coronavirus vaccine

Abstract

The present invention relates to the field of RNA for the prevention or treatment of coronavirus infection. In particular, the present invention relates to methods and agents for vaccination against coronavirus infection and for inducing effective coronavirus antigen-specific immune responses, such as antibody and / or T cell responses. These methods and agents are particularly useful for the prevention or treatment of coronavirus infection. Administration of the RNA disclosed herein to a subject can protect the subject from coronavirus infection. In particular, in certain embodiments, the present invention relates to a method for inducing an immune response in a subject against coronavirus spike protein (S protein), particularly the S protein of SARS-CoV-2, by administering to the subject RNA encoding a peptide or protein containing an epitope of the SARS-CoV-2 S protein, i.e., vaccine RNA encoding a vaccine antigen. Administration of the RNA encoding the vaccine antigen to the subject can provide the vaccine antigen (after expression of the RNA by appropriate target cells) for inducing an immune response in the subject against the vaccine antigen (and disease-associated antigens). In December 2019, a pneumonia outbreak of unknown cause occurred in Wuhan, China, and it was revealed that the underlying cause was a novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2; SARS-CoV-2). The genetic sequence of SARS-Cov-2 has been made available to the WHO and the public (MN908947.3), and the virus has been classified as a member of the Betacoronavirus subfamily. Sequence analysis revealed a phylogenetic relationship to Severe Acute Respiratory Syndrome (SARS) virus isolates, compared to other coronaviruses that infect humans (i.e., Middle East Respiratory Syndrome (MERS) virus). As of 2 March, a total of 14,557 cases had been confirmed globally in 24 countries, including Germany, and subsequent autonomous human-to-human spread of the virus led to SARS-Cov-2 becoming a global pandemic.

Description

[Technical Field]

[0001] The present invention relates to the field of RNA for preventing or treating coronavirus infection. In particular, the present invention relates to methods and agents for vaccinating against coronavirus infection and inducing an effective coronavirus antigen-specific immune response, such as an antibody and / or T cell response. These methods and agents are particularly useful for preventing or treating coronavirus infection. Administration of the RNA disclosed herein to a subject can protect the subject against coronavirus infection. In particular, in certain embodiments, the present invention relates to a method for inducing an immune response in a subject against coronavirus spike protein (S protein), particularly the S protein of SARS-CoV-2, comprising administering to the subject RNA encoding a peptide or protein comprising an epitope of the SARS-CoV-2 S protein, i.e., vaccine RNA encoding a vaccine antigen. Administration of the RNA encoding the vaccine antigen to a subject can provide the vaccine antigen (after expression of the RNA by appropriate target cells) for inducing an immune response against the vaccine antigen (and disease-associated antigens) in the subject. [Background technology]

[0002] In December 2019, a pneumonia outbreak of unknown cause occurred in Wuhan, China, and a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was identified as the underlying cause. The genetic sequence of SARS-CoV-2 was made available to the WHO and published (MN908947.3), and the virus was classified as a member of the Betacoronavirus subfamily. Sequence analysis revealed a phylogenetic tree that revealed a closer relationship to severe acute respiratory syndrome (SARS) virus isolates, namely the Middle East respiratory syndrome (MERS) virus, than to other coronaviruses infecting humans. By February 2, a total of 14,557 cases had been confirmed worldwide in 24 countries, including Germany, and subsequent autonomous human-to-human spread of the virus led to the global spread of SARS-CoV-2.

[0003] Coronaviruses are positive-sense, single-stranded RNA ((+)ssRNA) enveloped viruses that encode four structural proteins: spike protein, envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The spike protein (S protein) is responsible for receptor recognition, cell attachment, infection via the endosomal pathway, and genome release driven by fusion of the viral and endosomal membranes. Although the sequence varies among different family members, conserved regions and motifs within the S protein allow it to be divided into two subdomains, S1 and S2. The S2 domain, together with its transmembrane domain, is responsible for membrane fusion, while the S1 domain recognizes virus-specific receptors and binds to target host cells. A receptor-binding domain (RBD) has been identified in several coronavirus isolates, and the general structure of the S protein has been defined (Figure 1). Summary of the Invention [Problem to be solved by the invention]

[0004] Vaccine approaches and therapeutic agents against SARS-CoV-2 are not currently available and are urgently needed.

[0005] Due to the importance of the S protein in host cell recognition and entry and the induction of virus-neutralizing antibodies by the host immune system, we targeted the SARS-CoV-2 S protein and its subdomains, such as S1 or RBD, for vaccine development. Mutations within regions critical for conformation should be beneficial for inducing stronger protective immune responses. Therefore, we envisioned testing several constructs (Figure 2). Because the native S protein is a trimer, and this trimeric structure most likely affects protein stability and antigenicity, we included a strategy based on a stabilized construct incorporating the T4 bacteriophage fibritin domain, which is also used in HIV to generate stable gp140 trimers and is functional in the SARS RBD construct. [Means for solving the problem]

[0006] overview The present invention generally encompasses immunotherapeutic treatment of a subject, comprising administering RNA encoding an amino acid sequence, i.e., a vaccine antigen, comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant, i.e., an antigenic peptide or protein. Thus, the vaccine antigen comprises an epitope of the SARS-CoV-2 S protein to induce an immune response in a subject against the coronavirus S protein, particularly the SARS-CoV-2 S protein. Upon administration, the RNA encoding the vaccine antigen provides the antigen (after expression of the polynucleotide by the appropriate target cell) for the induction, i.e., stimulation, priming, and / or proliferation, of an immune response, e.g., antibody and / or immune effector cells, targeted by the target antigen (coronavirus S protein, particularly the SARS-CoV-2 S protein) or its proliferative products. In certain embodiments, the immune response induced by the present invention is a B cell-mediated immune response, i.e., an antibody-mediated immune response. Additionally or alternatively, in certain embodiments, the immune response induced by the present invention is a T cell-mediated immune response. In certain embodiments, the immune response is an anti-coronavirus, particularly an anti-SARS-CoV-2, immune response.

[0007] The vaccines described herein contain single-stranded RNA as an active ingredient, which can be translated into various proteins once it enters the recipient's cells. In addition to the wild-type or codon-optimized sequence encoding the antigen sequence, the RNA can contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized for maximum RNA effectiveness in terms of stability and translation efficiency. In some embodiments, the RNA contains all of these elements. In some embodiments, the RNA contains beta-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O) ApG can be used as a specific capping structure at the 5' end of an RNA drug substance. The 5'-UTR sequence can be the 5'-UTR sequence of human alpha-globin mRNA, optionally with an optimized "Kozak sequence" to increase translation efficiency. The 3'-UTR sequence can be a combination of two sequence elements (FI elements) from the "amino terminal enhancer of split gene" (AES) mRNA (designated F) and the mitochondrial-encoded 12S ribosomal RNA (designated I), located between the coding sequence and the poly(A)-tail, to ensure high maximum protein levels and long-term mRNA persistence. These were identified through an ex vivo selection process for sequences that contribute to enhanced RNA stability and total protein expression (see WO2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR can be two repeated 3'-UTRs of human beta-globin mRNA. Additionally, a 110-nucleotide-long poly(A)-tail can be used, consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence (of random nucleotides) and an additional 70 adenosine residues. This poly(A)-tail sequence was designed to enhance RNA stability and translation efficiency.

[0008] Additionally, a secretory signal peptide (sec) can be fused to the antigen-encoding region, preferably in such a way that the sec is translated as an N-terminal tag. In one embodiment, the sec corresponds to the secretory signal peptide of the S protein. As commonly used in fusion proteins, a sequence encoding a short linker peptide consisting mostly of the amino acids glycine (G) and serine (S) can be used as the GS / linker.

[0009] The vaccine RNAs described herein can be combined with proteins and / or lipids, preferably lipids, to produce RNA particles for administration. If different RNA combinations are used, these RNAs can be combined together or separately with proteins and / or lipids to produce RNA particles for administration.

[0010] In certain aspects, the present invention relates to a composition or pharmaceutical formulation comprising RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0011] In certain embodiments, the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 subunit of the SARS-CoV-2 S protein or the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

[0012] In certain embodiments, an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant can form a multimeric complex, particularly a trimeric complex. To this end, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant can comprise a domain that allows the formation of a multimeric complex, particularly a trimeric complex, of the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In certain embodiments, the domain that allows the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain described herein.

[0013] In certain embodiments, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is encoded by a coding sequence that is codon-optimized and / or has an increased G / C content compared to the wild-type coding sequence, wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0014] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1.

[0015] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1.

[0016] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0017] In certain embodiments, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a secretory signal peptide.

[0018] In one embodiment, the secretory signal peptide is fused, preferably at the N-terminus, to the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0019] In one embodiment, (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9 or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9; and / or (ii) The secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

[0020] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof has the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5.

[0021] In some embodiments, the RNA is modified RNA, particularly stabilized mRNA. In some embodiments, the RNA comprises a modified nucleoside instead of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside instead of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0022] In some embodiments, the RNA contains modified nucleosides in place of uridine.

[0023] In certain embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0024] In certain embodiments, the RNA comprises a 5' cap.

[0025] In certain embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0026] In certain embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0027] In certain embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a polyA sequence.

[0028] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0029] In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:14.

[0030] In certain embodiments, the RNA is or is to be formulated as a liquid, a solid, or a combination thereof.

[0031] In certain embodiments, the RNA is or is to be formulated for injection.

[0032] In certain embodiments, the RNA is or should be formulated for intramuscular administration.

[0033] In certain embodiments, the RNA is or is to be formulated as particles.

[0034] In certain embodiments, the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

[0035] In some embodiments, the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0036] In some embodiments, RNA lipoplex particles can be obtained by mixing RNA with liposomes. In some embodiments, RNA lipoplex particles can be obtained by mixing RNA with lipids.

[0037] In some embodiments, the RNA is or should be formulated as a colloid. In some embodiments, the RNA is or should be formulated as particles that form the dispersed phase of the colloid. In some embodiments, 50% or more, 75% or more, or 85% or more of the RNA is present in the dispersed phase. In some embodiments, the RNA is or should be formulated as particles comprising RNA and lipids. In some embodiments, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In some embodiments, the organic phase comprises ethanol. In some embodiments, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In some embodiments, the lipids dispersed in the aqueous phase form liposomes.

[0038] In certain embodiments, the RNA is mRNA or saRNA.

[0039] In certain embodiments, the composition or pharmaceutical formulation is a pharmaceutical composition.

[0040] In certain embodiments, the composition or pharmaceutical preparation is a vaccine.

[0041] In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0042] In certain embodiments, the composition or pharmaceutical preparation is a kit.

[0043] In some embodiments, the RNA and optionally the particle-forming components are contained in separate vials.

[0044] In certain embodiments, the kit further comprises instructions for using the composition or pharmaceutical preparation to induce an immune response against coronavirus in a subject.

[0045] In certain aspects, the present invention relates to a composition or pharmaceutical formulation described herein for pharmaceutical use.

[0046] In some embodiments, the medical use includes inducing an immune response to coronavirus in a subject.

[0047] In some embodiments, the medical uses include therapeutic or prophylactic treatment of coronavirus infections.

[0048] In certain embodiments, the compositions or pharmaceutical formulations described herein are for administration to humans.

[0049] In some embodiments, the coronavirus is a betacoronavirus.

[0050] In some embodiments, the coronavirus is a sarbecovirus.

[0051] In some embodiments, the coronavirus is SARS-CoV-2.

[0052] In one aspect, the present invention relates to a method of inducing an immune response against coronavirus in a subject, comprising administering to the subject a composition comprising RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0053] In certain embodiments, the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 subunit of the SARS-CoV-2 S protein or the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

[0054] In certain embodiments, an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant can form a multimeric complex, particularly a trimeric complex. To this end, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant can comprise a domain that allows the formation of a multimeric complex, particularly a trimeric complex, of the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In certain embodiments, the domain that allows the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain described herein.

[0055] In certain embodiments, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is encoded by a coding sequence that is codon-optimized and / or has an increased G / C content compared to the wild-type coding sequence, wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0056] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1.

[0057] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:1.

[0058] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0059] In certain embodiments, the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a secretory signal peptide.

[0060] In one embodiment, the secretory signal peptide is fused, preferably at the N-terminus, to the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0061] In one embodiment, (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9 or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9; and / or (ii) The secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

[0062] In one embodiment, (i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6; and / or (ii) The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants, includes the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5.

[0063] In some embodiments, the RNA is modified RNA, particularly stabilized mRNA. In some embodiments, the RNA comprises a modified nucleoside instead of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside instead of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0064] In some embodiments, the RNA contains modified nucleosides in place of uridine.

[0065] In certain embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0066] In some embodiments, the RNA includes a cap.

[0067] In certain embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0068] In certain embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0069] In certain embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a polyA sequence.

[0070] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0071] In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:14.

[0072] In certain embodiments, the RNA is formulated as a liquid, a solid, or a combination thereof.

[0073] In certain embodiments, the RNA is administered by injection.

[0074] In certain embodiments, the RNA is administered by intramuscular administration.

[0075] In certain embodiments, the RNA is formulated as particles.

[0076] In certain embodiments, the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

[0077] In some embodiments, the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0078] In some embodiments, RNA lipoplex particles can be obtained by mixing RNA with liposomes. In some embodiments, RNA lipoplex particles can be obtained by mixing RNA with lipids.

[0079] In some embodiments, the RNA is formulated as a colloid. In some embodiments, the RNA is formulated as particles that form the dispersed phase of the colloid. In some embodiments, 50% or more, 75% or more, or 85% or more of the RNA is present in the dispersed phase. In some embodiments, the RNA is formulated as particles comprising RNA and lipids. In some embodiments, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In some embodiments, the organic phase comprises ethanol. In some embodiments, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In some embodiments, the lipids dispersed in the aqueous phase form liposomes.

[0080] In certain embodiments, the RNA is mRNA or saRNA.

[0081] In some embodiments, the method is a method for vaccination against a coronavirus.

[0082] In certain embodiments, the method is for the therapeutic or prophylactic treatment of a coronavirus infection.

[0083] In certain embodiments, the subject is a human.

[0084] In some embodiments, the coronavirus is a betacoronavirus.

[0085] In some embodiments, the coronavirus is a sarbecovirus.

[0086] In some embodiments, the coronavirus is SARS-CoV-2.

[0087] In some embodiments of the methods described herein, the composition is a composition described herein.

[0088] In certain aspects, the invention relates to a composition or pharmaceutical formulation described herein for use in a method described herein.

[0089] Among other things, the present invention demonstrates that a composition comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., the SARS-CoV-2-encoded S protein) can achieve detectable antibody titers to the epitope in serum within seven days of administration to an adult subject population in a regimen comprising at least one administration of the vaccine composition. Furthermore, the present invention demonstrates the persistence of such antibody titers. In certain embodiments, the present invention demonstrates an increase in such antibody titers when using modified mRNA compared to that achieved with the corresponding unmodified mRNA.

[0090] In some embodiments, the provided regimen comprises at least one administration. In some embodiments, the provided regimen comprises a first administration and at least one subsequent administration. In some embodiments, the first administration is the same amount as at least one subsequent administration. In some embodiments, the first administration is the same amount as all subsequent administrations. In some embodiments, the first administration is different from the amount of at least one subsequent administration. In some embodiments, the first administration is different from the amount of all subsequent administrations. In some embodiments, the provided regimen comprises two administrations. In some embodiments, the provided regimen consists of two administrations.

[0091] In a specific embodiment, the immunogenic composition is formulated as a single dose in a container, for example, a vial. In some embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation contains at least two doses per vial. In some embodiments, the multi-dose formulation contains a total of 2 to 20 doses per vial, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in a vial is of equal volume. In some embodiments, the first dose has a different volume from subsequent doses.

[0092] A "stable" multi-dose formulation does not exhibit unacceptable levels of microbial growth and exhibits substantial or no degradation or degradation of the active biological molecule components. As used herein, a "stable" immunogenic composition includes a formulation that maintains the ability to elicit a desired immune response when administered to a subject.

[0093] In some embodiments, the multi-dose formulation remains stable for a specified period of time after multiple or repeated inoculations / insertion into a multi-dose container. For example, in some embodiments, the multi-dose formulation may be stable for at least 3 days and up to 10 uses when placed in a multi-dose container. In some embodiments, the multi-dose formulation remains stable for 2 to 20 inoculations / insertion.

[0094] In certain embodiments, administration of a composition comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope of or including an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein), e.g., according to a regimen described herein, may result in lymphopenia in some subjects (e.g., all subjects, a majority of subjects, about 50% or less, about 40% or less, about 40% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, etc.). Among other things, the present invention demonstrates that such lymphopenia can resolve over time. For example, in certain embodiments, lymphopenia resolves within about 14 days, about 10 days, about 9 days, about 8 days, about 7 days, or fewer. In certain embodiments, lymphopenia is Grade 3, Grade 2, or less.

[0095] Thus, among other things, the present invention provides compositions comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope of or including an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., SARS-CoV-2-encoded S protein), characterized by exhibiting a certain characteristic (e.g., achieving a certain effect) described herein when administered to an appropriate population of adults. In certain embodiments, the provided compositions can be manufactured, stored, transported, characterized, and / or used under conditions in which the temperature does not exceed a certain threshold. Alternatively or additionally, in certain embodiments, the provided compositions can be protected from light (e.g., from certain wavelengths) during some or all of the manufacturing, storage, transport, characterization, and / or use. In certain embodiments, one or more characteristics of the provided compositions (e.g., one or more of mRNA stability; lipid nanoparticle stability or aggregation; pH; etc., which may be assessed, e.g., by size, the presence of a particular moiety or modification, etc.) can be or have been assessed at one or more time points prior to administration, manufacturing, storage, transport, and / or use.

[0096] Among other things, the present invention demonstrates that certain provided compositions and / or methods provided regarding such compositions, in which the nucleotides in the mRNA are unmodified (e.g., naturally occurring A, U, C, G), are characterized by an intrinsic adjuvant effect (e.g., when administered to a suitable population, which in some embodiments may be or include an adult population). In some embodiments, such compositions and / or methods can induce antibody and / or T cell responses. In some embodiments, such compositions and / or methods can induce enhanced T cell responses compared to conventional vaccines (e.g., non-mRNA vaccines such as protein vaccines).

[0097] Alternatively or additionally, the present invention provides that provided compositions (e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., an epitope of or including an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein)) in which nucleotides within the mRNA are modified, and / or provided methods relating to such compositions, are characterized by a reduced intrinsic adjuvant effect (e.g., when administered to a suitable population, which in certain embodiments may be or include an adult population) compared to an otherwise equivalent composition (or method) having no intrinsic adjuvant effect or an unmodified result. Alternatively or additionally, in certain embodiments, such compositions (or methods) are characterized by a reduced antibody response and / or CD4 + and characterized by the induction of a T cell response (e.g., when administered to a suitable population, which in some embodiments may be or include an adult population). Yet further alternatively or additionally, in some embodiments, such compositions (or methods) may induce a higher CD4 T cell response than observed with other vaccine formats (e.g., peptide vaccines). +and characterized by inducing a T cell response (e.g., when administered to a suitable population, which in some embodiments may be or include an adult population). In some embodiments involving modified nucleotides, such modified nucleotides may be present, for example, in the 3'UTR sequence, the antigen-encoding sequence, and / or the 5'UTR sequence. In some embodiments, the modified nucleotides are or include one or more modified uracil residues and / or one or more modified cytosine residues.

[0098] Among other things, the present invention provides that the provided (e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding at least a portion (e.g., that is or includes an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein)) and / or methods are characterized (e.g., when administered to a suitable population, which may be or may include, in certain embodiments, an adult population) by sustained expression of the encoded polypeptide (e.g., a SARS-CoV-2-encoded protein [e.g., S protein] or a portion thereof that may be or include, in certain embodiments, an epitope thereof). For example, in certain embodiments, such compositions and / or methods, when administered to a human, achieve detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and are characterized, in certain embodiments, by such expression persisting for a period of at least at least 36 hours or more, including, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours or more.

[0099] Those of skill in the art having access to this specification will understand that this specification describes various mRNA constructs that encode at least a portion (e.g., that is or includes an epitope of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein)). Those of skill in the art having access to this specification will particularly understand that this specification describes various mRNA constructs that encode at least a portion of a SARS-CoV-2 S protein, e.g., at least the RBD portion of the SARS-CoV-2 S protein. Still further, those of skill in the art having access to this specification will understand that this specification describes particular features and / or advantages of mRNA constructs that encode at least a portion (e.g., that is or includes an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., a SARS-CoV-2-encoded S protein). Among other things, this specification particularly demonstrates the surprising and useful features and / or advantages of certain mRNA constructs that encode a portion of the SARS-CoV-2 RBD that, in certain embodiments, does not encode the full-length SARS-CoV-2 S protein. Without wishing to be bound by any particular theory, the present specification provides evidence that the provided mRNA constructs encoding less than the full-length SARS-CoV-2 S protein, and in particular encoding at least the RBD portion of such SARS-CoV-2 S protein, can be used as or in immunogenic compositions (e.g., vaccines) and / or to induce the immunological effects described herein (e.g., SARS-CoV-2 neutralizing antibody and / or T cell responses (e.g., CD4 + and / or CD8 + This suggests that the use of agonists may be particularly useful and / or effective in achieving (the generation of) T cell responses.

[0100] In certain embodiments, the present invention provides RNA (e.g., mRNA) comprising an open reading frame encoding a polypeptide comprising a receptor-binding portion of the SARS-CoV-2 S protein, wherein the RNA is suitable for intracellular expression of the polypeptide. In certain embodiments, such encoded polypeptide does not comprise the entire S protein. In certain embodiments, the encoded polypeptide comprises a receptor-binding domain (RBD), e.g., as set forth in SEQ ID NO: 5. In certain embodiments, the encoded polypeptide comprises the peptide of SEQ ID NO: 29 or 31. In certain embodiments, such RNA (e.g., mRNA) can be complexed with a polycationic polymer, polyplex, protein, or peptide. In certain embodiments, such RNA can be formulated into lipid nanoparticles (e.g., those described herein). In certain embodiments, such RNA (e.g., mRNA) is suitable for use as or in an immunogenic composition (e.g., vaccine) and / or to induce an immunological effect (e.g., SARS-CoV-2 neutralizing antibody and / or T cell response (e.g., CD4 + and / or CD8 + In certain embodiments, such RNA (e.g., mRNA) may be useful for vaccinating humans (e.g., including humans known to have been exposed to and / or infected with SARS-CoV-2 and / or humans with no known exposure to SARS-CoV-2).

[0101] Those of skill in the art having access to this specification will further recognize that this specification describes various mRNA constructs comprising nucleic acid sequences encoding a full-length SARS-CoV-2 spike protein (including, e.g., embodiments in which such encoded SARS-CoV-2 spike protein can include at least one or more amino acid substitutions, e.g., a proline substitution as described herein, and / or embodiments in which the mRNA sequence has been codon-optimized, e.g., for a mammalian, e.g., human, subject). In certain embodiments, such a full-length SARS-CoV-2 spike protein can have an amino acid sequence that is set forth in or includes SEQ ID NO: 7. Still further, those of skill in the art having access to this specification will recognize that this specification describes, among other things, certain features and / or advantages of certain mRNA constructs comprising nucleic acid sequences encoding a full-length SARS-CoV-2 spike protein. Without wishing to be bound by any particular theory, the present invention suggests that the provided mRNA constructs encoding a full-length SARS-CoV-2 S protein may be particularly useful and / or effective for use as or for immunogenic compositions (e.g., vaccines), particularly in subject populations (e.g., particular age groups). For example, in certain embodiments, such mRNA compositions may be particularly useful for younger subjects (e.g., under 25, 20, 18, 15, 10, or younger); alternatively or additionally, in certain embodiments, such mRNA compositions may be particularly useful for older subjects (e.g., over 55, 60, 65, 70, 75, 80, 85, or older).In specific embodiments, immunogenic compositions comprising such mRNA constructs provided herein exhibit, at least in some subjects (e.g., in subjects of a certain age group), minimal to moderate increases (e.g., no more than a 30% increase, no more than a 20% increase, or no more than a 10% increase or less) in dose-level- and / or dose-number-dependent systemic reactogenicity (e.g., fever, fatigue, headache, chills, diarrhea, muscle and / or joint pain, etc.) and / or local tolerance (e.g., pain, redness and / or swelling, etc.); in certain embodiments, such reactogenicity and / or local tolerance is observed particularly in subjects of younger age groups (e.g., under 25 years old, 20 years old, 18 years old or younger) and / or older (e.g., elderly) age groups (e.g., 65-85 years old). In certain embodiments, the provided mRNA constructs encoding the full-length SARS-CoV-2 S protein may be particularly useful and / or effective for inducing SARS-CoV-2 neutralizing antibody response levels in subject populations at high risk for severe disease associated with SARS-CoV-2 infection (e.g., elderly populations, e.g., the 65-85 age group), particularly for use as or in immunogenic compositions (e.g., vaccines). In certain embodiments, one of skill in the art having access to this specification will understand that the provided mRNA constructs encoding the full-length SARS-CoV-2 S protein, which exhibit, among other things, favorable reactogenicity profiles (e.g., those described herein) in younger and older age populations, can induce the immunological effects described herein (e.g., SARS-CoV-2 neutralizing antibodies and / or T cell responses (e.g., CD4 + and / or CD8 +The present invention also recognizes that the mRNA constructs provided encoding full-length SARS-CoV-2 S proteins may be particularly useful and / or effective for use as or in immunogenic compositions (e.g., vaccines) to achieve a SARS-CoV-2 S protein (e.g., the generation of a T cell response). In certain embodiments, the present invention also recognizes that the provided mRNA constructs encoding full-length SARS-CoV-2 S proteins may be particularly effective in protecting against SARS-CoV-2 infection, as characterized by early clearance of SARS-CoV-2 viral RNA in non-human mammalian subjects (e.g., rhesus macaques) immunized with an immunogenic composition comprising such mRNA constructs and subsequently exposed to a SARS-CoV-2 strain. In certain embodiments, such early clearance of SARS-CoV-2 viral RNA can be observed in the noses of non-human mammalian subjects (e.g., rhesus macaques) immunized with an immunogenic composition comprising such mRNA constructs and subsequently exposed to a SARS-CoV-2 strain.

[0102] In certain embodiments, the present invention provides RNA (e.g., mRNA) comprising an open reading frame encoding a full-length SARS-CoV-2 S protein (e.g., a full-length SARS-CoV-2 S protein with one or more amino acid substitutions), wherein the RNA is suitable for intracellular expression of a polypeptide. In certain embodiments, the encoded polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, such RNA (e.g., mRNA) can be complexed with a polycationic polymer, polyplex, protein, or peptide. In certain embodiments, such RNA can be formulated into lipid nanoparticles (e.g., those described herein).

[0103] In certain embodiments, the immunogenic compositions provided herein may comprise multiple (e.g., at least two or more, including, e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, etc.) immunoreactive epitopes of a SARS-CoV-2 polypeptide or variants thereof. In certain such embodiments, such multiple immunoreactive epitopes may be encoded by multiple RNAs (e.g., mRNAs). In certain such embodiments, such multiple immunoreactive epitopes may be encoded by a single RNA (e.g., mRNA). In certain embodiments, nucleic acid sequences encoding multiple immunoreactive epitopes may be separated from each other in a single RNA (e.g., mRNA) by linkers (e.g., peptide linkers, in certain embodiments). Without wishing to be bound by any particular theory, in certain embodiments, provided polyepitopic immunogenic compositions (including, e.g., those encoding a full-length SARS-CoV-2 spike protein) may be particularly useful in providing protection against multiple viral variants, given the genetic diversity of SARS-CoV-2 variants, and / or in eliciting diverse and / or otherwise robust (e.g., durable, e.g., detectable for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days after one or more doses) neutralizing antibody and / or T cell responses, particularly particularly robust T H Type 1 T cells (e.g., CD4 + and / or CD8 + This may increase the chances of obtaining a T cell response.

[0104] In certain embodiments, the present invention demonstrates that the provided compositions and / or methods are characterized (e.g., when administered to a suitable population, which in certain embodiments may be or include an adult population) by achieving one or more particular therapeutic results (e.g., an effective immune response described herein and / or detectable expression of the encoded SARS-CoV-2 S protein or immunogenic fragment thereof) with a single dose; in certain such embodiments, the results may be evaluated, for example, as compared to those observed in the absence of an mRNA vaccine described herein. In certain embodiments, the particular results may be achieved at lower doses than would be required with one or more alternative strategies.

[0105] In some embodiments, the present invention provides immunogenic compositions comprising an isolated messenger ribonucleic acid (mRNA) polynucleotide, wherein the isolated mRNA polynucleotide comprises an open reading frame encoding a polypeptide comprising a receptor-binding portion of the SARS-CoV-2 S protein, and the isolated mRNA polynucleotide is formulated into at least one lipid nanoparticle. For example, in some embodiments, such lipid nanoparticles may comprise a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol or steroid, and 0.5-15% polymer-conjugated lipid (e.g., PEG-modified lipid). In some embodiments, the sterol or steroid contained in the lipid nanoparticles may be or may comprise cholesterol. In some embodiments, the neutral lipid may be or may comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the polymer-conjugated lipid may be or may comprise PEG2000 DMG. In certain embodiments, such immunogenic compositions may contain a total lipid content of about 1 mg to 10 mg, 3 mg to 8 mg, or 4 mg to 6 mg. In certain embodiments, such immunogenic compositions may contain a total lipid content of about 5 mg / mL to 15 mg / mL, 7.5 mg / mL to 12.5 mg / mL, or 9 to 11 mg / mL. In certain embodiments, such isolated mRNA polynucleotides are provided in an amount effective for inducing an immune response in a subject administered the immunogenic composition at least once. In certain embodiments, the polypeptides encoded by the provided isolated mRNA polynucleotides do not include the complete S protein. In certain embodiments, such isolated mRNA polynucleotides provided in the immunogenic compositions are not self-replicating RNA.

[0106] In certain embodiments, the immune response can include the production of binding antibody titers against a SARS-CoV-2 protein (including, in certain embodiments, for example, a stabilized pre-fusion spike trimer) or a fragment thereof. In certain embodiments, the immune response can include the production of binding antibody titers against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In certain embodiments, the provided immunogenic compositions are established to achieve detectable binding antibody titers after the first administration, e.g., at least 70% (e.g., at least 80%, at least 90%, at least 95%, and up to 100%) seroconversion in a subject population receiving such provided immunogenic compositions, for example, in about two weeks.

[0107] In certain embodiments, the immune response may include the production of neutralizing antibody titers against a SARS-CoV-2 protein (including, in certain embodiments, for example, a stabilized pre-fusion spike trimer) or a fragment thereof. In certain embodiments, the immune response may include the production of neutralizing antibody titers against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. In certain embodiments, the provided immunogenic compositions have been established to achieve neutralizing antibody titers in an appropriate system (e.g., humans infected with SARS-CoV-2, humans infected with SARS-CoV-2, and / or populations thereof and / or model systems therefor). For example, in certain embodiments, such neutralizing antibody titers have been demonstrated in one or more human populations, non-human primate models (e.g., rhesus macaques), and / or mouse models.

[0108] In certain embodiments, the neutralizing antibody titer is sufficient (e.g., established to be sufficient) to reduce viral infection of B cells relative to that observed in an appropriate control (e.g., a non-vaccinated control subject or a subject vaccinated with a live-attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In certain such embodiments, such reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0109] In certain embodiments, the neutralizing antibody titer is sufficient (e.g., established to be) to reduce the rate of asymptomatic viral infection relative to that observed in an appropriate control (e.g., non-vaccinated control subjects or subjects vaccinated with a live-attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In certain such embodiments, such a reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, such a reduction can be characterized by evaluation of SARS-CoV-2 N protein serology. Significant protection against asymptomatic infection was also confirmed through real-life observations (Dagan N. et al., N Engl J Med. 2021, doi: 10.1056 / NEJMoa2101765. See also Epub ahead of print. PMID: 33626250).

[0110] In certain embodiments, the neutralizing antibody titer is sufficient (e.g., established to be sufficient) to reduce or block fusion of the virus with epithelial cells and / or B cells in the vaccinated subject relative to that observed in an appropriate control (e.g., a non-vaccinated control subject or a subject vaccinated with a live-attenuated virus vaccine, an inactivated virus vaccine, or a protein subunit virus vaccine, or a combination thereof). In certain such embodiments, such reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0111] In certain embodiments, the induction of neutralizing antibody titers can be characterized by an increase in B cell numbers, which in certain embodiments can include plasma cells, class-switched IgG1- and IgG2-positive B cells, and / or germinal center B cells. In certain embodiments, provided immunogenic compositions have been shown to achieve such an increase in B cell numbers in an appropriate system (e.g., humans infected with SARS-CoV-2, humans infected with SARS-CoV-2, and / or populations thereof and / or model systems therefor). For example, in certain embodiments, such an increase in B cell numbers has been demonstrated in one or more human populations, non-human primate models (e.g., rhesus macaques), and / or mouse models. In certain embodiments, such an increase in B cell numbers can be demonstrated in the draining lymph nodes and / or spleen of mouse models after immunization of such mouse models with the provided immunogenic compositions (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after immunization).

[0112] In certain embodiments, the induction of neutralizing antibody titers can be characterized by a reduction in the number of circulating B cells in the blood. In certain embodiments, the provided immunogenic compositions have been established to achieve such a reduction in the number of circulating B cells in an appropriate system (e.g., humans infected with SARS-CoV-2, humans infected with SARS-CoV-2, and / or populations and / or model systems therefor). For example, in certain embodiments, such a reduction in the number of circulating B cells in the blood has been demonstrated in one or more human populations, non-human primate models (e.g., rhesus macaques), and / or mouse models. In certain embodiments, such a reduction in the number of circulating B cells in the blood can be demonstrated in a mouse model (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days) after immunization of such mouse model with the provided immunogenic composition. Without wishing to be bound by theory, the reduction in the number of circulating B cells in the blood may be due to B cell homing to the lymphoid compartment.

[0113] In certain embodiments, the immune response induced by the provided immunogenic compositions can include an increase in the number of T cells. In certain embodiments, such an increase in the number of T cells can include an increase in the number of T follicular helper (T FH ) cell number, which in some embodiments may include one or more subsets with upregulated ICOS. FH Proliferation is essential for adaptive B cell responses and, in humans, T cells that arise in the circulation after vaccination. FH It is understood that a high frequency of T cells generally correlates with a high frequency of antigen-specific antibodies. In certain embodiments, the immunogenic compositions provided demonstrate an increase in such T cell numbers (e.g., T cells) in an appropriate system (e.g., SARS-CoV-2 infected humans and / or populations thereof and / or model systems therefor). FH For example, in one embodiment, such an expansion of T cell numbers (e.g., T FHcells) can be demonstrated in one or more human populations, non-human primate models (e.g., rhesus monkeys), and / or mouse models. In certain embodiments, such increased T cell numbers (e.g., T FH Cells) may be present in the draining lymph nodes, spleen, and / or blood of a mouse model after immunization of such mouse model with a provided immunogenic composition (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days after).

[0114] In certain embodiments, a protective response against SARS-CoV-2 induced by a provided immunogenic composition has been established in an appropriate model system for SARS-CoV-2. For example, in certain embodiments, such a protective response can be demonstrated in an animal model, such as a non-human primate model (e.g., rhesus macaque) and / or a mouse model. In certain embodiments, a non-human primate (e.g., rhesus macaque), or a population thereof, that has been immunized at least once with a provided immunogenic composition is exposed to SARS-CoV-2, for example, via the intranasal and / or intratracheal route. In certain embodiments, such exposure can be performed several weeks (e.g., 5 to 10 weeks) after at least one immunization (e.g., including at least two immunizations) with a provided immunogenic composition. In certain embodiments, such exposure may occur when a detectable level of SARS-CoV-2 neutralizing titer (e.g., an antibody response against the SARS-CoV-2 spike protein and / or fragments thereof, including, but not limited to, the stabilized prefusion spike trimer, S-2P, and / or an antibody response against the receptor-binding portion of SARS-CoV-2) is achieved in a non-human primate (e.g., rhesus macaque) that has been immunized at least once (e.g., including at least two immunizations) with a provided immunogenic composition. In certain embodiments, a protective response is characterized by the absence or reduction of detectable viral RNA in bronchoalveolar lavage fluid (BAL) and / or nasal swabs of the exposed non-human primate (e.g., rhesus macaque). In certain embodiments, the immunogenic compositions described herein may be characterized by a higher percentage of exposed animals, e.g., non-human primates (e.g., rhesus macaques), that have received at least one immunization (e.g., including at least two immunizations) with a provided immunogenic composition, showing an absence of detectable RNA in BAL and / or nasal swabs compared to a population of non-immunized animals, e.g., non-human primates (e.g., rhesus macaques).In certain embodiments, the immunogenic compositions described herein can be characterized in that a population of exposed animals, e.g., non-human (e.g., rhesus macaques), that have received at least one immunization (e.g., including at least two immunizations) with a provided immunogenic composition can exhibit clearance of viral RNA in nasal swabs within 10 days, including, e.g., within 8 days, within 6 days, within 4 days, etc., compared to a population of non-immunized animals, e.g., non-human primates (e.g., rhesus macaques).

[0115] In some embodiments, the immunogenic compositions described herein do not substantially increase the risk of enhanced respiratory disease when administered to a subject in need thereof. In some embodiments, such vaccine-associated enhanced respiratory disease may be associated with antibody-dependent enhancement of replication and / or vaccine antigens that induce insufficient neutralizing activity and a Th2-biased response. In some embodiments, the immunogenic compositions described herein do not substantially increase the risk of antibody-dependent enhancement of replication when administered to a subject in need thereof.

[0116] In some embodiments, a single administration of an mRNA composition (e.g., formulated in lipid nanoparticles) can induce a therapeutic antibody response within 10 days of vaccination. In some embodiments, such a therapeutic antibody response can be characterized when such an mRNA vaccine can induce the production of approximately 10-100 μg / mL IgG in an animal model at a dose of 0.1-10 μg or 0.2-5 μg, measured 10 days after vaccination. In some embodiments, such a therapeutic antibody response can be characterized when such an mRNA vaccine can induce approximately 100-1000 μg / mL IgG in an animal model at a dose of 0.1-10 μg or 0.2-5 μg, measured 20 days after vaccination. In some embodiments, a single administration can induce a pseudovirus neutralization titer of 10-200 pVN50 titers, measured 15 days after vaccination in an animal model. In certain embodiments, a single dose can induce pseudovirus neutralization titers of 50-500 pVN50 titers 15 days after vaccination, as measured in an animal model.

[0117] In certain embodiments, a single administration of an mRNA composition can expand an antigen-specific CD8 and / or CD4 T cell response by at least 50% or more (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) compared to that observed in the absence of such an mRNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain). In certain embodiments, a single administration of an mRNA composition can expand an antigen-specific CD8 and / or CD4 T cell response by at least 1.5-fold or more (e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more) compared to that observed in the absence of such an mRNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain).

[0118] In certain embodiments, the regimen (e.g., a single administration of an mRNA composition) can expand T cells exhibiting a Th1 phenotype (e.g., as characterized by expression of IFN-gamma, IL-2, IL-4, and / or IL-5) at least 50% or more (e.g., including at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) compared to that observed in the absence of such an mRNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain). In certain embodiments, the regimen (e.g., a single administration of an mRNA composition) can expand, for example, at least 1.5-fold or more (including, for example, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more) T cells exhibiting a Th1 phenotype (e.g., characterized by expression of IFN-gamma, IL-2, IL-4, and / or IL-5) compared to that observed in the absence of such an mRNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain). In certain embodiments, the T cell phenotype can or may include a predominantly Th1 cytokine profile (e.g., characterized by IFN-gamma positivity and / or IL-2 positivity) and / or absent or biologically insignificant IL-4 secretion.

[0119] In certain embodiments, the regimens described herein (e.g., one or more administrations of an mRNA composition) induce CD4 + Among other things, the present invention provides an mRNA composition encoding an RBD-containing portion of the SARS-CoV-2 spike protein (e.g., that does not additionally encode the full-length SARS-CoV-2 spike protein) that induces and / or achieves the production of RBD-specific CD4 T cells. +In some embodiments, the RBD-specific CD4 T cells induced by the mRNA compositions described herein (e.g., mRNA compositions encoding an RBD-containing portion of the SARS-CoV-2 spike protein, and in some embodiments, not encoding the full-length SARS-CoV-2 spike protein) may be particularly useful and / or effective for such induction and / or generation of T cells. + The T cells exhibit a predominantly Th1 cytokine profile (eg, characterized by INF-gamma positivity and / or IL-2 positivity) and / or exhibit absent or biologically insignificant IL-4 secretion.

[0120] In one embodiment, CD4 in a subject receiving an mRNA composition (e.g., one described herein) + and / or CD8 + Characterization of T cell responses (eg, as described herein) may be performed using ex vivo assays using PBMCs obtained from a subject, such as the assays described in the Examples.

[0121] In certain embodiments, the immunogenicity of the mRNA compositions described herein may be assessed by one or more serum immunogenicity assays that detect IgG, IgM, and / or IgA against the SARS-CoV-2 S protein present in a blood sample from a subject who received a provided mRNA composition and / or a neutralization assay using a SARS-CoV-2 pseudovirus and / or wild-type SARS-CoV-2 virus.

[0122] In some embodiments, mRNA compositions (e.g., those described herein) at doses of 10 μg to 100 μg or 1 μg to 50 μg result in relatively low adverse effects (e.g., grade 1 to grade 2 pain, redness, and / or swelling) within 7 days after vaccination. In some embodiments, mRNA compositions (e.g., those described herein) at doses of 10 μg to 100 μg result in relatively low observation of systemic events (e.g., grade 1 to grade 2 fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain, medication, and combinations thereof) within 7 days after vaccination.

[0123] In certain embodiments, the mRNA composition is characterized by being capable of producing IgG against a SARS-CoV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain) at levels of 100 to 100,000 U / mL or 500 to 50,000 U / mL 21 days after vaccination when administered to a subject at a dose of 10 to 100 μg or 1 μg to 50 μg.

[0124] In some embodiments, the mRNA encodes a naturally folded trimeric receptor-binding protein of SARS-CoV-2. In some embodiments, the mRNA encodes a variant of such a receptor-binding protein such that the encoded variant binds to ACE2 with a Kd of 10 pM or less, including, for example, a Kd of 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM or less. In some embodiments, the mRNA encodes a variant of such a receptor-binding protein such that the encoded variant binds to ACE2 with a Kd of 5 pM. In some embodiments, the mRNA encodes a receptor-binding portion of trimeric SARS-CoV-2 that includes an ACE2 receptor-binding site. In some embodiments, the mRNA includes a coding sequence for a receptor-binding portion of SARS-CoV-2 and a trimerization domain (e.g., the native trimerization domain (foldon) of T4 fibritin) such that the coding sequence directs the expression of a trimeric protein that has an ACE2 receptor-binding site and binds to ACE2. In some embodiments, the mRNA encodes a receptor-binding portion of trimeric SARS-CoV-2 or a variant thereof such that the Kd is less than for the monomeric receptor binding domain (RBD) of SARS-CoV-2. For example, in some embodiments, the mRNA encodes a receptor-binding portion of trimeric SARS-CoV-2 or a variant thereof such that the Kd is at least 10-fold (including, e.g., at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, etc.) less than for the RBD of SARS-CoV-2.

[0125] In certain embodiments, the receptor-binding portion of trimeric SARS-CoV-2 encoded by an mRNA (e.g., as described herein) can be characterized by cryo-electron microscopy (cryoEM) and determined to have a size of approximately 3-4 angstroms when in a closed conformation and complexed with ACE2 and the B0AT1 neutral amino acid transporter. In certain embodiments, the geometric mean SARS-CoV-2 neutralization titer characterized and / or achieved by the mRNA compositions or methods described herein can reach at least 1.5-fold, including at least 2-fold, at least 2.5-fold, at least 3-fold, or more, of a COVID-19 convalescent human panel (e.g., a serum panel obtained from COVID-19 convalescent humans 20-40 days after onset of symptom onset and at least 14 days after the onset of the asymptomatic convalescent phase).

[0126] In certain embodiments, the mRNA compositions provided herein may be characterized in that subjects treated with such compositions (e.g., at least one dose, at least two doses, etc.) may exhibit reduced and / or shorter-lasting presence of viral RNA in relevant sites (e.g., nose and / or lungs, etc., and / or any other tissue susceptible to infection) compared to appropriate controls (e.g., established expected levels for comparable subjects or populations not so treated and exposed to the virus under reasonably comparable exposure conditions).

[0127] In certain embodiments, the RBD antigen expressed by an mRNA construct (e.g., one described herein) can be modified, for example, by the addition of the T4-fibritin-derived "Foldon" trimerization domain to enhance immunogenicity.

[0128] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by certain local reactions (e.g., pain, redness, and / or swelling, etc.) and / or systemic events (e.g., fever, fatigue, headache, etc.) that may appear and / or peak on day 2 after vaccination. In certain embodiments, the mRNA compositions described herein are characterized by certain local reactions (e.g., pain, redness, and / or swelling, etc.) and / or systemic events (e.g., fever, fatigue, headache, etc.) that may resolve on day 7 after vaccination.

[0129] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by the absence of Grade 1 or higher changes or laboratory abnormalities in routine laboratory tests in subjects receiving an mRNA composition (e.g., one described herein). Examples of such laboratory assays can include lymphocyte counts, hematological changes, and the like.

[0130] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by being capable of achieving a geometric mean concentration (GMC) of IgG against a SARS-CoV-2 S polypeptide or immunogenic fragment thereof (e.g., RBD) of 200-3000 Units / mL, or 500-3000 Units / mL, or 500-2000 Units / mL, compared to 602 Units / mL in a panel of COVID-19 convalescent human sera, by 21 days after the first administration (e.g., containing 10-100 μg or containing 1 μg-50 μg). In certain embodiments, the mRNA compositions described herein are characterized by the fact that by 7 days after the second administration (e.g., comprising 10-30 μg; or comprising 1 μg-50 μg), the geometric mean concentration (GMC) of IgG against the SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD) can be increased by at least 8-fold or more, including, for example, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold or more. In certain embodiments, the mRNA compositions described herein are characterized by an increase in the geometric mean concentration (GMC) of IgG against a SARS-CoV-2 S polypeptide or immunogenic fragment thereof (e.g., RBD) of 1500 Units / mL to 40,000 Units / mL or 4000 Units / mL to 40,000 Units / mL by 7 days after the second administration (e.g., containing 10 to 30 μg; or containing 1 μg to 50 μg). In certain embodiments, the antibody concentrations described herein can persist for at least 20 days or more, including at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days, after the first administration, or for at least 10 days or more, including at least 15 days, at least 20 days, at least 25 days, or more, after the second administration. In certain embodiments, the antibody concentrations can persist for 35 days after the first administration or for at least 14 days after the second administration.

[0131] In certain embodiments, the mRNA compositions described herein are characterized by a GMC of IgG against a SARS-CoV-2 S polypeptide or immunogenic fragment thereof (e.g., RBD) that is at least 30% higher, including, for example, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, or at least 95% higher, when measured 7 days after the second dose (e.g., comprising 1-50 μg), compared to antibody concentrations observed in a panel of COVID-19 convalescent human sera. In many embodiments, the geometric mean concentrations (GMCs) of IgG described herein are GMCs of RBD-binding IgG.

[0132] In certain embodiments, the mRNA compositions described herein are characterized by a GMC of IgG against a SARS-CoV-2 S polypeptide or immunogenic fragment thereof (e.g., RBD) that is at least 1.1-fold higher (including, e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, or at least 30-fold higher) compared to antibody concentrations observed in a panel of COVID-19 convalescent human sera when measured 7 days after the second dose (e.g., containing 10-50 μg). In many embodiments, the geometric mean concentration (GMC) of IgG described herein is the GMC of RBD-binding IgG.

[0133] In certain embodiments, the mRNA compositions described herein are characterized by a GMC of IgG against a SARS-CoV-2 S polypeptide or immunogenic fragment thereof (e.g., RBD) that is at least 5-fold higher (including, e.g., at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, or at least 30-fold higher) compared to antibody concentrations observed in a panel of COVID-19 convalescent human sera, measured 21 days after the second dose. In many embodiments, the geometric mean concentration (GMC) of IgG described herein is the GMC of RBD-binding IgG.

[0134] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by an increase in SARS-CoV-2 neutralization geometric mean titer (GMT) (e.g., at least 30%, at least 40%, at least 50% or more) observed 21 days after the first dose. In certain embodiments, the mRNA compositions described herein are characterized by a significantly greater serum neutralization GMT achieved 7 days after receiving a second dose (e.g., comprising 10 μg to 30 μg), reaching 150 to 300 compared to 94 in a COVID-19 convalescent serum panel.

[0135] In some embodiments, the mRNA compositions and / or methods described herein are characterized by a preventive efficacy of at least 60%, e.g., at least 70%, at least 80%, at least 90%, or at least 95%, 7 days after the second administration. In some embodiments, the mRNA compositions and / or methods described herein are characterized by a preventive efficacy of at least 70% 7 days after the second administration. In some embodiments, the mRNA compositions and / or methods described herein are characterized by a preventive efficacy of at least 80% 7 days after the second administration. In some embodiments, the mRNA compositions and / or methods described herein are characterized by a preventive efficacy of at least 90% 7 days after the second administration. In some embodiments, the mRNA compositions and / or methods described herein are characterized by a preventive efficacy of at least 95% 7 days after the second administration.

[0136] In some embodiments, the RNA compositions provided herein are characterized by inducing an immune response against SARS-CoV-2 at least 7 days after administration (e.g., after the second administration). In some embodiments, the RNA compositions provided herein are characterized by inducing an immune response against SARS-CoV-2 within 14 days after administration (e.g., after the second administration). In some embodiments, the RNA compositions provided herein are characterized by inducing an immune response against SARS-CoV-2 at least 7 days after a vaccination regimen. In some embodiments, the vaccination regimen includes a first administration and a second administration. In some embodiments, the first administration and the second administration are administered at least 21 days apart. In some such embodiments, the immune response against SARS-CoV-2 is induced at least 28 days after the first administration.

[0137] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by a substantially higher geometric mean concentration (GMC) of antibodies to a SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD) as measured in serum from a subject who received an mRNA composition of the invention (e.g., a dose of 10-30 μg, inclusive) than in a convalescent serum panel (e.g., one described herein). In certain embodiments, when a subject may have received a second dose (e.g., 21 days after the first dose), the geometric mean concentration (GMC) of antibodies to a SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD) as measured in the serum of the subject may be 8.0-fold to 50-fold higher than the convalescent serum panel GMC. In certain embodiments, when a subject may have received a second dose (e.g., 21 days after the first initial dose), the geometric mean concentration (GMC) of antibodies to a SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD), as measured in the subject's serum, may be at least 8.0-fold or more, including at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold or more, compared to the convalescent serum panel GMC.

[0138] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by the SARS-CoV-2 neutralization geometric mean titer, measured 28 days after the first dose or 7 days after the second dose, can be at least 1.5-fold or greater (including, e.g., at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold or greater) compared to the neutralization GMT of a convalescent serum panel.

[0139] In some embodiments, the regimen administered to a subject can be or can include a single administration. In some embodiments, the regimen administered to a subject can include multiple administrations (for example, at least two administrations, at least three administrations or more). In some embodiments, the regimen administered to a subject can include a first administration and a second administration, which are given at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart or more. In some embodiments, such administrations can be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months or more apart. In certain embodiments, administrations can be separated by a number of days, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days or more. In some embodiments, administrations can be about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeks apart, or about 1 to 6 weeks or more apart. In some embodiments, administrations can be separated by a period of about 7 to about 60 days, e.g., about 14 to about 48 days. In some embodiments, the minimum number of days between administrations can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more. In certain embodiments, the maximum number of days between doses can be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or less.In some embodiments, administrations can be about 21 to about 28 days apart. In some embodiments, administrations can be about 19 to about 42 days apart. In some embodiments, administrations can be about 7 to about 28 days apart. In some embodiments, administrations can be about 14 to about 24 days apart. In some embodiments, administrations can be about 21 to about 42 days apart.

[0140] In certain embodiments, particularly for compositions established to achieve antibody and / or T cell titer elevations for periods of greater than about three weeks—for example, in certain embodiments, provided compositions are established to achieve antibody and / or T cell titer elevations (e.g., specific to the relevant portion of the SARS-CoV-2 spike protein) for periods of greater than about three weeks—in certain such embodiments, the dosing regimen may include only a single administration or may include two administrations, which, in certain embodiments, may be separated from each other by periods of greater than about 21 days or 3 weeks. For example, in certain such embodiments, such periods may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or in certain embodiments, about 1 year or more.

[0141] In some embodiments, the first and second doses (and / or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first and second doses may be administered in the deltoid muscle. In some embodiments, the first and second doses may be administered in the same arm. In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 mL each) spaced 21 days apart. In some embodiments, each dose is about 30 μg. In some embodiments, each dose may be greater than 30 μg, e.g., about 40 μg, about 50 μg, or about 60 μg. In some embodiments, each dose may be less than 30 μg, e.g., about 20 μg, about 10 μg, about 5 μg, etc. In some embodiments, each dose is about 3 μg or less, e.g., about 1 μg. In some such embodiments, the mRNA compositions described herein are administered to subjects aged 16 years or older (e.g., including those aged 16 to 85 years). In some such embodiments, the mRNA compositions described herein are administered to subjects between the ages of 18 and 55. In some such embodiments, the mRNA compositions described herein are administered to subjects between the ages of 56 and 85. In some embodiments, the mRNA compositions described herein are administered as a single dose (e.g., by intramuscular injection).

[0142] In certain embodiments, the mRNA compositions and / or methods described herein are characterized in that the RBD-specific IgG (e.g., polyclonal response) induced by such mRNA compositions and / or methods exhibits higher binding affinity to the RBD compared to a control human monoclonal antibody with SARS-CoV-2 RBD binding affinity (e.g., CR3022 described in J. ter Meulen et al., PLOS Med. 3, e237 (2006)).

[0143] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity across a panel of SARs-CoV-2 spike variants (e.g., at least 10, at least 15, or more). In certain embodiments, such SARs-CoV-2 spike variants include mutations in the RBD (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO: 1) and / or mutations in the spike protein (e.g., but not limited to, D614G, etc., compared to SEQ ID NO: 1). Those skilled in the art will be aware of the various spike variants and / or resources describing them (e.g., the Table of mutating sites in Spike maintained by the COVID-19 Viral Genome Analysis Pipeline and found at https: / / cov.lanl.gov / components / sequence / COV / int_sites_tbls.com) and will, upon reading this specification, know that the mRNA compositions and / or methods described herein may be characterized by their ability to induce sera in vaccinated subjects that exhibit neutralizing activity with respect to any or all of such variants and / or combinations thereof.

[0144] In a specific embodiment, the mRNA composition encoding the RBD of the SARS-CoV-2 spike protein is characterized by sera from vaccinated subjects exhibiting neutralizing activity across a panel (e.g., at least 10, at least 15, or more) of SARS-CoV-2 spike variants, including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO: 1) and spike protein variants (e.g., but not limited to, D614G, compared to SEQ ID NO: 1).

[0145] In a specific embodiment, an mRNA composition encoding a SARS-CoV-2 spike protein variant comprising two consecutive proline substitutions at amino acid positions 986 and 987 at the apex of the central helix in the S2 subunit is characterized in that sera from vaccinated subjects exhibit neutralizing activity across a panel (e.g., at least 10, at least 15, or more) of SARs-CoV-2 spike variants, including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO: 1) and spike protein variants (e.g., but not limited to, D614G, compared to SEQ ID NO: 1). For example, in certain embodiments, an mRNA composition encoding SEQ ID NO:7 (SP2) elicits an immune response against any one of the SARs-CoV-2 spike variants, including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., compared to SEQ ID NO:1) and spike protein variants (e.g., but not limited to, D614G, compared to SEQ ID NO:1).

[0146] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a mutation at position 501 of the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a N501Y mutation in the spike protein compared to SEQ ID NO: 1.

[0147] The one or more SARs-CoV-2 spike variants comprising a mutation at position 501 of the spike protein compared to SEQ ID NO: 1 or the one or more SARs-CoV-2 spike variants comprising a N501Y mutation in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (such as, but not limited to, an H69 / V70 deletion, a Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, or an L242 / A243 / L244 deletion compared to SEQ ID NO: 1).

[0148] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARS-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01; also known as lineage B.1.1.7). The variant was previously designated by Public Health England as Variant Under Investigation in December 2020 (VUI - 202012 / 01) but has since been reclassified as Variant of Concern (VOC-202012 / 01). VOC-202012 / 01 is a variant of SARS-CoV-2 that was first detected in the UK in October 2020 during the COVID-19 pandemic in samples collected the previous month and began spreading rapidly by mid-December. This correlates with a significant increase in COVID-19 infection rates in the UK; this increase is thought to be due, at least in part, to the N501Y mutation within the receptor-binding domain of the spike glycoprotein, which is required for binding to ACE2 in human cells. The VOC-202012 / 01 variant is defined by 23 mutations: 13 nonsynonymous, 4 deletions, and 6 synonymous mutations (i.e., 17 protein-altering mutations and 6 non-altering mutations). Spike protein changes in VOC-202012 / 01 include deletions 69-70, 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. One of the most significant changes in VOC-202012 / 01 involves N501Y, an asparagine (N) to tyrosine (Y) change at amino acid position 501. This mutation, alone or together with the deletion at positions 69 / 70 in the N-terminal domain (NTD), can enhance viral infectivity.

[0149] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0150] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "501.V2." This variant was first observed in samples in October 2020; since then, over 300 cases of the 501.V2 variant have been confirmed by whole genome sequencing (WGS) in South Africa, where it became the dominant form of virus by December 2020. Preliminary results indicate that this variant may have increased transmissibility. The 501.V2 variant is defined by multiple spike protein changes, including D80A, D215G, E484K, N501Y, and A701V; more recently collected viruses harbor additional changes: L18F, R246I, K417N, and deletion 242-244.

[0151] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising mutations D80A, D215G, E484K, N501Y, and A701V compared to SEQ ID NO: 1, and optionally L18F, R246I, K417N, and deletion 242-244 compared to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation compared to SEQ ID NO: 1.

[0152] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising an H69 / V70 deletion in the spike protein compared to SEQ ID NO:1.

[0153] In certain embodiments, one or more SARs-CoV-2 spike variants comprising an H69 / V70 deletion in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., but not limited to, a Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, an L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, etc. compared to SEQ ID NO: 1).

[0154] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01; also known as lineage B.1.1.7).

[0155] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0156] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "Cluster 5," also designated ΔFVI-spike by the Danish Serum Institute (SSI). It was discovered in North Denmark and is believed to have spread from mink to humans on mink farms. Several different mutations in the viral spike protein have been identified in Cluster 5. Specific mutations include 69-70 deltaHV (deletion of histidine and valine residues at positions 69 and 79 of the protein), Y453F (a tyrosine to phenylalanine change at position tyrosine), I692V (isoleucine to valine at position isoleucine), M1229I (methionine to isoleucine at position methionine), and, optionally, S1147L (serine to leucine at position serine).

[0157] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising deletions 69-70, Y453F, I692V, M1229I, and optionally the mutation S1147L compared to SEQ ID NO:1.

[0158] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a mutation at position 614 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a D614G mutation in the spike protein compared to SEQ ID NO: 1.

[0159] In certain embodiments, one or more SARs-CoV-2 spike variants comprising a mutation at position 614 in the spike protein compared to SEQ ID NO: 1 or one or more SARs-CoV-2 spike variants comprising a D614G mutation in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., but not limited to, H69 / V70 deletion, Y144 deletion, N501Y, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, etc., compared to SEQ ID NO: 1).

[0160] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01; also known as lineage B.1.1.7).

[0161] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0162] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally L18F, R246I, K417N, and deletion 242-244, relative to SEQ ID NO:1.

[0163] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 614 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions N501Y and D614G in the spike protein compared to SEQ ID NO: 1.

[0164] In certain embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 614 in the spike protein compared to SEQ ID NO: 1 or one or more SARs-CoV-2 spike variants comprising N501Y and D614G mutations in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., but not limited to, H69 / V70 deletion, Y144 deletion, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, etc., compared to SEQ ID NO: 1).

[0165] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "Variant of Concern 202012 / 01" (VOC-202012 / 01; also known as lineage B.1.1.7).

[0166] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against SARs-CoV-2 spike variants comprising the following mutations compared to SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0167] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally L18F, R246I, K417N, and deletion 242-244, relative to SEQ ID NO:1.

[0168] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a mutation at position 484 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising an E484K mutation in the spike protein compared to SEQ ID NO: 1.

[0169] In certain embodiments, one or more SARs-CoV-2 spike variants comprising a mutation at position 484 in the spike protein compared to SEQ ID NO: 1 or one or more SARs-CoV-2 spike variants comprising an E484K mutation in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., a H69 / V70 deletion, a Y144 deletion, a Examples of such deletions include, but are not limited to, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, and V1176F.

[0170] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against SARs-CoV-2 spike variant "501.V2."

[0171] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, and A701V, and optionally L18F, R246I, K417N, and deletion 242-244, relative to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise the D614G mutation relative to SEQ ID NO: 1.

[0172] The lineage B.1.1.248, also known as the Brazilian variant, is one of the SARS-CoV-2 variants designated the P.1 lineage. It contains 17 unique amino acid changes, 10 of which are in its spike protein, including N501Y and E484K. B.1.1.248 originated from B.1.1.28. E484K is present in both B.1.1.28 and B.1.1.248. B.1.1.248 contains multiple S-protein polymorphisms [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F] and is similar to the variant reported from South Africa at certain critical RBD positions (K417, E484, and N501).

[0173] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.28."

[0174] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0175] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0176] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 484 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions N501Y and E484K in the spike protein compared to SEQ ID NO: 1.

[0177] In certain embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 501 and 484 in the spike protein compared to SEQ ID NO: 1 or one or more SARs-CoV-2 spike variants comprising N501Y and E484K mutations in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., H69 / V compared to SEQ ID NO: 1). 70 deletion, Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F, etc.).

[0178] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against SARs-CoV-2 spike variant "501.V2."

[0179] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising mutations D80A, D215G, E484K, N501Y, and A701V compared to SEQ ID NO: 1, and optionally L18F, R246I, K417N, and deletion 242-244 compared to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation compared to SEQ ID NO: 1.

[0180] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0181] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0182] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 501, 484, and 614 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions N501Y, E484K, and D614G in the spike protein compared to SEQ ID NO: 1.

[0183] In certain embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 501, 484, and 614 in the spike protein compared to SEQ ID NO: 1 or one or more SARs-CoV-2 spike variants comprising N501Y, E484K, and D614G mutations in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., In comparison, mutations such as, but not limited to, H69 / V70 deletion, Y144 deletion, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, and V1176F.

[0184] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally L18F, R246I, K417N, and deletion 242-244, relative to SEQ ID NO:1.

[0185] In certain embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against one or more SARs-CoV-2 spike variants comprising an L242 / A243 / L244 deletion in the spike protein compared to SEQ ID NO:1.

[0186] In certain embodiments, one or more SARs-CoV-2 spike variants comprising an L242 / A243 / L244 deletion in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., but not limited to, an H69 / V70 deletion, a Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F, etc., compared to SEQ ID NO: 1).

[0187] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against SARs-CoV-2 spike variant "501.V2."

[0188] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising mutations at D80A, D215G, E484K, N501Y, A701V, and deletions 242-244 relative to SEQ ID NO: 1, and optionally L18F, R246I, and K417N relative to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation relative to SEQ ID NO: 1.

[0189] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a mutation at position 417 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising a K417N or K417T mutation in the spike protein compared to SEQ ID NO: 1.

[0190] In certain embodiments, one or more SARs-CoV-2 spike variants comprising a mutation at position 417 in the spike protein compared to SEQ ID NO: 1 or one or more SARs-CoV-2 spike variants comprising a K417N or K417T mutation in the spike protein compared to SEQ ID NO: 1 may comprise one or more additional mutations compared to SEQ ID NO: 1 (e.g., a H69 / V70 deletion, Examples include, but are not limited to, Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I, and V1176F.

[0191] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against SARs-CoV-2 spike variant "501.V2."

[0192] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising D80A, D215G, E484K, N501Y, A701V, and K417N compared to SEQ ID NO: 1, and optionally L18F, R246I, and deletions 242-244 compared to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise a D614G mutation compared to SEQ ID NO: 1.

[0193] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0194] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0195] In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 417 and 484 and / or 501 in the spike protein compared to SEQ ID NO: 1. In certain embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against one or more SARs-CoV-2 spike variants comprising mutations at positions 417 and 484 and / or 501 in the spike protein compared to SEQ ID NO: 1.

[0196] In some embodiments, one or more SARs-CoV-2 spike variants comprising mutations at positions 417 and 484 and / or 501 in the spike protein compared to SEQ ID NO: 1 or comprising a K417N or K417T mutation and an E484K and / or N501Y mutation in the spike protein compared to SEQ ID NO: 1 may comprise one or more further mutations compared to SEQ ID NO: 1. (For example, compared to SEQ ID NO: 1, H69 / V70 deletion, Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, L242 / A243 / L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F, etc., but are not limited to these.)

[0197] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against SARs-CoV-2 spike variant "501.V2."

[0198] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations relative to SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V, and K417N, and optionally L18F, R246I, and deletion 242-244, relative to SEQ ID NO: 1. The SARs-CoV-2 spike variant may also comprise the D614G mutation relative to SEQ ID NO: 1.

[0199] In specific embodiments, the mRNA compositions and / or methods described herein are characterized by sera from vaccinated subjects exhibiting neutralizing activity against the SARs-CoV-2 spike variant "B.1.1.248."

[0200] In specific embodiments, the mRNA compositions and / or methods described herein are characterized in that sera from vaccinated subjects exhibit neutralizing activity against a SARs-CoV-2 spike variant comprising the following mutations compared to SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

[0201] The SARs-CoV-2 spike variants described herein may or may not contain the D614G mutation compared to SEQ ID NO:1.

[0202] In certain embodiments, the mRNA compositions and / or methods described herein can provide protection against SARS-CoV-2 and / or reduce the severity of SARS-CoV-2 infection in at least 50% of subjects receiving such mRNA compositions and / or methods.

[0203] In some embodiments, the population treated with the mRNA compositions described herein includes subjects aged 18-55. In some embodiments, the population treated with the mRNA compositions described herein includes subjects aged 56-85. In some embodiments, the population treated with the mRNA compositions described herein includes older subjects (e.g., subjects over 60, 65, 70, 75, 80, 85, etc., e.g., subjects aged 65-85). In some embodiments, the population treated with the mRNA compositions described herein includes subjects aged 18-85. In some embodiments, the population treated with the mRNA compositions described herein includes subjects under 18. In some embodiments, the population treated with the mRNA compositions described herein includes subjects under 12. In some embodiments, the population treated with the mRNA compositions described herein includes subjects under 10. In some embodiments, the population treated with the mRNA compositions described herein can include an adolescent population (e.g., individuals between about 12 and about 17 years of age). In some embodiments, the population treated with the mRNA compositions described herein includes infants (e.g., under 1 year old). In some embodiments, the population treated with the mRNA compositions described herein does not include infants (e.g., under 1 year old) whose mothers received such mRNA compositions described herein during pregnancy. Without wishing to be bound by any particular theory, the rat study shown in Example 31 suggests that SARS-CoV-2 neutralizing antibody responses induced in female rats given such mRNA compositions during pregnancy can be inherited by the fetus. In some embodiments, the population treated with the mRNA compositions described herein includes infants (e.g., under 1 year old) whose mothers did not receive such mRNA compositions described herein during pregnancy.In certain embodiments, populations treated with the mRNA compositions described herein can include pregnant women; in certain embodiments, infants whose mothers were vaccinated during pregnancy (e.g., only those who received at least one dose or both doses) are not vaccinated for several weeks, months, or years after birth (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more, or 1 year, 2 years, 3 years, 4 years, 5 years or more). Alternatively or additionally, in certain embodiments, infants whose mothers were vaccinated during pregnancy (e.g., only those who received at least one dose or both doses) may require reduced vaccinations (e.g., lower doses and / or fewer administrations - e.g., booster - times and / or lower total exposure over a period of time) or reduced vaccinations (e.g., lower doses and / or fewer administrations - e.g., booster - times) after birth, e.g., within weeks, months, or years of age (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more, or 1 year, 2 years, 3 years, 4 years, 5 years or more). In some embodiments, the compositions provided herein are administered to a population that does not include pregnant women.

[0204] In certain embodiments, the compositions provided herein are administered to a pregnant woman using a regimen that includes a first administration after about 24 weeks of gestation (e.g., after about 22, 23, 24, 25, 26, 27, 28, or later weeks of gestation); in certain embodiments, the compositions provided herein are administered to a pregnant woman using a regimen that includes a first administration before about 34 weeks of gestation (e.g., before about 30, 31, 32, 33, 34, 35, 36, 37, 38 weeks of gestation). In certain embodiments, the compositions provided herein are administered to a pregnant woman using a regimen that includes a first administration after about 24 weeks of gestation (e.g., after about 27 weeks of gestation, e.g., between about 24 and 34 weeks or between about 27 and 34 weeks of gestation) and a second administration about 21 days later; in certain embodiments, both administrations are administered before birth. Without wishing to be bound by any particular theory, such a regimen (e.g., comprising a first administration after about 24 or 27 weeks of gestation and optionally before about 34 weeks of gestation), ideally before birth, and optionally a second administration within about 21 days, may have certain advantages in terms of safety (e.g., reduced risk of premature birth or fetal morbidity or mortality) and / or efficacy (e.g., deferred vaccination of the infant) compared to alternative administration regimens (e.g., administration at any time during pregnancy, withholding administration during pregnancy, and / or administration later in pregnancy, e.g., allowing for only one administration during pregnancy). In certain embodiments, as described herein (see also Example 34), for example, with certain regimens described herein, infants born to mothers vaccinated during pregnancy may not require further vaccinations or may require reduced vaccinations (e.g., lower doses and / or fewer administrations—e.g., boosters—and / or reduced overall exposure over a period of time) for a period of time after birth (e.g., as described herein).

[0205] In some embodiments, the compositions provided herein are administered to a population in which women are instructed not to become pregnant for a period of time after receiving the vaccine (e.g., after receiving the first dose of the vaccine, after receiving the last dose of the vaccine, etc.); in some such embodiments, the period of time can be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks or more, or at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or more.

[0206] In some embodiments, the population treated with the mRNA compositions described herein includes one or more populations with a history of one or more particularly high-risk conditions or behaviors, e.g., as described herein. For example, in some embodiments, the population treated with the mRNA compositions described herein can include subjects whose job expertise and / or environmental exposure may dramatically increase their risk of SARS-CoV-2 infection (e.g., public transportation workers, prisoners, grocery store clerks, long-term care facility residents, meat processors or other meat processing workers, healthcare workers and / or first responders, e.g., emergency responders). In specific embodiments, the population treated with the mRNA compositions described herein can include healthcare workers and / or first responders, e.g., emergency responders. In some embodiments, the population treated with the mRNA compositions described herein can include those with a history of smoking or vaping (e.g., including chronic smoking or vaping, for 6 months, 12 months, or more). In certain embodiments, the populations treated with the mRNA compositions described herein may include certain ethnic groups that have been identified as being more susceptible to SARS-CoV-2 infection.

[0207] In certain embodiments, populations treated with the mRNA compositions described herein may include populations with blood types that may be identified as more susceptible to SARS-CoV-2 infection. In certain embodiments, populations treated with the mRNA compositions described herein may include immunocompromised subjects (e.g., those with HIV / AIDS; cancer and transplant patients taking certain immunosuppressants; autoimmune diseases or other physiological conditions for which immunosuppressive treatment is predicted to be warranted (e.g., within three months, six months, or more); and those with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency)). In certain embodiments, populations treated with the mRNA compositions described herein may include those with infectious diseases. For example, in certain embodiments, populations treated with the mRNA compositions described herein may include those infected with human immunodeficiency virus (HIV) and / or hepatitis viruses (e.g., HBV, HCV). In certain embodiments, populations treated with the mRNA compositions described herein may include those with underlying diseases. Examples of such underlying diseases include hypertension, cardiovascular disease, diabetes, chronic respiratory diseases such as chronic lung disease, asthma, cancer, and conditions such as lupus, rheumatoid arthritis, chronic liver disease, and chronic kidney disease (e.g., in one embodiment, 60 mL / min / 1.73 m 2 In certain embodiments, the populations treated with the mRNA compositions described herein include, but are not limited to, other chronic diseases such as stage 3 or worse, as characterized by a glomerular filtration rate (GFR) of less than about 30 kg / m. 2 The population may include overweight or obese subjects, including those with a body mass index (BMI) greater than 100. In certain embodiments, the population treated with the mRNA compositions described herein may include subjects who have been previously diagnosed with COVID-19 or have evidence of current or prior SARS-CoV-2 infection, for example, based on serology or nasal swabs. In certain embodiments, the population treated includes white and / or non-Hispanic / non-Latino individuals.

[0208] In certain embodiments, certain mRNA compositions described herein (e.g., BNT162b1) can be selected for administration to Asian populations (e.g., Chinese populations), or in specific embodiments, to elderly Asian populations (e.g., 60 years of age or older, e.g., 60-85 or 65-85 years of age).

[0209] In certain embodiments, the mRNA compositions provided herein are administered to and / or evaluated in subjects who are found to have no evidence of prior and / or current infection prior to administration; in certain embodiments, evidence of prior and / or current infection can be or may include evidence of intact virus or any viral nucleic acid, protein, lipid, etc. present in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and / or tissue) and / or evidence of the subject's immune response thereto. In certain embodiments, the mRNA compositions provided herein are administered to and / or evaluated in subjects who are found to have evidence of prior and / or current infection prior to administration; in certain embodiments, evidence of prior and / or current infection can be or may include evidence of intact virus or any viral nucleic acid, protein, lipid, etc. present in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and / or tissue) and / or evidence of the subject's immune response thereto. In certain embodiments, a subject is considered to have had a prior infection based on a positive N-linked antibody test result or a positive nucleic acid amplification test (NAAT) result on day 1 of administration.

[0210] In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been informed of the risk of side effects, which may include, for example, one or more of chills, fever, headache, injection site pain, muscle pain, and fatigue; in some embodiments, the RNA (e.g., mRNA) compositions are administered to subjects who have been requested to notify a medical professional if one or more such side effects occur, are more than mild or moderate, and persist for a period of one or more days, or if the subject experiences any severe or unpredictable event that the subject reasonably believes may be associated with inoculation with the composition. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been requested to notify a medical professional of a particular health condition, which may include, for example, one or more of: allergies, bleeding disorders or anticoagulant medication, breastfeeding, fever, immunocompromised state or medication that affects the immune system, pregnancy or planning a pregnancy, etc. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been administered another COVID-19 vaccine and have been requested to notify a medical professional. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who does not experience a febrile illness, receive immunosuppressant therapy, receive anticoagulant therapy, suffer from a bleeding disorder (e.g., one that contraindicates intramuscular injection), or have one of the following health conditions: pregnancy and / or breastfeeding. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who has not received another COVID-19 vaccine. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who does not have an allergic reaction to any component of the RNA (e.g., mRNA) composition. Examples of such allergic reactions include, but are not limited to, shortness of breath, facial and / or pharyngeal swelling, palpitations, rash, dizziness, and / or weakness. In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who has received an initial dose and has not had an allergic reaction (e.g., one described herein) to the initial dose.In certain embodiments, if a subject experiences an allergic reaction after receiving an RNA (e.g., mRNA) composition provided herein, such subject may be administered one or more interventions, such as treatments to manage and / or reduce such allergic reaction, e.g., antipyretic and / or anti-inflammatory agents.

[0211] In certain embodiments, a subject who has received at least one administration of an RNA (e.g., mRNA) composition provided herein is instructed to avoid exposure to coronavirus (e.g., SARS-CoV-2) unless and until several days (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, etc.) have passed since the second administration. For example, a subject who has received at least one administration of an RNA (e.g., mRNA) composition provided herein is instructed to take preventative measures against SARS-CoV-2 infection (e.g., maintaining social distancing, wearing a mask, washing hands frequently, etc.) unless and until several days (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, etc.) have passed since the second administration. Thus, in some embodiments, the methods of administering an RNA (e.g., mRNA) composition provided herein include administering a second dose of such an RNA (e.g., mRNA) composition provided herein to a subject who has received an initial dose and has taken precautions to avoid exposure to a coronavirus (e.g., SARS-CoV-2).

[0212] In some embodiments, the mRNA compositions described herein can be delivered to the draining lymph nodes of a subject in need thereof, for example, for vaccine priming. In some embodiments, such delivery can be performed by intramuscular administration of the provided mRNA compositions.

[0213] In some embodiments, different specific mRNA compositions can be administered to different subject populations; alternatively or additionally, in some embodiments, different dosing regimens can be administered to different subject populations. For example, in some embodiments, the mRNA compositions administered to specific subject populations can be characterized by one or more specific effects (e.g., incidence and / or degree of effect) in these subject populations. In some embodiments, such effects can be measured, for example, by titer and / or neutralizing antibody and / or T cell (e.g., CD4 + and / or CD8 + T cells, such as T H The efficacy of the antibody may be or may include the persistence of type 1 T cells), protection against exposure (e.g., injection and / or intranasal exposure), incidence, severity and / or persistence of side effects (e.g., reactogenicity), etc.

[0214] In certain embodiments, one or more mRNA compositions described herein may be administered according to a regimen established to reduce the incidence of COVID-19 per 1000 person-years, based on a clinical test, such as a nucleic acid amplification test (NAAT). In certain embodiments, one or more mRNA compositions described herein may be administered according to a regimen established to reduce the incidence of COVID-19 per 1000 person-years, based on a clinical test, such as a nucleic acid amplification test (NAAT), in subjects who have received at least one dose of a provided mRNA composition and who have no serological or virological evidence of prior SARS-CoV-2 infection (e.g., up to 7 days after receiving their last dose). In certain embodiments, one or more mRNA compositions described herein may be administered according to a regimen established to reduce the incidence of confirmed severe COVID-19 per 1000 person-years. In certain embodiments, one or more of the mRNA compositions described herein may be administered according to a regimen established to reduce the incidence of confirmed severe COVID-19 per 1000 person-years in subjects who receive at least one dose of a provided mRNA composition and who have no serological or virological evidence of previous SARS-CoV-2 infection.

[0215] In certain embodiments, one or more mRNA compositions described herein may be administered in a regimen established to produce neutralizing antibodies to a SARS-CoV-2 spike polypeptide and / or immunogenic fragment thereof (e.g., RBD) that meet or exceed a control level (e.g., a control level determined based on human SARS-CoV-2 infected / COVID-19 convalescent sera) for a period of time as measured in serum from the subject, and / or to induce a cell-mediated immune response (e.g., a T cell response to SARS-CoV-2) for a period of time, e.g., in certain embodiments, including the induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and / or immunogenic fragment thereof (e.g., RBD). In certain such embodiments, the period may be at least 2 months, 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer. In certain embodiments, the period of time may include the induction of vaccine-induced T cells (e.g., CD8 + The one or more epitopes recognized by the HLA-B*T cells can be presented on an MHC class I allele present in at least 50%, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects in a population; in certain such embodiments, the MHC class I allele can be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. In certain embodiments, the epitope may include HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA-B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0216] In certain embodiments, efficacy is assessed as the incidence of COVID-19 per 1000 person-years in individuals without serological or virological evidence of previous SARS-CoV-2 infection before and during the vaccination regimen; alternatively or additionally, in certain embodiments, efficacy is assessed as the incidence of COVID-19 per 1000 person-years in subjects with and without serological or virological evidence of previous SARS-CoV-2 infection before and during the vaccination regimen. In certain such embodiments, such incidence is confirmed COVID-19 cases within a certain period of time after the final vaccination (e.g., the first dose in a single-dose regimen; the second dose in a two-dose regimen, etc.); in certain embodiments, such period can be within a certain number of days (e.g., 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 or more days) (i.e., up to and including 7 days). In certain embodiments, such period can be within 7 days, or within 14 days, or within 21 days, or within 28 days. In certain embodiments, such period can be within 7 days. In certain embodiments, such period can be within 14 days.

[0217] In some embodiments (e.g., in some embodiments evaluating efficacy), a subject is determined to have experienced COVID-19 infection if one or more of the following is established in a sample from the subject: detection of SARS-CoV-2 nucleic acid; detection of an antibody that specifically recognizes SARS-CoV-2 (e.g., SARS-CoV-2 spike protein); one or more symptoms of COVID-19 infection; and combinations thereof. In some such embodiments, detection of SARS-CoV-2 nucleic acid may include, for example, NAAT testing of a middle turbinate swab sample. In some such embodiments, detection of relevant antibodies may include serology testing of a blood sample or portion thereof. In some such embodiments, symptoms of COVID-19 infection may include fever, a new or increased cough, a new or increased shortness of breath, chills, a new or increased muscle pain, a new loss of taste or smell, sore throat, diarrhea, vomiting, and combinations thereof. In some such embodiments, symptoms of COVID-19 infection may include or include fever, a new or increased cough, a new or increased shortness of breath, chills, a new or increased muscle pain, a new or increased loss of taste or smell, sore throat, diarrhea, vomiting, fatigue, headache, nasal congestion or runny nose, nausea, and combinations thereof. In some such embodiments, a subject is determined to have experienced a COVID-19 infection if such subject experiences one of these symptoms and tests positive for SARS-CoV-2 nucleic acid or antibody, or both. In some such embodiments, a subject is determined to have experienced a COVID-19 infection if such subject experiences one of these symptoms and tests positive for SARS-CoV-2 nucleic acid. In some such embodiments, a subject is determined to have experienced a COVID-19 infection if such subject experiences one of these symptoms and tests positive for SARS-CoV-2 antibody.

[0218] In certain embodiments (e.g., in certain embodiments evaluating efficacy), a subject is determined to have experienced a severe COVID-19 infection if such subject experiences one or more of the following clinical signs of severe systemic illness at rest (e.g., one or more of a respiratory rate of 30 or more per minute, a heart rate of 125 or more beats per minute, an SpO2 of 93% or less at sea level on room air, or a PaO2 / FiO2 of less than 300 mHg), respiratory failure (e.g., one or more of high oxygen flow, noninvasive ventilation, mechanical ventilation, need for ECMO), evidence of shock (systolic blood pressure less than 90 mmHg, diastolic blood pressure less than 60 mmHg, need for vasopressors), significant acute renal, hepatic, or neurological dysfunction, intensive care unit admission, death, and combinations thereof.

[0219] In certain embodiments, one or more mRNA compositions described herein may be administered using a regimen established to reduce the percentage of subjects reporting at least one of: (i) one or more local reactions (e.g., those described herein) up to 7 days after each dose; (ii) one or more systemic events up to 7 days after each dose; (iii) an adverse event (e.g., those described herein) from the first dose up to 1 month after the last dose; and / or (iv) a severe adverse event (e.g., those described herein) from the first dose up to 6 months after the last dose.

[0220] In certain embodiments, one or more subjects who receive an RNA (e.g., mRNA) composition described herein can be monitored (e.g., for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 years, 8 years, 9 years, 10 years, or more), e.g., to assess the presence of an immune response to components of the administered composition, evidence of exposure to and / or an immune response to SARS-CoV-2 or other coronaviruses, any adverse events, etc. (For example, for a period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 weeks, 12 weeks or more, including 1 month, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more). In some embodiments, monitoring can be by remote visit. Alternatively or additionally, in some embodiments, monitoring can be in person.

[0221] In certain embodiments, the therapeutic effect provided by one or more mRNA compositions described herein may be characterized by (i) a SARS-CoV-2 anti-S1-binding antibody level above a predetermined threshold; (ii) a SARS-CoV-2 anti-RBD-binding antibody level above a predetermined threshold; and / or (iii) a SARS-CoV-2 serum neutralizing titer above a threshold level, e.g., baseline, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and / or 24 months post-vaccination. In certain embodiments, the anti-S1-binding antibody and / or anti-RBD-binding antibody level and / or serum neutralizing titer may be characterized by the geometric mean concentration (GMC), geometric mean titer (GMT), or geometric mean fold increase (GMFR).

[0222] In certain embodiments, the therapeutic effect provided by one or more mRNA compositions described herein can be characterized, for example, by a higher percentage of treated subjects exhibiting SARS-CoV-2 serum neutralizing titers above a predetermined threshold at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and / or 24 months post-vaccination than the percentage of untreated subjects exhibiting SARS-CoV-2 serum neutralizing titers above such predetermined threshold (e.g., those described herein). In certain embodiments, serum neutralizing titers can be characterized by geometric mean concentration (GMC), geometric mean titer (GMT), or geometric mean fold increase (GMFR).

[0223] In certain embodiments, the therapeutic effect provided by one or more of the mRNA compositions described herein can be characterized by the detection of SARS-CoV-2 NVA-specific binding antibodies.

[0224] In certain embodiments, the therapeutic effect provided by one or more of the mRNA compositions described herein can be characterized by detection of SARS-CoV-2 by nucleic acid amplification testing.

[0225] In certain embodiments, the therapeutic effect provided by one or more of the mRNA compositions described herein can be characterized by the induction of a cell-mediated immune response (e.g., a T cell response to SARS-CoV-2), including, for example, in certain embodiments, the induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and / or immunogenic fragments thereof (e.g., RBD). In certain embodiments, vaccine-induced T cells (e.g., CD8 +One or more epitopes recognized by a population of subjects (e.g., T cells) can be presented on an MHC class I allele present in at least 50%, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects in the population; in certain such embodiments, the MHC class I allele can be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. In certain embodiments, the epitope may include HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA-B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0226] In certain embodiments, the primary vaccine efficacy (VE) of one or more mRNA compositions described herein can be established when there is sufficient evidence (posterior probability) that primary VE1 or both primary VE1 and primary VE2 are >30% or higher (including, for example, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or higher), where primary VE is defined as primary VE = 100 × (1 - IRR); IRR is calculated as the ratio of COVID-19 disease rate in the vaccine group to the corresponding disease rate in the placebo group. Primary VE1 represents the VE of a prophylactic mRNA composition described herein against confirmed COVID-19 in participants who had no evidence of pre-vaccination infection, and primary VE2 represents the VE of a prophylactic mRNA composition described herein against confirmed COVID-19 in all participants after vaccination. In some embodiments, the primary VE1 and VE2 can be evaluated sequentially to control a 2.5% overall type I error (tiered testing). In some embodiments, when one or more RNA (e.g., mRNA) compositions described herein are shown to achieve the primary VE endpoint, secondary VE endpoints (e.g., confirmed severe COVID-19 in participants without evidence of pre-vaccination infection and confirmed severe COVID-19 in all participants) can be evaluated sequentially, for example, using the same method as used for the primary VE endpoint evaluation (tiered testing). In some embodiments, the evaluation of the primary and / or secondary VE endpoints can be based on at least 20,000 or more subjects (e.g., at least 25,000 or more subjects) randomized in a 1:1 ratio to the vaccine or placebo group, based on the following hypotheses: (i) a 1.0% incidence rate per year in the placebo group; and (ii) 20% of participants will be unevaluable or have serologic evidence of prior SARS-CoV-2 infection and may be immune to further infection.

[0227] In certain embodiments, one or more mRNA compositions described herein may be administered according to a regimen established to achieve a sustained and / or enhanced immune response. For example, in certain embodiments, the administration regimen may include an initial administration, followed, optionally, by one or more subsequent administrations; in certain embodiments, the necessity, timing, and / or intensity of any such subsequent administrations may be selected to maintain, enhance, and / or modify one or more immune responses or characteristics thereof. In certain embodiments, the number, timing, and / or amount of administrations may be established to be effective when administered to an appropriate population. In certain embodiments, the number, timing, and / or amount of administrations may be adjusted for individual subjects; for example, in certain embodiments, one or more characteristics of the immune response in an individual subject may be assessed at least once (and optionally more than once, e.g., often at preselected intervals, generally multiple times apart) after receiving the initial administration. For example, antibodies, B cells, and / or T cells (e.g., CD4 + and / or CD8 + The presence of T cells and / or cytokines secreted thereby and / or the nature and / or extent of the response to a particular antigen and / or epitope can be assessed. In certain embodiments, the need for, timing and / or amount of subsequent administration can be determined in light of such assessment.

[0228] As noted above, in certain embodiments, one or more subjects who receive the RNA (e.g., mRNA) compositions described herein are treated with antibodies, B cells and / or T cells (e.g., CD4 + and / or CD8 +The subject may be monitored (e.g., for 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more) after receiving any particular dose to assess, for example, the presence of an immune response to components of the administered composition, evidence of exposure to and / or immune response to SARS-CoV-2 or other coronaviruses, any adverse events, etc., including conducting an assessment of the presence of immune responses to specific antigens and / or epitopes (e.g., T cells) and / or cytokines secreted thereby and / or the nature and / or extent of the response to specific antigens and / or epitopes. (e.g., for a period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, including 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more). Administration of the compositions described herein can be administered in a regimen that includes one or more such monitoring steps.

[0229] For example, in certain embodiments, the necessity, timing, and / or amount of a second administration relative to a first administration (and / or a subsequent administration relative to a prior administration) is evaluated, determined, and / or selected such that such second (or subsequent) administration achieves an amplification or modification of the immune response (e.g., one described herein) observed with the first (or other prior) administration. In certain embodiments, such amplification of the immune response (e.g., one described herein) can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more compared to the level of the immune response observed after the first administration. In certain embodiments, such amplification of the immune response can be at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold or more compared to the level of the immune response observed after the first administration.

[0230] In certain embodiments, the necessity, timing, and / or amount of a second administration relative to a first administration (and / or subsequent administration relative to a prior administration) is assessed, determined, and / or selected such that the subsequent administration extends the durability of an immune response (e.g., one described herein) observed after the prior administration; in certain such embodiments, the durability may be extended for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, or more. In certain embodiments, the immune response observed after the first administration can be characterized by the production of neutralizing antibodies and / or a cell-mediated immune response (e.g., induction of a T cell response against SARS-CoV-2, including, in certain embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and / or immunogenic fragment thereof (e.g., RBD)) as measured in serum from the subject. In certain embodiments, vaccine-induced T cells (e.g., CD8 + One or more epitopes recognized by a population of subjects (e.g., T cells) can be presented on an MHC class I allele present in at least 50%, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects in the population; in certain such embodiments, the MHC class I allele can be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. In certain embodiments, the epitope may include HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA-B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0231] In certain embodiments, the necessity, timing, and / or amount of a second administration relative to a first administration (or other subsequent administration relative to a prior administration) is assessed, determined, and / or selected such that such second (or subsequent) administration maintains or exceeds a control level of immune response; in certain such embodiments, the control level is determined based on human SARS-CoV-2 infected / COVID-19 convalescent serum and / or PBMC samples taken from the subject (e.g., at least a period of time, such as at least 14 days or more, including, e.g., 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days after PCR-confirmed diagnosis if the subject was asymptomatic). In certain embodiments, the immune response can be characterized by the production of neutralizing antibodies and / or a cell-mediated immune response (e.g., induction of a T cell response against SARS-CoV-2, including, in certain embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and / or immunogenic fragment thereof (e.g., RBD)) as measured in serum from the subject. In certain embodiments, vaccine-induced T cells (e.g., CD8 +One or more epitopes recognized by a population of subjects (e.g., T cells) can be presented on an MHC class I allele present in at least 50%, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects in the population; in certain such embodiments, the MHC class I allele can be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. In certain embodiments, the epitope may include HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA-B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0232] In certain embodiments, determining the need, timing, and / or amount of a second (or subsequent) administration may include one or more steps of evaluating the presence and / or expression level of neutralizing antibodies to the SARS-CoV-2 spike polypeptide and / or immunogenic fragment thereof (e.g., RBD) and / or a cell-mediated immune response (e.g., induction of a T cell response against SARS-CoV-2, including, in certain embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and / or immunogenic fragment thereof (e.g., RBD)) measured in serum from the subject after the first (or other prior) administration (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more days later). In certain embodiments, vaccine-induced T cells (e.g., CD8 +One or more epitopes recognized by a population of subjects (e.g., T cells) can be presented on an MHC class I allele present in at least 50%, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects in the population; in certain such embodiments, the MHC class I allele can be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. In certain embodiments, the epitope may include HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA-B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0233] In certain embodiments, the kits provided herein can include a device for real-time transport monitoring logging, which can provide, for example, in certain embodiments, shipping temperature, shipping time and / or location.

[0234] In certain embodiments, the RNA (e.g., mRNA) compositions described herein can be transported, stored, and / or utilized in a container (e.g., a vial or syringe), e.g., a glass container (e.g., a glass vial or syringe), which, in certain embodiments, can be a single-dose container or a multi-dose container (e.g., arranged and constructed to hold, and / or in certain embodiments, can hold, a single dose or multiple doses for administration). In certain embodiments, a multi-dose container (e.g., a multi-dose vial or syringe) can hold and / or can hold 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses; in certain embodiments, it can be designed to hold and / or can hold 5 doses. In certain embodiments, a single-dose or multi-dose container (e.g., a single-dose or multi-dose vial or syringe) can be arranged and constructed to hold and / or hold a volume or amount greater than the indicated dose, e.g., to allow for some loss during transfer and / or administration. In certain embodiments, the RNA (e.g., mRNA) compositions described herein may be transported, stored, and / or utilized in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single-dose or multi-dose preservative-free glass vial or syringe). In certain embodiments, the RNA (e.g., mRNA) compositions described herein may be transported, stored, and / or utilized in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single-dose or multi-dose preservative-free glass vial or syringe) containing 0.45 mL of frozen liquid (e.g., containing 5 doses). In certain embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained, transported, stored, and / or utilized may be maintained below room temperature, such as at 4°C or below, 0°C or below, -20°C or below, -60°C or below, -70°C or below, -80°C or below, -90°C or below, etc. In certain embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., viruses or syringes) in which they are contained, transported, stored, and / or utilized can be maintained at a temperature of -80°C to -60°C and, in certain embodiments, are protected from light.In certain embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained, transported, stored, and / or utilized can be maintained at a temperature of less than about 25° C. and, in certain embodiments, protected from light. In certain embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained, transported, stored, and / or utilized can be maintained at a temperature of less than about 5° C. (e.g., less than about 4° C.) and, in certain embodiments, protected from light. In certain embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained, transported, stored, and / or utilized can be maintained at a temperature of about −20° C. and, in certain embodiments, protected from light. In certain embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained, transported, stored, and / or utilized can be maintained at a temperature of about -60°C or higher (e.g., in certain embodiments, about -20°C or higher, and in certain embodiments, about 4-5°C or higher, in either case optionally below about 25°C), and in certain embodiments are protected from light, or conversely, no active steps (e.g., cooling measures) are taken to achieve a storage temperature substantially below about -20°C.

[0235] In some embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained are transported, stored, and / or used with and / or under conditions of heat-resistant materials or containers and / or temperature-regulating materials. For example, in some embodiments, the RNA (e.g., mRNA) compositions described herein and / or the containers (e.g., vials or syringes) in which they are contained are transported, stored, and / or used with ice and / or dry ice and / or cold packs. In certain embodiments, the containers (e.g., vials or syringes) in which the RNA (e.g., mRNA) compositions are contained are placed in trays or other maintenance devices, and are further in contact with (or are otherwise in the presence of) temperature-regulating (e.g., ice and / or dry ice) materials and / or cold packs. In certain embodiments, multiple containers (e.g., multiple vials or syringes, e.g., single-use or multi-use vials or syringes described herein) containing the provided RNA (e.g., mRNA) compositions are co-present (e.g., on a common tray, rack, box, etc.) and packaged together (or otherwise in the presence of) temperature-regulating (e.g., ice and / or dry ice) and / or cold packs. By way of example only, in certain embodiments, multiple containers (e.g., multiple vials or syringes, e.g., single-use or multi-use vials or syringes described herein) containing the RNA (e.g., mRNA) compositions are placed in a common tray or rack, and multiple such trays or racks are arranged in a bin surrounded by temperature-regulating (e.g., dry ice) in an insulated (e.g., thermally insulated) shipper. In some embodiments, the temperature-modulating material is replenished periodically (e.g., within 24 hours of arrival at the site and / or every 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, etc.). Preferably, reintroduction to the insulated shipper is infrequent, desirably no more than twice a day.In some embodiments, the insulated shipper must be reclosed within 5, 4, 3, 2, or 1 minute or less after being opened. In some embodiments, a provided RNA (e.g., mRNA) composition stored in an insulated shipper for a period of time, optionally within a particular temperature range, remains useful. For example, in some embodiments, if an insulated shipper described herein containing a provided RNA (e.g., mRNA) composition is at or maintained (e.g., stored) at a temperature within a range of about 15°C to about 25°C, the RNA (e.g., mRNA) composition can be used for up to 10 days; i.e., in some embodiments, a provided RNA (e.g., mRNA) composition maintained in an insulated shipper at a temperature within a range of about 15°C to about 25°C for a period of not more than 10 days is administered to a subject. Alternatively or additionally, in certain embodiments, the provided RNA (e.g., mRNA) compositions can be used for up to 10 days if they are in or maintained (e.g., stored) in an insulated shipper maintained at a temperature within the range of about 15°C to about 25°C; i.e., in certain embodiments, the provided RNA (e.g., mRNA) compositions maintained in an insulated shipper maintained at a temperature within the range of about 15°C to about 25°C for a period not exceeding 10 days are administered to a subject.

[0236] In certain embodiments, the provided RNA (e.g., mRNA) compositions are shipped and / or stored in a frozen state. In certain embodiments, the provided RNA (e.g., mRNA) compositions are shipped and / or stored as a frozen suspension, which, in certain embodiments, does not contain a preservative. In certain embodiments, the frozen RNA (e.g., mRNA) composition is thawed. In certain embodiments, the thawed RNA (e.g., mRNA) composition (e.g., suspension) may comprise white to off-white opaque amorphous particles. In certain embodiments, the thawed RNA (e.g., mRNA) composition can be used up to several days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days) after thawing, provided that it is maintained (e.g., stored) at a temperature below room temperature (e.g., about 30°C, 25°C, 20°C, 15°C, 10°C, 8°C, less than 4°C, etc.). In certain embodiments, the thawed RNA (e.g., mRNA) composition can be used after storage (e.g., several days) at a temperature of about 2°C to about 8°C; alternatively or additionally, the thawed RNA (e.g., mRNA) composition can be used after several hours (e.g., 1 hour, The composition may be used within 2, 3, 4, 5, or 6 hours (e.g., 2, 3, 4, 5, or 6 hours). Thus, in certain embodiments, a provided RNA (e.g., mRNA) composition is administered to a subject after thawing and maintaining a temperature below room temperature, and in certain embodiments, at about 2°C to about 8°C, for a period not exceeding 6, 5, 4, 3, 2, or 1 day. Alternatively or additionally, in certain embodiments, a provided RNA (e.g., mRNA) composition is administered to a subject after thawing and maintaining a temperature at room temperature for not exceeding 6, 5, 4, 3, 2, or 1 hour. In certain embodiments, a provided RNA (e.g., mRNA) composition is transported and / or stored in a concentrated state. In certain embodiments, such concentrated compositions are diluted prior to administration. In certain embodiments, a diluted composition is administered within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour after dilution; in certain embodiments, such administration is within 6 hours after dilution.Thus, in certain embodiments, diluted preparations of provided RNA (e.g., mRNA) compositions are administered to a subject within 6 hours after dilution (e.g., after being maintained at an appropriate temperature, as described herein, e.g., below room temperature, 4°C or below, 0°C or below, -20°C or below, -60°C or below, -70°C or below, -80°C or below, etc., and generally at about 2°C or above, e.g., about 2°C to about 8°C or about 2°C to about 25°C). In certain embodiments, unused composition is discarded within several hours (e.g., about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, or less) after dilution; in certain embodiments, unused composition is discarded within 6 hours of dilution.

[0237] In certain embodiments, an RNA (e.g., mRNA) composition (e.g., frozen composition, liquid concentrated composition, diluted liquid composition, etc.) to be stored, transported, or utilized is maintained at a temperature substantially above −60° C. for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, or for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more, or for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more; in certain such embodiments, such compositions can be maintained at a temperature above about −20° C. for such periods and / or at a temperature up to about 4-5° C. for such periods and / or can be maintained at a temperature above about 4-5° C., and optionally about 25° C., for a period of less than two (2) months and / or optionally up to about one (1) month. In certain embodiments, such compositions cannot be stored, transported, or used (or otherwise exposed to) temperatures substantially above about 4-5° C., particularly at or about 25° C., for a period of about 2 weeks, or in some embodiments, 1 week. In certain embodiments, such compositions cannot be stored, transported, or used (or otherwise exposed to) temperatures substantially above about −20° C., particularly at or about 4-5° C., for a period of about 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or, in some embodiments, for a period of about 8 weeks or 6 weeks, or substantially more than about 2 months, or, in some embodiments, 3 months, or, in some embodiments, more than 4 months.

[0238] In certain embodiments, RNA (e.g., mRNA) compositions (e.g., frozen compositions, liquid concentrated compositions, diluted liquid compositions, etc.) stored, transported, or used can be protected from light. In certain embodiments, one or more steps can be taken to reduce or minimize exposure of such compositions to light (e.g., they can be placed in a container such as a vial or syringe). In certain embodiments, exposure to direct sunlight and / or UV light is avoided. In certain embodiments, diluted solutions can be handled and / or used under normal room lighting conditions (e.g., without taking specific steps to minimize or reduce exposure to room lighting). It should be understood that strict adherence to aseptic technique is desirable during handling (e.g., dilution and / or administration) of the RNA (e.g., mRNA) compositions described herein. In certain embodiments, the RNA (e.g., mRNA) compositions described herein are not administered intravenously (e.g., injected). In certain embodiments, the RNA (e.g., mRNA) compositions described herein are not administered intradermally (e.g., injected). In certain embodiments, the RNA (e.g., mRNA) compositions described herein are not administered subcutaneously (e.g., injected). In certain embodiments, the RNA (e.g., mRNA) compositions described herein are not administered (e.g., injected) intravenously, intradermally, or subcutaneously. In certain embodiments, the RNA (e.g., mRNA) compositions described herein are not administered to subjects known to be hypersensitive to any of their components. In certain embodiments, the subject to whom the RNA (e.g., mRNA) composition is administered is monitored for one or more signs of anaphylaxis. In certain embodiments, the subject to whom the RNA (e.g., mRNA) composition is administered has previously received at least one dose of a different vaccine against SARS-CoV-2; in certain embodiments, the subject to whom the RNA (e.g., mRNA) composition is administered has not previously received a different vaccine against SARS-CoV-2. In certain embodiments, the subject's temperature is taken immediately prior to administration of the RNA (e.g., mRNA) composition (e.g., immediately before or after thawing, diluting, and / or administering such a composition); in certain embodiments, administration is delayed or canceled if such a subject develops a fever.In some embodiments, the RNA (e.g., mRNA) compositions described herein are not administered to subjects receiving anticoagulant therapy or who are suffering from or suspected of having a bleeding disorder or a condition for which intramuscular injection is contraindicated. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered by a medical professional whom the subject contacts to obtain composition information regarding side effects and risks. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered by a medical professional who has agreed to provide adverse event reporting for any serious adverse events, which may include, for example, one or more of the following: occurrence of death, disability, or congenital anomaly / birth defect (e.g., in the subject's child), inpatient hospitalization (including an extension of a current hospitalization), a life-threatening event, medical or surgical intervention to prevent death, a persistent or significant or substantial disruption of the ability to perform normal life functions; or other significant medical events that may endanger the individual and may require medical or surgical intervention (treatment) to prevent one of the other consequences.

[0239] In certain embodiments, provided RNA compositions are administered to a population of individuals under the age of 18, or under the age of 17, or under the age of 16, or under the age of 15, or under the age of 14, or under the age of 13, e.g., by a regimen that has been established to result in an incidence of one or more local reaction events that does not exceed the incidence set forth below: · Pain at the injection site (75% after the first and / or second dose and / or low incidence after the second dose, e.g., 65% after the second dose); injection site redness (less than 5% after the first and / or second dose); and / or · Swelling at the injection site (less than 5% after the first and / or second dose).

[0240] In certain embodiments, provided RNA compositions are administered to a population of individuals under the age of 18, or under the age of 17, or under the age of 16, or under the age of 15, or under the age of 14, or under the age of 13, e.g., by a regimen that has been established to result in an incidence of one or more systemic reaction events that does not exceed the incidence set forth below: fatigue (55% after the first and / or second dose); Headache (50% after the first and / or second dose); · Myalgia (40% after the first and / or second dose); · Chills (40% after the first and / or second dose); · arthralgia (20% after the first and / or second dose); · Fever (25% after the first and / or second dose); Vomiting (10% after the first and / or second dose); and / or Diarrhea (10% after the first and / or second dose).

[0241] In certain embodiments, a medication that alleviates one or more symptoms (e.g., as described herein) of one or more local and / or systemic reaction events is administered to an individual under the age of 18, or under 17, or under 16, or under 15, or under 14, or under 13 who has been administered a provided RNA composition and experienced one or more local and / or systemic reaction events (e.g., as described herein). In certain embodiments, antipyretic and / or pain medication may be administered to such individuals. [Brief explanation of the drawings]

[0242] [Figure 1] Schematic representation of the SARS-CoV-2 S protein. The S1 subunit sequence consists of a signal sequence (SS) and a receptor-binding domain (RBD), which is an important subunit within the S protein involved in binding to the human cell receptor ACE2. The S2 subunit contains the S2 protease cleavage site (S2'), a fusion peptide (FP) for subsequent membrane fusion, heptad repeat regions (HR1 and HR2) with a central helix (CH) domain, a transmembrane domain (TM), and a cytoplasmic tail (CT). [Figure 2]Predictive constructs for SARS-CoV-2 vaccine development. Based on the complete and wild-type S protein, different constructs were designed encoding (1) the complete protein with mutations in the first seventh repeat (HRP1) containing stabilizing mutations that preserve the neutralization-sensitive site, (2) the S1 domain, or (3) only the RB domain (RBD). Additionally, a fibritin domain (F) was fused to the C-terminus to stabilize the protein fragment. All constructs began with a signal peptide (SP) to ensure Golgi transport to the plasma membrane. [Figure 3] Antibody immune responses to influenza HA using LNP-formulated modRNA. BALB / c mice were immunized twice with 1 μg of vaccine candidate. Total viral antigen-specific immunoglobulin G (IgG) was measured by ELISA. Antibody functionality was assessed by VNT. [Figure 4] T cell responses to influenza HA using an LNP-formulated modRNA platform. BALB / c mice were immunized twice IM with 1 μg of vaccine candidate. T cell responses were analyzed using antigen-specific peptides to stimulate T cells harvested from the spleen. IFNγ release was measured using an ELISpot assay after peptide stimulation. [Figure 5] Anti-S protein IgG responses 7, 14, 21, and 28 days after immunization with BNT162a1. BALB / c mice were immunized IM once with 1 μg, 5 μg, or 10 μg of LNP-formulated RBL063.3. At 7, 14, 21, and 28 days post-immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG), as measured by ELISA. Values ​​for 1:100 serum dilutions were included on the graph for days 7, 14, 21, and 28. Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for group included). [Figure 6]Anti-S protein IgG responses 7, 14, 21, and 28 days after immunization with BNT162b1. BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBP020.3. At 7, 14, 21, and 28 days post-immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions were included on the graph for days 7 (1:100), 14 (1:300), 21 (1:900), and 28 (1:2700). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for group is included). [Figure 7] Neutralization of SARS-CoV-2 pseudovirus 14, 21, and 28 days after immunization with SARS-CoV-2 pseudovirus. BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBP020.3. 14, 21, and 28 days after immunization, animals were bled and serum was tested for SARS-CoV-2 pseudovirus neutralization. Graph shows pVN50 serum dilution (50% reduction in infection events compared to the no serum positive control). Each point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean + SEM is shown by the horizontal bar with whiskers for each group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 8]Anti-S protein IgG responses 7, 14, and 21 days after immunization with BNT162c1. BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBS004.3. Seven, 14, and 21 days after immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions were included on the graph for days 7 (1:100), 14 (1:300), and 21 (1:900). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for group is included). [Figure 9] Neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with BNT162c1. BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBS004.3. 14 and 21 days after immunization, animals were bled and serum was tested for SARS-CoV-2 pseudovirus neutralization. Graph shows pVN50 serum dilution (50% reduction in infection events compared to the no serum positive control). One point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean + SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 10] Anti-S protein IgG responses 7, 14, 21, and 28 days after immunization with LNP-formulated RBL063.1. BALB / c mice were immunized IM once with 1 μg, 5 μg, or 10 μg of LNP-formulated RBL063.1. At 7, 14, 21, and 28 days after immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) levels, as measured by ELISA. Different serum dilutions were included on the graph for days 7 (1:100), 14 (1:100), 21 (1:300), and 28 (1:900). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for the group is included). [Figure 11] Neutralization of SARS-CoV-2 pseudovirus 14, 21, and 28 days after immunization with SARS-CoV-2 pseudovirus. BALB / c mice were immunized IM once with 1 μg, 5 μg, or 10 μg of LNP-formulated RBL063.1. 14, 21, and 28 days after immunization, animals were bled and serum was tested for SARS-CoV-2 pseudovirus neutralization. Graph shows pVN50 serum dilution (50% reduction in infection events compared to the no serum positive control). Each point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean + SEM is shown by the horizontal bar with whiskers for each group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 12] Anti-S protein IgG responses 7, 14, and 21 days after immunization with BNT162b2 (LNP-formulated RBP020.1). BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated tedRBP020.1. Seven, 14, and 21 days after immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) levels, as measured by ELISA. Different serum dilutions were included on the graph for days 7 (1:100), 14 (1:300), and 21 (1:1100). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for the group is included). [Figure 13]Neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with BNT162b2 (LNP-formulated RBP020.1). BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBP020.1. 14 and 21 days after immunization, animals were bled and serum was tested for SARS-CoV-2 pseudovirus neutralization. Graph shows pVN50 serum dilution (50% reduction in infection events compared to the no serum positive control). Each point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean + SEM is shown by the horizontal bar with whiskers for each group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 14] Anti-S protein IgG responses 7, 14, and 21 days after immunization with LNP-formulated RBS004.2. BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBS004.2. Seven, 14, and 21 days after immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) levels, as measured by ELISA. Different serum dilutions were included on the graph for days 7 (1:100), 14 (1:300), and 21 (1:900). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for the group is included). [Figure 15] Neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with LNP-formulated RBS004.2. BALB / c mice were immunized IM once with 0.2 μg, 1 μg, or 5 μg of LNP-formulated RBS004.2. 14 and 21 days after immunization, animals were bled and serum was tested for SARS-CoV-2 pseudovirus neutralization. Graph shows pVN50 serum dilution (50% reduction in infection events compared to the no serum positive control). One point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean + SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 16]ALC-0315 activity during the screening process. [Figure 17] After intramuscular injection of wild-type (WT) or ApoE knockout (ApoE) C57Bl / 6 mice with or without ApoE3, luciferase expression was monitored on the right (injection site), dorsal (injection site), and abdominal (liver drainage) sides of the animals. Luciferase expression was detected using XenoLight D-Luciferin Rediject at 4, 24, 72, and 96 hours after injection. [Figure 18] Luciferase activity after intravenous (IV) and intramuscular (IM) administration of ApoE3 in wild-type (WT) or ApoE knockout (ApoE) C57Bl / 6 mice with (KO+) or without (KO) ApoE3. Luciferase expression was detected 4 hours after administration using XenoLight D-Luciferin Rediject. [Figure 19] General structure of RNA. Schematic representation of the general structure of an RNA vaccine with 5' cap, 5' and 3' untranslated regions, coding sequence with endogenous secretory signal peptide and GS linker, and poly(A) tail. Note that individual elements are not listed with exact lengths for each sequence length. UTR = untranslated region; sec = secretory signal peptide; RBD = receptor binding domain; GS = glycine-serine linker. [Figure 20] General structure of RNA. Schematic representation of the general structure of drug substance RNA with 5' cap, 5' and 3' untranslated regions, coding sequence with endogenous secretory signal peptide and GS linker, and poly(A) tail. Note that individual elements are not listed with exact lengths for each sequence length. GS = glycine-serine linker; UTR = untranslated region; Sec = secretory signal peptide; RBD = receptor binding domain. [Figure 21]General structure of RNA. Schematic representation of the general structure of an RNA vaccine: 5' cap, 5' and 3' untranslated regions, Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicase, and coding sequence for SARS-CoV-2 antigens with endogenous secretory signal peptide and GS linker, and poly(A) tail. Note that individual elements are not listed with exact lengths for each sequence length. UTR = untranslated region; Sec = secretory signal peptide; RBD = receptor-binding domain; GS = glycine-serine linker. [Figure 22] ELISpot analysis 28 days after immunization with BNT162b1. BALB / c mice were immunized IM once with 1 μg of LNP-formulated RBP020.3. 28 days after immunization, mice were sacrificed and splenocytes were prepared. ELISpot assays were performed using MACS-sorted CD4+ and CD8+ T cells. T cells were stimulated with S protein- or RBD-specific overlapping peptide pools, and IFN-γ secretion was measured to assess T cell responses. Each point on the graph represents an individual spot count from one mouse, and all mouse samples were measured in duplicate (group size n=8; group averages are included). [Figure 23] Cytokine concentrations in restimulated splenocyte supernatants 12 days after immunization with BNT162b1. BALB / c mice were immunized IM once with 5 μg of LNP-formulated RBP020.3. 12 days after immunization, mice were sacrificed. Splenocytes were prepared and stimulated with an S protein-specific overlapping peptide pool. 48 hours after stimulation, supernatants were collected and cytokine concentrations were determined. Each point on the graph represents the individual cytokine concentration of one mouse, and all mouse samples were measured in duplicate (group size n=8; group means are included). [Figure 24]T cell immunophenotyping in PBMCs 7 days after immunization with BNT162b1. BALB / c mice were immunized IM once with 5 μg of LNP-formulated RBP020.3. Seven days after immunization, mice were bled. Flow cytometry analysis of PBMCs was performed for T cells. T cells were defined as viable CD3+CD4+ and CD3+CD8+ T cells. Additional phenotyping markers are included in the figure. Tfh cells were gated from CD4+ T cells and defined as CD4+T-bet-GATA3-CD44+CD62L-PD-1+CXCR5+ cells. Each point on the graph represents an individual cell fraction from one mouse (group size n=8; group means are included). [Figure 25] B cell immunophenotyping in draining lymph nodes 12 days after immunization with BNT162b1. BALB / c mice were immunized IM once with 5 μg of LNP-formulated RBP020.3. 12 days after immunization, mice were sacrificed. Flow cytometry analysis of lymphocytes was performed on B cells. Activated B cells were gated within single, viable lymphocytes and defined as IgD-Dump (CD4, CD8, F4 / 80, GR-1)- cells. Plasma cells were defined as CD138+B220low / - cells. Switched B cells were gated from non-plasma cells and defined as CD19+CD138-IgM-. Germinal center (GC) B cells were gated from switched B cells and defined as CD19+IgM-CD38-CD95+ cells, gated for IgG1 and IgG2a. One point on the graph represents an individual cell fraction from one mouse (group size n=8; mean for group is included). [Figure 26]ELISpot analysis 28 days after immunization with LNP-formulated modRNA RBP020.1. BALB / c mice were immunized IM once with 5 μg of LNP-formulated RBP020.1. 28 days after immunization, mice were sacrificed and splenocytes were prepared. ELISpot assays were performed using MACS-sorted CD4+ and CD8+ T cells. T cells were stimulated with an S protein-specific overlapping peptide pool, and IFN-γ secretion was measured to assess T cell responses. Each point on the graph represents an individual spot count from one mouse, and all mouse samples were measured in duplicate (group size n=8; group averages are included). [Figure 27] Cytokine concentrations in restimulated splenocyte supernatants 28 days after immunization with LNP-formulated modRNA RBP020.1. BALB / c mice were immunized IM once with 5 μg of LNP-formulated RBP020.1. 28 days after immunization, mice were sacrificed. Splenocytes were prepared and stimulated with an S protein-specific overlapping peptide pool. 48 hours after stimulation, supernatants were collected and cytokine concentrations were determined. Each point on the graph represents the individual cytokine concentration of one mouse, and all mouse samples were measured in duplicate (group size n=8; group means are included). [Figure 28] ELISpot analysis 28 days after immunization with LNP-formulated saRNA RBS004.2. BALB / c mice were immunized IM once with 5 μg of LNP-formulated RBS004.2. 28 days after immunization, mice were sacrificed and splenocytes were prepared. ELISpot assays were performed using MACS-sorted CD4+ and CD8+ T cells. T cells were stimulated with an S protein-specific overlapping peptide pool, and IFN-γ secretion was measured to assess T cell responses. Each point on the graph represents an individual spot count from one mouse, and all mouse samples were measured in duplicate (group size n = 8; group averages are included). [Figure 29]Cytokine concentrations in restimulated splenocyte supernatants 28 days after immunization with LNP-formulated saRNA RBS004.2. BALB / c mice were immunized IM once with 1 μg of LNP-formulated RBS004.2. 28 days after immunization, mice were sacrificed. Splenocytes were prepared and stimulated with an S protein-specific overlapping peptide pool. 48 hours after stimulation, supernatants were collected and cytokine concentrations were determined. Each point on the graph represents the individual cytokine concentration of one mouse, and all mouse samples were measured in duplicate (group size n=8; group averages are included). [Figure 30] Schematic overview of the S protein organization of the SARS-CoV-2 S protein and novel constructs for SARS-CoV-2 vaccine development. Based on the wild-type S protein, two different transmembrane-anchored RBD-based vaccine constructs were designed, encoding RBD fragments fused to the T4 fibritin trimerization domain (F) and the spontaneous transmembrane domain (TM). Construct (1) starts with the SARS-CoV-2-S signal peptide (SP; AA1-19 of the S protein), while construct (2) starts with the human Ig heavy chain signal peptide (huSec) to ensure Golgi transport to the plasma membrane. [Figure 31] Anti-S protein IgG responses 6, 14, and 21 days after immunization with LNP-C12-formulated modRNA encoding a transmembrane-anchored RBD-based vaccine construct. BALB / c mice were immunized IM once with 4 μg of LNP-C12-formulated transmembrane-anchored RBD-based vaccine construct (surrogate for BNT162b3c / BNT162b3d). At 6, 14, and 21 days post-immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions were included on the graph for days 6 (1:50), 14 (1:300), and 21 (1:900). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n = 8; mean + SEM for group is included). [Figure 32]Neutralization of SARS-CoV-2 pseudovirus 6, 14, and 21 days after immunization with LNP-C12-formulated modRNA encoding a transmembrane-anchored RBD-based vaccine construct. BALB / c mice were immunized IM once with 4 μg of LNP-C12-formulated transmembrane-anchored RBD-based vaccine construct (surrogate for BNT162b3c / BNT162b3d). Six, 14, and 21 days after immunization, animals were bled and serum was tested for SARS-CoV-2 pseudovirus neutralization. Graph shows pVN50 serum dilution (50% reduction in infection events compared to the no-serum positive control). Each point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean + SEM is shown by the horizontal bar with whiskers for each group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 33] Immunogenicity of BNT162b1 in rhesus macaques and comparison with human convalescent-phase sera. Rhesus macaques were immunized IM with 30 μg or 100 μg of BNT162b1 or placebo (0.9% NaCl) on days 0 and 21. Serum was obtained pre-immunization and 14, 21, 28, and 35 days post-immunization; PBMCs were obtained pre-immunization and 14 and 42 days post-immunization. Serum from COVID-19 patients was obtained 20–40 days after onset of symptoms and at least 14 days after asymptomatic convalescence. a) Geometric mean concentrations of IgG binding to the recombinant S1 protease fragment of SARS-CoV-2 S in rhesus macaque sera (n = 6 / group; all time points for the placebo group are listed under 'Control') and human convalescent-phase sera (n = 62) drawn at the indicated times post-immunization. b, SARS-CoV-2 geometric mean 50% neutralization titers of rhesus sera (n = 6 / group; all measurement time points for the placebo group are shown under 'Control') and human convalescent sera (n = 38). P values ​​were determined by two-sided one-way ANOVA and Dunnett's multiple comparison test. c, Flow cytometry analysis of CD4+ T cells producing IFNγ, IL-2, TNF (TH1), IL-21, or IL-4 (TH2) cytokines in rhesus PBMCs on day 42. P values ​​were determined by two-sided Kruskal-Wallis test followed by Dunn's multiple comparison test. Each data point corresponds to an individual animal. [Figure 34]Overview of the clinical trial population [Figure 35] Local reactions reported within 7 days of vaccination at all dose levels. Solicited injection site (local) reactions were injection site pain (mild = not interfering with activity; moderate = interfering with activity; severe = prevented from daily activities; grade 4 = emergency room visit or hospitalization) and redness and swelling (mild = 2.5-5.0 cm in diameter; moderate = 5.5-10.0 cm in diameter; severe = >10.0 cm in diameter; grade 4 = necrotic for redness or exfoliative dermatitis and necrotic for swelling). Data were collected 14 days after each vaccination using an electronic diary. [Figure 36] a: Systemic events reported within 7 days after vaccination 1: all dose levels; b: Systemic events reported within 7 days after vaccination 2: 10 μg and 30 μg dose levels. Responsive systemic events were nausea / vomiting (mild = not interfering with activity or 1-2 times in 24 hours; moderate = somewhat interfering with activity or >2 times in 24 hours; severe = prevented daily activities or required intravenous fluids; Grade 4 = emergency room visit or hospitalization for hypotensive shock), diarrhea (mild, 2-3 loose stools in 24 hours; moderate, 4-5 loose stools in 24 hours; severe, ≥6 loose stools in 24 hours; Grade 4 = emergency room visit or hospitalization), headache (mild = not interfering with activity; moderate = >repeated use of non-narcotic analgesics or somewhat interfering with activity in 24 hours; severe = significant, prevented any use of narcotic analgesics or prevented daily activities; Grade 4 = emergency room visit or hospitalization), fatigue / fatigue (mild = not interfering with activity; moderate = prevented daily activities). Grade 4 = emergency room visit or hospitalization), muscle pain (pain occurring in an area other than the injection site; mild = does not interfere with activities; moderate = some interference with activities; severe = noticeable; interference with daily activities; Grade 4 = emergency room visit or hospitalization), joint pain (mild = does not interfere with activities; moderate = some interference with activities; severe = noticeable; interference with daily activities; Grade 4 = emergency room visit or hospitalization), and fever (mild = 100.4°F to 101.1°F [38.0°C to 38.4°C]; moderate = 101.2°F to 102.0°F [38.5°C to 38.9°C]; severe = 102.1°F to 104.0°F [39.0°C to 40.0°C]; Grade 4 = >104.0°F [>40.0°C]). [Figure 37] Immunogenicity of BNT162b1 - RBD-binding IgG GMC and SARS-CoV-2 50% neutralization titers after one or two doses. Groups of 15 subjects were immunized with the indicated dose levels of BNT162b1 (n=12) or placebo (P, n=3) on day 1 (all dose levels and placebo) and day 21 (10 μg and 30 μg dose levels and placebo). Serum was obtained pre-immunization (day 1) and 7, 21, and 28 days after the first immunization. Human COVID-19 convalescent sera (HCS) (n=38) were obtained 20–40 days after symptom onset and at least 14 days after asymptomatic convalescence. a, GMC of recombinant RBD-binding IgG. Lower limit of quantification (LLOQ) 1.15 (dotted line). b, 50% SARS-CoV-2 neutralization GMT. Each data point represents one serum sample, and each vertical bar represents the geometric mean and 95% confidence interval. [Figure 38] BNT162b1 induces strong CD4 and CD8 T cell responses in humans. BNT162 induced T cell responses in 8 of 8 subjects tested ex vivo using the T cell:IFNγ ELISpot. Subjects were primed / boost vaccinated with 10 μg BNT162b1; CEF:CMV, EBV, and influenza CD8 T cell epitope mix; CEFT:CMV, EBV, influenza, and tetanus CD4 T cell epitope mix. [Figure 39]BNT162b1 induced IgG concentrations. Subjects were immunized with BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 60 μg) (n=12 / group, with n=11 for the 10 μg and 50 μg cohorts starting on day 22). Sera were obtained on days 1 (preprime), 8, 22 (preboost), 29, and 43. Predose responses were combined for all dose levels. Human COVID-19 convalescent sera (HCS, n=38) were obtained at least 14 days after PCR-confirmed diagnosis and when donors were no longer symptomatic. LLOQ / 2 values ​​were plotted for RBD-binding IgG concentrations below the lower limit of quantitation (LLOQ=1.15). Arrowheads indicate vaccination. Checkered bars indicate no booster immunization was administered. Values ​​above the bars are geometric means and 95% confidence intervals. At the time of submission, day 43 data were withheld for 5 subjects in the 50 μg cohort and all subjects in the 60 μg cohort. [Figure 40] BNT162b1-induced virus neutralization titers. Vaccination schedule and serum sampling are the same as in Figure 39. a, SARS-CoV-2 50% neutralization titers (VNT50) in immunized subjects and COVID-19 convalescent patients (HCS). For values ​​below the lower limit of quantification (LLOQ) = 20, the LLOQ / 2 value is plotted. Arrowheads indicate the day of immunization. Checkered bars indicate that no boost immunization was administered. Geometric means (values ​​above the bars) and 95% confidence intervals. At the time of submission, day 43 data were not yet available for five subjects in the 50 μg cohort and all subjects in the 60 μg cohort. b, Correlation between RBD-binding IgG geometric mean concentrations (GMC) (as in Figure 39) and day 29 VNT50 (all evaluable subject sera). Nonparametric Spearman correlation. c, Pseudovirus 50% neutralization titers (pVNT50) across a pseudovirus panel representing 17 SARS-CoV-2 spike protein variants, including 16 RBD mutants and the dominant spike protein variant D614G (dose levels 10 μg, 30 μg, and 50 μg, n = 1–2 each; day 29). Lower limit of quantification (LLOQ) = 40. Geometric mean. [Figure 41]Frequency and magnitude of BNT162b1-induced CD4+ and CD8+ T cell responses. The vaccination schedule is as shown in Figure 39. PBMCs obtained on days 1 (Pre) and 29 (Post, 7 days post-boost) (1 μg and 50 μg, n = 8 each; 10 μg and 30 μg, n = 10 each) were enriched for CD4+ or CD8+ T cell effectors and separately stimulated overnight with overlapping peptide pools representing the vaccine-encoded RBD for direct ex vivo IFNγ ELISpot assessment. The common pathogen T cell epitope pools CEF (CMV, EBV, and influenza virus HLA class I epitopes) and CEFT (CMV, EBV, influenza virus, and tetanus toxin HLA class II epitopes) served to assess general T cell reactivity, and medium served as a negative control. Each dot represents the normalized mean spot count from duplicate wells of one subject after subtraction from the medium-only control. a, Percentages above post-vaccination data points represent the number of subjects with detectable CD4+ or CD8+ T cell responses within the total number of subjects tested per dose cohort. b, Exemplary CD4+ and CD8+ ELISpot for 10μg cohort subjects. c, RBD-specific CD4+ and CD8+ T cell responses in all prime / boost vaccinated subjects and their baseline CEFT- and CEF-specific T cell responses. d, Correlation between VNT50 (as in Figure 40a) and CD4+ T cell responses (as in Figure 41) for dose cohorts 10μg to 50μg (1μg and 50μg, n=8 each; 10μg and 30μg, n=10 each). Non-parametric Spearman correlation. [Figure 42]Cytokine polarization of BNT162b1-induced T cells. Vaccination schedule and PBMC sampling are as in Figure 41. PBMCs from vaccinated and COVID-19-recovered donors (HCS n=6; in (c)) were stimulated overnight with overlapping peptide pools representing the vaccine-encoded RBD and assessed by flow cytometry (a-c) and bead-based immunoassay (d). a, Exemplary pseudocolor flow cytometry plots of cytokine-producing CD4+ and CD8+ T cells from 10 μg cohort subjects. b, Cytokine-producing RBD-specific CD4+ T cells shown as a fraction of total cytokine-producing RBD-specific CD4+ T cells, and c, cytokine-producing RBD-specific CD8+ (left) or CD4+ (right) T cells shown as a fraction of total circulating T cells from the same subsets. One CD4 non-responder (<0.02% total cytokine-producing T cells) and one CD8 non-responder (<0.01% total cytokine-producing T cells) from the 30 μg cohort were excluded in (b). Values ​​above data points are the average fraction across all dose cohorts. d, PBMCs from the 50 μg cohort. Each dot represents the average from duplicate wells subtracted from the DMSO control for one subject. The lower limits of quantitation (LLOQ) were 6.3 pg / mL for TNF, 2.5 pg / mL for IL-1β, and 7.6 pg / mL for IL-12p70. Mean (b). [Figure 43] Vaccination and evaluation schedule [Figure 44]Solicited adverse events. Subjects were immunized with BNT162b1 at the dose levels indicated on Day 1 (all dose levels) and Day 22 (all dose levels except 60 μg) (n=12 / group, n=11 for the 10 μg and 50 μg cohorts starting on Day 22). a, b, Number of subjects with local (a) or systemic reactions (b) by day (days 1-9, days 22-30) and cohort. Adverse events were graded according to FDA recommendations (US Department of Health and Human Services, Administration, F. and D. & Research, C. for BE and. Toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials. (2007). Available at https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / toxicity-grading-scale-healthy-adult-and-adolescent-volunteers-enrolled-preventive-vaccine-clinical). [Figure 45] Pharmacodynamic markers. Subjects were immunized with BNT162b1 at the dose levels indicated on day 1 (all dose levels) and day 22 (all dose levels except 60 μg). a, Kinetics of C-reactive protein (CRP) levels and b, kinetics of lymphocyte counts. Dotted lines indicate the upper and lower limits of the control range. For values ​​below the lower limit of quantitation (LLOQ=0.3), the LLOQ / 2 value was plotted (a). [Figure 46]Correlation of antibody and T cell responses. Subjects were immunized with BNT162b1 at the dose levels indicated on day 1 (all dose levels) and day 22 (all dose levels except 60 μg). a, Correlation of RBD-specific IgG responses (from Figure 39a) with CD4+ T cell responses on day 29. (1 μg and 50 μg, n=8 each; 10 μg and 30 μg, n=10 each). Non-parametric Spearman correlation. b, Correlation of CD4+ vs. CD8+ T cell responses on day 29 for dose cohorts 10 μg to 50 μg (as in Figure 41) (1 μg and 50 μg, n=8 each; 10 μg and 30 μg, n=10 each). Parametric Pearson correlation. c, Correlation between RBD-specific IgG responses (from Figure 39a) and CD8+ T cell responses (1 μg and 50 μg, n = 8 each; 10 μg and 30 μg, n = 10 each) on day 29. Non-parametric Spearman correlation. [Figure 47] Gating strategy for flow cytometry analysis of the data shown in Figure 42. Flow cytometry gating strategy for identifying IFNγ, IL-2, and IL-4-secreting T cells in clinical trial PBMC samples. a, CD4+ and CD8+ T cells were gated within single, viable lymphocytes. b, c, Gating for IFNγ, IL-2, and IL-4 in CD4+ T cells (b) and IFNγ and IL-2 in CD8+ T cells (c). [Figure 48] BNT162b1 18-55 years old: Local reactions after each administration [Figure 49] BNT162b1 18-55 years: Systemic events after each administration [Figure 50] BNT162b1 65-85 years: RBD-binding IgG GMC [Figure 51] BNT162b1 65-85 years: 50% SARS-CoV-2 neutralization GMT [Figure 52] BNT162b2 18-55 years old: Local reactions after each administration [Figure 53] BNT162b2 18-55 years: Systemic events after each administration [Figure 54] BNT162b2 65-85 years old: local reactions after each administration [Figure 55]BNT162b2 65-85 years: Systemic events after each administration [Figure 56] BNT162b2 18-55 years: S1-binding IgG GMC [Figure 57] BNT162b2 18-55 years: 50% SARS-CoV-2 neutralization GMT [Figure 58] BNT162b2 65-85 years: S1-binding IgG GMC [Figure 59] BNT162b2 65-85 years: 50% SARS-CoV-2 neutralization GMT [Figure 60] BNT162b2-induced T cell responses in mice. Splenocytes from BALB / c mice immunized IM with BNT162b2 or buffer were restimulated ex vivo with the full-length S peptide mix or negative control (irrelevant peptide in a, right; (a, left) and (c) no peptide). P values ​​were determined by two-tailed paired t-test. (a) IFNγ ELISpot (left) of splenocytes collected 12 days after immunization of mice (n = 8 / group) with 5 μg BNT162b2. IFNγ ELISpot (center and right) of isolated splenic CD4+ or CD8+ T cells 28 days after immunization of mice (n = 8 / group) with 1 μg BNT162b2. (b) CD8+ T cell-specific cytokine release by splenocytes from mice (n = 8 / group) immunized with 5 μg BNT162b2 or buffer (control), as determined by flow cytometry. S-peptide-specific responses are corrected for background (no peptide). (c) Cytokine production by splenocytes obtained 28 days after immunization of mice with 1 μg BNT162b2 (n=8 / group, n=7 for IL-4, IL-5, and IL-13, due to removal of one outlier by Rout's test [Q=1%] for S-peptide-stimulated samples) as determined by bead-based multiplex analysis. [Figure 61]IFNγ ELISpot data from five subjects vaccinated with 10 μg BNT162b2. Background-subtracted spot counts from duplicates at pre-vaccination (Pre) and day 29 (Post - 7 days after boost) per 10 cells. T cell response analysis was performed by a GCLP-compliant method using a validated ex vivo IFNγ ELISpot assay. All experiments were performed in duplicate and included negative and positive controls (media only and anti-CD3). In addition, peptide epitopes derived from cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus were used as positive controls. CD4- or CD8-depleted PBMCs were stimulated for 16–20 h with overlapping peptides covering the N- and C-terminal portions of the spike glycoprotein on precoated ELISpot plates (Mabtech). For analysis of ex vivo T cell responses, bound IFNγ was visualized with alkaline phosphatase-conjugated secondary antibodies. Plates were scanned using a Robot ELISPOT Reader and analyzed using ImmunoCapture V6.3 or AID ELISPOT 7.0 software. Spot counts were summarized as the average for each duplicate. T cell counts were calculated as the sum of the number of spots detected after stimulation with Spool 1 and Spool 2. Peptide-stimulated T cell responses were compared to effectors incubated with medium-only negative controls using the ELISpot data analysis tool (EDA) based on two statistical tests (distribution-independent resampling) according to Moody et al. (Moodie Z. et al., J Immunol Methods 315, 2006, 121-32; Moody Z. et al., Cancer Immunol Immunother 59, 2010, 1489-501), thus maintaining sensitivity while controlling for false positive rates. No significant changes were observed in pre- and day 29 T cell responses to positive control peptides from CMV, EBV, and influenza virus (not shown). [Figure 62]Example CD4+ and CD8+ IFNγ ELISpot Data. IFNγ ELISpot was performed as in Figure 61 using PBMCs obtained from subjects pre-immunization and 29 days after dose 1 (7 days after dose 2) of 10 μg BNT162b2. HLA class I and class II peptide pools CEF (cytomegalovirus [CMV], Epstein-Barr virus [EBV] (7 days after dose 2), and influenza virus, HLA class I epitope mix) and CEFT (CMV, EBV, influenza virus, and tetanus toxoid HLA class II epitope mix) were used as benchmark controls for assessment of CD8+ and CD4+ T cell reactivity. [Figure 63] Comparison of BNT162b2-induced and benchmark IFNγ ELISpot responses. IFNγ spot counts in PBMC samples on day 29 (7 days after dose 2) from five subjects immunized with 10 μg of BNT162b2 on days 1 and 22. CEF (CMV, EBV, and influenza virus HLA class I epitope mix) and CEFT (CMV, EBV, influenza virus, and tetanus toxin HLA class II cell epitope mix) were used as benchmark controls for assessment of CD8+ and CD4+ T cell reactivity. Horizontal lines indicate medians. [Figure 64] Immunogen design and characterization. a, Structure of BNT162b1. Line diagram of RNA (left) and schematic of LNP (right). UTR, untranslated region; SP, signal peptide. b, Representative 2D class average of electron micrograph of negatively stained RBD-Foldon trimer. Box edge: 37 nm. c, Density map of the ACE2 / B0AT1 / RBD-Foldon trimer complex at 3.24 Å after focused refinement of ACE2 extracellular domain binding to the RBD monomer. Surface coloring by subunit. A ribbon model refined against the density shows the RBD-ACE2 binding interface, labeling residues likely mediating polar interactions. [Figure 65]Mouse immunogenicity. a–c, BALB / c mice (n = 8 / group) were immunized intramuscularly (IM) with 0.2 μg, 1 μg, or 5 μg of BNT162b1 or buffer. Each group's geometric mean ± 95% CI; P values ​​are compared to non-immunized (0 μg; n = 8) baseline serum on day 28 (multiple comparisons in mixed-effects analysis using Dunnett's multiple comparison test) (a, c). a, RBD-binding IgG responses in serum obtained 7, 14, 21, and 28 days after immunization, as determined by ELISA. Prescreening of randomized animals was performed for day 0 (n = 4). b, Representative surface plasmon resonance sensorgrams (n = 8) of the binding kinetics of His-tagged RBD to immobilized mouse IgG from serum 28 days after immunization with 5 μg BNT162b1. Actual binding (green) and best fit of the data to a 1:1 binding model (black) are shown. c, VSV-SARS-CoV-2 pseudovirus 50% serum neutralization titer (pVNT50). d-f, Splenocytes from BALB / c mice immunized IM with BNT162b1 or buffer (control) were restimulated ex vivo with the full-length S peptide mix or negative control (no peptide (d, left) and (e, f); irrelevant peptide (d, right)). P values ​​were determined by a two-tailed paired t-test. d, IFNγ ELISpot of splenocytes collected 12 days after immunization of mice (n = 8 / group) with 5 μg BNT162b1 (left). IFNγ ELISpot of isolated splenic CD4+ T cells (n = 7, Grubbs' test with one outlier removed, α = 0.05) or CD8+ T cells (n = 8) 28 days after immunization with 1 μg BNT162b1 (center and right). e, T cell-specific cytokine release by splenocytes from mice immunized with 5 μg BNT162b1 (n=8 / group) as determined by flow cytometry. S peptide-specific responses are corrected for background (no peptide). f, Cytokine production by splenocytes obtained 28 days after immunization of mice (n=8 / group) with 0.2 μg BNT162b1 as determined by bead-based multiplex analysis. [Figure 66]Immunogenicity of BNT162b1 in rhesus macaques and comparison with human convalescent-phase sera. a, b, Male rhesus macaques aged 2–4 years (n = 6 / group) were immunized IM with 30 μg or 100 μg of BNT162b1 or buffer on days 0 and 21, and sera were obtained before immunization and on days 14, 21, 28, 35, and 42 after immunization. Human convalescent-phase sera (HCS) were obtained from SARS-CoV-2-infected patients (n = 38) at least 14 days after PCR-confirmed diagnosis and when acute COVID-19 symptoms had resolved. Values ​​above the bar indicate the geometric mean. a, Geometric mean concentration (GMC) of IgG binding to recombinant SARS-CoV-2 RBD. The dotted line indicates the geometric mean of sera across all time points for the placebo group (1.72 U / mL). Group IgG titers at all time points were analyzed for statistical significance relative to HCS samples using one-way ANOVA with Dunnett's multiple comparisons correction, and statistical significance was confirmed for the 30 μg dose level group (day 28, p < 0.0001; day 35, p = 0.0016) and the 100 μg dose level group (days 28, 35, and 42, all p < 0.0001). b, SARS-CoV-2 50% neutralization titer (VNT50). The dotted line indicates the geometric mean of sera at all time points for the placebo group (10.31 U / mL). Group VNT50 for all time points was analyzed for statistical significance relative to HCS samples using one-way ANOVA with Dunnett's multiple comparisons correction, and statistical significance was confirmed for the 30 μg dose level group (day 28, p<0.0001) and the 100 μg dose level group (days 28 and 35, both p<0.0001; day 42, p=0.007). [Figure 67]Viral RNA in non-immunized and immunized rhesus macaques after SARS-CoV-2 challenge. Rhesus macaques (n = 6 / group) were immunized with 100 μg BNT162b1 or buffer (control) on days 0 and 21, as shown in Figure 66. 41–48 days after the second immunization, animals were challenged with 1 × 10 total pfu of SARS-CoV-2, equally divided between the IN and IT routes. Three non-immunized, age-matched male rhesus macaques were challenged with cell culture medium (Sentinel). Viral RNA levels were detected by RT-qPCR. The ratios above the data points represent the number of viral RNA-positive animals among all animals in the group. a, Viral RNA in bronchoalveolar lavage fluid (BAL) obtained before challenge and on days 3 and 6 after challenge. On day 6, the difference in viral load between control and BNT162b1-immunized animals was statistically significant (p = 0.0131). b, Viral RNA in nasal swabs obtained pre-exposure and 1, 3, and 6 days after exposure. On day 3, the difference in viral load between control and BNT162b1-immunized animals was statistically significant (p=0.0229). The dotted line indicates the lower limit of detection (LLOD). Negative samples were set at 1 / 2 the LLOD. P values ​​were determined by categorical analysis of a binary response (undetectable viral load after exposure was considered a success and measurable viral load after exposure was considered a failure). [Figure 68] BNT162b1 and b2 V8 immunization reduces viral RNA in rhesus macaques after SARS-CoV-2 challenge; b2 exhibits early clearance from the nose [Figure 69] Exemplary Pandemic Supply Product Packaging Overview [Figure 70] Exemplary Vaccine Storage and Handling at the Time of Vaccination [Figure 71] Examples of multi-dose formulations [Figure 72] Geometric Mean Titers and 95% CI: SARS-CoV-2 Neutralization Assay - NT50 - Phase 1, 2-Dose, 21 Days Apart - 18-55 Years - BNT162b1 - Evaluable Immunogenicity Population [Figure 73]Geometric Mean Titers and 95% CI: SARS-CoV-2 Neutralization Assay - NT50 - Phase 1, 2-Dose, 21 Days Apart - 65-85 Years Old - BNT162b1 - Evaluable Immunogenicity Population [Figure 74] Geometric Mean Titers and 95% CI: SARS-CoV-2 Neutralization Assay - NT50 - Phase 1, 2-Dose, 21 Days Apart - 18-55 Years - BNT162b2 - Evaluable Immunogenicity Population [Figure 75] Geometric Mean Titers and 95% CI: SARS-CoV-2 Neutralization Assay - NT50 - Phase 1, 2-Dose, 21 Days Apart - 65-85 Years Old - BNT162b2 - Evaluable Immunogenicity Population [Figure 76] Geometric mean concentrations and 95% CI: SARS-CoV-2 RBD-binding IgG level assay - Phase 1, 2 doses, 21 days apart - 18-55 years - BNT162b1 - Evaluable immunogenicity population [Figure 77] Geometric mean concentrations and 95% CI: SARS-CoV-2 RBD-binding IgG level assay - Phase 1, 2 doses, 21 days apart - 65-85 years, BNT162b1 - Evaluable immunogenicity population [Figure 78] Geometric mean concentrations and 95% CI: SARS-CoV-2 S1-binding IgG level assay - Phase 1, 2 doses, 21 days apart - 18-55 years - BNT162b1 - Evaluable immunogenicity population [Figure 79] Geometric mean concentrations and 95% CI: SARS-CoV-2 S1-binding IgG level assay - Phase 1, 2-dose, 21-day interval - 65-85 years - BNT162b1 - Evaluable immunogenicity population [Figure 80] Geometric mean concentrations and 95% CI: SARS-CoV-2 S1-binding IgG level assay - Phase 1, 2 doses, 21 days apart - 18-55 years - BNT162b2 - Evaluable immunogenicity population [Figure 81]Geometric mean concentrations and 95% CI: SARS-CoV-2 S1-binding IgG level assay - Phase 1, 2-dose, 21-day interval - 65-85 years - BNT162b2 - Evaluable immunogenicity population [Figure 82] Geometric mean concentrations and 95% CI: SARS-CoV-2 RBD-binding IgG level assay - Phase 1, 2 doses, 21 days apart - 18-55 years - BNT162b2 - Evaluable immunogenicity population [Figure 83] Geometric mean concentrations and 95% CI: SARS-CoV-2 RBD-binding IgG level assay - Phase 1, 2 doses, 21 days apart - 65-85 years - BNT162b2 - Evaluable immunogenicity population [Figure 84] Subject-reported local reactions by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 18-55 - BNT162b1 - Safety population [Figure 85] Subject-reported local reactions by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 65-85 - BNT162b1 - Safety population [Figure 86] Subject-reported local reactions by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 18-55 - BNT162b2 - Safety population [Figure 87] Subject-reported local reactions by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 65-85 - BNT162b2 - Safety population [Figure 88] Subject-reported systemic events by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 18-55 - BNT162b1 - Safety population [Figure 89] Subject-reported systemic events by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 65-85 - BNT162b1 - Safety population [Figure 90]Subject-reported systemic events by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 18-55 - BNT162b2 - Safety population [Figure 91] Subject-reported systemic events by maximum severity within 7 days after each dose - Phase 1, 2 doses, 21 days apart - Ages 65-85 - BNT162b2 - Safety population [Figure 92] Subject-reported local reactions by maximum severity within 7 days after each dose, age group 18-55 years - Phase 2 - Safety population [Figure 93] Subject-reported local reactions by maximum severity within 7 days after each dose, age group 56-85 years - Phase 2 - Safety population [Figure 94] Subject-reported systemic events by maximum severity within 7 days after each dose, age group 18-55 years - Phase 2 - Safety population [Figure 95] Subject-reported systemic events by maximum severity within 7 days after each dose, age group 56-85 years - Phase 2 - Safety population [Figure 96] Subject-reported local reactions by maximum severity within 7 days after each dose, age group 18-55 years - Approximately 6,000 subjects in Phase 2 / 3 - Safety population [Figure 97] Subject-reported local reactions by maximum severity within 7 days after each dose, age group 56-85 years - Approximately 6,000 subjects in Phase 2 / 3 - Safety population [Figure 98] Subject-reported systemic events by maximum severity within 7 days after each dose, age group 18-55 years - Approximately 6,000 subjects in Phase 2 / 3 - Safety population [Figure 99] Subject-reported systemic events by maximum severity within 7 days after each dose, age group 56-85 years - Approximately 6,000 subjects in Phase 2 / 3 - Safety population [Figure 100] Cumulative incidence curve of first COVID-19 emergence after Dose 1 - Dose 1 all available efficacy population [Figure 101]BNT162b2 - Exemplary functional 50% SARS-CoV-2 neutralizing antibody titers (VN50). Young adults (18-55 years old) and older adults (56-85 years old) were immunized with BNT162b2 on days 1 and 22 (n=12 / group). Serum was obtained from young adults on days 1 (baseline), 8, 22 (pre-boost), 29, 43, 50, and 85. Serum was obtained from older adults on days 1 (baseline), 8, 22, and 29. Human COVID-19 convalescent serum (HSC, n=38) was obtained at least 14 days after diagnosis confirmation and when the donor was no longer symptomatic. SARS-CoV-2 50% neutralizing titers (VN50 titers) and 95% confidence intervals are shown for young adults immunized with 1 μg, 3 μg, 10 μg, 20 μg, or 30 μg BNT162b2 and older adults immunized with 20 μg BNT162b2. Values ​​below the limit of detection (LOD) are plotted as 0.5*LOD. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). Horizontal dotted lines represent LOD. VN50 = 50% SARS-CoV-2 neutralizing antibody titer; HCS = human COVID-19 convalescent sera. [Figure 102] BNT162b1 - Exemplary fold increase from baseline in functional 50% SARS-CoV-2 neutralizing antibody titers (VN50). Vaccination schedule and serum sampling are the same as in Figure 39 (n=12 / group). The geometric mean fold increase (GMFI) from baseline in VN50 titers and 95% confidence intervals are shown for young participants (18-55 years old) immunized with 1 μg, 10 μg, 30 μg, 50 μg, or 60 μg BNT162b1. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). Dose 2 was not administered in the 60 μg dose group. The horizontal dotted line represents the threshold for seroconversion (fold increase ≥ 4). VN50 = 50% SARS-CoV-2 neutralizing antibody titer. [Figure 103]BNT162b2 - Exemplary fold increase from baseline in functional 50% SARS-CoV-2 neutralizing antibody titers (VN50). Vaccination schedule and serum sampling are the same as in Figure 101. The geometric mean fold increase (GMFI) from baseline in VN50 titers and 95% confidence intervals are shown for (A) younger participants (18-55 years old) immunized with 1 μg, 3 μg, 10 μg, 20 μg, or 30 μg BNT162b2 and (B) older participants (56-85 years old) immunized with 20 μg BNT162b2. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). The horizontal dotted line represents the threshold for seroconversion (fold increase ≥ 4). VN50 = 50% SARS-CoV-2 neutralizing antibody titer. [Figure 104] Exemplary frequency of participants with SARS-CoV-2 GMT seroconversion after immunization with BNT162b1. Vaccination schedule and serum sampling are the same as in Figure 39 (n=12 / group). Seroconversion for 50% SARS-CoV-2 neutralizing antibody titers (VN50) is shown for young participants (18-55 years old) immunized with 1 μg, 10 μg, 30 μg, 50 μg, or 60 μg BNT162b1. Seroconversion is defined as a minimum 4-fold increase in functional antibody response compared to baseline. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). Dose 2 was not performed in the 60 μg dose group. GMT = geometric mean titer. [Figure 105] Exemplary frequency of participants with SARS-CoV-2 GMT seroconversion after immunization with BNT162b2. Vaccination schedule and serum sampling are the same as in Figure 101. Seroconversion in terms of 50% SARS-CoV-2 neutralizing antibody titers (VN50) is shown for (A) younger participants (18-55 years old) receiving 1 μg, 3 μg, 10 μg, 20 μg, or 30 μg BNT162b2 and (B) older participants (56-85 years old) receiving 20 μg BNT162b2. Seroconversion is defined as a minimum 4-fold increase in functional antibody response compared to baseline. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). GMT = geometric mean titer. [Figure 106]Exemplary fold increase from baseline in S1-binding antibody concentrations after immunization with BNT162b1. Vaccination schedule and serum sampling are the same as in Figure 39 (n=12 / group). The geometric mean fold increase (GMFI) from baseline in S1-binding antibody concentrations and 95% confidence intervals are shown for young participants (18-55 years old) immunized with 1 μg, 10 μg, 30 μg, 50 μg, or 60 μg BNT162b1. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). Dose 2 was not administered in the 60 μg dose group. The horizontal dotted line represents the threshold for seroconversion (fold increase ≧4). [Figure 107] Exemplary fold increase from baseline in S1-binding antibody concentrations after immunization with BNT162b2. Vaccination schedule and serum sampling are the same as in Figure 101. The geometric mean fold increase (GMFI) from baseline in S1-binding antibody concentrations and 95% confidence intervals are shown for (A) younger participants (18-55 years old) immunized with 1 μg, 3 μg, 10 μg, 20 μg, or 30 μg BNT162b2 and (B) older participants (56-85 years old) immunized with 20 μg BNT162b2. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). The horizontal dotted line represents the threshold for seroconversion (fold increase ≧4). [Figure 108] Exemplary frequency of participants who seroconverted to S1-binding IgG GMC after immunization with BNT162b1. Vaccination schedule and serum sampling were the same as in Figure 39 (n=12 / group). Seroconversion for S1-binding antibody GMC is shown for young participants (18-55 years old) immunized with 1 μg, 10 μg, 30 μg, 50 μg, or 60 μg BNT162b1. Seroconversion is defined as at least a 4-fold increase in S1-binding IgG GMC response compared to baseline. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). Dose 2 was not performed in the 60 μg dose group. GMC = geometric mean concentration. [Figure 109]Exemplary frequency of participants who seroconverted S1-binding IgG GMC after immunization with BNT162b2. Vaccination schedule and serum sampling were the same as in Figure 101. Seroconversion for S1-binding antibody GMC is shown for (A) younger participants (18-55 years old) immunized with 1 μg, 3 μg, 10 μg, 20 μg, or 30 μg BNT162b2 and (B) older participants (56-85 years old) who received 20 μg BNT162b2. Seroconversion is defined as at least a 4-fold increase in S1-binding IgG GMC response compared to baseline. Arrowheads indicate baseline (pre-dose 1, day 1) and dose 2 (day 22). GMC = geometric mean concentration [Figure 110] Exemplary results of cytokine production from S-specific CD4+ T cells from young participants immunized with BNT162b2. Peripheral blood mononuclear cell (PBMC) cell fractions isolated from the blood of participants treated with various doses of BNT162b2 were collected and analyzed at baseline (before dose 1) and 129 days (±3 days) after dose administration. Participants included young participants (ages 18-55) who received 1 μg (n=8), 3 μg (n=9), 10 μg (n=10), 20 μg (n=9), or 30 μg (n=10). Bar graphs show arithmetic means and 95% confidence intervals. Cytokine production was calculated by summing the fraction of total CD4+ T cells positive for IFNγ, IL-2, or IL-4, setting this sum to 100%, and calculating the fraction of each specific cytokine-producing subset. Two participants from the 1 μg cohort, one from the 3 μg cohort, and one from the 10 μg cohort were excluded from this analysis (total cytokine-producing CD4+ T cell frequency <0.03%). IFN = interferon; IL = interleukin; young participants = participants aged 18–55 years; S protein = SARS-CoV-2 spike protein. [Figure 111]Exemplary results of cytokine production from S-specific CD4+ T cells from elderly participants immunized with BNT162b2. Peripheral blood mononuclear cell (PBMC) cell fractions isolated from the blood of participants treated with various doses of BNT162b2 were collected and analyzed at baseline (before dose 1) and 129 days (±3 days) after dose administration. Participants included elderly participants (56-85 years old) who received 10 μg (n=11), 20 μg (n=8), or 30 μg (n=9). Bar graphs show arithmetic means and 95% CI. Cytokine production was calculated by summing the fraction of total CD4+ T cells positive for IFNγ, IL-2, or IL-4, setting this sum to 100%, and calculating the fraction of each specific cytokine-producing subset. Six participants from the 10 μg cohort and one participant from the 20 μg cohort were excluded from this analysis (total cytokine-producing CD4+ T cell frequency <0.03%). IFN = interferon; IL = interleukin; elderly participants = participants 56–85 years of age; S protein = SARS-CoV-2 spike protein. [Figure 112]Incidence and strength of BNT162b2-induced T cell responses. PBMCs (dose cohorts 1 μg, 10 μg, and 20 μg, n = 9 each; 30 μg, n = 10) obtained on day 1 (preprime) and day 29 (7 days postboost) were enriched for CD4+ or CD8+ T cell effectors and separately stimulated overnight with three overlapping peptide pools representing different portions of the wild-type sequence of SARS-CoV-2 S (N-terminal pool S pool 1 and RBD and C-terminal S pool 2) for direct ex vivo IFNγ ELISpot assessment. The common pathogenic T cell epitope pools CEF (immunodominant HLA class I epitopes of CMV, EBV, and influenza virus) and CEFT (immunodominant HLA class II epitopes of CMV, EBV, influenza virus, and tetanus toxin) were used as controls. Cell culture medium served as a negative control. Each dot represents the normalized mean spot count from duplicate wells for one study participant after subtraction from the medium-only control (a, c). a, Antigen-specific CD4+ and CD8+ T cell responses for each dose cohort. The number of participants with detectable T cell responses at day 29 relative to the total number of participants tested per dose cohort is provided. Spot count data for two participants from the 20 μg dose cohort could not be normalized and were not plotted. b, Example CD4+ and CD8+ ELISpots for participants in the 30 μg dose cohort. c, S-specific T cell responses and their baseline CEFT- and CEF-specific T cell responses in all participants who recognized either S peptide pool. Horizontal bars indicate median values. [Figure 113]BNT162b2-induced S-specific CD8+ and CD4+ T cells. Immunized participants' CD4+ or CD8+ T cell effector-enriched fractions (1 μg, 10 μg, and 20 μg dose cohorts, n = 9 each; 30 μg dose cohort, n = 10) from PBMCs obtained on day 1 (preprime) and day 29 (7 days postboost) were stimulated overnight with two overlapping peptide pools covering wild-type SARS-CoV-2 S (Spool 1 and Spool 2) for assessment by direct ex vivo IFNγ ELISpot (a–c). Each dot represents the normalized mean spot count from duplicate wells for one trial participant after subtraction from the media-only control. T cell responses to Spool 1 and Spool 2 per participant were combined. Spot count data from two participants from the 20 μg dose cohort could not be normalized and are not plotted. PBMCs from day 29 (7 days post-boost) vaccinated participants (dose cohort 1 μg, n=7; 10 μg and 30 μg, n=10; 20 μg, n=9) were stimulated as described above and analyzed by flow cytometry (d, e). a, S-specific CD4+ and CD8+ T cell responses for each dose cohort. The number of participants with detectable day 29 T cell responses relative to the total number of participants tested per dose cohort is provided. b, Mapping of vaccine-induced responses for participants with evaluable baseline data for different parts of S (n=34 for CD4+ and n=37 for CD8+ T cell responses). De novo induced or amplified responses are classified as BNT162b2-induced responses; no response or existing response not amplified by vaccination is classified as no vaccine response (none). c, Response strength to S pool 1 for individuals with or without an existing response to S pool 2. Data from the 1 μg dose cohort are excluded due to the absence of a baseline response to S-pool 2 in this dose cohort. Horizontal bars represent the center of each group. d, Example pseudocolor flow cytometry plot of cytokine-producing CD4+ and CD8+ T cells from participants prime / boost vaccinated with 30 μg BNT162b2. e, Vaccine-induced S-specific IFNγ+ CD4+ T cell frequency versus IL4+ CD4+ T cells. ICS stimulation was performed using a peptide mixture of S-pool 1 and S-pool 2.Each data point represents one trial participant (1 μg dose cohort, n=8; 20 μg dose cohort, n=8; 10 μg and 30 μg, n=10 each). One participant from the 20 μg dose cohort was excluded due to a strong existing CD4+ T cell response to S-pool 2. f, Antigen-specific CD8+ T cell frequencies were determined by pMHC class I multimer staining (% multimer+ of CD8+), ICS, and ELISpot (% IFNγ+ of CD8+) for the three participants analyzed in Figure 116. Signals from S-pool 1 and S-pool 2 were combined. [Figure 114] Correlation between antibody and T-cell responses. Data are plotted for all prime / boost vaccinated participants (dose cohort 1, 10 μg, 20 μg, and 30 μg) from day 29 onwards, and data points from participants with undetectable T-cell responses (open circles; b, c) were excluded from the correlation analysis. a, Correlation between S1-specific IgG responses and S-specific CD4+ T-cell responses. b, Correlation between S-specific CD4+ and CD8+ T-cell responses. c, Correlation between S1-specific IgG responses and S-specific CD8+ T-cell responses. [Figure 115]Cytokine polarization of BNT162b2-induced T cells. PBMCs obtained on days 1 (before prime) and 29 (7 days after boost) (dose cohorts 1 μg, n = 8; 10 μg and 30 μg, n = 10 each; 20 μg, n = 9) from COVID-19 convalescent donors (HCS, n = 18; c, d) were stimulated overnight with three overlapping peptide pools representing different portions of the wild-type sequence of SARS-CoV-2 S (N-terminal pool S pool 1 [aa 1-643] and RBD [aa 1-16 fused to aa 327-528 of S] and C-terminal S pool 2 [aa 633-1273]) and analyzed by flow cytometry. a, Example pseudocolor flow cytometry plot of cytokine-producing CD4+ and CD8+ T cells from a 30 μg dose cohort participant responding to S pool 1. b) S-specific CD4+ T cells producing the indicated cytokines as a fraction of total cytokine-producing S-specific CD4+ T cells responding to S-pool 1 and S-pool 2. CD4 non-responders (<0.03% total cytokine-producing T cells: 1 μg, n = 2 [S-pool 1] and n = 1 [S-pool 2]; 10 μg, n = 1) were excluded. Arithmetic means and 95% confidence intervals. c) S-specific CD4+ (S-pool 1, S-pool 2, and RBD) and d) CD8+ T cells producing the indicated cytokines as a fraction of total circulating T cells from the same subsets (S-pool 1, S-pool 2, and RBD). Values ​​above data points indicate the average fraction per dose cohort. Participant PBMCs were tested as a single instance (b–d). [Figure 116] Characterization of BNT162b2-induced T cells at the single epitope level. PBMCs from three vaccinated participants (dose cohorts 10 μg, n=1; 30 μg, n=2) obtained on day 1 (preprime) and day 29 (7 days postboost) were stained with each individual pMHC class I multimer cocktail and analyzed by flow cytometry for T cell epitope specificity (a) and phenotype (b; example from participant 3; YLQPRTFLL). Peptide sequences on dot plots indicate pMHC class I multimer epitope specificity, and numbers on dot plots indicate amino acids corresponding to epitopes within S. c, Localization of identified MHC class I-restricted epitopes within S. [Figure 117]ELISA screening analysis of exemplary cohort sera to detect antibody responses to recombinant SARS-CoV-2 spike protein 1 domain. ELISA was performed using serum samples collected 10 days after two immunizations with BNT162c1 (prime / boost on days 1 and 8) or 17 days after three doses of BNT162a1, BNT162b1, or BNT162b2 (prime / boost on days 1, 8, and 15) to analyze the induction of antibody responses. Serum samples were tested against the S1 protein. Group mean ΔOD values ​​for n=20 mice / group are indicated by dots representing serum dilutions ranging from 1:100 to 1:24,300. [Figure 118] ELISA screening analysis of exemplary cohort sera to detect antibody responses to the recombinant SARS-CoV-2 spike protein RBD domain. ELISA was performed using serum samples collected 10 days after two immunizations with BNT162c1 (prime / boost on days 1 and 8) or 17 days after three doses of BNT162a1, BNT162b1, or BNT162b2 (prime / boost on days 1, 8, and 15) to analyze the induction of antibody responses. Serum samples were tested against the RBD domain. Group mean ΔOD values ​​for n=20 mice per group are indicated by dots representing serum dilutions ranging from 1:100 to 1:24,300. [Figure 119] Pseudovirus neutralization activity of representative cohort sera plotted as pVN50 titers. Serum samples were collected 10 days (BNT162c1, saRNA) or 17 days (all other cohorts) after the animals' first immunization, and virus-neutralizing antibody titers were determined by pseudovirus-based neutralization test (pVNT). Individual VNT titers (pVN50) resulting in 50% pseudovirus neutralization are indicated by dots; group means are indicated by horizontal bars (±SEM, standard error of the mean). [Figure 120]Virus-neutralizing and specific binding antibody responses to RBD and S1 in participants. RBD = receptor-binding domain. GMT = geometric mean titer. Serum samples were obtained pre-vaccination (day 1) and 8, 22, 29, and 43 days after prime vaccination in the young adult group and pre-vaccination (day 1) and 22, 29, and 43 days after prime vaccination in the older adult group. A panel of human COVID-19 convalescent sera (n=24) was obtained from COVID-19 patients at least 14 days after PCR-confirmed diagnosis. (A) GMT of SARS-CoV-2 neutralizing antibodies. (B) GMT of binding antibodies to RBD measured by ELISA. (C) ELISA GMT of antibodies to S1. Each point represents one serum sample, and each vertical bar represents the geometric mean and 95% CI. [Figure 121] T cell responses measured by IFN-γ ELISpot in participants before and after vaccination. IFN = interferon. PBMC = peripheral blood mononuclear cells. The S1 peptide pool covers the N-terminal half of the SARS-CoV-2 spike, including the RBD. The S2 peptide pool covers the C-terminus of the SARS-CoV-2 spike, excluding the RBD. The CEF peptide pool consists of 32 MHC class I-restricted viral peptides from human cytomegalovirus, Epstein-Barr virus, and influenza virus. Panel A shows the number of IFN-γ-secreting specific T cells on days 1, 29, and 43 in young participants aged 18-55 years. Panel B shows the number of IFN-γ-secreting specific T cells on days 1, 29, and 43 in older participants aged 65-85 years. [Figure 122] 50% pseudovirus neutralization titers of 16 sera from BNT162b2 vaccine recipients against VSV-SARS-CoV-2-S pseudoviruses carrying the Wuhan or lineage B.1.1.7 spike protein. N=8. Representative sera from young adults (18-55 years old; triangles) and older adults (56-85 years old; circles) each collected on day 43 (21 days after dose 2). [Figure 123]Schematic representation of the production of VSV pseudoviruses carrying SARS-CoV-2 S protein. (1) Transfection of HEK293 / T17 cells with a SARS-CoV-2-S expression plasmid. (2) Infection of SARS-CoV-2 S-expressing cells with a VSV-G-complemented input virus lacking VSV-G in its genome (VSVΔG) and encoding a reporter gene. (3) Neutralization of the remaining VSV-G-complemented input virus by addition of anti-VSV-G antibodies yields SARS-CoV-2 S-pseudotyped VSVΔG as a surrogate for live SARS-CoV-2. [Figure 124] Titering of SARS-CoV-2 Wuhan control strain and lineage B.1.1.7 spike-pseudotyped VSV on Vero 76 cells using GFP-infected cells as readout. [Figure 125] Scheme of BNT162b2 vaccination and serum sampling. [Figure 126] Plot of pVNT50 ratios between SARS-CoV-2 lineage B.1.1.7 and Wuhan control strain spike-pseudotyped VSV. Triangles represent sera from young adults (18-55 years old), and circles represent sera from older adults (56-85 years old). Serum was collected on day 43 (21 days after dose 2). [Figure 127] Pseudovirus 50% neutralization titers (pVNT50) of 12 sera from BNT162b2 vaccine recipients against VSV-SARS-CoV-2-S pseudoviruses carrying the Wuhan Hu-1 control, lineage B.1.1.298, or lineage B.1.351 spike protein. N=12. Sera from young adults immunized with 30 μg BNT162b2 taken on day 29 or day 43 (7 or 21 days after dose 2) were tested. Geometric mean titers are shown. Statistical significance of differences in neutralization between the Wuhan Hu-1 control pseudovirus and lineage B.1.1.298 or lineage B.1.351 pseudoviruses was calculated by Wilcoxon paired signed-rank test. Two-tailed p-values ​​are reported. ns, not significant; ***, P<0.001; LLOQ, lower limit of quantification. [Figure 128]50% plaque reduction neutralization titers of 20 sera from BNT162b2 vaccine recipients against N501 and Y501 SARS-CoV-2. Seven sera (indicated by triangles) were taken 2 weeks after the second dose of vaccine; 13 sera (indicated by circles) were taken 4 weeks after the second dose. [Figure 129] Diagram of the N501Y substitution. L - leader sequence; ORF - open reading frame; RBD - receptor binding domain; S - spike glycoprotein; S1 - N-terminal furin cleavage fragment of S; S2 - C-terminal furin cleavage fragment of S; E - envelope protein; M - membrane protein; N - nucleoprotein; UTR - untranslated region. [Figure 130] Plaque morphology of N501 and Y501 SARS-CoV-2 in Vero E6 cells. [Figure 131] Scheme of BNT162 vaccination and serum sampling. [Figure 132] Plot of PRNT50 ratio between Y501 and N501 viruses. Triangles represent sera taken 2 weeks after the second dose; circles represent sera taken 4 weeks after the second dose. [Figure 133] Engineered mutations. Nucleotide and amino acid positions are indicated. Deletions are represented by dotted lines. Mutant nucleotides are in red. L, leader sequence; ORF, open reading frame; RBD, receptor-binding domain; S, spike glycoprotein; S1, N-terminal furin cleavage fragment of S; S2, C-terminal furin cleavage fragment of S; E, envelope protein; M, membrane protein; N, nucleoprotein; UTR, untranslated region. [Figure 134] Plaque morphology of WT (USA-WA1 / 2020), mutant N501Y, Δ69 / 70+N501Y+D614G, and E484K+N501Y+D614G SARS-CoV-2 on Vero E6 cells. [Figure 135] Scheme of BNT162 vaccination and serum sampling. [Figure 136]PRNT50 of 20 BNT162b2-vaccinated human sera against wild-type (WT) and mutant ARS-CoV-2. (a) WT (USA-WA1 / 2020) and mutant N501Y. (b) WT and Δ69 / 70 + N501Y + D614G. (c) WT and E484K + N501Y + D614G. Seven (triangles) and 13 (circles) sera were collected 2 and 4 weeks after the second dose of vaccination, respectively. Lines connect sera with different PRNT50s against WT and mutant viruses. Results in (a) are from one experiment; results in (b) and (c) are from another set of experiments. Each data point is the average of duplicate assay results. [Figure 137] Ratio of neutralization GMTs for mutant virus to GMTs for WT virus. Triangles represent sera taken 2 weeks after the second vaccination dose; circles represent sera taken 4 weeks after the second vaccination dose. [Figure 138] Schematic representation of engineered spike substitutions and deletions. The genome and sequence of clinical isolate USA-WA1 / 2020 was used as the wild-type virus in this study. Mutations from the UK B.1.1.7, Brazil P.1, and South Africa B.1.351 lineages are shown. Deletions are indicated by dotted lines. Mutated nucleotides are in red. Nucleotide and amino acid positions are indicated. L - leader sequence; ORF - open reading frame; RBD - receptor binding domain; S - spike glycoprotein; S1 - N-terminal furin cleavage fragment of S; S2 - C-terminal furin cleavage fragment of S; E - envelope protein; M - membrane protein; N - nucleoprotein; UTR - untranslated region. [Figure 139] Plaque morphology of USA-WA1 / 2020 and mutant SARS-CoV-2s. Plaque assays were performed on Vero E6 cells in 6-well plates. [Figure 140] Scheme of BNT162 immunization and serum collection. [Figure 141]Serum neutralization of variant strains of SARS-CoV-2 after two doses of the BNT162b2 vaccine. Results of a 50% plaque reduction neutralization test (PRNT50) using 20 samples from 15 trial participants are shown 2 weeks (circles) or 4 weeks (triangles) after the second dose of the BNT162b2 vaccine. Mutant viruses were derived from USA-WA1 / 2020 by engineering the full set of mutations in the B.1.1.7, P.1., or B.1.351 lineage or a subset of S gene mutations in the B.1.351 lineage (B.1.351-Δ242-244+D614G and B.1.351-RBD-D614G). Each data point represents the geometric mean PRNT50 obtained with serum samples for the indicated virus and includes data from replicate experiments as detailed in Table 31. Data for USA-WA1 / 2020 are from three experiments; one experiment each for B.1.1.7-spike, B.1.351-Δ242-244+D614G, and B.1.351-RBD-D614G viruses; and two experiments each for P.1-spike and B.1.351-spike viruses. For each experiment, neutralization titers were determined in duplicate assays and the geometric mean was taken. LOD: limit of detection. [Figure 142] Durability of BNT162b2-induced T cell responses. PBMCs obtained on days 1 (preprime), 29, 85, and 184 (7 days, 9 weeks, and 23 weeks after the boost, respectively) were analyzed by ex vivo IFNγ ELISpot (see GA-RB-022-01A for details). The common pathogen T cell epitope pools CEF (CMV, EBV, and influenza virus HLA class I epitopes) and CEFT (CMV, EBV, influenza virus, and tetanus toxin HLA class II epitopes) served to assess general T cell reactivity, and cell culture medium served as a negative control. Each dot represents the sum of the normalized mean spot counts from duplicate wells stimulated with two peptide pools corresponding to the full-length wt S protein for one subject, after subtraction from the media-only control. The ratio above the post-vaccination data point is the number of controls with a detectable CD4+ or CD8+ T cell response within the total number of test controls per dose cohort and time point. [Figure 143]Specific vaccine mRNA signal (red) is detected in LNs 6 hours post-injection using a modV9 probe in a dual IHC-ISH assay. The vaccine is primarily localized to the subcapsular sinus (LNs at positions 9 and 5) and B cell follicles (LNs at positions 12 and 1). Dendritic cells are visualized by CD11c staining (turquoise, top panel), only a portion of which internalize the vaccine. The majority of CD169+ macrophages (subcapsular sinus macrophages, turquoise, middle panel) are positive for the vaccine. B cells (CD19+, turquoise, bottom panel) are the second most abundant population showing vaccine signal. [Figure 144] Specific vaccine mRNA signal (red) is detected in the spleen 6 hours after injection using a modV9 probe in a dual IHC-ISH assay. The majority of the vaccine signal is detected in the white pulp. Dendritic cells are visualized by CD11c staining (turquoise, top panel), only a portion of which take up the vaccine. A small proportion of F4 / 80+ macrophages (turquoise, middle panel) take up the vaccine. B cells (CD19+, turquoise, bottom panel) are the major population that show vaccine signal. [Figure 145] Example Stability Data. Example data from a stability study (see, e.g., Example 42) is shown for preparations of BNT162b2 LNP at the concentrations and temperature conditions indicated, as assessed by ELISA characterizing antibodies reactive to the S1 spike protein.

[0243] Sequence description The following table provides a list of the specific sequences described herein.

[0244] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] DETAILED DESCRIPTION OF THE INVENTION

[0245] Detailed Description Although the present invention is described in detail below, it is understood that this invention is not limited to particular methodologies, protocols, and reagents, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. 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.

[0246] Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0247] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0248] Below, elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed as limiting the invention to only the specifically described embodiments. The description should be understood to disclose and encompass embodiments combining any number of the disclosed elements with the specifically described embodiments. Furthermore, all permutations and combinations of all described elements should be considered disclosed by this description, unless the context dictates otherwise.

[0249] The term "about" means approximately or approximation, and in the context of a numerical value or range herein, means, in certain embodiments, ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

[0250] In the context of describing the present invention (particularly in the context of the claims), the singular term "a," "an," or "the" is to be construed as including both the singular and the plural unless otherwise stated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended merely as a shorthand method of referring to each individual value within the range. Unless otherwise stated herein, each individual value is encompassed herein as if it were individually described herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or clearly contradicted by context. Any and all examples or exemplary language (e.g., "etc.") provided herein are intended merely to better describe the present invention and do not pose a limitation on the scope of the claims. No language herein should be construed as indicating any non-claimed element necessary to the practice of the invention.

[0251] Unless otherwise indicated, the term "comprising" is used herein to indicate that additional members may optionally be present in addition to the listed members introduced by "comprising." However, it is contemplated that the term "comprising" may be a specific embodiment of the present invention that does not exclude the possibility of additional members being present, i.e., for the purposes of this embodiment, "comprising" is understood to have the meaning of "consisting of" or "consisting essentially of."

[0252] Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0253] definition The following definitions are provided which apply to all aspects of the present invention. The following terms have the following meanings unless otherwise specified: Any undefined terms have their art-recognized meanings.

[0254] As used herein, terms such as "reduce," "reduce," "inhibit," or "impair" refer to an overall decrease or the ability to cause an overall decrease in a level, preferably at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or more. These terms include complete or essentially complete inhibition, i.e., a decrease to or essentially to zero.

[0255] Terms such as "increase," "enhancement," or "exceed" preferably relate to an increase or enhancement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500% or more.

[0256] According to this disclosure, the term "peptide" includes oligo- and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids joined together by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 150 amino acids, although the terms "peptide," "protein," and "polypeptide" are generally used interchangeably herein.

[0257] A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or disease state. In certain embodiments, a therapeutic protein has therapeutic or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of such a disease or reduce the severity of such a disease or condition. The term "therapeutic protein" includes whole proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, vaccination antigens and immunostimulants such as cytokines.

[0258] A "fragment" in relation to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains a start codon that serves for translation initiation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of the amino acid sequence.

[0259] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence or may be a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications compared to the parent, and preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0260] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic versions. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0261] For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes total mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0262] Amino acid insertion variants include the insertion of one, two, or more amino acids into a specific amino acid sequence. In amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants that include deletions at the N-terminus and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications that substitute amino acid positions and / or amino acids that are not conserved in homologous proteins or peptides with others having similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of members of a family of amino acids related in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0263] Preferably, the degree of similarity, preferably identity, between an amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably displayed over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably expressed for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is expressed for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0264] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0265] The terms "% identical", "% identity" or similar terms refer specifically to the percentage of nucleotides or amino acids that are identical in optimal alignment of the compared sequences. The percentage is purely statistical, and the differences between the two sequences are not necessarily randomly distributed throughout the compared sequences. Comparison of two sequences is usually performed by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In one embodiment, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In one embodiment, the algorithm parameters used in the BLASTN algorithm on the NCBI website include (i) a prediction threshold setting of 10; (ii) a word size setting of 28; (iii) a maximum match within the query range setting of 0; (iv) a match / mismatch score setting of 1 or -2; (v) a gap cost setting of linear; and (vi) use of a filter for low complexity regions. In one embodiment, the algorithm parameters used in the BLASTP algorithm on the NCBI website include (i) a prediction threshold setting of 10; (ii) a word size setting of 3; (iii) a maximum match within the query range setting of 0; (iv) a matrix setting of BLOSUM62; (v) gap costs set to presence: 11 and extension: 1; and (vi) a conditional compositional score matrix adjustment.

[0266] Percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying the result by 100.

[0267] In some embodiments, the degree of similarity or identity is expressed over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is expressed over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is expressed over the entire length of the reference sequence.

[0268] Homologous amino acid sequences according to the present disclosure exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

[0269] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily produced with the aid of known peptide synthesis techniques, such as, for example, by solid-phase synthesis and similar methods.

[0270] In some embodiments, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigenic sequence, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" particularly refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been changed compared to the amino acid sequence of the parent molecule or sequence, yet still fulfills one or more functions of the parent molecule or sequence, such as inducing an immune response. In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced, but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0271] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be altered in sequence to differ from the naturally occurring or native sequence from which they are derived while maintaining the desired activity of the native sequence.

[0272] As used herein, "instruction material" or "instructions" includes publications, records, diagrams, or any other medium of representation that can be used to communicate the utility of the compositions and methods of the invention. The instruction material of the kits of the invention can be, for example, attached to or shipped with a container containing a composition of the invention. Alternatively, the instruction material can be shipped separately from the container, with the intention that the instruction material and the composition be used cooperatively by the recipient.

[0273] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0274] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, a "recombinant entity," such as a recombinant nucleic acid, in the context of the present invention does not occur in nature.

[0275] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus), can be isolated from a natural source, and has not been artificially modified in a laboratory is naturally occurring.

[0276] As used herein, "physiological pH" refers to a pH of about 7.5.

[0277] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of nucleic acids described herein can be present in vitro or in vivo; for example, the cells may form part of a patient's organ, tissue, and / or organism. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during transfection are usually not integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells capable of episomal amplification of nucleic acids have a greatly reduced dilution rate. If transfected nucleic acid is desired to remain in the genome of the cell and its daughter cells, stable transfection should be performed.Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding antigen is transiently transfected into cells.RNA can be transfected so that cells transiently express the protein it encodes.

[0278] The term "seroconversion" includes a 4-fold or greater increase from pre-vaccination to 1 month after dose 2.

[0279] coronavirus Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+)ssRNA) viruses. They possess the largest genomes (26–32 kb) among known RNA viruses and are phylogenetically classified into four genera (α, β, γ, and Δ), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory illness, although the severity of the illness can be greater in infants, the elderly, and immunocompromised individuals. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), which belong to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses have emerged from animal reservoirs into human populations within the last 15 years and caused highly lethal pandemics. A severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak, which causes atypical pneumonia (coronavirus disease 2019; COVID-19), emerged in China in mid-December 2019 and has developed into a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to the betacoronavirus lineage B and shares at least 70% sequence similarity with SARS-CoV.

[0280] Generally, coronaviruses have four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins have important functions in virus assembly, while the N protein is required for viral RNA synthesis. A key glycoprotein is responsible for virus binding and entry into target cells. The S protein is synthesized as a single-chain inactive precursor and is cleaved into two noncovalently linked subunits, S1 and S2, by a furin-like host protease in the producing cell. The S1 subunit contains a receptor-binding domain (RBD) that recognizes host cell receptors. The S2 subunit contains a fusion peptide, two heptad repeat regions, and a transmembrane domain, all of which undergo large conformational rearrangements to mediate fusion between the virus and host cell membranes. The S1 and S2 subunits trimerize to form the large prefusion spike.

[0281] The SARS-CoV-2 S precursor protein is proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit consists of a receptor-binding domain (RBD) that mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0282] antigen The present invention includes the use of RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, its immunogenic variants, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variants. Thus, the RNA encodes a peptide or protein comprising at least an epitope of the SARS-CoV-2 S protein or its immunogenic variants to induce an immune response in a subject against the coronavirus S protein, particularly the SARS-CoV-2 S protein. Amino acid sequences comprising the SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants (i.e., antigenic peptides or proteins) are also referred to herein as "vaccine antigens," "peptide and protein antigens," "antigenic molecules," or simply "antigens." The SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants are also referred to herein as "antigenic peptides or proteins" or "antigenic sequences."

[0283] The SARS-CoV-2 coronavirus full-length spike (S) protein consists of 1273 amino acids (see SEQ ID NO: 1). In certain embodiments, the full-length spike (S) protein of SEQ ID NO: 1 is modified in such a way that the prototype pre-fusion conformation is stabilized. Stabilization of the pre-fusion conformation can be obtained by introducing two consecutive proline substitutions at AS residues 986 and 987 of the full-length spike protein. In particular, a spike (S) protein stabilized protein variant is obtained in such a way that amino acid residue 986 is replaced with proline and amino acid residue 987 is also replaced with proline. In certain embodiments, the SARS-CoV-2 S protein variant comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0284] In certain embodiments, the vaccine antigens described herein comprise, consist essentially of, or consist of the spike protein of SARS-CoV-2, a variant thereof, or a fragment thereof.

[0285] In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0286] In some embodiments, the RNA encoding the vaccine antigen is (i) a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or ... and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

[0287] In certain embodiments, the vaccine antigen comprises, consists essentially of, or consists of the SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the spike protein of SARS-CoV-2), a variant thereof, or a fragment thereof.

[0288] In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1. In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.

[0289] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9 or the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.

[0290] In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1. In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.

[0291] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9 or the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.

[0292] In some embodiments, the vaccine antigen comprises, consists essentially of, or consists of the receptor binding domain (RBD) of the S1 subunit of the spike protein of SARS-CoV-2, a variant thereof, or a fragment thereof. The amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, a variant thereof, or a fragment thereof is also referred to herein as "RBD" or "RBD domain."

[0293] In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.

[0294] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9 or the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.

[0295] In some embodiments, the signal peptide is fused directly or via a linker to the SARS-CoV-2 S protein, its variants, or fragments thereof, i.e., antigenic peptide or protein. Thus, in some embodiments, the signal peptide is fused to the amino acid sequence derived from the SARS-CoV-2 S protein or its immunogenic fragment (antigenic peptide or protein) contained in the vaccine antigen.

[0296] Such signal peptides generally have a length of about 15-30 amino acids and are preferably, but not limited to, sequences located at the N-terminus of an antigenic peptide or protein. A signal peptide as defined herein preferably enables transport of an antigenic peptide or protein encoded by an RNA to a predetermined intracellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosomal-lysosomal compartment. In certain embodiments, a signal peptide sequence as defined herein includes, but is not limited to, the signal peptide sequence of the SARS-CoV-2 S protein, particularly a sequence comprising amino acids 1-16 or 1-19 of SEQ ID NO: 1, or a functional variant thereof.

[0297] In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1. In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1.

[0298] In certain embodiments, the RNA encoding the signal sequence comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1.

[0299] In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-19 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO:1. In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-19 of SEQ ID NO:1.

[0300] In certain embodiments, the RNA encoding the signal sequence comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9, or the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 1-57 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-19 of SEQ ID NO: 1.

[0301] The signal peptide sequence defined herein further includes, but is not limited to, an immunoglobulin signal peptide sequence, for example, the signal peptide sequence of an immunoglobulin heavy chain variable region, where the immunoglobulin may be a human immunoglobulin. In particular, the signal peptide sequence defined herein includes a sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functional variant thereof.

[0302] In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31. In certain embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31.

[0303] In certain embodiments, the RNA encoding the signal sequence is (i) nucleotides 54 to 119 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32 or the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31. In certain embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 54-119 of SEQ ID NO: 32; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31.

[0304] Such signal peptides are preferably used to facilitate secretion of the encoded antigenic peptide or protein. More preferably, a signal peptide as defined herein is fused to the encoded antigenic peptide or protein as defined herein.

[0305] Thus, in particularly preferred embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a signal peptide, the signal peptide preferably being fused to the N-terminus of the antigenic peptide or protein, more preferably an antigenic peptide or protein described herein.

[0306] In certain embodiments, the vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In certain embodiments, the vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7.

[0307] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) the nucleotide sequence of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9 or the nucleotide sequence of SEQ ID NO: 2, 8 or 9. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7.

[0308] In certain embodiments, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7, or a fragment of the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7. In certain embodiments, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:7.

[0309] In certain embodiments, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24 or 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24 or 25 or ... and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7, or a fragment of the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

[0310] In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1. In certain embodiments, the vaccine antigen comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.

[0311] In certain embodiments, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8, or 9, or to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8, or 9, or to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8, or 9. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1. In certain embodiments, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8, or 9; and / or (ii) encodes an amino ac...

Claims

(i) containing modified uridines in place of all uridines; and (ii) comprises a nucleotide sequence encoding a polypeptide; A pharmaceutical composition comprising RNA, wherein the polypeptide is (a) one or more fragments of the SARS-CoV-2 spike (S) protein, wherein one of the fragments comprises a receptor binding domain (RBD); (b) a secretion signal, and (c) one or more additional domains selected from a trimerization domain, a transmembrane domain, and combinations thereof. Including, A pharmaceutical composition, wherein the RBD is linked to one of the additional domains via a linker, and the linker comprises a GS linker.

2. The secretory signal, (i) the secretory signal peptide of the SARS-CoV-2 S protein; or (ii) human immunoglobulin secretory signal peptide The pharmaceutical composition of claim 1 , 3. The pharmaceutical composition of claim 2, wherein the secretory signal peptide of the SARS-CoV-2 S protein comprises an amino acid sequence that is at least 90% identical to amino acids 1 to 19 of SEQ ID NO:

1.

4. The pharmaceutical composition of claim 1, wherein one of the additional domains is a trimerization domain, and the trimerization domain is the trimerization domain of T4 fibritin.

5. The pharmaceutical composition of claim 4, wherein the trimerization domain of T4 fibritin comprises an amino acid sequence that is at least 90% identical to amino acids 3 to 29 of SEQ ID NO:

10.

6. One of the additional domains is a transmembrane domain, and The N-terminal to C-terminal orientation of the secretory signal, the fragment containing the RBD, and the transmembrane domain is secretory signal-fragment-transmembrane domain; The pharmaceutical composition of claim 1.

7. (i) the secretory signal is a secretory signal peptide of the SARS-CoV-2 S protein, and (ii) the transmembrane domain is the transmembrane domain of the SARS-CoV-2 S protein; The pharmaceutical composition according to claim 6.

8. The pharmaceutical composition of claim 1, wherein the RBD comprises a sequence that is at least 90% identical to amino acids 327-528 of SEQ ID NO:

1.

9. The pharmaceutical composition of claim 1, wherein the polypeptide comprises an array that is at least 90% identical to SEQ ID NO:

29.

10. The pharmaceutical composition of claim 1, wherein each of the modified uridines is N1-methyl-pseudouridine.

11. RNA, (i) a 5′ cap containing a cap1 structure; (ii) a modified human alpha-globin 5′-UTR; (iii) a 3′-UTR comprising a first sequence derived from an amino-terminal enhancer of splitting (AES) messenger RNA and a second sequence derived from a mitochondrially encoded 12S ribosomal RNA; and (iv) a polyA sequence, the polyA sequence comprising 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence. The pharmaceutical composition of claim 1 , comprising:

12. The pharmaceutical composition of claim 1, wherein the RNA comprises a nucleotide sequence that is codon-optimized for a human subject.

13. The pharmaceutical composition of claim 1, wherein the nucleotide sequences encoding the secretory signal, the fragment containing the RBD, and the one or more additional domains each have an increased G / C content compared to their respective wild-type nucleotide sequences.

14. The pharmaceutical composition of claim 1, wherein the RNA is formulated in lipid nanoparticles comprising a cationic ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)-lipid.

15. The pharmaceutical composition of claim 14, further comprising at least one salt and / or cryoprotectant, wherein the cryoprotectant comprises sucrose.

16. The pharmaceutical composition of claim 14, wherein the RNA is present in the pharmaceutical composition in an amount ranging from about 1 μg to about 100 μg per dose.

17. The pharmaceutical composition of claim 14, wherein the RNA is present in the pharmaceutical composition in an amount of about 1 μg, about 3 μg, about 10 μg, about 20 μg, or about 30 μg per dose.

18. The pharmaceutical composition of claim 14, formulated for intramuscular administration.

19. The pharmaceutical composition of claim 1, wherein the secretory signal comprises an amino acid sequence that is at least 90% identical to amino acids 1 to 16 of SEQ ID NO:

1.

20. The pharmaceutical composition of claim 1, wherein the one or more additional domains include a transmembrane domain.

21. The pharmaceutical composition of claim 20, wherein the transmembrane domain is the transmembrane domain of SARS-CoV-2 S protein.

22. The pharmaceutical composition of claim 21, wherein the transmembrane domain of the SARS-CoV-2 S protein comprises an amino acid sequence that is at least 90% identical to amino acids 1207 to 1254 of SEQ ID NO:

1.

23. The pharmaceutical composition of claim 1, wherein the RBD comprises a sequence comprising from 1 to about 20 amino acid modifications relative to amino acids 327 to 528 of SEQ ID NO:

1.

24. The pharmaceutical composition of claim 1, wherein the RBD comprises a sequence that is at least 85% identical to amino acids 327-528 of SEQ ID NO:1.