Vaccines and Related Methods

Abstract

Provided herein are SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and immunogenic variants thereof)) that comprise at least one set of amino acid substitutions, and nucleic acid molecules encoding them. Further provided herein are compositions (e.g., pharmaceutical compositions) and vaccines comprising same, e.g., for use in preventing, treating, and / or ameliorating SARS-CoV-2 infection.

Description

[Technical field]

[0001] 1. Related Applications This application claims priority to U.S. Patent Application No. 63 / 362,710, filed April 8, 2022, and U.S. Patent Application No. 63 / 476,310, filed December 20, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] 2. The present disclosure relates to SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and / or immunogenic variants thereof)) that comprise at least one set of amino acid substitutions described herein, and the nucleic acid molecules encoding them. The disclosure further relates to compositions (e.g., vaccine compositions, pharmaceutical compositions) comprising them, and methods of making and using them. [Background technology]

[0003] 3. Coronaviruses are a family of enveloped, single-stranded, positive-sense RNA viruses that infect a wide variety of mammalian and avian species. The viral genome is packaged into a capsid composed of viral nucleocapsid proteins and surrounded by a lipid envelope. Several proteins are incorporated into the lipid envelope, including membrane proteins, small envelope proteins, hemagglutinin-esterase, and spike proteins. Human coronaviruses usually cause respiratory diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV).

[0004] SARS-Cov-2 emerged in humans in 2019 and spread rapidly, causing an ongoing global pandemic. SARS-CoV-2 is the cause of coronavirus disease 2019 (COVID-19). COVID-19 has caused an ongoing public health crisis, with millions of deaths and severe illnesses attributed to COVID-19 worldwide. Protection against COVID-19 is, in large part, mediated by immune responses directed against the SARS-CoV-2 spike protein, the primary target of SARS-CoV-2 vaccines. The spike protein mediates binding and entry into host cells via binding of the receptor-binding domain (RBD) to the host cell receptor angiotensin-converting enzyme 2 (ACE2). Summary of the Invention [Means for solving the problem]

[0005] 4. Provided herein, inter alia, are SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein or polypeptide immunogens (as well as immunogenic fragments and / or immunogenic variants thereof)), nucleic acid molecules encoding them, compositions (e.g., vaccine compositions, pharmaceutical compositions) comprising SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein or polypeptide immunogens (as well as immunogenic fragments and / or immunogenic variants thereof)) or nucleic acid molecules encoding them, methods of making them, including, e.g., methods of preventing, ameliorating, or treating SARS-CoV-2 infection, methods of vaccinating against SARS-CoV-2 infection, etc., and methods of using them.

[0006] In one aspect, provided herein, inter alia, is a nucleic acid molecule that includes a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one set of amino acid substitutions set forth in Table 2.

[0007] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD).

[0008] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of the full-length SARS-CoV-2 spike protein.

[0009] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.

[0010] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, or 10-1300 amino acids.

[0011] In some embodiments, other than at least one set of amino acid substitutions set forth in Table 2, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90 ...20000, 30000, 40000, 50000, 60000, 70000, 80 , 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.

[0012] In some embodiments, other than at least one set of amino acid substitutions set forth in Table 2, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:1-4.

[0013] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises multiple sets of amino acid substitutions set forth in Table 2.

[0014] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least a set of 2, 3, 4, 5, or 6 or more of the amino acid substitutions set forth in Table 2.

[0015] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%, less than 5%) amino acid changes (e.g., substitutions, additions, deletions, etc.) not listed in Table 2.

[0016] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes (e.g., substitutions, additions, deletions, etc.) not set forth in Table 2 relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4, but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%, less than 5%).

[0017] In some embodiments, the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) is stabilized in the pre-fusion state.

[0018] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 that stabilizes the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) in the pre-fusion state.

[0019] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) includes a proline at amino acid position 986 and / or a proline at amino acid position 987, which are amino acids numbered relative to the amino acid positions set forth in SEQ ID NO:4.

[0020] In some embodiments, the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises one or more non-native N-glycosylation sites.

[0021] In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises the addition of one or more N-glycosylation sites relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

[0022] In some embodiments, the encoded SARS-CoV-2 spike protein is an immunogen (or an immunogenic fragment and / or immunogenic variant thereof).

[0023] In some embodiments, the nucleic acid molecule is RNA or DNA. In some embodiments, the RNA is messenger ribonucleic acid (mRNA).

[0024] In some embodiments, the nucleic acid molecule comprises at least one modified nucleotide, hi some embodiments, the nucleic acid molecule comprises N1-methyl-pseudouridine, cytosine, adenine, and guanine.

[0025] In some embodiments, the nucleic acid molecule comprises a heterologous 5'-untranslated region (UTR), a 3'-UTR, or both a 5'-UTR and a 3'-UTR. In some embodiments, the nucleic acid molecule comprises a poly(A) sequence. In some embodiments, the nucleic acid molecule comprises a 5' cap structure. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0026] In some embodiments, the nucleic acid molecule further encodes a heterologous polypeptide or protein.

[0027] In some embodiments, the nucleic acid molecule encodes a signal peptide. In some embodiments, the nucleic acid molecule encodes a homologous or heterologous signal peptide.

[0028] In one aspect, provided herein is a vector comprising a nucleic acid described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)). In some embodiments, the vector is a non-viral vector (e.g., a plasmid) or a viral vector.

[0029] In one aspect, provided herein is a conjugate that includes a nucleic acid molecule described herein (e.g., a nucleic acid molecule that includes a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) operably linked (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety (e.g., a heterologous polypeptide or protein).

[0030] In one aspect, provided herein is a composition comprising at least one nucleic acid molecule described herein. In some embodiments, the composition comprises a plurality of nucleic acid molecules described herein (e.g., a plurality of nucleic acid molecules each comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof))), wherein the amino acid sequence of each of the encoded SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) encoded by each of the plurality of nucleic acid molecules comprises a different set of amino acid substitutions as set forth in Table 2. In some embodiments, the composition comprises a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprising an amino acid sequence that does not include the set of amino acid substitutions as set forth in Table 2.

[0031] In one aspect, provided herein is a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one set of amino acid substitutions set forth in Table 2.

[0032] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

[0033] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of the full-length SARS-CoV-2 spike protein.

[0034] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids.

[0035] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, or 10-1300 amino acids.

[0036] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) other than at least one set of amino acid substitutions comprises at least a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 9 It is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or 1200 amino acids.

[0037] In some embodiments, other than at least one set of amino acid substitutions, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:1-4.

[0038] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises multiple amino acid substitutions as set forth in Table 2.

[0039] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least a set of 2, 3, 4, 5, or 6 or more of the amino acid substitutions set forth in Table 2.

[0040] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%, less than 5%) amino acid changes (e.g., substitutions, additions, deletions, etc.) that are not listed in Table 2.

[0041] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes (e.g., substitutions, additions, deletions, etc.) not listed in Table 2, relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4, but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%, less than 5%).

[0042] In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) is stabilized in the pre-fusion state.

[0043] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 that stabilizes the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) in the pre-fusion state.

[0044] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) includes a proline at amino acid position 986 and / or a proline at amino acid position 987, which are amino acids numbered relative to the amino acid positions set forth in SEQ ID NO:4.

[0045] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises one or more non-native N-glycosylation sites.

[0046] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises the addition of one or more N-glycosylation sites relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

[0047] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises an inactive furin cleavage site.

[0048] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change in the furin cleavage site that inactivates the furin cleavage site.

[0049] In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) further comprises a heterologous protein.

[0050] In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a signal peptide. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a homologous or heterologous signal peptide. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) does not comprise a signal peptide.

[0051] In some embodiments, the SARS-CoV-2 spike protein is the immunogen (or an immunogenic fragment and / or immunogenic variant thereof).

[0052] In one aspect, provided herein are compositions comprising at least one SARS-CoV-2 spike protein (e.g., at least one SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) described herein (e.g., a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof) comprising at least one set of amino acid substitutions described in Table 2). In some embodiments, the composition comprises multiple SARS-CoV-2 spike proteins (e.g., multiple SARS-CoV-2 spike protein immunogens (or immunogenic fragments and / or immunogenic variants thereof) described herein. In some embodiments, the composition comprises a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), wherein the amino acid sequence of each of the multiple SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a different set of amino acid substitutions set forth in Table 2. In some embodiments, the composition comprises at least one SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprising an amino acid sequence that does not include the set of amino acid substitutions set forth in Table 2.

[0053] In one aspect, provided herein is a fusion protein comprising a SARS-CoV-2 spike protein described herein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) operably linked (e.g., directly or indirectly (e.g., via a linker)) to a heterologous polypeptide or protein (e.g., a SARS-CoV-2 spike protein comprising at least one set of amino acid substitutions described in Table 2 (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

[0054] In one aspect, provided herein is a conjugate comprising a SARS-CoV-2 spike protein described herein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) operably linked (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety (e.g., a SARS-CoV-2 spike protein comprising at least one set of amino acid substitutions described in Table 2 (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

[0055] In one aspect, provided herein is a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) encoded by a nucleic acid molecule described herein.

[0056] In one aspect, provided herein is a nucleic acid molecule encoding a SARS-CoV-2 spike protein described herein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

[0057] In one aspect, provided herein is a carrier comprising a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, or a pharmaceutical composition described herein. In some embodiments, the carrier is a lipid nanoparticle (LNP), a liposome, a lipoplex, or a nanoliposome. In some embodiments, the carrier is an LNP. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and / or a PEG lipid. In some embodiments, the LNP has an average particle size between 80 nm and 160 nm.

[0058] In one aspect, described herein is a vaccine composition comprising a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, or a pharmaceutical composition described herein.

[0059] In some embodiments, the vaccine composition is a prime vaccine composition. In some embodiments, the vaccine composition is a boost vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition and a boost vaccine composition. In some embodiments, the vaccine composition may be utilized as a prime vaccine composition and / or a booster vaccine composition in a homologous or heterologous prime boost vaccine regimen. In some embodiments, the vaccine composition further comprises an adjuvant.

[0060] In one aspect, provided herein is a vaccine composition comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof) comprising at least one amino acid substitution as set forth in Table 2) formulated in a lipid nanoparticle, the vaccine composition having the following characteristics: (a) the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid; (b) the LNP has an average particle size between 80 nm and 160 nm; and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region.

[0061] In some embodiments, the vaccine composition is a prime vaccine composition. In some embodiments, the vaccine composition is a boost vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition and a boost vaccine composition. In some embodiments, the vaccine composition may be utilized as a prime vaccine composition and / or a booster vaccine composition in a homologous or heterologous prime boost vaccine regimen. In some embodiments, the vaccine composition further comprises an adjuvant.

[0062] In one aspect, provided herein is a pharmaceutical composition comprising a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, or a vaccine composition described herein, and a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an adjuvant.

[0063] In one aspect, provided herein is a pharmaceutical composition comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof) comprising at least one amino acid substitution as set forth in Table 2) formulated in a lipid nanoparticle, the pharmaceutical composition having the following characteristics: (a) the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, (b) the LNP has an average particle size between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region. In some embodiments, the pharmaceutical composition further comprises an adjuvant.

[0064] In one aspect, provided herein is a kit comprising a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0065] In some embodiments, the kit comprises instructions for use of the nucleic acid molecule, vector, protein (or an immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition.

[0066] In one aspect, described herein is a method of delivering a nucleic acid molecule, vector, protein (or an immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition to a subject in need thereof, comprising administering to the subject a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby delivering the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition to the subject.

[0067] In some embodiments, the subject is a human.

[0068] In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to a subject at least twice. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.

[0069] In one aspect, provided herein is a method of inducing or enhancing an immune response in a subject in need thereof, comprising administering to the subject a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby inducing or enhancing the immune response in the subject.

[0070] In some embodiments, the subject is a human.

[0071] In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to a subject at least twice. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.

[0072] In one aspect, provided herein is a method of preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby preventing, ameliorating, or treating a SARS-CoV-2 infection in the subject.

[0073] In some embodiments, the subject is a human.

[0074] In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to a subject at least twice. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.

[0075] In one aspect, provided herein is a method of vaccinating a subject against SARS-CoV-2, the method comprising administering to the subject a nucleic acid molecule described herein, a vector described herein, a composition described herein, a SARS-CoV-2 spike protein described herein, a vaccine composition described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby vaccinating the subject against SARS-CoV-2.

[0076] In some embodiments, the subject is a human.

[0077] In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to a subject at least twice. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.

[0078] In one aspect, provided herein is a method of vaccinating a subject against SARS-CoV-2, the method comprising administering to the subject (a) an mRNA molecule (e.g., an mRNA molecule described herein) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or a polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) described herein (or a conjugate or fusion protein thereof); (b) a vector comprising the mRNA molecule; (c) a carrier comprising the mRNA molecule or vector; (d) a vaccine composition comprising the mRNA molecule, vector, or carrier; or (e) a pharmaceutical composition comprising the mRNA molecule, vector, carrier, or vaccine composition, thereby vaccinating the subject against SARS-CoV-2.

[0079] In some embodiments, the subject is a human.

[0080] In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to a subject at least twice. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.

[0081] In one aspect, provided herein is a method of vaccinating a subject against SARS-CoV-2, comprising administering to the subject a vaccine composition described herein or a pharmaceutical composition described herein, thereby vaccinating the subject against SARS-CoV.

[0082] In some embodiments, the subject is a human.

[0083] In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to a subject at least twice. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen. In some embodiments, the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] 5. SARS-CoV-2 continues to evolve into novel variants that contain a variety of amino acid changes, e.g., substitutions, deletions, and additions. Many of the changes are found in the RBD of the spike protein, which is important for SARS-CoV-2 viral entry into host cells. Because the majority of SARS-CoV-2 vaccines and existing antibody therapies target the RBD of the spike protein, this creates the potential for the evolution of SARS-CoV-2 variants that escape vaccine-induced immunity, infection-induced immunity, or existing antibody therapies. The inventors have identified sequence changes in the SARS-CoV-2 spike protein (e.g., in the RBD) that, among others, counter and / or are promising SARS-CoV-2 variants that, e.g., resist SARS-CoV-2 resistance to vaccine-induced immunity. Thus, novel SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein or polypeptide immunogens (as well as immunogenic fragments and / or immunogenic variants thereof)) (or nucleic acid molecules, e.g., mRNAs, encoding such SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein or polypeptide immunogens (as well as immunogenic fragments and / or immunogenic variants thereof)) that comprise at least one such sequence change are good candidates for variant-insensitive or variant-resistant vaccines against COVID-19. As such, the present disclosure provides, inter alia, novel SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein or polypeptide immunogens) for use in pharmaceutical compositions and vaccines for inducing a desired immune response against one or more variants of SARS-CoV-2.

[0085] 5.1 Definition The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter.

[0087] In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, the use of the terms "including" as well as other forms such as "include," "includes," and "included" are not intended to be limiting.

[0088] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and "consisting essentially of" are also provided.

[0089] The term "and / or" as used herein should be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended herein to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0090] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the recited range, and fractions thereof (tenths and hundredths of integers), where appropriate.

[0091] The terms "about" or "consisting essentially of" refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When a particular value or composition is provided in the specification and claims, unless otherwise indicated, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value or composition.

[0092] Where proteins and / or polypeptides are described herein, it is understood that nucleic acid molecules (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) that encode the proteins or polypeptides are also provided herein.

[0093] Where a protein, polypeptide, nucleic acid molecule, vector, carrier, etc. are described herein, it is understood that isolated forms of the protein, polypeptide, nucleic acid molecule, vector, carrier, etc. are also provided herein.

[0094] Where a protein, polypeptide, nucleic acid molecule, vector, carrier, etc. are described herein, it is understood that recombinant forms of the protein, polypeptide, nucleic acid molecule, vector, carrier, etc. are also provided herein.

[0095] Where a polypeptide or set of polypeptides is described herein, proteins comprising the polypeptide or set of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein, and vice versa (i.e., where a protein is described herein, polypeptides comprising the amino acid sequence of the protein are also provided herein).

[0096] As used herein, the term "adjuvant" refers to a substance that, when administered to a subject, causes stimulation of the subject's immune system.

[0097] As used herein, the term "administration" refers to the physical introduction of an agent (e.g., a therapeutic agent, a vaccine) (or a precursor of the agent that is metabolized or modified (e.g., translation of a nucleic acid molecule) in the subject to produce the agent in vivo) to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0098] As used herein, the term "agent" is used generally to describe any macromolecule or small molecule. Exemplary moieties include, but are not limited to, polypeptides, proteins, peptides, nucleic acid molecules (e.g., DNA, RNA), small molecules, carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG).

[0099] As used herein, the term "derived" with respect to a nucleic acid molecule refers to a nucleic acid molecule having at least 70% sequence identity with a reference nucleic acid molecule (e.g., a naturally occurring nucleic acid molecule) or a fragment thereof. The term "derived" with respect to a polypeptide or protein refers to a polypeptide or protein comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term "derived" as used herein does not refer to any particular process or method for obtaining a nucleic acid molecule, polypeptide, or protein. For example, a nucleic acid molecule, polypeptide, or protein can be recombinantly produced or chemically synthesized.

[0100] As used herein, the term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, disease, disorder, pathology, disorder, condition, or syndrome in which physiological function is impair, regardless of the nature of the etiology. The term disease includes infectious diseases (e.g., viral (e.g., SARS-Cov-2 infection), bacterial, fungal, protozoan infections).

[0101] The terms "DNA" and "polydeoxyribonucleotide" are used interchangeably herein to refer to a polymer comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0102] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin (Ig) heavy chain that comprises at least one CH2 region operably linked to a CH3 region from the N-terminus to the C-terminus. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably linked to the N-terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region. Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (KO Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," 2019, Frontiers in Immunology, V.10, Art.1296, pp.1-20, the entire contents of which are incorporated herein by reference for all purposes).

[0103] As used herein, the term "full length" with respect to a SARS-CoV-2 spike protein refers to a SARS-CoV-2 spike protein in which the amino acid sequence of the SARS-CoV-2 spike protein contains substantially the same number of amino acids as a reference SARS-CoV-2 spike protein (e.g., a reference naturally-occurring SARS-CoV-2 spike protein).

[0104] As used herein, the term "fused" and its grammatical equivalents refer to the operative linkage of at least one first polypeptide or protein to a second polypeptide or protein, where the first and second polypeptides or proteins are not naturally operatively linked together. For example, the first and second polypeptides or proteins are derived from different proteins. The term fused encompasses both the direct linkage of at least two polypeptides or proteins via a peptide bond and the indirect linkage via a linker (e.g., a peptide linker).

[0105] As used herein, the term "fusion protein" and its grammatical equivalents refer to a protein comprising at least one polypeptide operably linked to another polypeptide, where the first and second polypeptides are different and are not naturally operably linked together. For example, the first and second polypeptides of the fusion protein are each derived from a different protein. The at least two polypeptides of the fusion protein can be directly operably linked via a peptide bond; or indirectly operably linked via a linker (e.g., a peptide linker). Thus, for example, the term fusion polypeptide encompasses an embodiment in which polypeptide A is directly operably linked to polypeptide B via a peptide bond (polypeptide A-polypeptide B), and an embodiment in which polypeptide A is operably linked to polypeptide B via a peptide linker (polypeptide A-peptide linker-polypeptide B).

[0106] As used herein, the term "half-life extending moiety" refers to a moiety (e.g., a small molecule, a polypeptide, a polynucleotide, a carbohydrate, a lipid, a synthetic polymer (e.g., a polymer of PEG), etc.) that, when conjugated or otherwise operably linked (e.g., fused) to another moiety (a moiety of interest) (e.g., a protein), increases the half-life of the moiety of interest (e.g., a protein) in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of a moiety of interest (e.g., a protein) can be evaluated utilizing in vivo models known in the art.

[0107] As used herein, the term "half-life extending protein" or "half-life extending polypeptide" refers to a polypeptide or protein that, when operably linked to another moiety (a moiety of interest) (e.g., a protein), increases the half-life of the moiety of interest (e.g., a protein of interest) in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of a protein can be evaluated utilizing in vivo models known in the art.

[0108] As used herein, the term "heterologous," when used to describe a first element in relation to a second element, means that the first element and the second element are not naturally occurring in the arrangement as described. For example, a polypeptide that includes a "heterologous moiety" refers to a polypeptide that is linked to a moiety (e.g., a small molecule, a polypeptide, a nucleic acid molecule, a carbohydrate, a lipid, a synthetic polymer (e.g., a polymer of PEG), etc.) that is not naturally linked to the polypeptide.

[0109] As used herein, the term "heterologous signal peptide" refers to a signal peptide that is not operably linked to a native subject polypeptide or protein. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably linked to human IL-12, the human IL-2 signal peptide would constitute a heterologous signal peptide.

[0110] As used herein, the term "homologous signal peptide" refers to a signal peptide that is operably linked to a native subject polypeptide or protein. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably linked to human IL-2, the human IL-2 signal peptide would constitute the homologous signal peptide.

[0111] As used herein, the term "prime boost" refers to a vaccine regimen that includes at least one initial vaccine dose and one or more subsequent vaccine doses. The initial vaccine dose includes a prime vaccine composition, and the one or more subsequent vaccine doses are referred to as boost (or booster) vaccine compositions. A prime boost vaccine regimen can include two or more boosters (e.g., 2, 3, 4, 5, 6, or more, etc.).

[0112] As used herein, the term "allogeneic prime boost" refers to a prime boost vaccine regimen, in which the prime vaccine composition and the boost (or booster) vaccine composition are the same.

[0113] As used herein, the term "heterologous prime boost" refers to a prime boost vaccine regimen, where the prime vaccine composition and the boost (or booster) vaccine composition are different (e.g., the immunogens are different, the form of the immunogen is different (e.g., a protein-based vaccine versus a vaccine based on a nucleic acid (e.g., mRNA) molecule), the immunogens are expressed from different vectors (e.g., plasmids, viral vectors), the method of delivery of the immunogen to the subject is different, etc.).

[0114] As used herein, the term "immunogen" refers to a substance that can induce an immune response (eg, an adaptive immune response) in a subject (eg, a human subject).

[0115] As used herein, the term "immunogenic fragment" refers to a fragment of a reference polypeptide or protein that retains the immunogen.

[0116] As used herein, the term "immunogenic variant" refers to a variant of a reference polypeptide or protein that retains its immunogenicity. In some embodiments, the polypeptide or protein contains at least one but no more than 25% (e.g., no more than 20%, no more than 15%, no more than 12%, no more than 10%, no more than 8%) amino acid change (e.g., substitution, deletion, addition) compared to the amino acid sequence of the reference polypeptide or protein.

[0117] As used herein, the term "in combination with" means that two (or more) different agents or treatments are administered to a subject as part of a predefined treatment regimen for a particular disease. The treatment regimen defines the dose and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or concomitant, and the agents may be combined. In other embodiments, the two or more agents are not combined, but are administered in a sequential manner as part of a prescribed regimen (e.g., a prime-boost vaccine regimen). In some embodiments, the administration of two or more agents or treatments in combination results in a reduction in symptoms, or a reduction in other parameters associated with the disease, that is greater than would be observed if one agent or treatment were delivered alone or in the absence of the other. The effect of the two treatments may be partially additive, wholly additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each agent may be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The agents may be administered by the same route or by different routes.

[0118] As used herein, the term "isolated" with respect to an agent (e.g., a polypeptide, protein, or nucleic acid molecule) refers to an agent (e.g., a polypeptide, protein, or nucleic acid molecule) that is substantially free from other cellular components with which it is naturally associated.

[0119] As used herein, the term "long COVID" is generally used to refer to signs and symptoms that continue or develop after acute COVID-19. Long COVID is also referred to in the art as Prolonged Post-Covid Syndrome (PPCS), Post-Acute Sequelae of COVID-19 (PASC), Long COVID, and Chronic COVID. The term long COVID encompasses any clinically accepted definition.

[0120] As used herein, the term "modified" with respect to a nucleic acid sequence refers to a nucleic acid molecule that includes at least one nucleotide that includes a chemical modification, such as a modified sugar moiety, a modified nucleobase, and / or a modified internucleotide linkage, or any combination thereof. Exemplary nucleotide modifications are provided herein, see, e.g., § 5.3 (e.g., § 5.3.2). In certain embodiments of the present disclosure, the inclusion of deoxynucleotides (recognized as the naturally occurring form of nucleotide) is considered to constitute a modified nucleotide when present in an RNA molecule (e.g., an mRNA molecule).

[0121] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymer of DNA or RNA. A nucleic acid molecule may be single-stranded or double-stranded; may contain natural, non-natural, or modified nucleotides; may contain modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiesters found between nucleotides of natural, non-natural, or unmodified nucleic acid molecules. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning nucleic acid molecules from recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reaction, and synthetic means. Those skilled in the art will understand that, unless otherwise stated, the nucleic acid sequences described herein will recite thymidine (T) in representative DNA sequences, but when the sequence represents RNA (e.g., mRNA), thymidine (T) will be replaced by uracil. Thus, any of the RNA nucleic acid molecules encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, in which each thymidine (T) in the DNA sequence is replaced with uracil (U).

[0122] As used herein, the term "operably linked" refers to the binding of two moieties in a functional relationship. For example, a polypeptide is operably linked to another polypeptide when they are linked in frame (directly or indirectly via a peptide linker) such that both polypeptides are functional (e.g., a fusion protein as described herein). Or, for example, a transcriptional regulatory polynucleotide, such as a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide encoding a protein if it affects the transcription of the polynucleotide encoding the protein. The term "operably linked" can also refer to the conjugation of a moiety, such as a polynucleotide or a polypeptide (e.g., the conjugation of a PEG polymer to a protein).

[0123] As used herein, the term "peptide" refers to a polymer of at least two amino acids linked by peptide bonds. The term "peptide" does not restrict the length of the polymeric chain of amino acids. It is common in the art to refer to short polymers of amino acids (e.g., approximately 2-50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., more than approximately 50 amino acids) as polypeptides. However, the terms "peptide" and "polypeptide" are used interchangeably herein.

[0124] The determination of "percent identity" between two sequences (e.g., peptide or protein (amino acid sequence) or nucleic acid sequence) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, each of which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with XBLAST program parameters set, for example, score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relatedness between molecules (Id.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0125] As used herein, the term "pharmaceutical composition" refers to a composition suitable for administration to an animal (e.g., a human subject) and comprising a therapeutic agent and a pharma- ceutically acceptable carrier or diluent. A "pharma- ceutically acceptable carrier or diluent" means a material for use in contact with the tissues of humans and / or non-human animals without undue toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable therapeutic benefit / risk ratio.

[0126] As used herein, the term "plurality" means two or more (e.g., three or more, four or more, five or more, six or more, seven or more, nine or more, or ten or more).

[0127] As used herein, the term "poly(A) sequence" refers to adenosine nucleotides, typically a sequence of up to about 1000 adenosine nucleotides located at the 3' end of a linear RNA (or a circular RNA). In some embodiments, the poly(A) sequence is essentially homopolymeric, e.g., a poly(A) sequence of 100 adenosine nucleotides has a length of essentially 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from adenosine nucleotides, e.g., a poly(A) sequence of 100 adenosine nucleotides may have a length of more than 100 nucleotides (including 100 adenosine nucleotides in addition to said at least one nucleotide (or stretch of nucleotides) different from adenosine nucleotides). Although "poly(A) sequence" as defined herein is usually related to RNA, in the context of the present invention, it should be understood that the term also relates to the corresponding sequence in a DNA molecule (e.g., "poly(T) sequence").

[0128] As used herein, the term "prime-boost" with respect to a vaccine regimen refers to a vaccine regimen that includes a first administration of a first immunogen to a subject (vaccine prime) and then at some time thereafter a vaccine booster (e.g., a second immunogen). In some embodiments, the vaccine booster includes a second immunogen. As described herein, the first immunogen of the vaccine prime and the second immunogen of the vaccine booster may be the same or different, may be administered via the same or different routes, etc.

[0129] A "prophylactic" treatment is a treatment administered to a subject who does not show signs of the disease, or who shows only early signs, to reduce the risk of developing a pathological condition.

[0130] As used herein, the term "protein" refers to one or more peptides folded into their three-dimensional structure.

[0131] As used herein, the term "receptor binding domain" or "RBD" with respect to the SARS-CoV-2 spike protein refers to the minimal amino acid sequence required for the SARS-CoV-2 spike protein to bind to ACE2. An exemplary reference amino acid sequence of the SARS-CoV-2 spike protein RBD is set forth in SEQ ID NO:1.

[0132] The terms "RNA" and "polyribonucleotide" are used interchangeably herein and refer to a polymer that comprises multiple ribonucleotides polymerized through phosphodiester bonds. Ribonucleotides are nucleotides whose sugar is ribose. RNA may contain modified nucleotides; and may contain modified internucleotide bonds, such as phosphoramidate or phosphorothioate bonds, instead of the phosphodiester found between nucleotides of natural, non-natural, or unmodified nucleic acid molecules.

[0133] As used herein, the term "SARS-CoV-2 spike protein" refers to a SARS-CoV-2 protein that mediates binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). The amino acid sequence of a first exemplary reference SARS-CoV-2 spike protein is set forth in SEQ ID NO:2. The term SARS-CoV-2 spike protein includes naturally occurring and engineered variants. SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike proteins and polypeptides) described herein include fragments and variants thereof (e.g., immunogenic fragments and / or immunogenic variants thereof).

[0134] As used herein, the term "set" with respect to amino acid changes (e.g., substitutions) does not require two or more amino acid changes (e.g., substitutions). For example, Table 2 herein describes "sets" of amino acid substitutions, some sets having only one amino acid substitution and some sets having two or more amino acid substitutions.

[0135] As used herein, the term "signal peptide" or "signal sequence" refers to a sequence (e.g., an amino acid sequence) that can direct the transport or localization of a protein to a particular organelle, cellular compartment, or extracellular export. The term encompasses both signal peptides (the amino acid sequence of the signal peptide) and nucleic acid sequences that encode the signal peptide. Thus, reference to a signal peptide in the context of a nucleic acid molecule refers to the nucleic acid sequence that encodes the signal peptide.

[0136] As used herein, the term "subject" includes any animal, such as a human or other animal. In embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In embodiments, the subject is a human. In embodiments, the subject of the method is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In embodiments, the subject is a bird, such as a member of the avian taxonomic group Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleognathidae (e.g., ostriches, emus), Columbidae (e.g., pigeons, doves), or Psittacidae (e.g., parrots).

[0137] A "therapeutically effective amount" of an agent (e.g., therapeutic agent, vaccine), when used alone or in combination with another agent (e.g., therapeutic agent, vaccine), refers to an amount of agent (e.g., therapeutic agent, vaccine) that protects a subject against the onset of a disease (e.g., infection), ameliorates the severity of a disease (e.g., infection), and / or promotes regression of a disease (e.g., infection) as evidenced by a reduction in the severity of disease (e.g., infection) symptoms, an increase in the frequency and duration of disease (e.g., infection) symptom-free periods, or prevention of impairment or disability due to the affliction of a disease (e.g., infection). The ability of an agent (e.g., therapeutic agent, vaccine) to do any of the foregoing (or any combination thereof) can be evaluated using a variety of methods known to those skilled in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0138] As used herein, the term "translatable RNA" refers to any RNA that encodes at least one polypeptide or protein and can be translated to produce the encoded polypeptide or protein in vitro, in vivo, in situ, or ex vivo. The translatable RNA can be an mRNA or a circular RNA that encodes a polypeptide or protein.

[0139] As used herein, the terms "treat", "treating", "treatment" and the like refer to reducing or ameliorating a disease (e.g., an infection) and / or symptoms associated therewith or obtaining a desired pharmacological and / or physiological effect. Although not precluded, it will be understood that treating a disease (e.g., an infection) does not require that the disease (e.g., an infection) or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, reduces, inhibits, alleviates, relieves, reduces the intensity of, or cures the disease (e.g., an infection) and / or adverse symptoms that may result from the disease (e.g., an infection). In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the onset or recurrence of a disease (e.g., an infection) or prevents severe disease (e.g., severe infection, severe disease associated with an infection).

[0140] As used herein, the term "alteration" or "variant" with respect to a nucleic acid molecule refers to a nucleic acid molecule that contains at least one nucleotide substitution, modification, inversion, addition, or deletion compared to a reference nucleic acid molecule. As used herein, the term "alteration" or "variant" with respect to a polypeptide or protein refers to a polypeptide or protein that contains at least one amino acid residue substitution, modification, inversion, addition, or deletion compared to a reference polypeptide or protein.

[0141] As used herein, the term "5'-untranslated region" or "5'-UTR" refers to a portion of a nucleic acid molecule located 5' (i.e., "upstream") of a coding sequence that is not translated into a protein or polypeptide. Typically, the 5'-UTR begins at the transcription initiation site and ends before the start codon of the coding sequence. The 5'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc. The 5'-UTR may be post-transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5'-cap structure.

[0142] As used herein, the term "3'-untranslated region" or "3'-UTR" refers to a portion of a nucleic acid molecule located 3' (i.e., "downstream") of a coding sequence that is not translated into a protein or polypeptide. The 3'-UTR may be located between the coding sequence and an (optional) terminal poly(A) sequence of a nucleic acid sequence. The 3'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc.

[0143] 5.2 SARS-CoV-2 Spike Protein (e.g., immunogen) The SARS-CoV-2 spike protein mediates viral entry into host cells. The spike protein contains two functional subunits involved in binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit). The SARS-CoV-2 spike protein is cleaved at the interface between the S1 and S2 subunits, which remain non-covalently associated in the prefusion conformation. The distal S1 subunit contains the RBD and contributes to stabilizing the prefusion state of the membrane-tethered S2 subunit, which contains the fusion machinery. The RBD mediates binding to the host cell receptor ACE2. The spike protein is cleaved by host proteases at the so-called S2' site, located immediately upstream of the fusion peptide. This cleavage is proposed to activate the protein for membrane fusion through an extensive irreversible conformational change. (See, e.g., Walls, Alexandra C et al. “Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.” Cell vol. 181,2 (2020):281-292.e6.doi:10.1016 / j.cell.2020.02.058, the entire contents of which are incorporated herein by reference for all purposes.)

[0144] The amino acid sequence of an exemplary reference SARS-CoV-2 spike protein is provided in SEQ ID NO:2. The signal sequence is boxed (amino acids 1-13 of SEQ ID NO:2), the S1 subunit is in bold (amino acids 14-682 of SEQ ID NO:2), the S2 subunit is in italics (amino acids 683-1270 of SEQ ID NO:2), the N-terminal domain comprises amino acids 14-300 of SEQ ID NO:2, and the transmembrane and cytoplasmic domains are italicized and underlined (amino acids 1209-1270 of SEQ ID NO:2). The amino acid sequence of the ectodomain of an exemplary reference SARS-CoV-2 spike protein (without the native signal sequence) is set forth in SEQ ID NO:3. The S1 subunit is in bold (amino acids 1-669 of SEQ ID NO:3), the S2 subunit is in italics (amino acids 670-1195 of SEQ ID NO:3), and the N-terminal domain comprises amino acids 1-287 of SEQ ID NO:3. The amino acid sequence of the RBD of an exemplary reference SARS-CoV-2 spike protein is set forth in SEQ ID NO: 1. The amino acid sequence of the immature SARS-CoV-2 Wuhan-Hu-1 spike reference protein is set forth in SEQ ID NO: 4. The signal peptide (amino acids 1-13 of SEQ ID NO: 4 are underlined).

[0145] [Table 1-1]

[0146] [Table 1-2]

[0147] [Table 1-3]

[0148] Provided herein, inter alia, are SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein or polypeptide immunogens (and immunogenic fragments and / or immunogenic variants thereof)) that comprise at least one amino acid substitution described herein (e.g., at least one amino acid substitution described in Table 2) (e.g., at least one set of amino acid substitutions described herein, e.g., at least one set of amino acid substitutions described in Table 2), and nucleic acid molecules encoding them.

[0149] While exemplary amino acid substitutions are provided herein, e.g., in Table 2, specifying the parent amino acid and the substituted amino acid, it should be understood that the present disclosure includes the replacement of any parent amino acid with the substituted amino acid. Thus, the substitutions described herein (e.g., in Table 2) include the replacement of any parent amino acid with the substituted amino acid. For example, the disclosure of the amino acid substitution I28Y described in Table 2 includes the replacement of any parent amino acid with a tyrosine at amino acid position 28 (amino acid numbering is with respect to SEQ ID NO:1).

[0150] While exemplary amino acid substitutions that designate a parent amino acid and a substituted amino acid are provided herein, e.g., in Table 2, it should be understood that the present disclosure includes the substitution of any parent amino acid with a substituted amino acid set forth in Table 2, or the substitution of a physiochemically similar amino acid for the substituted amino acid. One of skill in the art can determine which amino acids are considered physiochemically similar to any given substituted amino acid set forth in Table 2 using standard methods (e.g., based on the physiochemical properties (e.g., charge, polarity, etc.) of each amino acid).

[0151] The amino acid numbering utilized in Table 2 is with respect to the amino acid sequence set forth in SEQ ID NO: 1. One of skill in the art could readily determine whether any SARS-CoV-2 spike protein contained one or more of the amino acid substitutions (e.g., one or more of a set of substitutions) set forth in Table 2 through standard sequence comparison (e.g., including standard sequence alignment).

[0152] For clarity, while the amino acid numbering utilized in Table 2 is with respect to the amino acid sequence set forth in SEQ ID NO:1, which includes a portion of the reference spike protein (i.e., the RBD), it is clear that the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (as well as immunogenic fragments and / or immunogenic variants thereof)) provided herein may include an amino acid sequence longer than the RBD (e.g., the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (as well as immunogenic fragments and / or immunogenic variants thereof)) provided herein may include the full-length SARS-CoV-2 spike protein).

[0153] [Table 2-1]

[0154] [Table 2-2]

[0155] [Table 2-3]

[0156] [Table 2-4]

[0157] [Table 2-5]

[0158]

Table 2-6

[0159]

Table 2-7

[0160]

Table 2-8

[0161]

Table 2-9

[0162]

Table 2-10

[0163]

Table 2-11

[0164]

Table 2-12

[0165]

Table 2-13

[0166]

Table 2-14

[0167]

Table 2-15

[0168]

Table 2-16

[0169]

Table 2-17

[0170]

Table 2-18

[0171]

Table 2-19

[0172]

Table 2-20

[0173]

Table 2-21

[0174]

Table 2-22

[0175]

Table 2-23

[0176]

Table 2-24

[0177]

Table 2-25

[0178]

Table 2-26

[0179]

Table 2-27

[0180]

Table 2-28

[0181]

Table 2-29

[0182]

Table 2-30

[0183]

Table 2-31

[0184]

Table 2-32

[0185]

Table 2-33

[0186]

Table 2-34

[0187]

Table 2-35

[0188]

Table 2-36

[0189]

Table 2-37

[0190]

Table 2-38

[0191]

Table 2-39

[0192]

Table 2-40

[0193]

Table 2-41

[0194]

Table 2-42

[0195]

Table 2-43

[0196]

Table 2-44

[0197]

Table 2-45

[0198]

Table 2-46

[0199] In some embodiments, the SARS-CoV-2 spike protein or polypeptide is an immunogen (SARS-CoV-2 spike protein or polypeptide immunogen). In some embodiments, the SARS-CoV-2 spike protein or polypeptide comprises an immunogen (SARS-CoV-2 spike protein or polypeptide immunogen). In some embodiments, the SARS-CoV-2 spike protein or polypeptide comprises or consists of an immunogenic fragment of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide comprises or consists of an immunogenic variant of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic fragment of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic variant of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein immunogen. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein immunogen.

[0200] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains an amino acid substitution at any one or more of the amino acid positions set forth in Table 2 (e.g., amino acid positions N1, I2, T3, N4, L5, C6, P7, F8, D9, E 10, V11, F12, N13, A14, T15, R16, F17, A18, S19, V20, Y21, A22, W23, N24, R25, K26, R27, I28, S29, N30, C31, V32, A33, D24, Y25, S36, V37, L38, Y39, N40, L41, A42, P43, F44, F45, T46, F47, K48, C49, Y50, G51, V52, S53, P54, T55,K56, L57, N58, D59, L60, C61, F62, T63, N64, V65, Y66, A67, D68, S69, F70, V71, I72, R73, G74, D75, E76, V77, R7 8, Q79, I80, A81, P82, G83, Q84, T85, G86, N87, I88, A89, D90, Y91, N92, Y93, K94, L95, P96, D97, D98, F99, T100, G101, C102, V103, I104, A105, W106, N107, S108, N109, K110, L111, D112, S113, K114, V115, S116, G117, N118, Y 119, N120, Y121, L122, Y123, R124, L125, F126, R127, K128, S129, N130, L131, K132, P133, F124, E135, R136, D1 37, I138, S139, T140, E141, I142, Y143, Q144, A145, G146, N147, K148, P149, C150, N151, G152, V153, A154, G1 55, F156, N157, C158, Y159, F160, P161, L162, R163, S154, Y165, S166, F167, R168, P169, T170, Y171, G172, V17 3, G174, H175, Q176, P177, Y178, R179, V180, V181, V182, L183, S184, F185, E186, L187, L188, H189, A190, P191, A192, T193, V194, C195, G196, P197, K198, K199, S200, or T201 (amino acid numbering relative to SEQ ID NO: 1).

[0201] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50 or more of the amino acid substitutions set forth in Table 2. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of at least one, but not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 50, of the amino acid substitutions set forth in Table 2. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50 or more of the amino acid substitutions set forth in Table 2. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of about 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 of the amino acid substitutions set forth in Table 2.

[0202] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of a set of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50 or more amino acid substitutions set forth in Table 2. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of at least one, but not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 50, of the set of amino acid substitutions set forth in Table 2. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, or more, of the set of amino acid substitutions set forth in Table 2. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of about 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 sets of amino acid substitutions set forth in Table 2.

[0203] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) amino acid changes not set forth in Table 2. In some embodiments, at least one of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) amino acid changes is relative to an amino acid in a reference SARS-CoV-2 spike protein (e.g., a naturally occurring SARS-CoV-2 spike protein). In some embodiments, at least one of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) amino acid changes are relative to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, at least one of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) amino acid changes are relative to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, at least one of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) amino acid changes are found in one or more circulating variants of SARS-CoV-2. In some embodiments, at least one of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) amino acid changes is found in one or more variants of SARS-CoV-2 known to have previously circulated.

[0204] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 95 or 100% identical to 50, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 93 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids are at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0205] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 ... 00, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids are at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of the preceding claims. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 93 , 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids.

[0206] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 ... 00, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids are at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of the preceding claims. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 93 , 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids.

[0207] In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 4 or 100% identical to 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids. In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) other than those included in any set of amino acid substitutions set forth in Table 2, does not include at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, or 100% identical to 0, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids.In some embodiments, the amino acid sequence of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) other than those included in any set of amino acid substitutions set forth in Table 2, does not include at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 8 or 100% identical to 0, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids.

[0208] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is about 10-1300, 10-1200, 10-1100, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-500, 10-400, 10-300, 10-250, 10-200, 10-300, 10-400, 10-500, 10-400, 10-500, 10-400, 10-500, 10-400, 10-300, 10-250, 10-200, 10-500, 10-600, 10-700, 10-800, 10-800, 10-900, 10-10 ... The amino acid sequence may include 0-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-1300, 20-1300, 30-1300, 40-1300, 50-1300, 60-1300, 70-1300, 80-1300, 90-1300, 100-1300, 10-250, 20-250, 30-250, 40-250, 50-250, 60-250, 70-250, 80-250, 90-250, or 100-250 amino acids.In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, or 10-1300 amino acids.

[0209] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, 10- 1300, 20~30, 20~40, 20~50, 20~60, 20~70, 20~80, 20~90, 20~100, 20~200, 20~300, 20~400, 20~500, 20~600, 20~700, 20~800, 20~900, 20~1000, 20~1100 , 20~1200, 20~1300, 30~40, 30~50, 30~60, 30~70, 30~80, 30~90, 30~100, 30~200, 30~300, 30~400, 30~500, 30~600, 30~700, 30~800, 30~900, 30~1000, 3 0~1100, 30~1200, 30~1300, 40~50, 40~60, 40~70, 40~80, 40~90, 40~100, 40~200, 40~300, 40~400, 40~500, 40~600, 40~700, 40~800, 40~900, 40~1000, 4 0~1100, 40~1200, 40~1300, 50~60, 50~70, 50~80, 50~90, 50~100, 50~200, 50~300, 50~500, 50~500, 50~600, 50~700, 50~800, 50~900, 50~1000, 50~1100 , 50~1200, 50~1300, 60~70, 60~80, 60~90, 60~100, 60~200, 60~300, 60~400, 60~500, 60~600, 60~700, 60~800, 60~900, 60~1000, 60~1100, 60~1200, 60~ 1300, 70~80, 70~90, 70~100, 70~200, 70~300, 70~400, 70~500, 70~600, 70~700, 70~800, 70~900, 70~1000, 70~1100, 70~1200, 70~1300, 80~90, 80~100,80~200、80~300、80~400、80~500、80~600、80~700、80~800、80~900、80~1000、80~1100、80~1200、80~1300、90~100、90~200、90~300、90~400、90~500、90~600、90~700、90~800、90~900、90~1000、90~1100、90~1200、90~1300、100~200、100~300、100~400、100~500、100~600、100~700、100~800、100~900、100~1000、100~1100、100~1200、100~1300、200~300、200~400、200~500、200~600、200~700、200~800、200~900、200~1000、200~1100、200~1200、200~1300、300~400、300~500、300~600、300~700、300~800、300~900、300~1000、300~1100、300~1200、300~1300、400~500、400~600、400~700、400~800、400~900、400~1000、400~1100、400~1200、400~1300、500~600、500~700、500~800、500~900、500~1000、500~1100、500~1200、500~1300、600~50、600~60、600~70、600~80、600~90、600~100、600~200、600~300、600~6000、600~500、600~600、600~700、600~800、600~900、600~1000、600~1100、600~1200、600~1300、700~50、700~60、700~70、700~80、700~90、700~100、700~200、700~300、700~7000、700~500、700~600、700~700、700~800、700~900、700~1000、700~1100、700~1200、700~1300、800~900、800~1000、800~1100、800~1200、800~1300、900~50、900~60、900~70、900~80、900~90、900~100、900~200、900~300、900-9000, 900-500, 900-600, 900-700, 900-800, 900-900, 900-1000, 900-1100, 900-1200, 900-1300, 1000-1100, 1000-1200, 1000-1300, 1100-1200, 1100-1300, or 1200-1300.

[0210] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least a portion of the RBD of the SARS-CoV-2 spike protein. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises the RBD of the SARS-CoV-2 spike protein. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises the full-length SARS-CoV-2 spike protein. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises the ectodomain of a SARS-CoV-2 spike protein. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises the ectodomain of a SARS-CoV-2 spike protein and does not comprise the transmembrane or cytoplasmic domain of a SARS-CoV-2 spike protein. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a cognate signal peptide. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a heterologous signal peptide.In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) does not contain a signal peptide.

[0211] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) is stabilized in the pre-fusion state. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change (e.g., relative to the amino acid sequence set forth in SEQ ID NO:2, relative to a naturally occurring SARS-CoV-2 spike protein or polypeptide) that stabilizes the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) in the pre-fusion state. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a proline at amino acid position 983 and / or a proline at amino acid position 984 (amino acids with amino acid numbering relative to SEQ ID NO:2). In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a proline at amino acid position 986 and / or a proline at amino acid position 987 (amino acids with amino acid numbering relative to SEQ ID NO:4).

[0212] In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises an inactive furin cleavage site. To be able to induce fusion, the spike protein of SARS-Cov-2 needs to be cleaved into S1 and S2 subunits. The cleavage site in SARS-Cov-2 is a polybasic motif (RRAR) (e.g., amino acids 679-682 of SEQ ID NO:2 or amino acids 682-685 of SEQ ID NO:4, which can be activated by a furin-like protease). Modifications to the furin cleavage site that inactivate it are known in the art. See, e.g., Amanat F, Strohmeier S, Rathnasinghe R, et al. Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike-based SARS-CoV-2 vaccine in the mouse model. Preprint.bioRxiv.2020; 2020.09.16.300970. Published September 17, 2020. doi:10.1101 / 2020.09.16.300970 (describing replacement of the RRAR cleavage site with a single alanine); and WO 2022203963 (describing replacement of the RRAR cleavage site with the amino acid sequence QQAQ), the entire contents of each of which are incorporated herein by reference. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change in the furin cleavage site that inactivates the furin cleavage site (e.g., relative to the amino acid sequence set forth in SEQ ID NO:2, relative to a naturally occurring SARS-CoV-2 spike protein or polypeptide).In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a replacement of the RRAR cleavage site with a single alanine. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises a replacement of the RRAR cleavage site with the amino acid sequence QQAQ.

[0213] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises one or more non-naturally occurring glycosylation motifs (e.g., N-glycosylation motifs). See, e.g., Lin Wei-Shuo et al., Glycan Masking of Epitopes in the NTD and RBD of the Spike Protein Elicits Broadly Neutralizing Antibodies Against SARS-CoV-2 Variants, Frontiers in Immunology, (12) Article 795741, 02 December 2021, DOI=10.3389 / fimmu.2021.795741, the entire contents of which are incorporated herein by reference for all purposes. In some embodiments, the inclusion of one or more glycosylation motifs (e.g., N-glycosylation motifs) promotes glycan shielding of an immunodominant epitope of an immunogenic protein (or an immunogenic fragment or immunogenic variant thereof). In some embodiments, when a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or a SARS-CoV-2 spike protein or a polypeptide immunogenic fragment and / or immunogenic variant thereof)) is administered to a subject, the subject does not generate an effective amount of neutralizing antibodies that specifically bind to the immunodominant epitope.Thus, in some embodiments, inclusion of one or more N-glycosylation motifs in a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) alters the immune response generated from administration of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) to generate more neutralizing antibodies against the SARS-CoV-2 spike protein. Glycosylation motifs are known in the art. For example, NX1X2 (X1 can be any amino acid except proline and X2 can be S, T, or C) is known as a consensus N-glycosylation sequence.

[0214] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises one or more heterologous peptide or protein elements, or a nucleic acid molecule described herein encodes at least one heterologous peptide or protein element. In some embodiments, at least one heterologous peptide or protein element confers additional functionality to a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), such as to facilitate or enhance secretion or ( For example, via a secretory signal peptide), promote or enhance anchoring of an encoded SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein in a cell membrane (e.g., via a membrane-spanning element), promote or enhance formation of an antigen complex (e.g., via a multimerization domain or antigen clustering element), or promote or enhance virus-like particle formation (VLP-forming sequences). In some embodiments, the ectodomain of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is modified to improve the stability of the generated protein or polypeptide.

[0215] 5.3 Nucleic acid molecules encoding the SARS-CoV-2 spike protein (e.g., immunogens) In one aspect, provided herein is a nucleic acid molecule that includes a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) described herein (see, e.g., §5.2). In some embodiments, the nucleic acid molecule is RNA (e.g., mRNA or circular RNA) or DNA. In some embodiments, the nucleic acid (e.g., RNA) molecule is translatable RNA. In some embodiments, the nucleic acid (e.g., RNA) molecule is circular RNA. In some embodiments, the nucleic acid (e.g., RNA) molecule is mRNA.

[0216] In some embodiments, a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (e.g., as described herein) comprises about 30 to about 20,000 nucleotides, about 50 to about 20,000 nucleotides, about 500 to about 10,000 nucleotides, about 1,000 to about 10,000 nucleotides, about 1,000 to about 5,000 nucleotides, or about 2,000 to about 5,000 nucleotides. In some embodiments, a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least 30 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides.

[0217] In some embodiments, a segment of a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (e.g., as described herein) comprises from about 30 to about 20,000 nucleotides, from about 50 to about 20,000 nucleotides, from about 500 to about 10,000 nucleotides, from about 1,000 to about 10,000 nucleotides, from about 1,000 to about 5,000 nucleotides, or from about 2,000 to about 5,000 nucleotides. In some embodiments, a segment of a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least 30 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides.

[0218] In some embodiments, the nucleic acid molecule is modified (e.g., compared to the sequence of a reference nucleic acid molecule, a naturally occurring nucleic acid molecule) to confer, e.g., one or more of: (a) improved resistance to in vivo degradation, (b) improved in vivo stability, (c) reduced secondary structure, and / or (d) improved in vivo translatability, e.g., compared to the reference nucleic acid sequence. Modifications include, but are not limited to, e.g., codon optimization, nucleotide modifications (see, e.g., those described herein), and the like.

[0219] In some embodiments, the sequence of the nucleic acid molecule is codon-optimized, for example, for expression in humans. Codon optimization can be used in some embodiments to match codon frequencies in target and host organisms to ensure proper folding; bias guanosine (G) and / or cytosine (C) content to increase nucleic acid stability; minimize tandem repeat codons or stretches of bases that may impair gene assembly or expression; tailor transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rates to allow various domains of the protein to fold properly; and / or reduce or remove secondary structures (e.g., problematic secondary structures) within the nucleic acid molecule. In some embodiments, the codon-optimized nucleic acid sequence exhibits one or more of the above (compared to a reference nucleic acid sequence). In some embodiments, a codon-optimized nucleic acid sequence exhibits one or more of increased resistance to in vivo degradation, increased stability in vivo, reduced secondary structure, and / or increased translatability in vivo compared to a reference nucleic acid sequence. Codon optimization methods, tools, algorithms, and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park Calif.). In some embodiments, an open reading frame (ORF) sequence is optimized using an optimization algorithm (e.g., an optimization algorithm known in the art). In some embodiments, a nucleic acid sequence is modified to optimize the number of G and / or C nucleotides compared to a reference nucleic acid sequence. An increase in the number of G and C nucleotides can be brought about by the replacement of codons containing adenosine (A) or thymidine (T) (or uracil (U)) nucleotides with codons containing G or C nucleotides.

[0220] 5.3.1 DNA nucleic acids In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the DNA is a linear coding DNA construct. In some embodiments, the DNA is contained within a vector (e.g., a non-viral vector (e.g., a plasmid) or a viral vector). In some embodiments, the DNA is contained within a non-viral vector (e.g., a plasmid). In some embodiments, the DNA is contained within a viral vector (e.g., as described herein). A more detailed description of vectors for both RNA and DNA nucleic acids is provided in §5.6.

[0221] The coding DNA molecule may also contain one or more heterologous nucleic acid elements to mediate the expression of the coding region. These include, for example, promoters, enhancers, polyadenylation signals, synthetic introns, transcription termination signals, polyadenylation sequences, and other transcriptional regulatory elements. Those skilled in the art are familiar with the transcriptional regulatory elements required for the expression of coding DNA and can optimize the expression construct (e.g., linear DNA, plasmid DNA, etc.) accordingly.

[0222] In some embodiments, a promoter is operably linked to the respective coding nucleic acid sequence. Those skilled in the art are aware of various promoters that can be utilized, such as promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukosis virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter, Epstein-Barr virus (EBV) promoter, or Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a natural or synthetic tissue-specific promoter, such as a muscle or skin specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. 20040175727, the entire contents of which are incorporated herein by reference for all purposes. Exemplary polyadenylation signals include, but are not limited to, the bovine growth hormone (BGH) polyadenylation site, the SV40 polyadenylation signal, and the LTR polyadenylation signal.

[0223] 5.3.2 RNA nucleic acids In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the RNA molecule is a translatable RNA molecule. In some embodiments, the RNA molecule is selected from an mRNA, a self-replicating RNA, a circular RNA, a viral RNA, or a replicon RNA. In some embodiments, the RNA molecule is a circular RNA.

[0224] In some embodiments, the RNA molecule is an mRNA. The basic components of an mRNA molecule typically include at least one coding region (encoding at least one peptide or protein herein (e.g., a coding region encoding a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or a polypeptide immunogen (or an immunogenic fragment and / or variant thereof)) (e.g., as described herein), a 5'-untranslated region (UTR), a 3'-UTR, a 5' cap, and a poly-A tail.

[0225] In some embodiments, the RNA (e.g., mRNA) comprises at least one heterologous UTR. The UTR may have regulatory sequence elements that determine RNA (e.g., mRNA) turnover, stability, localization, and / or expression of an operably linked coding sequence. The heterologous UTR may be derived from a naturally occurring gene or may be synthetically designed. In some embodiments, the 5'-UTR comprises elements for controlling gene expression, e.g., ribosome binding sites, miRNA binding sites. The 5'-UTR may be post-transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5' cap structure. In some embodiments, the 3'-UTR comprises a polyadenylation signal. In some embodiments, the RNA (e.g., mRNA) comprises at least one coding region encoding a polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., SARS-CoV-2 spike protein or a polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (e.g., as described herein) and a 5'-UTR and / or a 3'-UTR. In some embodiments, the RNA (e.g., mRNA) comprises at least one coding sequence encoding a polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., SARS-CoV-2 spike protein or a polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (e.g., as described herein) operably linked to at least one heterologous 5'-UTR and at least one 3'-UTR.

[0226] In some embodiments, the RNA (e.g., mRNA) comprises a poly(A) sequence. The poly(A) sequence can comprise about 10-500 adenosine nucleotides, 10-200 adenosine nucleotides, 20-200 adenosine nucleotides, 30-200 adenosine nucleotides, 40-200 adenosine nucleotides, or 50-200 adenosine nucleotides. In some embodiments, the poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides. The poly(A) sequence may comprise about 10-500 adenosine nucleotides, 10-200 adenosine nucleotides, 20-200 adenosine nucleotides, 30-200 adenosine nucleotides, 40-200 adenosine nucleotides, or 50-200 adenosine nucleotides, and the 3' terminal nucleotide of the nucleic acid molecule is an adenosine. In some embodiments, the poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides, and the 3' terminal nucleotide of the nucleic acid molecule is an adenosine.

[0227] In some embodiments, the RNA (e.g., mRNA) comprises a 5'-cap structure. In some embodiments, the 5'-cap structure stabilizes the RNA (e.g., mRNA), enhances expression of an encoded polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or a polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (e.g., as described herein) and / or reduces stimulation of the innate immune system (e.g., following administration to a subject).

[0228] Exemplary 5'-cap structures include, but are not limited to, cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), cap2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the 5' cap structure comprises an m7G, cap0, cap1, cap2, a modified capO, or a modified cap1 structure.

[0229] In some embodiments, an RNA (e.g., an mRNA) comprises one or more modified nucleotides (e.g., a nucleotide analog, a backbone modification, a sugar modification, and / or a base modification). A backbone modification in the context of this disclosure is a modification in which the phosphate of the backbone of a nucleotide of an RNA (e.g., an mRNA) is chemically modified. A sugar modification in the context of this disclosure is a chemical modification of the sugar of a nucleotide of an RNA (e.g., an mRNA). A base modification in the context of this disclosure is a chemical modification of the base moiety of a nucleotide of an RNA (e.g., an mRNA).

[0230] In some embodiments, the RNA (e.g., mRNA) comprises at least one modified nucleotide. Exemplary nucleotide analogs / modifications include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate. 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodo 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5 -propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate '-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate,Examples of suitable uridine derivatives include, but are not limited to, xanthosine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-uridine, 1-taurinomethyl-2-thio ... linomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy Pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine cytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl arylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine , N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, aza-cytidine, 5'-O-(1-thiophosphate) ... rufa-thio-cytidine, pseudoiso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thioguanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine,Particularly preferred are nucleotides with respect to base modifications selected from the group of base-modified nucleotides consisting of N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thioadenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0231] In some embodiments, the RNA (e.g., mRNA) comprises pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and / or 2'-O-methyluridine.

[0232] In some embodiments, the RNA (e.g., mRNA) comprises one or more of pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. In some embodiments, essentially all, e.g., essentially 100%, of the uracils in the coding sequence of the RNA (e.g., mRNA) have a chemical modification, preferably the chemical modification is at the 5-position of the uracil. For example, incorporation of modified nucleotides such as pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and / or 5-methoxyuridine into the coding sequence may be advantageous because unwanted innate immune responses (upon administration of the coding RNA or vaccine) may be modulated or reduced (if desired).

[0233] In one embodiment, the RNA (e.g., mRNA) comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region.

[0234] The RNA (e.g., mRNA) described herein can be produced, for example, by in vitro transcription. In vitro transcription is a method well known to those skilled in the art for the production of RNA (e.g., mRNA). In general, RNA is obtained by DNA-dependent in vitro transcription of a suitable DNA template, for example, a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Examples of DNA-dependent RNA polymerases include 17, T3, SP6, or Syn5 RNA polymerase. In some examples, the DNA template is linearized with a suitable restriction enzyme before it is subjected to RNA in vitro transcription. Reagents used in RNA in vitro transcription typically include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has high binding affinity for its corresponding RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5) that can bind to the promoter sequence in the DNA template. RNA polymerase; optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphates that may inhibit RNA in vitro transcription; MgCh to provide Mg2+ ions as a cofactor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value that may also contain an antioxidant (e.g., DTT), and / or an optimal concentration of a polyamine such as spermidine, e.g., a buffer system including TRIS-citrate as disclosed in WO2017109161. The resulting RNA (e.g., mRNA) product may be purified according to methods known in the art.For example, using PureMessenger® (CureVac, Tubingen, Germany; RP-HPLC according to WO2008077592) and / or tangential flow filtration (as described in WO2016193206) and / or oligo d(T) purification (see WO2016180430); or using RP-HPLC, e.g., using reverse phase high pressure liquid chromatography (RP-HPLC), the entire contents of which are hereby incorporated by reference for all purposes.

[0235] 5.4 Fusions and Conjugates In some embodiments, a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) described herein (see, e.g., §5.2) or a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) described herein (see, e.g., §5.3) is operably linked to a heterologous moiety (e.g., a heterologous polypeptide) to form a fusion protein or polypeptide or conjugate.

[0236] In some embodiments, the heterologous moiety is a half-life extending moiety. Exemplary half-life extending moieties include, but are not limited to, human immunoglobulin (hIg), a fragment of hIg, a hIg constant region, a fragment of a hIg constant region, a hIg Fc region, human transferrin, human serum albumin (HSA), an HSA binding protein, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extending polypeptide. Exemplary half-life extending polypeptides include, but are not limited to, hIg, a fragment of hIg, one or more hIg heavy chain constant regions, a fragment of a hIg constant region, a hIg Fc region, human transferrin, human serum albumin (HSA), and an HSA binding protein. A SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) fused or conjugated to a half-life extending moiety (e.g., a half-life extending polypeptide) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) can be evaluated for their pharmacokinetic properties using standard in vivo methods known in the art. In some embodiments, the heterologous moiety is a detectable agent (e.g., a protein, e.g., a fluorescent protein).

[0237] A heterologous moiety (e.g., a heterologous polypeptide) can be directly operably linked or indirectly operably linked to a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

[0238] In some embodiments, a heterologous moiety is directly operably linked to a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)). In some embodiments, a heterologous moiety is indirectly operably linked to a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

[0239] Those skilled in the art can evaluate and select a suitable linker for the fusion or conjugation of a particular heterologous moiety. For example, in embodiments, the heterologous moiety comprises a heterologous polypeptide and a peptide linker can be utilized. Peptide linkers are known in the art and can be selected based on certain properties including, for example, length, flexibility, rigidity, cleavability, etc. The amino acid sequences of commonly utilized peptide linkers include glycine amino acid residues, serine amino acid residues, glycine and serine amino acid residues, or glycine, serine, and proline amino acid residues.

[0240] A heterologous moiety (e.g., a heterologous polypeptide) and a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) may be oriented in a reversed manner so long as each functional portion of the fusion protein or polypeptide or conjugate maintains the ability to mediate its function.

[0241] 5.5 Vaccine Compositions In some embodiments, a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) described herein (see, e.g., § 5.2) (or a fusion or conjugate thereof (see, e.g., § 5.4)) or a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) described herein (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)) forms the basis for a vaccine composition (e.g., a prime vaccine composition, a prime boost composition, a vaccine prime and booster composition). Thus, in one aspect, provided herein are vaccine compositions (e.g., prime vaccine compositions, prime boost compositions, vaccine prime and booster compositions) comprising a nucleic acid molecule comprising a coding region encoding at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof (see, e.g., § 5.4)) described herein (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)).

[0242] In some embodiments, the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a prime boost composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the prime boost composition may be utilized as a prime and / or booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster.

[0243] 5.5.1 Peptide and protein-based vaccines In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof (see, e.g., §5.4)) described herein (see, e.g., §5.2) forms the basis for a vaccine composition (e.g., a prime vaccine composition, a prime boost composition, a vaccine prime and booster composition). Thus, in one aspect, provided herein are vaccine compositions (e.g., a prime vaccine composition, a prime boost composition, a vaccine prime and booster composition) comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof (see, e.g., §5.4)) described herein (see, e.g., §5.2).

[0244] In some embodiments, the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a prime boost composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the prime boost composition may be utilized as a prime and / or booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster.

[0245] In some embodiments, the vaccine composition comprises multiple SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)).

[0246] In some embodiments, the plurality comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)). In some embodiments, the plurality is about 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 3 0-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)). In some embodiments, the plurality includes at least two, but not more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)).

[0247] In some embodiments, the amino acid sequence of each of the multiple SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) is different.

[0248] In some embodiments, the amino acid sequence of at least one of the multiple SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) is derived from a pandemic strain of SARS-CoV-2.

[0249] In some embodiments, the amino acid sequence of a first SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of the plurality comprises at least a first amino acid substitution (e.g., a set of amino acid substitutions) set forth in Table 2; and the amino acid sequence of a second SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of the plurality comprises at least a second amino acid substitution (e.g., a set of amino acid substitutions) set forth in Table 2, wherein the first and second amino acid substitutions (e.g., the first and second sets of amino acid substitutions) set forth in Table 2 are different. In some embodiments, the amino acid sequences of at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of the plurality contain at least one amino acid substitution (e.g., at least one set of amino acid substitutions) set forth in Table 2. In some embodiments, at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of the plurality contain at least one amino acid substitution (e.g., at least one set of amino acid substitutions) set forth in Table 2, and each amino acid substitution (e.g., each set of amino acid substitutions) is different.

[0250] In some embodiments, the amino acid sequence of at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more) of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of the plurality comprises or consists of one or more amino acid changes not set forth in Table 2. In some embodiments, the one or more amino acid changes are relative to the amino acid sequence of a reference SARS-CoV-2 spike protein or polypeptide (e.g., a naturally occurring SARS-CoV-2 spike protein). In some embodiments, the one or more amino acid changes are relative to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the one or more amino acid changes are relative to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the one or more changes are found in one or more circulating variants of SARS-CoV-2. In some embodiments, the one or more changes are found in one or more variants of SARS-CoV-2 known to have previously circulated.

[0251] In some embodiments, the vaccine composition further comprises at least one immunogen (or an immunogenic fragment and / or immunogenic variant thereof) from a non-SARS-CoV-2 virus, such as an influenza virus (e.g., influenza A, influenza B), respiratory syncytial virus (RSV), rhinovirus, parvovirus, parainfluenza virus, adenovirus. In some embodiments, the vaccine composition comprises one or more immunogens (or an immunogenic fragment or immunogenic variant thereof) from an influenza virus (e.g., influenza A, influenza B), respiratory syncytial virus (RSV), rhinovirus, parvovirus, parainfluenza virus, and / or adenovirus (or any combination thereof).

[0252] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) (e.g., the ectodomain of a SARS-CoV-2 spike protein or polypeptide) is modified to improve expression of the protein in a host cell (e.g., insect cells, mammalian cells, eggs) as described below.

[0253] The polypeptides and proteins (e.g., immunogens) described herein, e.g., SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (and immunogenic fragments and / or immunogenic variants thereof)), can be produced by recombinant techniques in host cells (e.g., insect cells, mammalian cells, bacteria) transfected or transduced with a nucleic acid expression vector (e.g., a plasmid, a viral vector (e.g., a baculovirus expression vector)) encoding a SARS-CoV-2 spike protein or polypeptide (e.g., an immunogen (or an immunogenic fragment and / or immunogenic variant thereof)). Such general methods are common knowledge in the art. An expression vector usually contains an expression cassette that includes nucleic acid sequences that can cause expression of a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., an immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), such as a promoter, enhancer, polyadenylation signal, etc. One skilled in the art will recognize that a variety of promoter and enhancer elements can be used to obtain expression of a nucleic acid molecule in a host cell. For example, promoters can be constitutive or regulatable and can be obtained from various sources (e.g., viral, prokaryotic, or eukaryotic sources) or can be artificially engineered. After transfection or transduction, host cells containing an expression vector encoding a SARS-CoV-2 spike protein or polypeptide (e.g., an immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) are cultured under conditions conducive to expression of the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., an immunogen (or an immunogenic fragment and / or immunogenic variant thereof)). Culture media are available from various vendors, and a suitable medium can be routinely selected for host cells expressing a polypeptide or protein of interest, the SARS-CoV-2 spike protein or polypeptide (e.g., an immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) herein.The host cells may be adherent or suspension cultures, and one skilled in the art may optimize the culture method for the particular host cells selected. For example, suspension cells may be cultured, for example, in a bioreactor, for example, in a batch or fed-batch process. The produced immunogenic peptides or proteins may be isolated from the cell culture, for example, by column chromatography, either in flow-through or bind-and-elute mode. Examples include, but are not limited to, ion exchange and affinity resins, such as lentil lectin sepharose, and mixed mode cation exchange-hydrophobic interaction columns (CEX-HIC). The peptides or proteins may be concentrated and buffer exchanged by ultrafiltration, and the retentate from the ultrafiltration may be filtered through a suitable filter, for example, a 0.22 μm filter. See, e.g., McPherson et al., "Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant," Chapter 4, in Sunil Thomas (ed.), Vaccine Design: Methods and Protocols: Volume 1: Vaccines for Human Diseases, Methods in Molecular Biology, Springer, New York, 2016. See also U.S. Patent No. 5,762,939, the entire contents of each of which are incorporated herein by reference for all purposes.

[0254] The SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) described herein can also be produced synthetically. The SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) described herein can be produced by using egg-based manufacturing methods.

[0255] In some embodiments, the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of the vaccine compositions are formulated in one or more carriers (e.g., a carrier described herein (see, e.g., §5.7)).

[0256] In some embodiments, the vaccine composition is a pharmaceutical composition (e.g., as described herein, see, e.g., §5.8). In some embodiments, the vaccine composition includes an adjuvant (e.g., as described herein, see, e.g., §5.9).

[0257] 5.5.2 Nucleic acid-based vaccines In some embodiments, nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof (see, e.g., §5.4)) described herein (see, e.g., §5.3) form the basis for a vaccine composition (e.g., a prime vaccine composition, a prime boost composition, a vaccine prime and booster composition). Thus, in one aspect, provided herein are vaccine compositions (e.g., a prime vaccine composition, a prime boost composition, a vaccine prime and booster composition) comprising a nucleic acid molecule comprising a coding region encoding at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof (see, e.g., §5.4)) described herein (see, e.g., §5.3).

[0258] In some embodiments, the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a prime boost composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the prime boost composition may be utilized as a prime and / or booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster.

[0259] In some embodiments, the vaccine composition comprises a plurality of nucleic acid moles, each of which comprises a coding region encoding at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof) (e.g., as described herein).

[0260] In some embodiments, each of the plurality of nucleic acid molecules is part of the same larger nucleic acid molecule. In some embodiments, each of the plurality of nucleic acid molecules is a separate (i.e., unlinked) nucleic acid molecule. In some embodiments, at least two of the plurality of nucleic acid molecules are part of the same larger nucleic acid molecule. In some embodiments, at least two of the plurality of nucleic acid molecules are separate (i.e., unlinked) nucleic acid molecules. In some embodiments, at least two of the plurality of nucleic acid molecules are part of the same larger nucleic acid molecule; at least one (e.g., at least 2, 3, 4, 5, etc.) of the plurality of nucleic acid molecules is a separate (i.e., unlinked) nucleic acid molecule.

[0261] In some embodiments the plurality comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid molecules. In some embodiments, the plurality is about 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20 The nucleic acid molecule comprises or consists of up to 50, 20 to 40, 20 to 30, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40, 40 to 100, 40 to 90, 40 to 80, 40 to 70, 40 to 60, 40 to 50, 50 to 100, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 60 to 100, 60 to 90, 60 to 80, 60 to 70, 70 to 100, 70 to 90, 70 to 80, 80 to 100, 80 to 90, or 90 to 100 nucleic acid molecules. In some embodiments, the plurality comprises at least two but no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 nucleic acid molecules.

[0262] In some embodiments, the amino acid sequence of each of the multiple encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) is different.

[0263] In some embodiments, the amino acid sequence of at least one of the multiple encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) is derived from a pandemic strain of SARS-CoV-2.

[0264] In some embodiments, the amino acid sequence of a first encoded SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of the plurality comprises at least a first amino acid substitution (e.g., a first set of amino acid substitutions) set forth in Table 2; and the amino acid sequence of a second encoded SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of the plurality comprises at least a second amino acid substitution (e.g., a second set of amino acid substitutions) set forth in Table 2, wherein the first and second amino acid substitutions (e.g., the first and second sets of amino acid substitutions) set forth in Table 2 are different. In some embodiments, the amino acid sequences of at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) of the encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of the plurality contain at least one amino acid substitution (e.g., at least one set of amino acid substitutions) set forth in Table 2. In some embodiments, the amino acid sequences of at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) of the encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of the plurality contain an amino acid substitution (e.g., a set of amino acid substitutions) set forth in Table 2, and each amino acid substitution (e.g., each set of amino acid substitutions) is different.

[0265] In some embodiments, the amino acid sequence of at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)) of the plurality comprises or consists of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50, or more) amino acid changes not set forth in Table 2. In some embodiments, the one or more amino acid changes are relative to the amino acid sequence of a reference SARS-CoV-2 spike protein or polypeptide (e.g., a naturally occurring SARS-CoV-2 spike protein). In some embodiments, the one or more amino acid changes are relative to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the one or more amino acid changes are relative to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the one or more amino acid changes are found in one or more circulating variants of SARS-CoV-2. In some embodiments, the one or more amino acid changes are found in one or more SARS-CoV-2 strains known to have previously circulated.

[0266] In some embodiments, the vaccine composition further comprises one or more nucleic acid molecules encoding one or more immunogens (e.g., one or more immunogenic peptides or proteins) from a non-SARS-CoV-2 virus, such as an influenza virus (e.g., influenza A, influenza B), respiratory syncytial virus (RSV), rhinovirus, parvovirus, parainfluenza virus, or adenovirus. In some embodiments, the vaccine composition comprises one or more nucleic acid molecules encoding one or more immunogens (e.g., one or more immunogenic peptides or proteins) from an influenza virus (e.g., influenza A, influenza B), respiratory syncytial virus (RSV), rhinovirus, parvovirus, parainfluenza virus, and / or adenovirus (or any combination thereof).

[0267] In some embodiments, the nucleic acid molecule is comprised within one or more vectors (e.g., vectors described herein) (see, e.g., §5.6). In some embodiments, the nucleic acid molecule or vector of the vaccine composition is formulated in one or more carriers (e.g., carriers described herein) (see, e.g., §5.7).

[0268] In some embodiments, the vaccine composition is a pharmaceutical composition (e.g., as described herein, see, e.g., §5.8). In some embodiments, the vaccine composition includes an adjuvant (e.g., as described herein, see, e.g., §5.9).

[0269] The nucleic acid molecules can be made using general methods known in the art and described above in §5.3.

[0270] 5.6 Vectors In some embodiments, a nucleic acid molecule (e.g., a DNA molecule, an RNA molecule (e.g., an mRNA molecule)) (see, e.g., §5.3) (or a fusion or conjugate thereof (see, e.g., §5.4)) described herein is contained in a vector (e.g., a non-viral vector, a viral vector). Thus, in one aspect, also provided herein is a vector (e.g., a viral vector, a non-viral vector (e.g., a plasmid, a minicircle)) comprising one or more nucleic acids (e.g., a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) described herein. Such vectors can be readily engineered by methods well known to those of skill in the art.

[0271] In some embodiments, the vector is a non-viral vector (e.g., plasmid, minicircle). In some embodiments, the vector is a plasmid. Those skilled in the art will recognize suitable plasmids for expression of DNA of interest. For example, suitable plasmid DNA can be generated to allow efficient production of encoded peptides or proteins (e.g., SARS-CoV-2 proteins or polypeptides (e.g., immunogens)) in cell lines, e.g., insect cell lines, using vectors as described in WO2009150222A2 and defined in PCT claims 1-33 (the present disclosure relates to claims 1-33 of WO2009150222A2, the entire contents of which are incorporated herein by reference for all purposes).

[0272] In some embodiments, the vector is a viral vector. Viral vectors include both RNA-based and DNA-based vectors. Vectors can be designed to meet various specifications. For example, viral vectors can be engineered to be capable or incapable of replication in prokaryotic and / or eukaryotic cells. In some embodiments, the vector is replication-deficient. In some embodiments, the vector is replication-competent. Viral vectors can be engineered or selected to fully or partially integrate (or not integrate) into the genome of the host cell, resulting in suitable host cells that contain the desired nucleic acid in their genome (or (e.g., episomal expression)).

[0273] Exemplary viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus vector, lentivirus vector, retrovirus vector, poxvirus vector, parapoxvirus vector, vaccinia virus vector, fowlpox virus vector, herpes virus vector, adeno-associated virus vector, alphavirus vector, lentivirus vector, rhabdovirus vector, measles virus, Newcastle disease virus vector, picornavirus vector, or lymphocytic choriomeningitis virus vector.In some embodiments, the viral vector is an adenovirus vector, an adeno-associated virus vector, or a lentivirus vector.

[0274] In some embodiments, the vector is an adenovirus vector (e.g., a human adenovirus vector, e.g., HAdV or AdHu). In some embodiments, the adenovirus vector is deleted of the E1 region, resulting in replication deficiency in human cells. Other regions of the adenovirus, such as E3 and E4, may also be deleted. Exemplary adenovirus vectors include, but are not limited to, those described in, for example, WO2005071093 or WO2006048215, the entire contents of each of which are incorporated herein by reference for all purposes. In some embodiments, the adenovirus-based vector used is a simian adenovirus, thereby avoiding suppression of the immune response after vaccination by pre-existing antibodies against common human entities such as AdHu5. Exemplary simian adenovirus vectors include AdCh63 (see, for example, WO2005071093, the entire contents of which are incorporated herein by reference for all purposes) or AdCh68.

[0275] Viral vectors can be produced through the use of packaging / producer cell lines (e.g., mammalian cell lines) using standard methods known to those of skill in the art. Generally, a nucleic acid construct (e.g., a plasmid) encoding a peptide or protein of interest (e.g., a peptide or protein described herein (e.g., a SARS-CoV-2 peptide or protein described herein (e.g., a SARS-CoV-2 immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (with additional elements, e.g., promoter, inverted terminal repeats (ITRs) flanking the transgene), e.g., a plasmid encoding viral replication and structural proteins (along with one or more helper plasmids), and a host cell (e.g., a host cell line) are transfected into the host cell line (i.e., a packaging / producer cell line). In some instances, depending on the viral vector, helper genes from another virus (e.g., in the example of an adeno-associated viral vector) may be used. A per-plasmid may also be required. Eukaryotic expression plasmids are commercially available from various suppliers, such as the plasmid series: pcDNA™, pCR3.1™, pCMV™, pFRT™, pVAX1™, pCI™, Nanoplasmid™, and Pcaggs. Those skilled in the art are aware of numerous transfection methods, and any suitable method of transfection may be utilized (e.g., by mechanical means, by electroporation, using biochemical substances (e.g., lipofectamine) as carriers). The cells are cultured under suitable conditions and for a sufficient time for plasmid expression. Viral particles may be purified from the cell culture medium using standard methods known to those skilled in the art, such as, for example, centrifugation followed by, for example, chromatography and / or ultrafiltration.

[0276] 5.7 Carriers In some embodiments, a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof), a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof), or a vector comprising a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) is formulated in one or more carriers. Accordingly, further provided herein is a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), or a vector comprising a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

[0277] Proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and vectors may be encapsulated within a carrier, chemically conjugated to a carrier, and / or associated with a carrier. In this context, the term "associated" refers to an essentially stable combination of a protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA, DNA), or vector with one or more molecules of a carrier (e.g., one or more lipids of a lipid-based carrier, e.g., a lipid nanoparticle (LNP), liposome, lipoplex, and / or nanoliposome) into a larger non-covalent complex or aggregate. In this context, the term "encapsulation" refers to the incorporation of a protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), or vector into a carrier (e.g., a lipid-based carrier, e.g., a LNP, liposome, lipoplex, and / or nanoliposome) such that the protein, peptide, nucleic acid molecule, e.g., RNA (e.g., mRNA, DNA), or vector is entirely contained within the interior space of the carrier (e.g., a lipid-based carrier, e.g., a LNP, liposome, lipoplex, and / or nanoliposome).

[0278] Exemplary carriers include, but are not limited to, lipid-based carriers (e.g., LNPs, liposomes, lipoplexes, and nanoliposomes). In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the carrier is an LNP. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and / or a PEG lipid. Lipid-based carriers are further described below in §5.7.1.

[0279] 5.7.1 Lipid-based carriers In some embodiments, a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), and / or a vector comprising a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), is encapsulated or associated with one or more lipids (e.g., cationic lipids, and / or neutral lipids), thereby forming a lipid-based carrier, such as a LNP, liposome, lipoplex, or nanoliposome.

[0280] In some embodiments, proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors are encapsulated in one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming a lipid-based carrier such as a LNP, liposome, lipoplex, or nanoliposome. In some embodiments, proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors are associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming a lipid-based carrier such as a LNP, liposome, lipoplex, or nanoliposome.

[0281] In some embodiments, proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors are encapsulated in LNPs (e.g., as described herein). In some embodiments, proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors are associated with LNPs (e.g., as described herein). LNPs are described in further detail in § 5.7.1.1. The use of LNPs for mRNA delivery is further detailed in, for example, Hou X et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078-1094. doi:10.1038 / s41578-021-00358-0. Epub 2021 Aug 10. PMID:34394960; PMCID:PMC8353930, the entire contents of which are incorporated herein by reference for all purposes.

[0282] The proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors are fully or partially located in the interior space of the LNPs, liposomes, lipoplexes, and / or nanoliposomes within the lipid layer / membrane or associated with the exterior space of the lipid layer / membrane. One purpose of incorporating proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors into LNPs, liposomes, lipoplexes, and / or nanoliposomes is to protect the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors from environments that may contain enzymes or chemicals or conditions that degrade the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors and / or systems or receptors that result in rapid excretion of the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors. Furthermore, incorporation of proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors into LNPs, liposomes, lipoplexes, and / or nanoliposomes may facilitate uptake of the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors, and thus enhance the therapeutic effect of the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors. Thus, incorporation of proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and / or vectors (e.g., as described herein) into LNPs, liposomes, lipoplexes, and / or nanoliposomes may be particularly suitable for pharmaceutical compositions described herein, e.g., for intramuscular and / or intradermal administration.

[0283] LNPs, liposomes, lipoplexes, and / or nanoliposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which can be between 50 nm and 500 nm in diameter. In some embodiments, LNPs, liposomes, lipoplexes, and / or nanoliposomes have a diameter of about 10-500 nm, 10-400 nm, 10-300 nm, 10-200 nm, 10-100 nm, or 10-50 nm. In some embodiments, the LNPs, liposomes, lipoplexes, and / or nanoliposomes have a diameter of at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm.

[0284] 5.7.1.1 Lipid Nanoparticles (LNPs) In some embodiments, a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), or a vector comprising a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)). In some embodiments, the LNPs include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as lipids conjugated to PEG or lipids conjugated to a polymer described in Table 5 of WO2019217941, the entire contents of which are incorporated by reference herein for all purposes); and one or more sterols (e.g., cholesterol). In embodiments, the LNP preparation comprises cationic lipids, neutral lipids, cholesterol, and PEG lipids and has an average particle size between 50-200 nm, e.g., 80 nm-160 nm.

[0285] Lipids (e.g., LNPs) that can be used in nanoparticle formation include, for example, those described in Table 4 of WO2019217941, which is incorporated herein by reference, e.g., the lipid-containing nanoparticles can include one or more of the lipids in Table 4 of WO2019217941. LNPs can include additional elements such as polymers, e.g., those described in Table 5 of WO2019217941, which is incorporated herein by reference in its entirety for all purposes.

[0286] In some embodiments, the conjugated lipid, if present, is a PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecanoyloxy)-1,1'-dimethylamino)-2,2'-dimethylamino ... )propyl-l-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and one or more of those listed in Table 2 of WO2019051289, the entire contents of which are incorporated herein by reference for all purposes, as well as combinations of the foregoing.

[0287] In some embodiments, sterols that may be incorporated into the lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO 2009 / 127060 or U.S. Patent Publication No. 2010 / 0130588, the entire contents of which are incorporated by reference herein for all purposes. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, the entire contents of which are incorporated by reference herein for all purposes.

[0288] In some embodiments, the lipid particles include an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amounts of these components may vary independently and to achieve the desired properties. For example, in some embodiments, the lipid particles include an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid (in other embodiments, it may be 20-70% (mol), 30-60% (mol), or 40-50% (mol); about 50 mol% to about 90 mol% of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid, a conjugated lipid in an amount of about 0.5 mol% to about 20 mol% of the total lipid, and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid. The ratio of total lipid to nucleic acid may vary as desired. For example, the ratio of total lipid to nucleic acid (mass or weight) may be about 10:1 to about 30:1.

[0289] In some embodiments, the ratio of lipid to nucleic acid (mass / mass ratio; w / w ratio) can range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and nucleic acid can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the overall lipid content of the lipid nanoparticle formulation can range from about 5 mg / ml to about 30 mg / mL.

[0290] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivery of the compositions described herein, e.g., the nucleic acids described herein (e.g., RNA (e.g., circular polyribonucleotides, linear polyribonucleotides)), include the following:

[0291] [ka] In some embodiments, an LNP comprising formula (i) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0292] [ka] In some embodiments, an LNP comprising formula (ii) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0293] [ka] In some embodiments, an LNP comprising formula (iii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0294] [ka] In some embodiments, an LNP comprising formula (v) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0295] [ka] In some embodiments, an LNP comprising formula (vi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0296] [ka] In some embodiments, an LNP comprising formula (viii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0297] [ka] In some embodiments, an LNP comprising formula (ix) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0298] [ka] (In the formula, X 1 , O, NR 1 , or a direct bond, X 2 is C2-5 alkylene, and X 3 is C(=O) or a direct bond, and R 1 is H or Me, R 3 is C1-3 alkyl, R 2 is C1-3 alkyl, or R 2 The nitrogen atom to which it is attached and X 2 or X 1 is NR 1 and R 1 and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring; Y 1 is C2-12 alkylene, and Y 2 teeth, [ka] is selected from n is 0 to 3; R 4is C1-C15 alkyl, Z 1 is a C1-6 alkylene or a direct bond; Z 2 teeth, [ka] (in any direction) or absent, except Z 1 If is a direct bond, then Z 2 shall not exist; R 5 is C5-9 alkyl or C6-C10 alkoxy, R 6 is C5-9 alkyl or C6-C10 alkoxy, W is methylene or a direct bond, and R 7 is H or Me), or a salt thereof, with the proviso that R 3 and R 2 is a C2 alkyl group; and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear Ce alkylene, (Y 2 )nR 4 but, [ka] and R 4 is a linear C5 alkyl; Z 1 is C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 is not a Cx alkoxy.

[0299] In some embodiments, an LNP comprising formula (xii) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0300] [ka] In some embodiments, an LNP comprising formula (xi) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0301] [ka]

[0302] In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv).

[0303] [ka] In some embodiments, an LNP comprising formula (xv) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0304] [ka] In some embodiments, an LNP comprising a formulation of formula (xvi) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

[0305] [ka]

[0306] In some embodiments, lipid compounds are used to form lipid nanoparticles for delivery of a composition described herein, e.g., a nucleic acid (e.g., RNA (e.g., circular polynucleotide, linear polyribonucleotide)) described herein, made by one of the following reactions: [ka]

[0307] In some embodiments, LNPs comprising formula (xxi) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxi) are LNPs described by WO2021113777 (e.g., lipids of formula (1), such as lipids in Table 1 of WO2021113777, the entire contents of which are incorporated herein by reference for all purposes). [ka] During the ceremony, Each n is independently an integer from 2 to 15; L 1 and L 3 are each independently -OC(O)-* or -C(O)O-*, and "*" represents R 1 or R 3 Indicates the attachment point to; R 1 and R 3are each independently oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyl A straight or branched chain C optionally substituted with one or more substituents selected from the group consisting of oxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl. 9 ~C 20 Alkyl or C 9 ~C 20 alkenyl; and R 2 teeth, [ka] is selected from the group consisting of:

[0308] In some embodiments, LNPs comprising formula (xxii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxii) are LNPs described by WO2021113777 (e.g., lipids of formula (2), such as lipids in Table 2 of WO2021113777). [ka] During the ceremony, each n is independently an integer from 1 to 15; R 1 and R 2 are each independently [ka] is selected from the group consisting of R 3 teeth, [ka] is selected from the group consisting of:

[0309] In some embodiments, LNPs comprising formula (xxiii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNPs of formula (xxiii) are LNPs described by WO2021113777 (e.g., lipids of formula (3), such as lipids in Table 3 of WO2021113777). [ka] During the ceremony, X is selected from -O-, -S-, or -OC(O)-*, * being R 1 Indicates the attachment point to; R 1 teeth, [ka] and R 2 teeth, [ka] is selected from the group consisting of:

[0310] In some embodiments, a composition described herein (e.g., a nucleic acid (e.g., a circular polyribonucleotide, a linear polyribonucleotide) or a protein) is provided in a LNP that includes an ionizable lipid. In some embodiments, the ionizable lipid is 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)heptadecan-9-yl octanoate (SM-102), e.g., as described in Example 1 of U.S. Pat. No. 9,867,888, which is incorporated by reference in its entirety. In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01), for example, as synthesized in Example 13 of WO 2015 / 095340, which is incorporated by reference in its entirety. In some embodiments, the ionizable lipid is 9-((4-dimethylamino)butanoyl)oxy)heptadecanedienoate di((Z)-non-2-en-1-yl) (L319), for example, as synthesized in Example 7, 8, or 9 of U.S. Patent Publication No. 2012 / 0027803, which is incorporated by reference in its entirety. In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO 2010 / 053572, which is incorporated by reference in its entirety.In some embodiments, the ionizable lipid is the imidazole cholesterol ester (ICE) lipid 3-(1H-imidazol-4-yl)propanoic acid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl, e.g., structure (I) from WO 2020 / 106946, the entire contents of which are incorporated herein by reference for all purposes.

[0311] In some embodiments, the ionizable lipid may be a cationic lipid that is an ionizable cationic lipid, for example, a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be easily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids include one or more amine groups that have a positive charge. In some embodiments, the lipid particle includes a cationic lipid in a formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structured lipids (e.g., sterols), PEG, cholesterol, and lipids conjugated to a polymer. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein may have an effective pKa of greater than 6.0. In embodiments, the lipid nanoparticle may include a second cationic lipid that has a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid. The lipid nanoparticles can include between 40-60 mol percent cationic lipids, neutral lipids, steroids, lipids conjugated with polymers, and therapeutic agents, such as nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)), encapsulated within or associated with the lipid nanoparticles. In some embodiments, the nucleic acid is combined with the cationic lipids. The nucleic acid can be adsorbed to the surface of the LNP, e.g., LNPs including cationic lipids. In some embodiments, the nucleic acid can be encapsulated in the LNP, e.g., LNPs including cationic lipids. In some embodiments, the lipid nanoparticles can include a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable.In some embodiments, lipid nanoparticles comprising one or more lipids described herein, e.g., formula (i), (ii), (vii) and / or (ix), encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of the RNA molecules.

[0312] Exemplary ionizable lipids that may be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of International Publication No. WO2019051289, the entire contents of which are incorporated herein by reference for all purposes. Additional exemplary lipids include, but are not limited to, one or more of the following formulas: X of US2016 / 0311759; I of US20150376115 or in US2016 / 0376224; I, II, or III of US20160151284; I, IA, II, or IIA of US20170210967; Ic of US Patent Application Publication No. 150140070; A of US Patent Application Publication No. 2013 / 0178541; I of US Patent Application Publication No. 2013 / 0303587 or US Patent Application Publication No. 2013 / 0123338; ​​I of US Patent Application Publication No. 2015 / 0141678; II, III, IV, or V of US Patent Application Publication No. 2015 / 0239926; I of US Patent Application Publication No. 2017 / 0119904; WO 2017 / 117528 I or II of the specification of the patent application; U.S. Patent Application Publication No. 2012 / 0149894 A; U.S. Patent Application Publication No. 2015 / 0057373 A; WO 2013 / 116126 A; U.S. Patent Application Publication No. 2013 / 0090372 A; U.S. Patent Application Publication No. 2013 / 0274523 A; U.S. Patent Application Publication No. 2013 / 0274504 A; U.S. Patent Application Publication No. 2013 / 0053572 A; WO 201 A of WO 2012 / 162210; A of U.S. Patent Application Publication No. 2008 / 042973; I, II, III, or IV of U.S. Patent Application Publication No. 2012 / 01287670; I or II of U.S. Patent Application Publication No. 2014 / 0200257; I, II, or III of U.S. Patent Application Publication No. 2015 / 0203446; I or III of U.S. Patent Application Publication No. 2015 / 0005363;U.S. Patent Application Publication No. 2014 / 0308304, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV; U.S. Patent Application Publication No. 2013 / 0338210, I, II, III, or IV of WO 2009 / 132131; U.S. Patent Application Publication No. 2012 / 01011478, A; U.S. Patent Application Publication No. 2012 / 0027796, I or XXXV; U.S. Patent Application Publication No. XIV or XVII of US Patent Application Publication No. 2012 / 0058144; XIV or XVII of US Patent Application Publication No. 2013 / 0323269; I of US Patent Application Publication No. 2011 / 0117125; I, II, or III of US Patent Application Publication No. 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII of US Patent Application Publication No. 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, or XII of US Patent Application Publication No. 2011 / 0076335 XII, XIII, XIV, XV, or XVI; I or II of U.S. Patent Application Publication No. 2006 / 008378; I of U.S. Patent Application Publication No. 2013 / 0123338; ​​I or XAYZ of U.S. Patent Application Publication No. 2015 / 0064242; XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013 / 0022649; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; No. 62967, I or II; U.S. Patent Application Publication No. 2013 / 0189351, IX; U.S. Patent Application Publication No. 2014 / 0039032, I; U.S. Patent Application Publication No. 2018 / 0028664, V; U.S. Patent Application Publication No. 2016 / 0317458, I; U.S. Patent Application Publication No. 2013 / 0195920, I; U.S. Patent Application Publication No. 10,221,127, 5, 6, or 10; WO 2018 / 081480, III-3; WO 2020 / 081938, I-5 or I-8;18 or 25 of U.S. Pat. No. 9,867,888; A of U.S. Pat. Appl. Pub. No. 2019 / 0136231; II of WO 2020 / 219876; 1 of U.S. Pat. Appl. Pub. No. 2012 / 0027803; OF-02 of U.S. Pat. Appl. Pub. No. 2019 / 0240349; 23 of U.S. Pat. No. 10,086,013; cKK-E12 / A6 of Miao et al. (2020); C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al. (2017); Whitehead et al. al., 304-O13 or 503-O13; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO 2020 / 106946; I of WO 2020 / 106946; and (1), (2), (3), or (4) of WO 2021 / 113777. Exemplary lipids further include any one of the lipids in Tables 1-16 of WO 2021 / 113777. The entire contents of each reference are incorporated herein by reference for all purposes.

[0313] In some embodiments, the ionizable lipid is MC3 butanoic acid (6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3l-tetraen-l9-yl-4-(dimethylamino) (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, for example, as described in Example 10 of WO2019051289A9 (incorporated herein by reference in its entirety for all purposes). In some embodiments, the ionizable lipid is, for example, (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-diene-1-amine (compound 32), as described in Example 11 of WO2019051289A9 (the entire contents of which are incorporated by reference for all purposes). In some embodiments, the ionizable lipid is, for example, compound 6 or compound 22, as described in Example 12 of WO2019051289A9 (the entire contents of which are incorporated by reference for all purposes).

[0314] Exemplary non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl dioleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g. 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g. 16-O-dimethyl PE), l8 -l-transPE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dielucoyl phosphatidylcholine (DEPC), palmitoyl oleoyl ... dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phosphatidylethanolamine (DOPE), dielucoyl oleoyl phospha These include, but are not limited to, sphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids include, but are not limited to, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, which is incorporated herein by reference, in certain embodiments. Such lipids include, in some embodiments, plant lipids (e.g., DGTS) that have been found to improve liver transfection with mRNA.

[0315] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as, for example, stearylamine, dodeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, the entire contents of which are incorporated herein by reference for all purposes.

[0316] In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II, or IV of US Patent Publication No. 2018 / 0028664, the entire contents of which are incorporated herein by reference for all purposes. The non-cationic lipid may comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0317] In some embodiments, the lipid nanoparticles do not comprise any phospholipids.

[0318] In some aspects, the lipid nanoparticles can further include components such as sterols to provide membrane integrity. One exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009 / 127060 and US Patent Publication US 2010 / 0130588, the entire contents of each of which are incorporated herein by reference for all purposes.

[0319] In some embodiments, components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%). In some embodiments, such components are 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0320] In some embodiments, the lipid nanoparticles may contain polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit lipid nanoparticle aggregation and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as a (methoxypolyethylene glycol)-conjugated lipid.

[0321] Exemplary PEG-lipid conjugates include PEG-diacylglycero (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy( Polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287, 6,311, 6,414, 6,511, 6,611, 6,287, 6,311, 6,411, 6,5 ... 59l, U.S. Patent Application Publication No. 2003 / 0077829, U.S. Patent Application Publication No. 2003 / 0077829, U.S. Patent Application Publication No. 2005 / 0175682, U.S. Patent Application Publication No. 2008 / 0020058, U.S. Patent Application Publication No. 2011 / 0117125, U.S. Patent Application Publication No. 2010 / 0130588, U.S. Patent Application Publication No. 2016 / 0376224, U.S. Patent Application Publication No. 2017 / 0119904, and U.S. Patent Application Publication No. 2017 / 0119904. In some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2, or V of U.S. Patent Application Publication No. 2018 / 0028664, the contents of which are incorporated herein by reference in their entireties. In some embodiments, the PEG-lipid is of formula II of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224, the entire contents of each of which are incorporated herein by reference for all purposes.In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmitoyloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be, for example, PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (l-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3, PEG-lipids may be one or more of PEG-DMG, 4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, ... [ka] The present invention includes a structure selected from the following:

[0322] In some embodiments, lipids conjugated with molecules other than PEG can also be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymeric lipid (GPL) conjugates can be used in place of or in addition to PEG-lipids.

[0323] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids, are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9, the entire contents of which are incorporated herein by reference for all purposes.

[0324] In some embodiments, the PEG or conjugated lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. The molar ratios of ionizable lipid, non-cationic lipid, sterol, and PEG / conjugated lipid may vary as needed. For example, the lipid particle may comprise 30-70% ionizable lipid by molar or total weight of the composition, 0-60% cholesterol by molar or total weight of the composition, 0-30% non-cationic lipid by molar or total weight of the composition, and 1-10% conjugated lipid by molar or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by molar or total weight of the composition, 40-50% cholesterol by molar or total weight of the composition, and 10-20% non-cationic lipid by molar or total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by molar or total weight of the composition, 20-40% cholesterol by molar or total weight of the composition, and 5-10% non-cationic lipid by molar or total weight of the composition and 1-10% conjugated lipid by molar or total weight of the composition. The composition may contain 60-70% ionizable lipid by molar or total weight of the composition, 25-35% cholesterol by molar or total weight of the composition, and 5-10% non-cationic lipid by molar or total weight of the composition. The composition may also contain up to 90% ionizable lipid by molar or total weight of the composition and 2-15% non-cationic lipid by molar or total weight of the composition.Formulations may also be used that contain, for example, 8-30% ionizable lipid by molar or total weight of the composition, 5-30% noncationic lipid by molar or total weight of the composition, and 0-20% cholesterol by molar or total weight of the composition; 4-25% ionizable lipid by molar or total weight of the composition, 4-25% noncationic lipid by molar or total weight of the composition, 2-25% cholesterol by molar or total weight of the composition, 10-35% conjugated lipid by molar or total weight of the composition, and 5% cholesterol by molar or total weight of the composition; The lipid nanoparticle formulation may comprise 2-30% ionizable lipid by molar or total weight of the composition, 2-30% non-cationic lipid by molar or total weight of the composition, 1-15% cholesterol by molar or total weight of the composition, 2-35% conjugated lipid by molar or total weight of the composition, and 1-20% cholesterol by molar or total weight of the composition; or up to 90% ionizable lipid by molar or total weight of the composition and 2-10% non-cationic lipid by molar or total weight of the composition, or 100% cationic lipid by molar or total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol, and PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol, and PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0325] In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, with the molar ratio of lipids ranging from 20-70 mole percent with respect to the ionizable lipid, with a target of 40-60; the molar percent of the non-cationic lipid ranging from 0-30, with a target of 0-15; the molar percent of the sterol ranging from 20-70, with a target of 30-50; and the molar percent of the PEGylated lipid ranging from 1-6, with a target of 2-5.

[0326] In some embodiments, the lipid particles comprise a molar ratio of ionizable lipid / non-cationic lipid / sterol / conjugated lipid of 50:10:38.5:1.5.

[0327] In some aspects, the disclosure provides lipid nanoparticle formulations comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0328] In some embodiments, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticles may contain other compounds in addition to the nucleic acid, or at least one second nucleic acid different from the first one. Without being limited thereto, the other additional compounds may be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0329] In some embodiments, the LNPs comprise a biodegradable, ionizable lipid. In some embodiments, the LNPs comprise octadeca-9,l2-dienoic acid (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl, also known as (9Z,l2Z)-octadeca-9,l2-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl, or another ionizable lipid. See, e.g., the lipids in WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, the entire contents of each of which are incorporated herein by reference for all purposes, and the references provided therein. In some embodiments, the terms cationic and ionizable in the context of LNP lipids are interchangeable, e.g., ionizable lipids are cationic depending on the pH.

[0330] In some embodiments, the mean LNP diameter of an LNP formulation can be between 10 nm and 100 nm, for example, as measured by dynamic light scattering (DLS). In some embodiments, the mean LNP diameter of an LNP formulation can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 70 nm to about 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation can be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

[0331] The LNPs may be relatively homogeneous in some instances. The polydispersity index may be used to indicate the uniformity of the LNPs, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The LNPs may have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNPs may be about 0.10 to about 0.20.

[0332] The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low positive or negative charge are generally desirable because highly charged species can undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about 10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0333] The efficiency of protein and / or nucleic acid encapsulation describes the amount of protein and / or nucleic acid encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disintegration of the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0334] The LNPs may optionally include one or more coatings. In some embodiments, the LNPs may be formulated in a capsule, film, or tablet with a coating. The capsule, film, or tablet containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0335] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO 2020 / 061457 and WO 2021 / 113777, the entire contents of each of which are incorporated herein by reference for all purposes. Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021).oi.org / 10.1038 / s41578-021-00358-0, the entire contents of which are incorporated herein by reference (see, e.g., exemplary lipids and lipid derivatives in FIG. 2 of Hou et al.), the entire contents of which are incorporated herein by reference for all purposes.

[0336] In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), formulations and in vivo uses as taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), the entire contents of which are incorporated herein by reference for all purposes.

[0337] LNP formulations optimized for delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, the entire contents of each of which are incorporated by reference herein for all purposes, and are useful for delivery of circular and linear polyribonucleotides as described herein.

[0338] Additional specific LNP formulations useful for delivery of nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) are described in U.S. Pat. Nos. 8,158,601 and 8,168,775, the entire contents of each of which are incorporated herein by reference for all purposes, including the formulations used in Patisiran, sold under the name ONPATTRO.

[0339] Exemplary administrations of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) LNPs can include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). Exemplary administrations of AAVs including polyribonucleotides (e.g., cyclic polyribonucleotide, linear polyribonucleotide) can include about 10 11 , 10 12 , 10 13 , and 10 14 The antibody may include an MOI of vg / kg.

[0340] 5.8 Pharmaceutical Compositions In one aspect, the present disclosure provides a method for the preparation of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof) described herein, a nucleic acid molecule (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof)) described herein, a vector (e.g., a nucleic acid molecule (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof) described herein) or ...) described herein) or a vector (e.g., a vector (e.g., a vector) described herein) Provided herein are pharmaceutical compositions comprising a vector comprising a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), or a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), and / or a composition described herein (e.g., a vaccine composition), and a pharma- ceutical composition comprising a pharma-ceutical composition comprising a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein ... and a pharma- ceutical composition comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2

[0341] In one aspect, also provided herein is a method of making a pharmaceutical composition described herein, comprising administering to a patient a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or variant thereof)), a nucleic acid molecule described herein (e.g., a nucleic acid molecule described herein comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or variant thereof)), a vector described herein (e.g., a nucleic acid molecule described herein ... and formulating it into a pharmaceutically acceptable composition by the addition of one or more pharmaceutically acceptable excipients. Methods are provided that include providing a vector comprising a nucleic acid molecule comprising a coding region encoding an immunogen (or immunogenic fragment and / or variant thereof)) as described herein, a carrier comprising a SARS-CoV-2 spike protein (or polypeptide, e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or variant thereof)) as described herein, or a nucleic acid molecule described herein (e.g., a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or variant thereof))) as described herein, and / or a composition (e.g., a vaccine composition) as described herein, and formulating it into a pharmaceutically acceptable composition by the addition of one or more pharmaceutically acceptable excipients.

[0342] Acceptable excipients (e.g., carriers and stabilizers) are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid or methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; or m-cresol); low molecular weight (less than about 10 residues) polypeptides; These include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0343] The pharmaceutical composition may be formulated for any route of administration to a subject. The skilled artisan is aware of the various possibilities of administering the pharmaceutical composition described herein to induce an immune response against the immunogen and / or antigen in the pharmaceutical composition. Non-limiting embodiments include intramuscular, intradermal, subcutaneous, transdermal, or mucosal administration, parenteral administration, such as inhalation, intranasal, oral, etc. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular, intradermal, or subcutaneous injection. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular injection. In one embodiment, the pharmaceutical composition is formulated for administration by intradermal injection. In one embodiment, the pharmaceutical composition is formulated for administration by subcutaneous injection. The injection may be prepared in conventional forms, either as a liquid solution or suspension. The injection may contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if necessary, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins. In some embodiments, the pharmaceutical composition is formulated as a single dose. In some embodiments, the pharmaceutical composition is formulated as a multiple dose.

[0344] Pharmaceutically acceptable excipients used in the parenteral preparations described herein include, for example, aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, or other pharma- ceutically acceptable substances.Examples of aqueous media that can be incorporated in one or more of the formulations described herein include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose or lactated Ringer's injection.Non-aqueous parenteral media that can be incorporated in one or more of the formulations described herein include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrates may be added to the parenteral preparations described herein and packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride or benzethonium chloride. Isotonicity agents that may be incorporated in one or more of the formulations described herein include sodium chloride or dextrose. Buffers that may be incorporated in one or more of the formulations described herein include phosphates or citrates. Antioxidants that may be incorporated in one or more of the formulations described herein include sodium hydrogen sulfate. Local anesthetics that may be incorporated in one or more of the formulations described herein include procaine hydrochloride. Suspending and dispersing agents that may be incorporated in one or more of the formulations described herein include sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone. An emulsifying agent that may be incorporated in one or more of the formulations described herein includes polysorbate 80 (TWEEN® 80). A suspending or chelating agent for metal ions that may be incorporated in one or more of the formulations described herein is EDTA.Pharmaceutical carriers that may be incorporated in one or more of the formulations described herein also include ethyl alcohol, polyethylene glycol or propylene glycol for water miscible vehicles; or sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0345] The exact dosage to be used in the pharmaceutical composition will also depend on the route of administration and the severity of the condition caused thereby, and should be determined according to the doctor's judgment and each subject's circumstances.For example, the effective dosage can also vary according to the means of administration, the target site, the subject's physiological condition (including age, weight, and health condition), other medications or therapies administered, whether the therapy is preventive or therapeutic.The dosage of the therapeutic agent is preferably titrated to optimize safety and effectiveness.

[0346] 5.9 Adjuvants Any of the foregoing, e.g., a SARS-CoV-2 spike protein or polypeptide (e.g., an immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof) described herein, a nucleic acid molecule (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (or a fusion or conjugate thereof)) described herein, a vector (e.g., a nucleic acid molecule (e.g., ... vector or a vector described herein) described herein, A vector comprising a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) as described herein, a carrier (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof) as described herein) or a nucleic acid molecule (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) as described herein, a composition (e.g., a vaccine composition), and / or a pharmaceutical composition described herein may be combined with and / or administered in combination with an adjuvant.

[0347] Adjuvants are known in the art to further enhance immune responses (e.g., to immunogens). Common classes of adjuvants include, but are not limited to, inorganic adjuvants, small molecule adjuvants, oil-in-water emulsions, lipids, polymers, peptides, peptidoglycans, carbohydrates, polysaccharides, RNA-based adjuvants, DNA-based adjuvants, viral particles, bacterial adjuvants, nanoparticles (e.g., inorganic nanoparticles), and multicomponent adjuvants. Exemplary adjuvants include aluminum salts, such as aluminum hydroxide and / or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions) including MF59 (see, e.g., WO 90 / 14837, the entire contents of which are incorporated herein by reference for all purposes), squalene-water emulsions such as MF59, AS03, and Montanide; immunostimulating complexes (ISCOMS), such as QS21 and immunostimulating complexes (ISCOMS) (see, e.g., U.S. Pat. No. 5,057,540, the entire contents of each of which are incorporated herein by reference for all purposes; WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2004 / 004763, WO 2004 / 004764, WO 2004 / 004765, WO 2004 / 004766, WO 2004 / 004767 ... 05 / 002620); protamine or protamine salts (e.g., protamine sulfate); calcium salts; bacterial or microbial derivatives (examples of which include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif-containing oligonucleotides, ADP-ribosylating bacterial toxins or variants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, etc.); eukaryotic proteins (e.g., antibodies or fragments thereof (e.g., directed against the antigen itself or CD1a, CD3, CD7, CD80) and ligands for receptors (e.g., CD40L, GMCSF, GCSF, etc.).

[0348] Exemplary RNA-based adjuvants include, but are not limited to, polyIC, polyIC:LC, hairpin RNA (e.g., with 5' PPP-containing sequences, viral sequences, poly U-containing sequences), dsRNA, natural or synthetic immunostimulatory RNA sequences, nucleic acid analogs, cyclic di-GMP and immunostimulatory base analogs, optionally cyclic GMP-AMP or cyclic dinucleotides, such as C8-substituted or N7,C8-disubstituted guanine ribonucleotides. Exemplary DNA-based adjuvants include, but are not limited to, CpG, dsDNA, or natural or synthetic immunostimulatory DNA sequences. Exemplary bacterial-based adjuvants include, but are not limited to, bacterial adjuvants that are flagellin, LPS, or bacterial toxins, such as enterotoxins, heat-labile toxins, and cholera toxins. Exemplary carbohydrate or polysaccharide adjuvants include, but are not limited to, dextran (branched bacterial polysaccharide), dextran-sulfate, lentinan, zymosan, beta-glucan, deltin, mannan, and chitin. Exemplary small molecule adjuvants include, but are not limited to, imiquimod, resiquimod, and gardquimod. Exemplary lipid or polymer adjuvants include, but are not limited to, polymeric nanoparticles (e.g., PLGA, PLG, PLA, PGA, or PHB), liposomes (e.g., virosomes and CAF01), LNP or components thereof, lipopolysaccharides (LPS) (e.g., monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA)), lipopeptides (e.g., Pam2 (Pam2CSK4) or Pam3 (Pam3CSK4)), and glycolipids (e.g., trehalose dimycolate). Exemplary peptides or peptidoglycans include, but are not limited to, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), flagellin-fusion proteins, mannose-binding lectin (MBL), cytokines, and chemokines. Exemplary inorganic nanoparticle adjuvants include, but are not limited to, gold nanorods, silica-based nanoparticles (eg, mesoporous silica nanoparticles (MSN)).Exemplary multicomponent adjuvants include, but are not limited to, AS01, AS03, AS04, Freund's complete adjuvant, and CAF01.

[0349] 5.10 Method of Use References herein include, but are not limited to, the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (and immunogenic fragments and / or immunogenic variants thereof)) (or fusions or conjugates thereof) described herein, the nucleic acid molecules (e.g., nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (or fusions or conjugates thereof) described herein, the vectors (e.g., nucleic acid molecules (e.g., coding regions encoding a SARS-CoV-2 spike protein, e.g., an immunogen (or immunogenic fragment and / or immunogenic variant thereof)) (or fusions or conjugates thereof) described herein ... Various methods are provided that utilize a vector comprising a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) as described herein, a carrier comprising a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) or a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof))), a vaccine composition (e.g., a vaccine composition comprising any of the foregoing), and / or a pharmaceutical composition (e.g., a pharmaceutical composition comprising any of the foregoing) as described herein.

[0350] In some of the following aspects, the method includes administering to a subject one or more of the foregoing (e.g., a protein (or a fusion or conjugate thereof), a polypeptide (or a fusion or conjugate thereof), an immunogen (or a fusion or conjugate thereof), a nucleic acid molecule (or a fusion or conjugate thereof), a vector, a carrier, a vaccine composition, a pharmaceutical composition). Exemplary subjects include mammals, e.g., humans, non-human mammals, e.g., non-human primates. In some embodiments, the subject is a human.

[0351] In some embodiments, the subject is elderly, pregnant, newborn, immunocompromised, or immunosuppressed. In some embodiments, the subject has one or more of the following: cancer, heart disease, obesity, diabetes, asthma, chronic lung disease, and / or sickle cell disease. In some embodiments, the subject has a weakened immune system or a weakened immune response (e.g., a weakened immune response to a vaccine). In some embodiments, the subject is immunocompromised or immunosuppressed. In some embodiments, the subject is clinically vulnerable to an infectious disease. In some embodiments, the subject has cancer, has an autoimmune disease, has an immunodeficiency, has undergone a bone marrow or organ transplant, is undergoing an immune cell depleting therapy, is undergoing chemotherapy, has a chronic viral infection, post-viral syndrome or post-viral fatigue syndrome (e.g., HIV infection or AIDS; long-term Covid or chronic post-Covid syndrome), is using or has had a long-term use of immunosuppressive medication, is a current smoker or has a history of smoking, or is at least 50 (e.g., at least 55, 60, 65, 70, 75, 80, 85, 90, or 100) years old. In some embodiments, the subject is at least 50, 60, 65, 70, or 75 years old. In some embodiments, the subject is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, or 120 years old. In some embodiments, the subject is about 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-120, 70-110, 70-100, 70-90, 70-80, 80-120, 80-110, 80-100, 80-90, 90-120, 90-110, or 90-100 years old.

[0352] The dosage of one or more of the foregoing (e.g., proteins, polypeptides, immunogens, nucleic acid molecules, vectors, carriers, vaccine compositions, pharmaceutical compositions) to be administered to a subject can be determined according to standard techniques well known to those of skill in the art, including the type of adjuvant (if any) used, the route of administration, and the age and weight of the subject. In some embodiments, a single dose of any one of the foregoing is administered to a subject in need thereof. In some embodiments, a series of doses of any one of the foregoing is administered to a subject in need thereof (e.g., two doses given at a range of set intervals (e.g., 2 weeks, 3 weeks) or within a range (e.g., 2-6 weeks apart)). In some embodiments, a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a vaccine composition described herein, or a pharmaceutical composition (e.g., any one of the foregoing) is administered in a therapeutically effective amount. In some embodiments, the dose of an mRNA molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)) described herein (e.g., a vaccine or pharmaceutical composition comprising same) is 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.

[0353] 5.10.1 Method of service In one aspect, provided herein is (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a nucleic acid molecule described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof). The present invention relates to a method of delivering a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) to a subject in need thereof, the method comprising: (d) a carrier described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof) described herein or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (e.g., a carrier comprising a nucleic acid molecule (e.g., an RNA molecule)); (e) a vaccine composition described herein (e.g., a vaccine composition comprising any of the foregoing); or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing) to a subject in need thereof, the method comprising:Methods are provided that include administering to a subject (a) a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), (b) a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), (c) a vector, (d) a carrier, (e) a vaccine composition, or (f) a pharmaceutical composition, thereby delivering a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, a carrier, a vaccine composition, or a pharmaceutical composition to the subject.

[0354] In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule, vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject in an amount and for a time sufficient to deliver the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition to the subject.

[0355] 5.10.2 Methods of Inducing or Enhancing an Immune Response In one aspect, provided herein is a method of inducing and / or enhancing an immune response in a subject in need thereof, comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) (or a fusion or conjugate thereof) described herein), (c) a vector described herein (e.g., a vector described herein comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (d) a vector comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (e) a vector comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (f) a vector comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (g) a vector comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (h) a vector comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immuno (d) a carrier described herein (e.g., a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a carrier comprising a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) that comprises a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein)), (e) a vaccine composition described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), thereby inducing and / or enhancing an immune response in a subject.

[0356] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject in an amount and for a time sufficient to induce and / or enhance an immune response in the subject.

[0357] The immune response in a subject can be measured by common methods known to those skilled in the art. For example, serological assays can be utilized to detect hormonal responses by measuring the titer of anti-antigen (e.g., anti-SARS-CoV-2 spike protein, anti-SARS-CoV-2 spike protein RBD) IgG antibodies after administration. For example, enzyme-linked immunosorbent assay (ELISA) is a standard clinical test for detecting and quantifying antibodies well known to those skilled in the art. Generally, blood is collected from a consenting subject, centrifuged, and serum is isolated according to standard procedures. Recombinant target antigens (e.g., SARS-CoV-2 spike protein, SARS-CoV-2 spike protein RBD) are immobilized in microplate wells. The microplate is blocked by incubation with an irrelevant antigen (e.g., bovine serum albumin). Serum samples from the subject are prepared and added to the blocked wells to allow binding of antigen-specific antibodies to the immobilized antigen. Bound antibodies are detected using a tagged secondary antibody that binds to the previously bound antibody (e.g., an anti-human IgG antibody). See, e.g., Front. Immunol., Forgacs David et al., SARS-CoV-2 mRNA Vaccines Elicit Different Responses in Immunologically Naieve and Pre-Immune Humans; Vol 12 (27 September 2021) https: / / doi.org / 10.3389 / fimmu.2021.728021, the entire contents of which are incorporated herein by reference for all purposes.

[0358] Cell-based assays can also be utilized to detect cell-based immune responses (e.g., T cell immune responses). For example, antigen-specific T cells (e.g., CD4+ or CD8+ T cells) can be measured using enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS) assays, or activation-induced marker assays (AIM). Each of these assays is commonly used to detect cell-based (e.g., T cell) immune responses to vaccines and is well known to those of skill in the art. See, for example, Bowyer, Georgina et al. "Activation-induced Markers Detect Vaccine-Specific CD4+ T cells," in J. Immunol. 1999, 143:1311-1323, the entire contents of which are incorporated herein by reference for all purposes. + Please refer to “T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials.” Vaccines vol.6,3 50.31 Jul.2018,doi:10.3390 / vaccines6030050.

[0359] 5.10.3 Methods for preventing, ameliorating, and / or treating SARS-CoV-2 infection In one aspect, provided herein is a method of preventing, ameliorating, and / or treating a SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) (or a fusion or conjugate thereof) described herein), (c) a vector described herein (e.g., a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (d) a vector comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (e) a vector comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (f) a vector comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (g) a vector comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (h) a vector comprising a coding region encoding a SARS-CoV-2 (d) a carrier described herein (e.g., a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a carrier comprising a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein)); (e) a vaccine composition described herein (e.g., a vaccine composition comprising any of the foregoing); or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), thereby preventing SARS-CoV-2 infection in the subject;Methods are provided that include ameliorating and / or treating

[0360] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 protein or peptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject in an amount and for a time sufficient to prevent, ameliorate, and / or treat a SARS-CoV-2 infection in the subject.

[0361] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, or pharmaceutical composition is administered to a subject as a prophylactic treatment. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, or pharmaceutical composition is administered as a treatment after the onset of at least one symptom of SARS-CoV-2 infection or SARS-CoV-2 infection-associated disease.

[0362] In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject after a determination that the subject has or does not have a SARS-CoV-2 infection.

[0363] In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS-CoV-2, reduces the likelihood of developing an established infection after challenge with SARS-CoV-2, reduces the duration of SARS-CoV-2 infection, prevents or delays the onset of one or more symptoms of COVID-19, reduces the frequency and / or severity of one or more symptoms of COVID-19, and / or reduces the risk of hospitalization or death associated with COVID-19, or any combination thereof.

[0364] Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than 20 breaths per minute, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.

[0365] The SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), vectors, carriers, vaccine compositions, and pharmaceutical compositions described herein can be administered as the prime and / or boost in an allogeneic or heterogeneous prime-boost regimen.

[0366] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a prime in an allogeneic or heterogeneous prime-boost regimen.

[0367] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a boost in an allogeneic or heterogeneous prime-boost regimen.

[0368] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a vaccine prime and a vaccine boost in an allogeneic prime-boost regimen.

[0369] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a prime in a heterologous prime-boost regimen. The boost vaccine composition in the regimen can be a vaccine based on mRNA, DNA, a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a vesicular stomatitis virus vector, a vaccinia virus vector, or a measles virus vector), a peptide or protein, a virus-like particle (VLP), a capsid-like particle (CLP), a live attenuated virus, an inactivated virus (killed vaccine), or the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen than the booster vaccine.

[0370] In some embodiments, the SARS-CoV-2 spike protein (e.g., an immunogen (or an immunogenic fragment or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, and pharmaceutical composition described herein are administered to a subject as a boost in a heterologous prime-boost regimen. The prime vaccine composition in the regimen can be a vaccine based on mRNA, DNA, a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a vesicular stomatitis virus vector, a vaccinia virus vector, or a measles virus vector), a peptide or protein, a virus-like particle (VLP), a capsid-like particle (CLP), a live attenuated virus, an inactivated virus (killed vaccine), or the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen than the booster vaccine.

[0371] In some embodiments, a single dose of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, carrier, vaccine composition, or pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, carrier, vaccine composition, or pharmaceutical composition is administered to the subject (e.g., two doses given at a range of set intervals (e.g., two weeks, three weeks apart) or within a range (e.g., two to six weeks apart)).

[0372] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject in a therapeutically effective amount. In some embodiments, an mRNA molecule encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) (e.g., a vaccine or pharmaceutical composition comprising same) is administered to a subject, and the mRNA molecule is administered at a dose of about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg).

[0373] 5.10.4 Methods of vaccination against SARS-CoV-2 Provided herein, inter alia, are various methods of vaccinating a subject (e.g., a human subject) against SARS-CoV-2. As such, in §§ 5.10.4.1 and 5.10.4.2, various methods of vaccinating a subject are provided below utilizing one or more of the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), vectors, carriers, vaccine compositions, and pharmaceutical compositions described herein.

[0374] 5.10.4.1 Methods for vaccinating subjects against SARS-CoV-2 In one aspect, provided herein is a method of vaccinating a subject against SARS-CoV-2, comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein (or a fusion or conjugate thereof)), (c) a vector described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (d) a vector described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (e) a vector described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein), (f) a vector described herein (f) a vector described herein (f) a vector described herein (f) a vector described herein (f) a vector described herein (f) a vector described herein (f) a vector described herein (f) a vector described herein (d) a carrier described herein (e.g., a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a carrier comprising a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof) described herein)), (e) a vaccine composition described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), thereby vaccinating the subject against SARS-CoV-2.

[0375] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject in an amount and for a time sufficient to vaccinate the subject against SARS-CoV-2.

[0376] In some embodiments, the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), vectors, carriers, vaccine compositions, and pharmaceutical compositions described herein are administered as the prime and / or boost in an allogeneic or heterogeneous prime-boost regimen.

[0377] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a prime in an allogeneic or heterogeneous prime-boost regimen.

[0378] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a boost in an allogeneic or heterogeneous prime-boost regimen.

[0379] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a vaccine prime and a vaccine boost in an allogeneic prime-boost regimen.

[0380] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a prime in a heterologous prime-boost regimen. The boost vaccine composition in the regimen can be a vaccine based on mRNA, DNA, a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a vesicular stomatitis virus vector, a vaccinia virus vector, or a measles virus vector), a peptide or protein, a virus-like particle (VLP), a capsid-like particle (CLP), a live attenuated virus, an inactivated virus (killed vaccine), or the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen than the booster vaccine.

[0381] In some embodiments, the SARS-CoV-2 spike protein (e.g., an immunogen (or an immunogenic fragment or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, and pharmaceutical composition described herein are administered to a subject as a boost in a heterologous prime-boost regimen. The prime vaccine composition in the regimen can be a vaccine based on mRNA, DNA, a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a vesicular stomatitis virus vector, a vaccinia virus vector, or a measles virus vector), a peptide or protein, a virus-like particle (VLP), a capsid-like particle (CLP), a live attenuated virus, an inactivated virus (killed vaccine), or the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen than the booster vaccine.

[0382] In some embodiments, a single dose of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, carrier, vaccine composition, or pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, carrier, vaccine composition, or pharmaceutical composition is administered to the subject (e.g., two doses given at a range of set intervals (e.g., two weeks, three weeks apart) or within a range (e.g., two to six weeks apart)).

[0383] In some embodiments, a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein (e.g., a vaccine or pharmaceutical composition comprising same) is administered at a dose of 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg).

[0384] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, or pharmaceutical composition is administered to a subject as a prophylactic treatment. In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, or pharmaceutical composition is administered as a treatment after the onset of at least one symptom of SARS-CoV-2 infection or SARS-CoV-2 infection-associated disease.

[0385] In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject after a determination that the subject has or does not have a SARS-CoV-2 infection.

[0386] In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS-CoV-2, reduces the likelihood of developing an established infection after challenge with SARS-CoV-2, reduces the duration of SARS-CoV-2 infection, prevents or delays the onset of one or more symptoms of COVID-19, reduces the frequency and / or severity of one or more symptoms of COVID-19, and / or reduces the risk of hospitalization or death associated with COVID-19, or any combination thereof.

[0387] Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than 20 breaths per minute, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.

[0388] 5.10.4.2 How to vaccinate subjects using SARS-CoV-2 mRNA vaccines In one aspect, provided herein is a method of vaccinating a subject against SARS-CoV-2, the method comprising administering to the subject (a) an mRNA molecule (e.g., an mRNA molecule described herein) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or a polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof); (b) a vector comprising the mRNA molecule; (c) a carrier comprising the mRNA molecule or vector; (d) a vaccine composition comprising the mRNA molecule, vector, or carrier; or (e) a pharmaceutical composition comprising the mRNA molecule, vector, carrier, or vaccine composition, thereby vaccinating the subject against SARS-CoV-2.

[0389] In some embodiments, the mRNA molecule is formulated in a lipid nanoparticle, and the vaccine composition has the following characteristics: (a) the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid, (b) the LNP has an average particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region.

[0390] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject in an amount and for a time sufficient to vaccinate the subject against SARS-CoV-2.

[0391] In some embodiments, the mRNA molecules, vectors, carriers, vaccine compositions, and pharmaceutical compositions described herein are administered as the prime and / or boost in an allogeneic or heterogeneous prime-boost regimen.

[0392] In some embodiments, an mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a prime in an allogeneic or heterogeneous prime-boost regimen.

[0393] In some embodiments, an mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a boost in an allogeneic or heterogeneous prime-boost regimen.

[0394] In some embodiments, the mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a vaccine prime and a vaccine boost in an allogeneic prime-boost regimen.

[0395] In some embodiments, the mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition described herein is administered to a subject as a prime in a heterologous prime-boost regimen. The boost vaccine composition in the regimen can be a vaccine based on mRNA, DNA, a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a vesicular stomatitis virus vector, a vaccinia virus vector, or a measles virus vector), a peptide or protein, a virus-like particle (VLP), a capsid-like particle (CLP), a live attenuated virus, an inactivated virus (killed vaccine), or the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen from the booster vaccine.

[0396] In some embodiments, the mRNA molecules, vectors, carriers, vaccine compositions, and pharmaceutical compositions described herein are administered to subjects as a boost in a heterologous prime-boost regimen. The prime vaccine composition in the regimen can be a vaccine based on mRNA, DNA, a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a vesicular stomatitis virus vector, a vaccinia virus vector, or a measles virus vector), a peptide or protein, a virus-like particle (VLP), a capsid-like particle (CLP), a live attenuated virus, an inactivated virus (killed vaccine), or the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen from the booster vaccine.

[0397] In some embodiments, a single dose of an mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of an mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition is administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)).

[0398] In some embodiments, the mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject in a therapeutically effective amount, hi some embodiments, the mRNA molecule is administered in a dose of about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg).

[0399] In some embodiments, the mRNA molecule, vector, carrier, or pharmaceutical composition is administered to a subject as a prophylactic treatment. In some embodiments, the mRNA molecule, vector, carrier, or pharmaceutical composition is administered as a treatment after the onset of at least one symptom of SARS-CoV-2 infection or SARS-CoV-2 infection-associated disease.

[0400] In some embodiments, the mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition is administered to a subject after a determination that the subject has or does not have a SARS-CoV-2 infection.

[0401] In some embodiments, the mRNA molecule, vector, carrier, vaccine composition, or pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS-CoV-2, reduces the likelihood of developing an established infection after challenge with SARS-CoV-2, reduces the duration of SARS-CoV-2 infection, prevents or delays the onset of one or more symptoms of COVID-19, reduces the frequency and / or severity of one or more symptoms of COVID-19, and / or reduces the risk of hospitalization or death associated with COVID-19, or any combination thereof.

[0402] Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than 20 breaths per minute, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.

[0403] 5.11 Kits In one aspect, provided herein is a kit comprising at least one SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule described herein (e.g., an RNA molecule, e.g., an mRNA molecule), a vector described herein, a carrier described herein, a vaccine composition described herein, and / or a pharmaceutical composition described herein. Additionally, the kit may include a liquid medium for solubilizing or diluting any one of the foregoing, and / or technical instructions. The technical instructions for the kit may contain information about administration and dosages and subjects (e.g., subject groups).

[0404] In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, a carrier, a vaccine composition, or a pharmaceutical composition described herein is provided in separate parts of a kit, and the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, a carrier, a vaccine composition, or a pharmaceutical composition described herein is optionally lyophilized, spray dried, or spray freeze dried. The kit may further contain a medium (e.g., a buffer solution) for stabilizing the dried or lyophilized pharmaceutical composition as part.

[0405] In some embodiments, the kit comprises a single-dose container of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, a carrier, a vaccine composition, or a pharmaceutical composition. In some embodiments, the kit comprises a multi-dose container and / or an administration device (e.g., a syringe for intradermal injection or a syringe for intramuscular injection) for administration of a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), a vector, a carrier, a vaccine composition, or a pharmaceutical composition described herein.

[0406] In some embodiments, the kit comprises an adjuvant in a separate container from the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein. The kit may further contain technical instructions for mixing the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule), vector, carrier, vaccine composition, or pharmaceutical composition described herein and the adjuvant prior to or for co-administration.

[0407] Any of the kits described herein may be used in any of the methods described herein, e.g., in § 5.10. Any of the kits described herein may be used in therapy or prophylaxis as defined herein (e.g., for the treatment, amelioration, and / or prevention of SARS-CoV-2 infection). EXAMPLES

[0408] 6. 6.1 Example 1. SARS-CoV-2 RNA Vaccine. The following examples provide exemplary methods of preparing an mRNA vaccine comprising mRNA encoding any one or more of the immunogens identified herein (e.g., immunogens comprising the amino acid substitutions described in Table 2).

[0409] 6.1.1 DNA preparation A DNA construct containing a SARS-CoV-2 protein (e.g., an immunogen) containing at least one of the amino acid substitutions listed in Table 2 is prepared and used for subsequent RNA in vitro transcription. Preparation of the DNA coding sequence may include codon optimization for stabilization and expression by introducing specific codons to create a DNA coding sequence (as discussed herein) with optimized G / C content. The optimized coding sequence is introduced into a DNA plasmid containing a 3'-UTR, a 5-UTR, and a polyadenylation sequence. The resulting DNA plasmid is transformed and propagated in bacteria using common protocols known in the art. The DNA plasmid is then extracted, purified, and used for RNA in vitro transcription.

[0410] 6.1.2 RNA in vitro transcription The DNA plasmid is enzymatically linearized using the restriction enzyme used for DNA-dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP / GTP / CTP / UTP) and a 5' cap (or analog) under suitable buffer conditions. The resulting RNA construct is purified using a suitable method known in the art, for example, RP-HPLC. RNA in vitro transcription is performed in the presence of modified nucleotides for incorporation in RNA in place of UTP, for example, pseudouridine or N1-methylpseudouridine (m1ψ). In some examples, a 5' cap is enzymatically added to the RNA after in vitro transcription using a capping enzyme as commonly known in the art.

[0411] 6.1.3 Preparation of LNPs and encapsulated RNA LNPs are prepared according to general procedures known in the art using, for example, cationic lipids, structured lipids, PEG-lipids, and cholesterol, see, for example, WO2015199952, WO2017004143, and WO2017075531, the entire contents of each of which are incorporated herein by reference for all purposes. The lipid solution (in ethanol) is mixed with the RNA (in aqueous solution) in a small T-piece to encapsulate the mRNA in the LNP, see, for example, Reichmuth, Andreas M et al. “mRNA vaccine delivery using lipid nanoparticles.” Therapeutic delivery vol.7,5 (2016):319-34. doi:10.4155 / tde-2016-0006, the entire contents of which are incorporated herein by reference for all purposes. The mRNA formulated in the LNPs is rebuffered as necessary via dialysis and concentrated to the target concentration using ultracentrifugation.

[0412] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing and accompanying figures. Such modifications are intended to be within the scope of the appended claims.

[0413] All references (e.g., publications or patents or patent applications) cited in this specification are herein incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0414] Other embodiments are within the scope of the following claims.

Claims

1. A nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), wherein the amino acid sequence of said SARS-CoV-2 spike protein (e.g., said SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one set of amino acid substitutions set forth in Table 2.

2. 2. The nucleic acid molecule of claim 1, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of a SARS-CoV-2 spike protein receptor binding domain (RBD).

3. 3. The nucleic acid molecule of claim 1 or 2, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein.

4. 4. The nucleic acid molecule of any one of claims 1-3, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.

5. 5. The nucleic acid molecule of any one of claims 1-4, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, or 10-1300 amino acids.

6. Other than at least one set of amino acid substitutions set forth in Table 2, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is at least one contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 15 ...

6. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.

7. 7. The nucleic acid molecule of any one of claims 1-6, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) other than at least one set of amino acid substitutions set forth in Table 2 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

8. 8. The nucleic acid molecule of any one of claims 1-7, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises a plurality of sets of amino acid substitutions set forth in Table 2.

9. 9. The nucleic acid molecule of any one of claims 1-8, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least two, three, four, five, six or more sets of amino acid substitutions set forth in Table 2.

10. 10. The nucleic acid molecule of any one of claims 1-9, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes (e.g., substitutions, additions, deletions, etc.) not set forth in Table 2, but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%, less than 5%).

11. 11. The nucleic acid molecule of any one of claims 1-10, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes (e.g., substitutions, additions, deletions, etc.) not set forth in Table 2 relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4, but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%, less than 5%).

12. 12. The nucleic acid molecule of any one of claims 1 to 11, wherein the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is stabilized in a pre-fusion state.

13. 13. The nucleic acid molecule of any one of claims 1-12, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 that stabilizes the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) in a pre-fusion state.

14. 14. The nucleic acid molecule of any one of claims 1-13, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises a proline at amino acid position 986 and / or a proline at amino acid position 987, which are amino acids numbered relative to the amino acid positions set forth in SEQ ID NO:

4.

15. 15. The nucleic acid molecule of any one of claims 1-14, wherein the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises one or more non-naturally occurring N-glycosylation sites.

16. 16. The nucleic acid molecule of any one of claims 1-15, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises the addition of one or more N-glycosylation sites relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

17. 17. The nucleic acid molecule of any one of claims 1 to 16, wherein the encoded SARS-CoV-2 spike protein is an immunogen (or an immunogenic fragment and / or immunogenic variant thereof).

18. The nucleic acid molecule according to any one of claims 1 to 17, wherein the nucleic acid molecule is RNA or DNA.

19. The nucleic acid molecule according to any one of claims 1 to 18, wherein the RNA is messenger ribonucleic acid (mRNA).

20. The nucleic acid molecule according to any one of claims 1 to 19, wherein the nucleic acid molecule comprises at least one modified nucleotide.

21. The nucleic acid molecule according to any one of claims 1 to 20, wherein the nucleic acid molecule comprises N1-methyl-pseudouridine, cytosine, adenine, and guanine.

22. The nucleic acid molecule of any one of claims 1 to 21, wherein the nucleic acid molecule comprises a heterologous 5'-untranslated region (UTR), a 3'-UTR, or both a 5'-UTR and a 3'-UTR.

23. The nucleic acid molecule according to any one of claims 1 to 22, wherein the nucleic acid molecule comprises a poly(A) sequence.

24. The nucleic acid molecule according to any one of claims 1 to 23, wherein the nucleic acid molecule comprises a 5' cap structure.

25. The nucleic acid molecule according to any one of claims 1 to 24, wherein the nucleotide sequence of the nucleic acid molecule is codon-optimized.

26. The nucleic acid molecule according to any one of claims 1 to 25, wherein the nucleic acid molecule further encodes a heterologous polypeptide or protein.

27. The nucleic acid molecule according to any one of claims 1 to 26, wherein the nucleic acid molecule encodes a signal peptide.

28. The nucleic acid molecule according to any one of claims 1 to 27, wherein the nucleic acid molecule encodes a homologous or heterologous signal peptide.

29. The nucleic acid molecule according to any one of claims 1 to 28, wherein the nucleic acid molecule does not encode a signal peptide.

30. A vector comprising the nucleic acid molecule according to any one of claims 1 to 29.

31. The vector of claim 30 , wherein the vector is a non-viral vector (e.g., a plasmid) or a viral vector.

32. 30. A conjugate comprising a nucleic acid molecule according to any one of claims 1 to 29 operably linked (e.g. directly or indirectly (e.g. via a linker)) to a heterologous moiety (e.g. a heterologous polypeptide or protein).

33. A composition comprising at least one nucleic acid molecule according to any one of claims 1 to 29.

34. 34. The composition of claim 33, comprising a plurality of nucleic acid molecules of any one of claims 1-29, wherein the amino acid sequence of each of the encoded SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and / or immunogenic variants thereof)) encoded by each of the plurality of nucleic acid molecules comprises a different set of amino acid substitutions as set forth in Table 2.

35. 35. The composition of claim 33 or 34, wherein the composition comprises a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprising an amino acid sequence that does not include the set of amino acid substitutions set forth in Table 2.

36. A SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), wherein the amino acid sequence of said SARS-CoV-2 spike protein (e.g., said SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one set of amino acid substitutions set forth in Table 2.

37. 37. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of claim 36, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

38. 38. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of claim 36 or 37, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein.

39. 39. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-38, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids.

40. The amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-90 ...

40. The SARS-CoV-2 spike protein of any one of claims 36-39 (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprising 0-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, or 10-1300 amino acids.

41. Other than the at least one set of amino acid substitutions, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 10 ...

41. The SARS-CoV-2 spike protein of any one of claims 36-40 (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)), which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 1,000, 1,100, or 1,200 amino acids of the SARS-CoV-2 spike protein.

42. 42. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) of any one of claims 36-41, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) other than said at least one set of amino acid substitutions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

43. 43. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-42, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprises a plurality of amino acid substitutions as set forth in Table 2.

44. 44. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) of any one of claims 36-43, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprises at least two, three, four, five, six or more sets of amino acid substitutions set forth in Table 2.

45. 45. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) of any one of claims 36-44, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes (e.g., substitutions, additions, deletions, etc.) not listed in Table 2.

46. 46. ​​The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) of any one of claims 36-45, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes (e.g., substitutions, additions, deletions, etc.) not listed in Table 2, relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

47. 47. The SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-46, wherein the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) is stabilized in a pre-fusion state.

48. 48. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-47, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises at least one amino acid change relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 that stabilizes the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) in a pre-fusion state.

49. 49. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-48, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprises a proline at amino acid position 986 and / or a proline at amino acid position 987, the amino acids numbering relative to the amino acid positions set forth in SEQ ID NO:

4.

50. 50. The SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-49, wherein the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises one or more non-native N-glycosylation sites.

51. 51. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-50, wherein the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises the addition of one or more N-glycosylation sites relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

52. 52. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-51, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprises an inactive furin cleavage site.

53. 53. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-52, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprises at least one amino acid change in the furin cleavage site that inactivates the furin cleavage site.

54. 54. The SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-53, wherein the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) further comprises a heterologous protein.

55. 55. The SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-54, wherein the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises a signal peptide.

56. 56. The SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-55, wherein the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) comprises a homologous or heterologous signal peptide.

57. 57. The SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-56, wherein the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and / or immunogenic variant thereof)) does not include a signal peptide.

58. 58. The SARS-CoV-2 spike protein of any one of claims 36-57, wherein the SARS-CoV-2 spike protein is an immunogen (or an immunogenic fragment and / or immunogenic variant thereof), such as a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof).

59. 59. A composition comprising at least one SARS-CoV-2 spike protein (e.g., at least one SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) according to any one of claims 36-58.

60. 60. The composition of claim 59, wherein the composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., a plurality of SARS-CoV-2 spike protein immunogens (or immunogenic fragments and / or immunogenic variants thereof)) of any one of claims 36-58, wherein the amino acid sequence of each of the plurality of SARS-CoV-2 spike proteins (e.g., the SARS-CoV-2 spike protein immunogens (or immunogenic fragments and / or immunogenic variants thereof)) comprises a different set of amino acid substitutions as set forth in Table 2.

61. 61. The composition of claim 59 or 60, wherein the composition comprises at least one SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) comprising an amino acid sequence that does not include the set of amino acid substitutions set forth in Table 2.

62. 59. A fusion protein comprising the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-58 operably linked (e.g., directly or indirectly (e.g., via a linker)) to a heterologous polypeptide or protein.

63. 59. A conjugate comprising the SARS-CoV-2 spike protein of any one of claims 36-58 (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) operably linked (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety.

64. A SARS-CoV-2 spike protein encoded by the nucleic acid molecule of any one of claims 1 to 29 (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

65. 59. A nucleic acid molecule encoding the SARS-CoV-2 spike protein of any one of claims 36 to 58 (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)).

66. 80. A carrier comprising a nucleic acid molecule according to any one of claims 1 to 29 or 65, a vector according to any one of claims 30 to 31, a composition according to any one of claims 33 to 35 or 59 to 61, a SARS-CoV-2 spike protein according to any one of claims 36 to 58 or 64 (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a conjugate according to any one of claims 32 or 63, a fusion protein according to claim 62, a vaccine composition according to any one of claims 71 to 77, or a pharmaceutical composition according to any one of claims 78 to 80.

67. 67. The carrier of claim 66, wherein the carrier is a lipid nanoparticle (LNP), a liposome, a lipoplex, or a nanoliposome.

68. The carrier of claim 67, wherein the carrier is an LNP.

69. 69. The carrier of claim 67 or 68, wherein the LNP comprises a cationic lipid, a neutral lipid, cholesterol, and / or a PEG lipid.

70. The carrier according to any one of claims 67 to 69, wherein the LNPs have an average particle size between 80 nm and 160 nm.

71. 80. A vaccine composition comprising the nucleic acid molecule of any one of claims 1 to 29 or 65, the vector of any one of claims 30 to 31, the composition of any one of claims 33 to 35 or 59 to 61, the SARS-CoV-2 spike protein of any one of claims 36 to 58 or 64 (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), the vaccine composition of any one of claims 71 to 77, the conjugate of any one of claims 32 or 63, the fusion protein of claim 62, the carrier of any one of claims 66 to 70, or the pharmaceutical composition of any one of claims 78 to 80.

72. 72. The vaccine composition of claim 71, wherein the vaccine composition is a prime vaccine composition.

73. 73. The vaccine composition of claim 71 or 72, wherein the vaccine composition is a boost vaccine composition.

74. 74. The vaccine composition according to any one of claims 71 to 73, wherein the vaccine composition is a prime vaccine composition and a boost vaccine composition.

75. The vaccine composition according to any one of claims 71 to 74, wherein said vaccine composition may be utilized as a prime vaccine composition and / or a booster vaccine composition in a homologous or heterologous prime boost vaccine regime.

76. 1. A vaccine composition comprising messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprising at least one amino acid substitution as set forth in Table 2, formulated in a lipid nanoparticle, the LNPs having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid; (b) the LNPs have an average particle size between 80 nm and 160 nm; and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region. A vaccine composition comprising:

77. The vaccine composition of any one of claims 71 to 76, wherein the vaccine composition further comprises an adjuvant.

78. 62. A pharmaceutical composition comprising a nucleic acid molecule of any one of claims 1 to 29 or 65, a vector of any one of claims 30 to 31, a composition of any one of claims 33 to 35 or 59 to 61, a SARS-CoV-2 spike protein of any one of claims 36 to 58 or 64 (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a vaccine composition of any one of claims 71 to 77, a conjugate of any one of claims 32 or 63, a fusion protein of claim 62, a carrier of any one of claims 66 to 70, and a pharma- ceutical acceptable excipient.

79. 1. A pharmaceutical composition comprising messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof)) comprising at least one amino acid substitution as set forth in Table 2, formulated in a lipid nanoparticle, the LNPs having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, cholesterol, and a PEG lipid; (b) the LNPs have an average particle size between 80 nm and 160 nm; and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region.

1. A pharmaceutical composition comprising:

80. The method of any one of claims 78 to 79, wherein the pharmaceutical composition further comprises an adjuvant.

81. 80. A kit comprising a nucleic acid molecule according to any one of claims 1 to 29 or 65, a vector according to any one of claims 30 to 31, a composition according to any one of claims 33 to 35 or 59 to 61, a SARS-CoV-2 spike protein according to any one of claims 36 to 58 or 64 (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), a vaccine composition according to any one of claims 71 to 77, a conjugate according to any one of claims 32 or 63, a fusion protein according to claim 62, a carrier according to any one of claims 66 to 70, or a pharmaceutical composition according to any one of claims 78 to 80.

82. 82. The kit of claim 81, comprising instructions for use of the nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition.

83. 20. A method of delivering a nucleic acid molecule, vector, protein (or an immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition or pharmaceutical composition to a subject in need thereof, comprising administering to said subject a nucleic acid molecule according to any one of claims 1 to 29 or 65, a vector according to any one of claims 30 to 31, a SARS-CoV-2 spike protein (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof) according to any one of claims 36 to 58 or 64), a conjugate according to claim 32 or 63, a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or variant thereof) according to claim 62, a fusion protein of any one of claims 66-70, a carrier of any one of claims 33-35 or 59-61, a vaccine composition of any one of claims 71-77, or a pharmaceutical composition of any one of claims 78-80, thereby delivering the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition to the subject.

84. 62. A method of inducing or enhancing an immune response in a subject in need thereof comprising administering to the subject a nucleic acid molecule of any one of claims 1 to 29 or 65, a vector of any one of claims 30 to 31, a SARS-CoV-2 spike protein (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof) of any one of claims 36 to 58 or 64), a conjugate of claim 32 or 63, a fusion protein of claim 62, a carrier of any one of claims 66 to 70, a composition of any one of claims 33 to 35 or 59 to 61, a vaccine composition of any one of claims 71 to 77, or a pharmaceutical composition of any one of claims 78 to 80, thereby inducing or enhancing an immune response in the subject.

85. 80. A method of preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject a nucleic acid molecule of any one of claims 1-29 or 65, a vector of any one of claims 30-31, a SARS-CoV-2 spike protein (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof) of any one of claims 36-58 or 64), a conjugate of claim 32 or 63, a fusion protein of claim 62, a carrier of any one of claims 66-70, a composition of any one of claims 33-35 or 59-61, a vaccine composition of any one of claims 71-77, or a pharmaceutical composition of any one of claims 78-80, thereby preventing, ameliorating, or treating said SARS-CoV-2 infection in the subject.

86. 80. A method of vaccinating a subject against SARS-CoV-2 comprising administering to the subject a nucleic acid molecule of any one of claims 1-29 or 65, a vector of any one of claims 30-31, a SARS-CoV-2 spike protein (e.g. a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof) of any one of claims 36-58 or 64), a conjugate of claim 32 or 63, a fusion protein of claim 62, a carrier of any one of claims 66-70, a composition of any one of claims 33-35 or 59-61, a vaccine composition of any one of claims 71-77, or a pharmaceutical composition of any one of claims 78-80, thereby vaccinating the subject against SARS-CoV-2.

87. 13. A method of vaccinating a subject against SARS-CoV-2 comprising administering to the subject (a) an mRNA molecule (e.g., an mRNA molecule described herein) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and / or immunogenic variant thereof)) of any one of claims 36-58 or 64 (or a conjugate or fusion protein thereof); (b) a vector comprising said mRNA molecule; (c) a carrier comprising said mRNA molecule or said vector; (d) a vaccine composition comprising said mRNA molecule, said vector, or said carrier; or (e) a pharmaceutical composition comprising said mRNA molecule, said vector, said carrier, or said vaccine composition, thereby vaccinating the subject against SARS-CoV-2.

88. A method of vaccinating a subject against SARS-CoV-2 comprising administering to said subject a vaccine composition according to any one of claims 76-77 or a pharmaceutical composition according to any one of claims 78-80, thereby vaccinating said subject against SARS-CoV-2.

89. 89. The method of any one of claims 83-88, wherein the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered to the subject at least twice.

90. The method of any one of claims 83 to 89, wherein the subject is a human.

91. 91. The method of any one of claims 83-90, wherein the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a prime and / or boost in an allogeneic or heterogeneous prime-boost regimen.

92. 92. The method of any one of claims 83-91, wherein the nucleic acid molecule, vector, SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and / or immunogenic variant thereof)), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.

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