an alternative translation initiation sequence, such as those described in paragraph

[0164] of WO 2019 / 118919 A1, the entire contents of which are incorporated herein by reference.

[0241] In one embodiment, translation is initiated by eukaryotic initiation factor 4A (eIF4A) processing by RocaGlate (translation is suppressed by blocking 43S scanning, causing premature upstream translation initiation and reduced protein expression from transcripts bearing RocA-eIF4A target sequences; see, e.g., nature.com / articles / nature17978).

[0242] Untranslated region In some embodiments, the circular or linear polyribonucleotide comprises an untranslated region (UTR). A UTR of a genomic region comprising a gene may be transcribed but not translated. In some embodiments, a UTR may be included upstream of the translation initiation sequence of an expression sequence described herein. In some embodiments, a UTR may be included downstream of an expression sequence described herein. In some cases, one UTR for a first expression sequence is the same as, contiguous with, or overlaps with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, such as ZKSCAN1.

[0243] Exemplary untranslated regions are described in paragraphs

[0197] to

[0201] of International Publication No. WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0244] In some embodiments, the cyclic polyribonucleotide comprises a polyA sequence. Exemplary polyA sequences are described in paragraphs

[0202] to

[0205] of International Publication No. 2019 / 118919, the entire contents of which are incorporated herein by reference. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence.

[0245] In certain embodiments, the cyclic or linear polyribonucleotide comprises a UTR with one or more stretches of adenosines and uridines embedded therein, and these AU-rich signatures may increase the turnover rate of the expression product.

[0246] Introduction, removal, or modification of UTR AU-rich elements (AREs) can be useful for modulating the stability or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of a cyclic or linear polyribonucleotide. When engineering a particular cyclic polyribonucleotide, one or more copies of an ARE can be introduced into the cyclic polyribonucleotide, and the copies of the ARE can modulate translation and / or production of the expression product. Similarly, AREs can be identified and removed or modified into cyclic polyribonucleotides to modulate intracellular stability and thus affect the translation and production of the resulting protein.

[0247] It should be understood that any UTR from any gene can be incorporated into each of the flanking regions of the circular polyribonucleotide.

[0248] In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic or linear polyribonucleotide lacks a 3'-UTR and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic or linear polyribonucleotide lacks a poly-A sequence and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic or linear polyribonucleotide lacks a termination element and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic or linear polyribonucleotide lacks an internal ribosome entry site and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the cyclic or linear polyribonucleotide lacks a cap and is capable of expressing a protein from one or more expression sequences thereof. In some embodiments, the circular or linear polyribonucleotide lacks a 5'-UTR, a 3'-UTR, and an IRES and is capable of expressing a protein from its one or more expression sequences. In some embodiments, the circular or linear polyribonucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic agent, a regulatory element (e.g., a translation modulator, e.g., a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids targeting an endogenous gene (e.g., an siRNA, lncRNA, shRNA), and a sequence encoding a therapeutic mRNA or protein.

[0249] In some embodiments, the cyclic or linear polyribonucleotide lacks a 5'-UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'-UTR. In some embodiments, the cyclic polyribonucleotide lacks a poly-A sequence. In some embodiments, the cyclic or linear polyribonucleotide lacks a termination element. In some embodiments, the cyclic or linear polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic or linear polyribonucleotide lacks susceptibility to degradation by exonucleases. In some embodiments, the lack of susceptibility to degradation of a cyclic polyribonucleotide can mean that the cyclic polyribonucleotide is not degraded by exonucleases, or is degraded only in the presence of exonucleases, for example, to a limited extent equivalent to or similar to that in the absence of exonucleases. In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, the cyclic polyribonucleotide has reduced degradation when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks a linkage to a cap-binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

[0250] Termination element In some embodiments, the polyribonucleotide described herein comprises at least one terminator. In some embodiments, the polyribonucleotide comprises a terminator operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a terminator.

[0251] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element. In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which lacks a termination element such that the polyribonucleotide is translated continuously. Omission of the termination element can result in rolling circle translation or continuous expression of the expression product.

[0252] In some embodiments, a cyclic polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element. In some embodiments, a cyclic polyribonucleotide comprises one or more expression sequences, each of which lacks a termination element, allowing the cyclic polyribonucleotide to be translated continuously. The omission of a termination element can result in rolling circle translation or continuous expression of expression products, such as peptides or polypeptides, because there is no pausing or shedding of ribosomes. In such embodiments, rolling circle translation expresses continuous expression products through each expression sequence. In certain other embodiments, the termination element of an expression sequence can be part of a staggered element. In some embodiments, one or more expression sequences in a cyclic polyribonucleotide contain a termination element. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the cyclic polyribonucleotide occurs. In such cases, the expression product may cause the ribosome to shed and stop translation when it encounters a termination element, such as a stop codon. In certain embodiments, translation is halted while the ribosome, eg, at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.

[0253] In some embodiments, the cyclic polyribonucleotide contains a termination element at the end of one or more expression sequences. In some embodiments, the one or more expression sequences contain two or more consecutive termination elements. In such embodiments, translation is stopped and rolling circle translation is terminated. In some embodiments, the ribosome is completely disengaged by the cyclic polyribonucleotide. In some such embodiments, generation of a subsequent (e.g., second, third, fourth, fifth, etc.) expression sequence in the cyclic polyribonucleotide may require the ribosome to re-engage with the cyclic polyribonucleotide before translation initiation. Generally, the termination element contains an in-frame nucleotide triplet (e.g., UAA, UGA, UAG) that signals a translation stop. In some embodiments, one or more termination elements in the cyclic polyribonucleotide are off-frame or frame-shifted termination elements, such as -1 and +1 shifted reading frames (e.g., cryptic terminations), which may terminate translation. Frameshifted termination elements include the nucleotide triplets TAA, TAG, and TGA that appear in the second and third reading frames of an expressed sequence. Frameshifted termination elements can be important in preventing misreading of mRNA, which is often harmful to the cell. In one embodiment, the termination element is a stop codon.

[0254] In certain embodiments, the expression sequence includes a poly-A sequence (e.g., at the 3' end of the expression sequence, e.g., 3' to the termination element). In certain embodiments, the length of the poly-A sequence is greater than 10 nucleotides in length. In one embodiment, the poly-A sequence is more than 15 nucleotides in length (e.g., at least or more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequence is designed according to the description of poly-A sequences in paragraphs

[0202] to

[0204] of WO 2019 / 118919 A1, the entire contents of which are incorporated herein by reference. In some embodiments, the expression sequence lacks a poly-A sequence (e.g., at the 3' end of the expression sequence).

[0255] In some embodiments, the cyclic polyribonucleotide comprises a poly A, lacks a poly A, or has a modified poly A to adjust one or more properties of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacks a poly A or has a modified poly A to improve one or more functional properties, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency.

[0256] Further examples of termination elements are described in paragraphs

[0169] to

[0170] of WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0257] Spacer sequence In some embodiments, the polyribonucleotides described herein comprise a spacer sequence. In some embodiments, the polyribonucleotides described herein comprise one or more spacer sequences. A spacer refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. A spacer can be present between any of the nucleic acid elements described herein. A spacer can also be present within a nucleic acid element described herein.

[0258] The spacer may be, for example, at least 5 (e.g., at least 10, at least 15, or at least 20) ribonucleotides in length. In certain embodiments, each spacer region is at least 5 (e.g., at least 10, at least 15, or at least 20) ribonucleotides in length. Each spacer region may be, for example, 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may comprise a polyA sequence. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may comprise a polyAC sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly-AG sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly-AT sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a random sequence.

[0259] In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides in length, at least 15 nucleotides in length, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the spacer sequence is not more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the spacer sequence is 20 to 50 nucleotides in length. In certain embodiments, the spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

[0260] The spacer sequence can be a poly-A sequence, a poly-AC sequence, a poly-C sequence, or a poly-U sequence.

[0261] In certain embodiments, the spacer sequence can be polyAT, polyAC, polyAG, or a random sequence.

[0262] Exemplary spacer sequences are described in paragraphs

[0293] to

[0302] of International Publication No. WO 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0263] qualification A polyribonucleotide may contain one or more substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly a parent polyribonucleotide, and is included within the scope of the present disclosure.

[0264] In some embodiments, the polyribonucleotide comprises one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequences, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than 100 different nucleoside modifications identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucleic Acids Res 27:196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the polyribonucleotide comprises at least one nucleoside selected from a group such as those described in paragraph

[0311] of International Publication No. WO 2019 / 118919 A1, the entire contents of which are incorporated herein by reference.

[0265] Polyribonucleotides can include any useful modifications, for example, to the sugar, nucleobase, or internucleoside linkage (e.g., linking phosphate / phosphodiester bond / phosphodiester backbone). One or more atoms of a pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. The modifications can be modifications of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. Further modifications are described herein.

[0266] In some embodiments, the polyribonucleotide comprises at least one N(6) methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification can reduce the immunogenicity of the polyribonucleotide (e.g., reduce the level of one or more markers of an immune or inflammatory response).

[0267] In some embodiments, the modification may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0268] In some embodiments, chemical modifications to the ribonucleotides of polyribonucleotides can enhance immune evasion. Polyribonucleotides can be synthesized and / or modified by methods well established in the art, such as those described in CURRENT PROTOCOLS IN NUCLEIC ACID CHEMISTRY, Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation (mono-, di-, and tri-), conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), base modifications (e.g., substitution with a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanded repertoire of partners), base removal (abasic nucleotide), or conjugated bases. Modified ribonucleotide bases can also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can regulate the expression, immune response, stability, and intracellular localization of polyribonucleotides, among other functional effects. In some embodiments, the modifications include bi-orthogonal nucleotides, such as unnatural bases. See, for example, Kimoto et al., Chem Commun (Camb), 2017, 53:12309, DOI:10.1039 / c7cc06661a, which is incorporated herein by reference.

[0269] In certain embodiments, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions of one or more ribonucleotides of a polyribonucleotide can include not only backbone modifications but also modifications or substitutions of phosphodiester linkages. Specific examples of polyribonucleotides include polyribonucleotides containing modified backbones or non-natural internucleoside linkages, such as internucleoside modifications, including, but not limited to, modifications or substitutions of phosphodiester linkages. Polyribonucleotides with modified backbones specifically include those that do not have a phosphorus atom in the backbone. For purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In certain embodiments, a polyribonucleotide will include ribonucleotides that have a phosphorus atom in their internucleoside backbone.

[0270] Modified polyribonucleotide backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments, polyribonucleotides can be negatively or positively charged.

[0271] Modified nucleotides that can be incorporated into polyribonucleotides can be modified in the internucleoside linkage (e.g., the phosphate backbone). In this specification, the terms "phosphate" and "phosphodiester" are used interchangeably in reference to the polynucleotide backbone. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can contain extensive replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, phosphorodiamidate, alkyl or aryl phosphonate, and phosphotriester. Phosphorodithioate has both non-linked oxygens replaced with sulfur. Phosphate linkers can also be modified by replacement of the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates).

[0272] The a-thio-substituted phosphate moieties are provided by the non-natural phosphorothioate backbone linkage to provide stability to RNA and DNA polymers. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently have a longer half-life in the cellular environment. Phosphorothioates linked to polyribonucleotides are expected to reduce the innate immune response by weaker binding / activation of cellular innate immune molecules.

[0273] In certain embodiments, the modified nucleoside comprises an α-thio-nucleoside (e.g., 5'-0-(l-thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine (α-thio-cytidine), 5'-0-(l-thiophosphate)-guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine).

[0274] Other internucleoside linkages that can be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain a phosphorus atom.

[0275] In certain embodiments, a polyribonucleotide can include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into a polyribonucleotide, such as a bifunctional modification. Cytotoxic nucleosides may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Further examples include fludarabine phosphate, N4-behenoyl-l-β-D-arabinofuranosylcytosine, N4-octadecyl-l-β-D-arabinofuranosylcytosine, N4-palmitoyl-l-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidate).

[0276] A polyribonucleotide may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., natural nucleotides, purines or pyrimidines, or one or more or all of A, G, U, C, I, and pU) may or may not be uniformly modified in a polyribonucleotide or a given predetermined sequence region thereof. In some embodiments, a polyribonucleotide contains pseudouridine. In some embodiments, a polyribonucleotide contains inosine, which may assist the immune system in characterizing polyribonucleotides as endogenous to viral RNA. Incorporation of inosine may also mediate improved RNA stability / reduced degradation. See, for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as "self." Cell Res. 25, 1283-1284, incorporated by reference in its entirety.

[0277] In some embodiments, all nucleotides in a polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, modifications may include m6A, which may enhance expression; inosine, which may attenuate immune responses; pseudouridine, which may increase RNA stability or translational readthrough (staggered elements), m5C, which may increase stability; and 2,2,7-trimethylguanosine, which aids in intracellular translocation (e.g., nuclear localization).

[0278] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a polyribonucleotide. Those skilled in the art will understand that nucleotide analogs or other modifications can be placed at any position in a polyribonucleotide so that the function of the polyribonucleotide is not substantially impaired. Modifications can also be non-coding region modifications. Polyribonucleotides may be present in an amount ranging from about 1% to about 100% (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any percentage therebetween (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 1% to 10 ... The modified nucleotides may be 0% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%.

[0279] Multimerization In certain embodiments, the cyclic polyribonucleotide may contain a multimerization domain. For example, the cyclic polyribonucleotide may encode a first polypeptide that is an immunogen (e.g., a coronavirus immunogen) and a second polypeptide that is a multimerization domain. For example, the multimerization domain may be encoded in the same open reading frame as the immunogen (e.g., a coronavirus immunogen) and expressed as a fusion protein with the immunogen. In certain embodiments, the cyclic polyribonucleotide may encode two or more immunogens, each of which may optionally be fused to a multimerization domain. The multimerization domain may promote the formation of an immunogen complex (e.g., a complex comprising multiple immunogens).

[0280] Multimerization of the encoded immunogen can be beneficial in inducing an immune response. Fusion of an immunogen to one or more multimerization elements (e.g., dimerization elements, trimerization elements, tetramerization elements, and oligomerization elements) can result in the formation of a multimeric immunogen complex (e.g., formation of a multimeric immunogen complex after expression in an immunized subject). In certain embodiments, the formation of a multimeric immunogen complex increases the immunogenicity of the immunogen. For example, the formation of a multimeric immunogen complex can increase the immunogenicity of an immunogen by mimicking infection with an exogenous pathogen (e.g., a virus) in which multiple potential immunogens are typically located in the envelope of the pathogen (e.g., the hemagglutinin (HA) immunogen of influenza virus). In some embodiments, the multimerized complex comprises at least 2, 3, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 immunogens. In some embodiments, the immunogen complex comprises 2-10, 2-50, 2-100, 5-10, 5-15, 5-20, 5-50, 5-100, 10-20, 10-30, 10-40, 10-50, 10-60, 10-100, 20-50, or 20-100 immunogens. In some embodiments, the immunogen complex comprises 6 copies of the immunogen (e.g., the cyclic polyribonucleotide encodes an immunogen-foldon-immunogen fusion protein). In some embodiments, the immunogen complex comprises 24 copies of the immunogen (e.g., the cyclic polyribonucleotide encodes an immunogen-ferritin fusion protein), hi some embodiments, the immunogen complex comprises 60 copies of the immunogen (e.g., the cyclic polyribonucleotide encodes an immunogen-AaLS fusion protein or encodes an immunogen-β-annulus peptide).

[0281] When used in conjunction with a polypeptide immunogen of interest in the context of the present disclosure, such multimerizing elements can be positioned N-terminal or C-terminal to the polypeptide of interest. At the nucleic acid level, the coding sequence for such multimerizing elements is typically positioned 5' or 3' to the coding sequence for the polypeptide or protein of interest, in the same reading frame.

[0282] The multimerization domain may have 10 to 500 amino acid residues (e.g., 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500 residues). In certain embodiments, the multimerization domain may comprise 20 to 2500 amino acid residues (e.g., 20 to 250, 20 to 225, 20 to 200, 20 to 175, 20 to 150, 20 to 150, 20 to 125, 20 to 100, 20 to 75, 20 to 50, 50 to 250, 75 to 250, 100 to 250, 125 to 250, 150 to 250, 175 to 250, 200 to 250, and 225 to 250 residues).

[0283] In some embodiments, the immunogen fused to the multimerization domain is at least 2-fold, 5-fold, or 10-fold more immunogenic (e.g., in a human subject) than the immunogen. In some embodiments, the immunogen fused to the multimerization domain is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% more immunogenic (e.g., in a human subject) than the immunogen not fused to the multimerization domain.

[0284] Specific multimerization elements are oligomerization elements, tetramerization elements, trimerization elements, or dimerization elements. Dimerization elements can be selected from, for example, heat shock proteins, immunoglobulin Fc domains, and leucine zipper dimerization elements / domains (dimerization domains of the basic region leucine zipper class of transcription factors). Trimerization and tetramerization elements can be selected from, for example, engineered leucine zippers (engineered α-helical coiled-coil peptides that adopt a parallel trimer state), fibritin foldon domains from enterobacteriaceae phage T4, GCN4p11, CCN4-p11, and p53. In certain embodiments, the cyclic polyribonucleotide comprises a T4-foldon domain. In certain embodiments, the T4-foldon domain has an amino acid sequence at least 95% identical to GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 204). In certain embodiments, the T4-foldon has the amino acid sequence of SEQ ID NO: 204. In some embodiments, the multimerization domain is a β-annulus peptide (see Matsuura et al. (2010), ANGEW. CHEM. INT. ED., 49:9662-65). In some embodiments, the β-annulus peptide has the amino acid sequence of INHVGGTGGAIMAPVAVTRQLVGS (SEQ ID NO: 205), wherein the C-terminal serine residue is optionally present or absent, or has an amino acid sequence at least 95% identical to SEQ ID NO: 205. In some embodiments, the cyclic polyribonucleotide comprises an AaLS peptide. In certain embodiments, the AaLS peptide has an amino acid sequence at least 95% identical to TDILGKYVINYLNKLKKKEDIFKEFLKW (SEQ ID NO: 282). In some embodiments, the AaLS peptide has the amino acid sequence of SEQ ID NO: 282.

[0285] The oligomerization element may be selected from, for example, ferritin, surfactant D, paramyxovirus phosphoprotein oligomerization domain, complement inhibitor C4 binding protein (C4bp) oligomerization domain, viral infectivity factor (Vif) oligomerization domain, sterile alpha motif (SAM) domain, and von Willebrand factor type D domain.

[0286] Ferritin is a highly conserved protein that forms oligomers and is found in all animals, bacteria, and plants. Ferritin is a protein that naturally forms nanoparticles of 24 identical subunits. Ferritin-immunogen fusion constructs potentially form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses. In one embodiment, the cyclic polyribonucleotide comprises a ferritin domain. In one embodiment, the cyclic polyribonucleotide comprises: [ka] The ferritin domain has the amino acid sequence:

[0287] Surfactant D protein (SPD) is a hydrophilic glycoprotein that spontaneously self-assembles to form oligomers. SPD-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses.

[0288] The phosphoproteins of paramyxoviruses (negative-strand RNA viruses) function as transcriptional transactivators of the viral polymerase. Phosphoprotein oligomerization is important for viral genome replication. Phosphoprotein-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses.

[0289] The complement inhibitor C4-binding protein (C4bp) can also be used as a fusion partner to generate oligomeric immunogen aggregates. Both C-terminal domains of C4bp (57 amino acid residues in humans and 54 amino acid residues in mice) are necessary and sufficient for oligomerization of C4bp or other polypeptides fused to it. C4bp-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses. The viral infectivity factor (Vif) multimerization domain has been shown to form oligomers both in vitro and in vivo. Vif oligomerization involves residues 151-164 in the C-terminal domain, which sequence maps to the 161PPLP164 motif (for human HIV-1: TPKKIKPPLP (SEQ ID NO: 205)). Vif-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses.

[0290] The sterile alpha motif (SAM) domain is a protein interaction module present in a wide variety of proteins involved in many biological processes. SAM domains, spanning approximately 70 residues, are found in diverse eukaryotic organisms. SAM domains have been shown to homo- and hetero-oligomerize to form multiple self-associated oligomeric structures. SAM-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses. von Willebrand factor (vWF) contains several types of D domains: D1 and D2 are present within the N-terminal propeptide, while the remaining D domains are required for oligomerization. vWF domains are found in a variety of plasma proteins, including complement factors B, C2, C3, and CR4; integrins (I-domains); collagen types VI, VII, XII, and XIV; and other extracellular proteins. vWF-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can enhance immune responses.

[0291] In certain embodiments, the cyclic polyribonucleotide may contain one or more multimerization domains. For example, the cyclic polyribonucleotide may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 multimerization domains. In certain embodiments, the cyclic polyribonucleotide contains two multimerization domains. The two or more multimerization domains may be adjacent to each other. Alternatively, the two or more multimerization domains may be separated by one or more other elements. For example, the two multimerization domains may be separated by an immunogen. In certain embodiments, the cyclic polyribonucleotide contains a ferritin domain and a T4-foldon domain. The ferritin and T4-foldon domains may be linked by a Gly-Ser linker. In certain embodiments, the ferritin domain linked to the T4-foldon domain is [ka] It has the amino acid sequence:

[0292] In some embodiments, the multimerization domain is a lumazine synthase domain. Lumazine synthase can assemble into a complex comprising 60 copies of the lumazine synthase domain, where each lumazine synthase domain can be fused to one or more immunogens. In some embodiments, the lumazine synthase domain comprises the amino acid sequence of any of SEQ ID NOs: 206-209 and 325, or an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 206-209 and 325. SEQ ID NO: 206 [ka] SEQ ID NO: 207 [ka] SEQ ID NO: 208 [ka] SEQ ID NO: 209 [ka] SEQ ID NO: 325 [ka]

[0293] The lumazine synthase domain is provided with one or more cysteine ​​substitutions to introduce non-natural disulfide bonds that stabilize the lumazine synthase complex formed by self-assembled subunits.In some embodiments, the non-natural disulfide bonds are introduced with L121C-K131C, L121CG-K131C, L121GC-K131C, K7C-R40C, I3C-L50C, I82C-K131CG, E5C-R52C, or E95C-A101C substitutions, or combinations thereof (such as I3C-L50C and I82C-K131CG; E5C-R52C and I82C-K131CG; or E95C-A101C and I82C-K131CG).Residue numbering is performed with reference to the lumazine synthase subunit set forth in SEQ ID NO:206. Non-limiting examples include: SEQ ID NO: 210 (L121C-K131C) [ka] SEQ ID NO: 211 (L121CG-K131C) [ka] SEQ ID NO: 212 (L121GC-K131C) [ka] SEQ ID NO: 213 (K7C-R40C) [ka] SEQ ID NO: 214 (I3C-L50C, I82C-K131CG) [ka] SEQ ID NO: 215 (E5C-R52C, I82C-K131CG) [ka] SEQ ID NO: 216 (E95C-A101C, I82C-K131CG) [ka]

[0294] Various methods for multimerizing polypeptides are described in International Publication No. WO 2020 / 061564, page 25, line 1 to page 26, line 20, which is incorporated herein by reference.

[0295] In some embodiments, the multimerization domain is a riboflavin synthase domain. For example, the riboflavin synthase domain may have an amino acid sequence having at least 95% sequence identity with TDILGKYVINYLNKLKKKEDIFKEFLKW (SEQ ID NO: 326). In some embodiments, the riboflavin synthase domain may have the amino acid sequence of SEQ ID NO: 326.

[0296] Suitable multimerization domains may be selected, for example, from the list of amino acid sequences set forth in SEQ ID NOs: 1116 to 1167 of International Patent Application WO 2017 / 081082, or fragments or variants of these sequences.

[0297] Generation method The present disclosure provides methods for generating circular polyribonucleotides, including, for example, recombinant techniques or chemical synthesis. For example, the DNA molecules used to generate RNA circles can contain the DNA sequence of a naturally occurring original nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide not normally found in nature (e.g., a chimeric molecule or fusion protein). DNA and RNA molecules can be modified using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification, or mutagenesis of selected regions of nucleic acid sequences, synthesis of mixtures of oligonucleotides and ligation of mixtures to "assemble" mixtures of nucleic acid molecules, and combinations thereof.

[0298] Circular polyribonucleotides can be prepared according to any available technique, including, but not limited to, chemical synthesis and enzymatic synthesis. In certain embodiments, a linear primary construct or linear RNA can be circularized or concatemerized to generate the circular RNA described herein. The circularization or concatemerization mechanism can be performed by methods such as chemical, enzymatic, splint ligation, or ribozyme-catalyzed methods. The newly formed 5'-3' bond can be an intramolecular or intermolecular bond. For example, a splint ligase, such as Splint® Ligase, can be used for splint ligation. According to this method, a single-stranded polynucleotide (splint), such as a single-stranded DNA or RNA, can be designed to hybridize with both ends of a linear polyribonucleotide so that the two ends can be juxtaposed upon hybridization with the single-stranded splint. Thus, the splint ligase can catalyze the ligation of the juxtaposed two ends of a linear polyribonucleotide to generate a circular RNA. In certain embodiments, a DNA or RNA ligase may be used in synthesizing the circular polynucleotide. As a non-limiting example, the ligase may be a circ ligase or a circular ligase.

[0299] In another example, either the 5' or 3' end of a linear polyribonucleotide can encode a ligase ribozyme sequence such that during in vitro transcription, the resulting linear circular RNA contains an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide to the 3' end of the linear polyribonucleotide. The ligase ribozyme can be derived from a group I intron, hepatitis delta virus, a hairpin ribozyme, or selected by SELEX (systematic evolution of ligands by exponential enrichment).

[0300] In another example, a linear polyribonucleotide can be circularized or concatemerized by using at least one non-nucleic acid moiety. For example, the at least one non-nucleic acid moiety can react with a region or feature near the 5' end or near the 3' end of the linear polyribonucleotide to circularize or concatemerize the linear polyribonucleotide. In another example, the at least one non-nucleic acid moiety can be located at, linked to, or near the 5' end or 3' end of the linear polyribonucleotide. The non-nucleic acid moiety can be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety can be a bond such as a hydrophobic bond, an ionic bond, a biodegradable bond, or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety can be a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety can be an oligonucleotide or peptide moiety, such as an aptamer or a non-nucleic acid linker described herein.

[0301] In another example, a linear polyribonucleotide can be circularized or concatemerized by self-splicing. In certain embodiments, the linear polyribonucleotide can include a loop E sequence for self-ligation. In other embodiments, the linear polyribonucleotide can include a self-circularizing intron, e.g., a 5' and 3' splice junction, or a self-circularizing catalytic intron, e.g., a group I, group II, or group III intron. Non-limiting examples of group I intron self-splicing sequences can include the self-splicing replacement intron-exon sequence derived from the T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena, the cyanobacterium Anabaena pre-tRNA-Leu gene, or Tetrahymena pre-rRNA.

[0302] In some embodiments, the polyribonucleotide can include a catalytic intron fragment, such as the 3' half of a group I catalytic intron fragment and the 5' half of a group I catalytic intron fragment. The first and second annealing regions can be positioned within the catalytic intron fragment. The group I catalytic intron is a self-splicing ribozyme that catalyzes its own excision from mRNA, tRNA, and rRNA precursors via two metal ion phosphoryl transfer mechanisms. Importantly, the RNA itself self-catalyzes intron removal without the recruitment of exogenous enzymes such as ligases.

[0303] In certain embodiments, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from the cyanobacterial Anabaena pre-tRNA-Leu gene or Tetrahymena pre-rRNA.

[0304] In one embodiment, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from the cyanobacterium Anabaena pre-tRNA-Leu gene, the 3' exon fragment comprises a first annealing region, and the 5' exon fragment comprises a second annealing region. The first annealing region can comprise, for example, 5 to 50, e.g., 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides, and the second annealing region can comprise, for example, 5 to 50, e.g., 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides.

[0305] In one embodiment, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from Tetrahymena pre-rRNA, and the 3' half of the group I catalytic intron fragment comprises a first annealing region, and the 5' exon fragment comprises a second annealing region. In one embodiment, the 3' exon comprises the first annealing region, and the 5' half of the group I catalytic intron fragment comprises the second annealing region. The first annealing region can comprise, for example, 6 to 50, e.g., 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region can comprise, for example, 6 to 50, e.g., 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0306] In certain embodiments, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from the cyanobacterial Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, or T4 phage td gene.

[0307] In one embodiment, the 3' half of the group I catalytic intron fragment and the 5' group I catalytic intron fragment are derived from the T4 phage td gene. The 3' exon fragment can comprise a first annealing region, and the 5' half of the group I catalytic intron fragment can comprise a second annealing region. The first annealing region can comprise, for example, 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region can comprise, for example, 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0308] In certain embodiments, the 3' half of the Group I catalytic intron fragment is the 5' end of the linear polynucleotide.

[0309] In certain embodiments, the 5' half of the Group I catalytic intron fragment is the 3' end of the linear polyribonucleotide.

[0310] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0311] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0312] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:307 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:308.

[0313] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0314] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0315] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:309 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:310.

[0316] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0317] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0318] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:311 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:312.

[0319] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0320] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0321] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:313 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:314.

[0322] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0323] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0324] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:315 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:316.

[0325] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0326] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0327] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:317 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:318.

[0328] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0329] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0330] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:319 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:320.

[0331] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0332] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0333] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:321 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:322.

[0334] In one embodiment, the 3' half of the Group I catalytic intron fragment is [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0335] In one embodiment, the 5' half of the Group I catalytic intron fragment comprises: [ka] has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0336] In one embodiment, the 3' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:323 and the 5' half of the Group I catalytic intron fragment has the sequence of SEQ ID NO:324.

[0337] In another example, linear polyribonucleotides can be circularized or concatemerized by non-nucleic acid moieties that induce attractive forces between atoms or molecular surfaces at, near, or attached to the 5' and 3' ends of the linear polyribonucleotide. One or more linear polyribonucleotides can be circularized or concatemerized by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, agnostic bonds, dipole bonds, conjugation, hyperconjugation, and antibonding.

[0338] In another example, a linear polyribonucleotide can contain ribozyme RNA sequences near the 5' end and near the 3' end. The ribozyme RNA sequences can be covalently linked to a peptide when the sequence is exposed to the remainder of the ribozyme. Peptides covalently linked to the ribozyme RNA sequences near the 5' and 3' ends can bind to each other, thereby circularizing or concatemerizing the linear polyribonucleotide. In another example, peptides covalently linked to the ribozyme RNA near the 5' and 3' ends can circularize or concatemerize a linear primary construct or linear mRNA after subjecting them to ligation using various methods known in the art, including, but not limited to, protein ligation. A non-limiting list of non-limiting examples of ribozymes or methods for incorporating or covalently attaching peptides for use in the linear primary constructs or linear polyribonucleotides of the invention is described in U.S. Patent Application Publication No. 20030082768, the entire contents of which are incorporated herein by reference.

[0339] In yet another example, chemical methods of cyclization can be used to generate cyclic polyribonucleotides, including, but not limited to, click chemistry (e.g., alkyne and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.

[0340] In another example, circular polyribonucleotides can be produced using a deoxyribonucleotide template that is transcribed in a cell-free system (e.g., by in vitro transcription) to produce linear RNA. The linear polyribonucleotide produces a splicing-competent polyribonucleotide, which can self-splice to produce the circular polyribonucleotide.

[0341] In certain embodiments, the present disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide; thereby producing a circular polyribonucleotide.

[0342] In certain embodiments, the present disclosure provides a method of producing a circular polyribonucleotide by providing deoxyribonucleotides encoding a linear polyribonucleotide; transcribing the deoxyribonucleotides in a cell-free system to produce the linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.

[0343] In some embodiments, the present disclosure provides a method for producing a cyclic polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide, and allowing the transcription to occur in solution under conditions suitable for splicing the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a cyclic polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a 5' split-intron and a 3' split-intron (e.g., a self-splicing construct for producing a cyclic polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5' annealing region and a 3' annealing region.

[0344] Suitable conditions for in vitro transcription and / or self-splicing can include any conditions (e.g., a solution or buffer, such as an aqueous buffer or solution) that mimic physiological conditions in one or more respects. In certain embodiments, suitable conditions include 0.1 to 100 mM Mg ions or a salt thereof (e.g., 1 to 100 mM, 1 to 50 mM, 1 to 20 mM, 5 to 50 mM, 5 to 20 mM, or 5 to 15 mM). In certain embodiments, suitable conditions include 1 to 1000 mM K ions or a salt thereof, such as KCl (e.g., 1 to 1000 mM, 1 to 500 mM, 1 to 200 mM, 50 to 500 mM, 100 to 500 mM, or 100 to 300 mM). In certain embodiments, suitable conditions include 1 to 1000 mM Cl ions or a salt thereof, such as KCl (e.g., 1 to 1000 mM, 1 to 500 mM, 1 to 200 mM, 50 to 500 mM, 100 to 500 mM, or 100 to 300 mM). In certain embodiments, suitable conditions include 0.1 to 100 mM Mn ions or a salt thereof, such as MnCl (e.g., 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 20 mM, 0.1 to 10 mM, 0.1 to 5 mM, 0.1 to 2 mM, 0.5 to 50 mM, 0.5 to 20 mM, 0.5 to 15 mM, 0.5 to 5 mM, 0.5 to 2 mM, or 0.1 to 10 mM). In certain embodiments, suitable conditions include dithiothreitol (DTT) (e.g., 1 to 1000 μM, 1 to 500 μM, 1 to 200 μM, 50 to 500 μM, 100 to 500 μM, 100 to 300 μM, 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 20 mM, 0.1 to 10 mM, 0.1 to 5 mM, 0.1 to 2 mM, 0.5 to 50 mM, 0.5 to 20 mM, 0.5 to 15 mM, 0.5 to 5 mM, 0.5 to 2 mM, or 0.1 to 10 mM). In some embodiments, suitable conditions include 0.1 mM to 100 mM ribonucleoside triphosphate (NTP) (e.g., 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 10 mM, 1 to 100 mM, 1 to 50 mM, or 1 to 10 mM). In some embodiments, suitable conditions include a pH of 4 to 10 (e.g., a pH of 5 to 9, a pH of 6 to 9, or a pH of 6.5 to 8.5).In certain embodiments, suitable conditions include a temperature of 4°C to 50°C (eg, 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, or 30°C to 40°C).

[0345] In some embodiments, the linear polyribonucleotide is generated from a deoxyribonucleic acid, e.g., a deoxyribonucleic acid described herein, e.g., a DNA vector, a linearized DNA vector, or a cDNA. In some embodiments, the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).

[0346] In another example, a circular polyribonucleotide can be produced within a cell, e.g., a prokaryotic or eukaryotic cell. In some embodiments, an exogenous polyribonucleotide is provided to a cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding the transcription of a linear polyribonucleotide described herein). The linear polyribonucleotide can be transcribed within the cell from an exogenous DNA molecule provided to the cell. The linear polyribonucleotide can be transcribed within the cell from an exogenous recombinant DNA molecule transiently provided to the cell. In some embodiments, the exogenous DNA molecule does not integrate into the genome of the cell. In some embodiments, the linear polyribonucleotide is transcribed within the cell from a recombinant DNA molecule that is integrated into the genome of the cell.

[0347] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell comprising the polyribonucleotides described herein can be a bacterial cell or an archaeal cell. For example, prokaryotic cells comprising the polyribonucleotides described herein include Escherichia coli, halophilic archaea (e.g., Haloferax volcanii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina spp. (Arthrospira spp.), and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis), and the like. The prokaryotic cells may be Bacillus subtilis, Bacillus anthracis, Bacillus cereus, Betaproteobacteria (e.g., Burkholderia), Alphaproteobacteria (e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), and Enterobacteriaceae. The prokaryotic cells may be grown in a culture medium. The prokaryotic cells may be contained in a bioreactor.

[0348] The cell can be a eukaryotic cell. In some embodiments, the eukaryotic cell is a unicellular eukaryotic cell. In some embodiments, the eukaryotic unicellular cell is a unicellular fungal cell, such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp., and Pichia spp.). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. The unicellular animal cell can be a cell isolated from a multicellular animal and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular animal cell can be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. The unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular plant cell may be dedifferentiated. In some embodiments, the unicellular plant cell is derived from plant callus. In some embodiments, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algal cell, such as a unicellular green alga, diatom, euglenid, or dinoflagellate.Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cell.

[0349] In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryotic organism. For example, the multicellular eukaryotic organism may be selected from the group consisting of a vertebrate, an invertebrate, a multicellular fungus, a multicellular alga, and a multicellular plant. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryotic organism is an invertebrate. In some embodiments, the eukaryotic organism is a multicellular fungus. In some embodiments, the eukaryotic organism is a multicellular plant. In embodiments, the eukaryotic cell is a human cell or a cell of a non-human mammal, such as a non-human primate (e.g., monkey, ape), an ungulate (e.g., bovine, including cow, buffalo, bison, sheep, goat, and muskox; pig; camelid, including camel, llama, and alpaca; deer, antelope; and equid, including horse and donkey), a carnivore (e.g., dog, cat), a rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or a lagomorph (e.g., rabbit, hare). In embodiments, the eukaryotic cell is a cell of an avian, e.g., a member of the avian taxa Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, geese), Paleognatha (e.g., ostrich, emu), Columbiformes (e.g., pigeon, dove), or Psittaciformes (e.g., parrot). In embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, parasitic worm, or mollusk. In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm (which may be a dicotyledon or a monocotyledon) or a gymnosperm (e.g., a conifer, cycad, gnetophyte, ginkgo), a fern, a horsetail, a clubmoss, or a bryophyte. In embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular alga.

[0350] The eukaryotic cells may be grown in a culture medium. The eukaryotic cells may be contained in a bioreactor.

[0351] Examples of bioreactors include, but are not limited to, stirred tank (e.g., well-mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of a bioreactor can be batch or continuous. A bioreactor is continuous when reagent and product streams are continuously supplied and withdrawn from the system. A batch bioreactor may have a continuous recycle stream, but does not have a continuous supply of reagents or product. Some methods of the present disclosure are directed to large-scale production of cyclic polyribonucleotides. For large-scale production methods, the methods can be carried out in volumes of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more). In certain embodiments, the method can be carried out in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 L to 50 L. In certain embodiments, the bioreactor can produce at least 1 g of circular RNA. In some embodiments, a bioreactor can produce 1-200 g of circular RNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, or 50-200 g of circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200 g per liter), per batch or reaction (e.g., 1-200 g per batch or reaction), or per unit of time (e.g., 1-200 g per hour or day). In some embodiments, two or more bioreactors can be used in series to increase production capacity (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 bioreactors can be used in series).

[0352] Methods for producing the circular polyribonucleotides described herein are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012).

[0353] Various methods for synthesizing cyclic polyribonucleotides have also been described elsewhere (see, e.g., U.S. Pat. No. 6,210,931, U.S. Pat. No. 5,773,244, U.S. Pat. No. 5,766,903, U.S. Pat. No. 5,712,128, U.S. Pat. No. 5,426,180, U.S. Patent Application Publication No. 20100137407, WO 1992001813, WO 2010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015), the contents of each of which are incorporated herein by reference in their entirety).

[0354] In some embodiments, the cyclic polyribonucleotides are purified, e.g., free ribonucleic acid, linear or nicked RNA, DNA, proteins, etc. In some embodiments, the cyclic polyribonucleotides can be purified by any known method commonly used in the art. Non-limiting examples of purification methods include column chromatography, gel exclusion, size exclusion, etc.

[0355] Linear Polyribonucleotides The linear polyribonucleotides disclosed herein contain one or more expressible sequences encoding one or more immunogens and / or epitopes from a coronavirus. The linear polyribonucleotides express the sequences encoding one or more immunogens and / or epitopes from a coronavirus in a subject. In some embodiments, linear polyribonucleotides containing one or more coronavirus immunogens and / or epitopes are used to generate an immune response in a subject. In some embodiments, linear polyribonucleotides containing one or more coronavirus immunogens and / or epitopes are used to generate polyclonal antibodies described herein.

[0356] Coronavirus immunogens and epitopes The linear polyribonucleotide comprises a sequence encoding a coronavirus immunogen or epitope. The immunogens and / or epitopes disclosed herein are associated with coronaviruses. In certain embodiments, the immunogens and / or epitopes are expressed by coronaviruses or are derived from immunogens and / or epitopes expressed by coronaviruses.

[0357] In some embodiments, the immunogens and / or epitopes of the present disclosure are derived from predicted transcripts from the SARS-CoV genome. In some embodiments, the immunogens and / or epitopes of the present disclosure are derived from proteins encoded by open reading frames from the SARS-CoV genome. Non-limiting examples of open reading frames in the SARS-CoV genome can include ORF1a, ORF1b, spike (S), ORF3a, ORF3b, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF8a, ORF8b, ORF9a, ORF9b, nucleocapsid (N), and ORF10. In some embodiments, the open reading frame from the SARS-CoV genome comprises SEQ ID NO: 11.

[0358] In certain embodiments, the linear polyribonucleotide comprises a SARS-CoV-2 immunogen listed in Table 6.

[0359] [Table 6]

[0360] In Table 6, "Proline Substitution" indicates a proline substitution at residues 986 and 987 and a "GSAS" substitution at the furin cleavage site (residues 682-685). For cloning optimization, a single base substitution was made at coordinate 2541 to destroy a BsaI site that aids in Golden Gate Cloning construction of the plasmid DNA template. For circularization optimization, four single nucleotides at positions 2307, 2709, 159, and 315 were substituted to destroy potential binding sites for the circularization element of the splint nucleic acid sequence, thereby potentially inhibiting efficient ligation. All single bp substitutions were designed to be translationally silent. Furthermore, in Table 6, the 5' element is globin (SEQ ID NO: 32); the 3' element is globin (SEQ ID NO: 33).

[0361] In some embodiments, the linear polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-111 and 293-295. In some embodiments, the linear polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-111 and 293-295. In some embodiments, the linear polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 63-111 and 293-295. In some embodiments, the linear polyribonucleotide comprises an open reading frame encoding a SARS-CoV-2 immunogen having an amino acid sequence that is any one of SEQ ID NOs: 63-111 and 293-295. In some embodiments, the SARS-CoV-2 immunogen is an immunogenic fragment comprising a contiguous stretch of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 amino acids of any one of SEQ ID NOs: 63-111 and 293-295. In some embodiments, the SARS-CoV-2 immunogen is an immunogenic fragment comprising a contiguous stretch of at least 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids of any one of the sequences of SEQ ID NOs: 63-111 and 293-295.

[0362] In certain embodiments, the linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-174 and 292-300. In some embodiments, the linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 112-174 and 292-300. In certain embodiments, the linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 112-174 and 292-300. In certain embodiments, the polyribonucleotide sequence encoding a SARS-CoV-2 immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 112-174 and 292-300. In one embodiment, the polyribonucleotide sequence encoding the SARS-CoV-2 immunogen is a fragment comprising at least a 50%, 60%, 70%, 80%, 90%, or 95% contiguous stretch of the amino acids of any one of SEQ ID NOs: 112-174 and 292-300.

[0363] In certain embodiments, the linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 80% (e.g., about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 219-281. In certain embodiments, a linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 90% (e.g., about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 219-281. In certain embodiments, a linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence has at least about 95% (e.g., about 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 219-281. In certain embodiments, a linear polyribonucleotide comprises an open reading frame having a nucleic acid sequence encoding a SARS-CoV-2 immunogen, wherein the nucleic acid sequence is any one of SEQ ID NOs: 219-281. In some embodiments, the polyribonucleotide sequence encoding the SARS-CoV-2 immunogen is a fragment comprising a contiguous stretch of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides of any one of SEQ ID NOs: 219 to 281. In some embodiments, the polyribonucleotide sequence encoding the SARS-CoV-2 immunogen is a fragment comprising a contiguous stretch of at least 50%, 60%, 70%, 80%, 90%, or 95% of the amino acids of any one of SEQ ID NOs: 219 to 281.

[0364] The present disclosure specifically contemplates that any of the DNA sequences described herein can be converted into a corresponding RNA sequence and included in the RNA molecules described herein.

[0365] In certain embodiments, a coronavirus epitope comprises or contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids, or more. In certain embodiments, a coronavirus epitope comprises or contains 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, or 30 or less amino acids, or fewer. In some embodiments, a coronavirus epitope comprises or contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, a coronavirus epitope contains 5 amino acids. In some embodiments, a coronavirus epitope contains 6 amino acids. In some embodiments, an epitope contains 7 amino acids. In some embodiments, a coronavirus epitope contains 8 amino acids. In some embodiments, an epitope can be about 8 to about 11 amino acids. In some embodiments, an epitope can be about 9 to about 22 amino acids.

[0366] The coronavirus immunogen may comprise an immunogen recognized by B cells, an immunogen recognized by T cells, or a combination thereof. In some embodiments, the immunogen comprises an immunogen recognized by B cells. In some embodiments, the coronavirus immunogen is an immunogen recognized by B cells. In some embodiments, the coronavirus immunogen comprises an immunogen recognized by T cells. In some embodiments, the immunogen is an immunogen recognized by T cells.

[0367] Coronavirus epitopes include epitopes recognized by B cells, epitopes recognized by T cells, or a combination thereof. In some embodiments, coronavirus epitopes include epitopes recognized by B cells. In some embodiments, the epitopes are epitopes recognized by B cells. In some embodiments, coronavirus epitopes include epitopes recognized by T cells. In some embodiments, coronavirus epitopes are epitopes recognized by T cells.

[0368] Techniques for identifying immunogens and epitopes in silico have been described, for example, in Sanchez-Trincado, et al. (2017), Fundamentals and methods for T- and B-cell epitope prediction, Journal of immunology research; Grifoni, Alba, et al., A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2. Cell host & microbe (2020); Russi et al., In silico prediction of T- and B-cell epitopes in PmpD: First step towards the design of a Chlamydia trachomatis vaccine. BIOMEDICAL JOURNAL 41.2(2018):109-17; Baruah, et al., Immunoinformatics-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV. Journal of Medical Virology (2020); each of which is incorporated herein by reference in its entirety.

[0369] The linear polyribonucleotides of the present disclosure can comprise sequences of a variety of coronavirus immunogens and / or epitopes, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more coronavirus immunogens or epitopes. In certain embodiments, the linear polyribonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more immunogenic or epitope sequences, e.g., derived from a target other than a coronavirus.

[0370] In certain embodiments, the linear polyribonucleotide comprises the sequence of, for example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or fewer coronavirus immunogens or epitopes. In certain embodiments, the linear polyribonucleotide comprises, e.g., 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or fewer sequences of immunogens or epitopes derived from a target other than a coronavirus.

[0371] In certain embodiments, the linear polyribonucleotide comprises the sequence of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus immunogens or epitopes. In certain embodiments, the linear polyribonucleotide comprises, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 sequences of immunogens or epitopes derived from a source other than a coronavirus.

[0372] The linear polyribonucleotide can include the sequence of one or more coronavirus epitopes from a coronavirus immunogen. For example, a coronavirus immunogen can include an amino acid sequence that contains multiple coronavirus epitopes therein (e.g., epitopes recognized by B cells and / or T cells), and the linear polyribonucleotide can include or encode one or more of those coronavirus epitopes.

[0373] A linear polyribonucleotide may comprise, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more epitope sequences from one coronavirus immunogen.

[0374] In certain embodiments, the linear polyribonucleotide comprises a sequence of, e.g., 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, or 500 or less coronavirus epitopes from one coronavirus immunogen.

[0375] In one embodiment, the linear polyribonucleotide comprises a sequence of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus epitopes from one coronavirus immunogen.

[0376] The linear polyribonucleotide can encode a variant of a coronavirus immunogen or epitope. The variant can be a naturally occurring variant (e.g., a variant identified in sequence data from different coronavirus genera, species, isolates, or quasispecies) or can be a derivative sequence disclosed herein that has been generated in silico (e.g., an immunogen or epitope having one or more amino acid insertions, deletions, substitutions, or a combination thereof compared to the wild-type immunogen or epitope).

[0377] A linear polyribonucleotide may, for example, comprise the sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more variants of a coronavirus immunogen or epitope.

[0378] In certain embodiments, the linear polyribonucleotide comprises a sequence of, for example, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or fewer variants of a coronavirus immunogen or epitope.

[0379] In some embodiments, the linear polyribonucleotide comprises the sequence of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 variants of a coronavirus immunogen or epitope.

[0380] Coronavirus immunogen and / or epitope sequences of linear polyribonucleotides may also be referred to as coronavirus expression sequences. In certain embodiments, a linear polyribonucleotide comprises one or more coronavirus expression sequences, each of which may encode a coronavirus polypeptide. Coronavirus polypeptides may be produced in significant quantities. Coronavirus polypeptides may be secreted from cells or localized to the cytoplasm, nucleus, or membrane compartment of a cell. Some coronavirus polypeptides include, but are not limited to, at least a portion of an immunogen disclosed herein, an epitope disclosed herein, or a coronavirus protein (e.g., a viral envelope protein, a viral matrix protein, a viral spike protein, a viral membrane protein, a viral nucleocapsid protein, a viral accessory protein, a fragment thereof, or a combination thereof). In certain embodiments, a coronavirus polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a fragment of a coronavirus immunogen disclosed herein. In certain embodiments, a coronavirus polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus immunogens disclosed herein, or a fragment thereof. In some embodiments, a coronavirus polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a coronavirus epitope. In some embodiments, a polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus epitopes disclosed herein, e.g., an artificial peptide sequence comprising multiple predicted epitopes from one or more coronaviruses of the present disclosure.

[0381] In certain embodiments, exemplary coronavirus proteins expressed from the linear polyribonucleotides disclosed herein include secreted proteins, e.g., proteins (e.g., immunogens and / or epitopes) that naturally include a signal peptide, or those that do not normally encode a signal peptide but have been modified to include one.

[0382] Linear polyribonucleotide elements The linear polyribonucleotide comprises the elements described below as well as the coronavirus immunogens or epitopes described herein.

[0383] A linear polyribonucleotide as described herein is a polyribonucleotide molecule having a 5'-end and a 3'-end. In some embodiments, the linear RNA has a free 5'-end or a free 3'-end. In some embodiments, the linear RNA has a 5'-end or a free 3'-end that is modified or protected from degradation. In some embodiments, the linear RNA has a non-covalently linked 5'-end or a non-covalently linked 3'-end. In some embodiments, the linear RNA is an mRNA.

[0384] In certain embodiments, a linear polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6 ,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0385] A linear polyribonucleotide of the present disclosure can include any element or combination of elements described herein, for example, any element or combination of elements described above with respect to circular polyribonucleotides. A linear polyribonucleotide can include any one or more of the IRES, signal sequence, regulatory element, cleavage domain, translation initiation sequence, untranslated region, termination element, or modification described herein (e.g., with respect to circular polyribonucleotides described above). A linear polyribonucleotide can include such elements in any number or structure described herein (e.g., with respect to circular polyribonucleotides described above).

[0386] Methods for generating an immune response The present disclosure provides immunogenic compositions comprising the above-described cyclic polyribonucleotides. The present disclosure provides immunogenic compositions comprising the above-described linear polyribonucleotides. The immunogenic compositions of the present invention may comprise a diluent or carrier, an adjuvant, or any combination thereof. The immunogenic compositions of the present invention may also comprise one or more immunomodulatory agents, for example, one or more adjuvants. The adjuvant may comprise a TH1 adjuvant and / or a TH2 adjuvant, as further described below. In some embodiments, the immunogenic composition comprises a diluent without a carrier and is used for naked delivery of a cyclic polyribonucleotide to a subject (e.g., a subject for immunization). In some embodiments, the immunogenic composition comprises a diluent without a carrier and is used for naked delivery of a linear polyribonucleotide to a subject.

[0387] The immunogenic compositions of the present invention are used to raise an immune response in a subject (e.g., a subject for immunization). The immune response can include an antibody response (usually including IgG) and / or a cell-mediated immune response. In some embodiments, the immunogenic compositions are used to generate polyclonal antibodies described herein. For example, a subject is immunized with an immunogenic composition comprising a cyclic polyribonucleotide comprising a coronavirus immunogen and / or epitope to stimulate the production of polyclonal antibodies that bind to the coronavirus immunogen and / or epitope. In another example, a subject is immunized with an immunogenic composition comprising a linear polyribonucleotide comprising a coronavirus immunogen and / or epitope to stimulate the production of polyclonal antibodies that bind to the coronavirus immunogen and / or epitope. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human animal has a humanized immune system. In some embodiments, the subject is further immunized with an adjuvant. In some embodiments, the subject is further immunized with a vaccine. Optionally, after immunization with an immunogenic composition comprising cyclic polyribonucleotides, the polyclonal antibodies generated are collected from the subject and purified. Optionally, after immunization with an immunogenic composition comprising linear polyribonucleotides, the polyclonal antibodies generated are collected from the subject and purified. In certain embodiments, the composition comprises plasma collected after administration of the immunogenic compositions described herein.

[0388] immunization In some embodiments, the methods of the present disclosure include immunizing a subject (e.g., a subject for immunization) with an immunogenic composition comprising a cyclic polyribonucleotide disclosed herein. In some embodiments, a coronavirus immunogen and / or epitope is expressed from the cyclic polyribonucleotide. In some embodiments, immunization induces an immune response in the subject against the coronavirus immunogen and / or epitope expressed from the cyclic polyribonucleotide. In some embodiments, immunization induces the production of polyclonal antibodies that bind to the coronavirus immunogen and / or epitope expressed from the immunogenic composition. In some embodiments, the immunogenic composition comprises a cyclic polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof in a single composition. In some embodiments, the subject is further immunized with a second adjuvant. In some embodiments, the subject is further immunized with a vaccine.

[0389] In some embodiments, the methods of the present disclosure include immunizing a subject (e.g., a subject for immunization) with an immunogenic composition comprising a linear polyribonucleotide disclosed herein. In some embodiments, the coronavirus immunogen and / or epitope is expressed from the linear polyribonucleotide. In some embodiments, the immunization induces an immune response in the subject against the coronavirus immunogen and / or epitope expressed from the linear polyribonucleotide. In some embodiments, the immunization induces the production of polyclonal antibodies that bind to the coronavirus immunogen and / or epitope expressed from the linear polyribonucleotide. In some embodiments, the immunogenic composition comprises the linear polyribonucleotide and a diluent, carrier, first adjuvant, or a combination thereof in a single composition. In some embodiments, the subject is further immunized with a second adjuvant. In some embodiments, the subject is further immunized with a vaccine.

[0390] The cyclic polyribonucleotides disclosed herein stimulate the production of human polyclonal antibodies by stimulating an adaptive immune response after immunization of a subject (e.g., a subject for immunization). In certain embodiments, the subject's adaptive immune response comprises stimulating B lymphocytes to release polyclonal antibodies that specifically bind to the coronavirus immunogen expressed by the cyclic polyribonucleotide. The linear polyribonucleotides disclosed herein stimulate the production of human polyclonal antibodies by stimulating an adaptive immune response after immunization of a subject. In certain embodiments, the subject's adaptive immune response comprises stimulating B lymphocytes to release polyclonal antibodies that specifically bind to the coronavirus immunogen expressed by the linear polyribonucleotide. In certain embodiments, the subject's adaptive immune response comprises stimulating a cell-mediated immune response.

[0391] A subject (e.g., a subject for immunization) is immunized with one or more immunogenic compositions comprising several cyclic polyribonucleotides. The subject is, for example, immunized with one or more immunogenic compositions comprising at least one cyclic polyribonucleotide. A non-human animal having a non-humanized immune system is immunized with one or more immunogenic compositions comprising, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or more different cyclic polyribonucleotides. In some embodiments, a subject is immunized with one or more immunogenic compositions comprising no more than one cyclic polyribonucleotide. In some embodiments, a subject is immunized with one or more immunogenic compositions comprising about one cyclic polyribonucleotide. In certain embodiments, a subject is immunized with one or more immunogenic compositions comprising about 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different cyclic polyribonucleotides. Different cyclic polyribonucleotides have different sequences from each other. For example, they may contain or encode different immunogens and / or epitopes, overlapping immunogens and / or epitopes, similar immunogens and / or epitopes, or the same immunogen and / or epitope (e.g., with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the present disclosure). When a subject is immunized with one or more immunogenic compositions comprising two or more different cyclic polyribonucleotides, the two or more different cyclic polyribonucleotides may be in the same or different immunogenic compositions and may be immunized at the same time or at different times.Immunogenic compositions comprising two or more different cyclic polyribonucleotides can be administered to the same anatomical location or to different anatomical locations.

[0392] The two or more different cyclic polyribonucleotides may comprise or encode immunogens and / or epitopes from the same coronavirus, different coronaviruses, or different combinations of coronaviruses disclosed herein. The two or more different cyclic polyribonucleotides may comprise or encode immunogens and / or epitopes from the same coronavirus or different coronaviruses, e.g., different isolates.

[0393] A subject (e.g., a subject for immunization) is immunized with one or more immunogenic compositions comprising several linear polyribonucleotides. The subject is, for example, immunized with one or more immunogenic compositions comprising at least one linear polyribonucleotide. A non-human animal having a non-humanized immune system is immunized with one or more immunogenic compositions comprising, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or more different linear polyribonucleotides. In one embodiment, the subject is immunized with one or more immunogenic compositions comprising no more than one linear polyribonucleotide. In one embodiment, the subject is immunized with one or more immunogenic compositions comprising about one linear polyribonucleotide. In certain embodiments, the subject receives a blood glucose level of about 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3 Immunization is with one or more immunogenic compositions comprising from 5, 3 to 4, 4 to 20, 4 to 15, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 4 to 4, 4 to 3, 5 to 20, 5 to 15, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 5 to 10, 10 to 15, or 15 to 20 different linear polyribonucleotides. The different linear polyribonucleotides have different sequences from each other. For example, they may contain or encode different immunogens and / or epitopes, overlapping immunogens and / or epitopes, similar immunogens and / or epitopes, or the same immunogen and / or epitope (e.g., with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the present disclosure). When a subject is immunized with one or more immunogenic compositions comprising two or more different linear polyribonucleotides, the two or more different linear polyribonucleotides may be in the same or different immunogenic compositions and may be immunized at the same time or at different times.Immunogenic compositions comprising two or more different linear polyribonucleotides can be administered to the same anatomical location or to different anatomical locations.

[0394] The two or more different linear polyribonucleotides can comprise or encode immunogens and / or epitopes from the same coronavirus, different coronaviruses, or different combinations of coronaviruses disclosed herein. The two or more different linear polyribonucleotides can comprise or encode immunogens and / or epitopes from the same coronavirus or different coronaviruses, e.g., different isolates.

[0395] In some embodiments, a subject (e.g., a subject for immunization) is immunized with one or more immunogenic compositions comprising a number of cyclic polyribonucleotides and one or more immunogenic compositions comprising a number of linear polyribonucleotides disclosed herein. In some embodiments, the immunogenic compositions disclosed herein comprise one or more cyclic polyribonucleotides and one or more linear polyribonucleotides disclosed herein.

[0396] In some embodiments, the immunogenic composition comprises a cyclic polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof. In certain embodiments, the immunogenic composition comprises a cyclic polyribonucleotide described herein and a carrier, or a diluent without a carrier. In some embodiments, an immunogenic composition comprising a cyclic polyribonucleotide together with a diluent without a carrier is used for naked delivery of the cyclic polyribonucleotide to a subject. In another specific embodiment, the immunogenic composition comprises a cyclic polyribonucleotide described herein and a first adjuvant.

[0397] In certain embodiments, a second adjuvant is further administered to a subject (e.g., a subject for immunization). The adjuvant promotes the innate immune response, which in turn promotes the adaptive immune response for the production of polyclonal antibodies in the subject. The adjuvant can be any adjuvant described below. In certain embodiments, the adjuvant is formulated with the cyclic polyribonucleotide as part of the immunogenic composition. In certain embodiments, the adjuvant is not part of the immunogenic composition comprising the cyclic polyribonucleotide. In certain embodiments, the adjuvant is administered separately from the immunogenic composition comprising the cyclic polyribonucleotide. In this aspect, the adjuvant is co-administered (e.g., administered simultaneously) or at different times with the immunogenic composition comprising the cyclic polyribonucleotide to the subject. For example, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minutes or hours therebetween, after the immunogenic composition comprising a cyclic polyribonucleotide. In one embodiment, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minutes or hours therebetween, before the immunogenic composition comprising a cyclic polyribonucleotide. For example, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days after the immunogenic composition comprising the cyclic polyribonucleotide, or any number of days therebetween. In one embodiment, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days before the immunogenic composition comprising the cyclic polyribonucleotide, or any number of days therebetween.The adjuvant may be administered in the same anatomical location as the immunogenic composition comprising the cyclic polyribonucleotide or in a different anatomical location.

[0398] In some embodiments, the immunogenic composition comprises a linear polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof. In certain embodiments, the immunogenic composition comprises a linear polyribonucleotide described herein and a carrier, or a diluent without a carrier. In some embodiments, an immunogenic composition comprising a linear polyribonucleotide together with a diluent with...

Claims

1. 1. A circular polyribonucleotide comprising an open reading frame encoding a coronavirus immunogen, wherein the circular polyribonucleotide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291, wherein the circular polyribonucleotide further comprises a sequence encoding an influenza immunogen.

2. 2. The cyclic polyribonucleotide of claim 1, wherein the coronavirus immunogen is a spike immunogen having at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, or 283-286.

3. 2. The cyclic polyribonucleotide of claim 1, wherein the coronavirus immunogen is an RBD immunogen having at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 63-68, 74, 79, 81-86, or 98-111.

4. 2. The cyclic polyribonucleotide of claim 1, wherein the coronavirus immunogen is a nonstructural protein (nsp) having at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 287-291.

5. 2. The cyclic polyribonucleotide of claim 1, wherein the coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291.

6. 2. The circular polyribonucleotide of claim 1, wherein the open reading frame comprises a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 112-174 and 292-300.

7. 7. The cyclic polyribonucleotide of claim 6, wherein the coronavirus immunogen is an RBD immunogen having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 112-117, 123, 128, 133-138, and 163-174.

8. 7. The cyclic polyribonucleotide of claim 6, wherein the coronavirus immunogen is a spike immunogen having at least 95% identity to any one of the nucleic acid sequences of SEQ ID NOs: 118-122, 124-127, 129-132, 139-162, and 292-295.

9. 7. The circular polyribonucleotide of claim 6, wherein the coronavirus immunogen is an nsp having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 296-300.

10. A cyclic polyribonucleotide comprising a first sequence encoding a coronavirus immunogen having at least 95% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291, and a second sequence encoding a polypeptide adjuvant.

11. A circular polyribonucleotide comprising a first sequence encoding a coronavirus immunogen having at least 95% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291, and a second sequence encoding a multimerization domain.

12. An immunogenic composition comprising a first cyclic polyribonucleotide comprising an open reading frame encoding a coronavirus immunogen, a second cyclic polyribonucleotide, and a pharmaceutically acceptable carrier or excipient, wherein the coronavirus immunogen comprises an amino acid sequence having at least 95% sequence identity with any one of the amino acid sequences set forth in SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291.

13. The immunogenic composition of claim 12, wherein the second circular polyribonucleotide comprises an open reading frame encoding a polypeptide immunogen.

14. The immunogenic composition of claim 12, wherein the second cyclic polyribonucleotide comprises an open reading frame encoding a polypeptide adjuvant.

15. 1. A linear polyribonucleotide comprising an open reading frame encoding a coronavirus immunogen, wherein the coronavirus immunogen comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291, and wherein the linear polyribonucleotide further comprises a sequence encoding an influenza immunogen.

16. 16. The linear polyribonucleotide of claim 15, wherein the coronavirus immunogen comprises the amino acid sequence of any one of SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291.

17. 16. The linear polyribonucleotide of claim 15, wherein the open reading frame comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300.

18. 18. The linear polyribonucleotide of claim 17, wherein the open reading frame comprises the nucleic acid sequence of any one of SEQ ID NOs: 112-174 and 292-300.

19. A linear polyribonucleotide comprising: a first sequence encoding a coronavirus immunogen having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 69-73, 75-78, 80, 87-97, 283-286, 63-68, 74, 79, 81-86, 98-111, or 287-291; and a second sequence encoding a multimerization domain, a second coronavirus immunogen, and / or a polypeptide adjuvant.

20. 16. An immunogenic composition comprising the linear polyribonucleotide of claim 15 and a pharmaceutically acceptable carrier or excipient.

21. 21. The immunogenic composition of claim 20, wherein the composition further comprises a second linear polyribonucleotide.

22. 22. The immunogenic composition of claim 21, wherein the second linear polyribonucleotide comprises an open reading frame encoding a polypeptide immunogen.

23. 22. The immunogenic composition of claim 21, wherein the second linear polyribonucleotide comprises an open reading frame encoding a polypeptide adjuvant.

24. 12. A composition comprising the cyclic polyribonucleotide of any one of claims 1 to 11 for use in a method of inducing an immune response to SARS-CoV-2 or preventing SARS-CoV-2 infection in a subject, said method comprising administering said composition to said subject.

25. A composition comprising a linear polyribonucleotide according to any one of claims 15 to 19 for use in a method for inducing an immune response to SARS-CoV-2 in a subject or for preventing SARS-CoV-2 infection in a subject, the method comprising administering the composition to the subject.

26. An immunogenic composition for use in a method for inducing an immune response to SARS-CoV-2 in a subject or for preventing SARS-CoV-2 infection in a subject, said method comprising administering to said subject an immunogenic composition according to any one of claims 12 to 14 and 20 to 23.

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