SARS-CoV-2 RNA vaccine and its use

Abstract

This disclosure relates to SARS-CoV-2 RNA vaccines and their use. This disclosure also relates to conventional mRNA vaccines and self-replicating RNA vaccines for the treatment of SARS-CoV-2 infection or COVID-19.

Description

Technical Field

[0001] Related Application Data This application claims the priority of U.S. Patent Application No. 63 / 511,340, filed on June 30, 2023, entitled "SARS-CoV-2 RNA vaccines and uses thereof", the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application is filed with a sequence listing in electronic format. The entire content of the sequence listing is incorporated herein by reference. …

[0003] Field This disclosure relates to SARS-CoV-2 RNA vaccines and their use. This disclosure also relates to conventional mRNA vaccines and self-replicating RNA vaccines for the treatment of SARS-CoV-2 infections or COVID-19.

Background Art

[0004] Respiratory virus infections are a major threat to human health and life. Infections such as those caused by influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV) are known to have caused worldwide pandemics and led to the deaths of millions of people around the world. More recently, SARS-CoV-2 has caused a global pandemic of severe infectious coronavirus disease 2019 (COVID-19).

[0005] SARS-CoV-2, belonging to the Coronaviridae family and Orthocoronavirinae subfamily, is an enclosed single-stranded positive-chain RNA virus that encodes non-structural proteins that play a role in viral replication and translation, as well as structural proteins including the spike protein (S protein), membrane protein (M protein), envelope protein (E protein), and nucleocapsid protein (N protein). The S protein is a transmembrane glycoprotein that forms a prominent homotrimer on the surface of the virus and consists of two functional subunits, S1 and S2, which are the main targets of current genetically engineered vaccine development.

[0006] SARS-CoV-2 is highly prone to mutation, and a significant number of variant strains have now been identified globally. Dominant variants include the alpha (B.1.1.7), beta (B.1.351), gamma (P1), epsilon (B.1.429), delta (B.1.617.2), kappa (B.1.617.1), and omicron (B.1.1.529) variants, which differ in their transmissibility, pathogenicity, and / or immune evasion capabilities. Typically, these variants are identified based on the number and location of mutations in the viral genome, particularly in the genome encoding the S protein of the variant SARS-CoV-2.

[0007] The Omicron strain contains up to 36 amino acid mutation sites, and this strain has been shown to have an improved affinity for the ACE2 target receptor, thereby increasing its toxicity and infectivity and accelerating viral escape. Currently, there are few available vaccines that target specific SARS-CoV-2 strains such as the Omicron strain. Currently available vaccines, such as those developed against the Wuhan (original) strain, which do not specifically target evolving mutant strains, are known to offer reduced protective efficacy and / or immunogenicity.

[0008] Therefore, there is a need to develop new vaccines that can specifically target SARS-CoV-2 strains, such as the Omicron strain. [Overview of the initiative]

[0009] This disclosure is based on the inventors' identification of RNA suitable for the treatment of SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19), comprising antigens from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. In particular, the inventors' research results provide a basis for RNA suitable for the treatment of SARS-CoV-2 infection or COVID-19, comprising antigens from SARS-CoV-2, for example, the spike (S) protein from the omicron strain (or its variant) of SARS-CoV-2. Furthermore, the inventors' research results provide a basis for methods to treat, prevent, or delay the progression of diseases or disorders such as SARS-CoV-2 infection or COVID-19 in subjects, as well as their complications, including pneumonia, sepsis, and acute respiratory distress syndrome (ARDS).

[0010] Accordingly, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding an antigen operably linked to a regulatory element, wherein the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0011] In this specification, when discussing the “Omicron strain” of SARS-CoV-2, this term will be understood to include variants and sublineages arising from the Omicron strain of SARS-CoV-2. For example, the Omicron strain of SARS-CoV-2 includes strains BA.1, BA.2, XB, XBB, JN.1, JN.2, JN.3, KP.1, and KP.2.

[0012] For example, the polynucleotide contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in SEQ ID NO: 17.

[0013] In one example, the polynucleotide further comprises a nucleotide sequence encoding a second antigen, operably linked to a regulatory element. In one example, the second antigen is the nucleocapsid (N) protein from SARS-CoV-2.

[0014] For example, the polynucleotide contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in SEQ ID NO: 18.

[0015] For example, the polynucleotide contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in SEQ ID NO: 19.

[0016] The disclosure also provides RNA comprising a first nucleotide sequence encoding an antigen, operably ligated to a regulatory element, wherein the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0017] In one example, the RNA further includes a nucleotide sequence encoding a second antigen, operably ligated to a regulatory element. In one example, the second antigen is the nucleocapsid (N) protein from SARS-CoV-2.

[0018] The disclosure also provides a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, wherein the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0019] In one example, the self-replicating RNA is encoded by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in Sequence ID No. 17.

[0020] In one example, the self-replicating RNA further includes a nucleotide sequence encoding a second antigen, operably ligated to a regulatory element. In one example, the second antigen is the nucleocapsid (N) protein from SARS-CoV-2.

[0021] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) Nucleotide sequences encoding the S protein antigen from the SARS-CoV-2 Omicron strain, b) A nucleotide sequence encoding the N protein antigen from SARS-CoV-2, and

[0022] In another example, self-replicating RNA is in the order from 5' to 3'. a) Nucleotide sequence encoding the N protein antigen from SARS-CoV-2, b) A nucleotide sequence encoding the S protein antigen from the Omicron strain of SARS-CoV-2, and

[0023] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) A nucleotide sequence encoding the S protein antigen from the Omicron strain of SARS-CoV-2, operably linked to the SG promoter, b) A nucleotide sequence encoding an N protein antigen from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0024] In another example, the self-replicating RNA, in order from 5' to 3', a) A nucleotide sequence encoding an N protein antigen from SARS-CoV-2, operably linked to an SG promoter, and b) A nucleotide sequence encoding an S protein antigen from the Omicron strain of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0025] In one example, the self-replicating RNA encodes a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or identical to the sequence shown in SEQ ID NO: 18.

[0026] In one example, the self-replicating RNA is encoded by a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or identical to the sequence shown in SEQ ID NO: 19.

[0027] The present disclosure further provides a polynucleotide comprising a nucleotide sequence encoding an antigen, operably linked to a regulatory element, wherein the antigen is a nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0028] In one example, the polynucleotide further comprises a nucleotide sequence encoding a second antigen, operably linked to the regulatory element. In one example, the second antigen is a spike (S) protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0029] The disclosure also provides RNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, wherein the antigen is a nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0030] In one example, the RNA further includes a nucleotide sequence encoding a second antigen, operably ligated to a regulatory element. In one example, the second antigen is the spike (S) protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0031] The disclosure also provides a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, wherein the antigen is a nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0032] In one example, the self-replicating RNA further includes a nucleotide sequence encoding a second antigen, operably ligated to a regulatory element. In one example, the second antigen is the spike (S) protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0033] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) Nucleotide sequences encoding the N protein antigen from the SARS-CoV-2 Omicron strain, b) A nucleotide sequence encoding the S protein antigen from SARS-CoV-2, and

[0034] In another example, self-replicating RNA is in the order from 5' to 3'. a) Nucleotide sequence encoding the S protein antigen from SARS-CoV-2, b) A nucleotide sequence encoding the N protein antigen from the Omicron strain of SARS-CoV-2, and

[0035] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) A nucleotide sequence encoding the N protein antigen from the Omicron strain of SARS-CoV-2, operably linked to the SG promoter, b) comprising a nucleotide sequence encoding the S protein antigen from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0036] In another example, self-replicating RNA is in the order from 5' to 3'. a) A nucleotide sequence encoding the S protein antigen from SARS-CoV-2, operably linked to the SG promoter, b) comprising a nucleotide sequence encoding the N protein antigen from the omicron strain of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0037] For example, the self-replicating RNA is encoded by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in Sequence ID No. 18.

[0038] For example, the self-replicating RNA is encoded by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in Sequence ID No. 19.

[0039] In one example, the regulatory element is selected from a group consisting of a subgenome (SG) promoter, an internal ribosome entry site (IRES), and a Kozac consensus sequence, or a combination thereof. In one example, the regulatory element is the SG promoter.

[0040] In one example, the nucleotide sequence encoding the second antigen is operably linked to the same regulatory element as the nucleotide sequence encoding the first antigen.

[0041] The disclosure also provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first polypeptide of interest; and b) a second nucleotide sequence encoding a second polypeptide of interest, operably linked to a regulatory element selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES), wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0042] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first polypeptide of interest, and b) a second nucleotide sequence encoding a second polypeptide of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0043] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first polypeptide of interest, and b) a second nucleotide sequence encoding a second polypeptide of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0044] The disclosure also provides a polynucleotide comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0045] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0046] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0047] For example, polynucleotides are RNA or DNA. For example, RNA is messenger RNA (mRNA). For example, mRNA is conventional mRNA (cRNA) or self-replicating RNA.

[0048] Accordingly, the present disclosure provides an RNA comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0049] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0050] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0051] The disclosure also provides a cRNA comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0052] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0053] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0054] The disclosure further provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0055] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0056] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a second antigen of interest, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0057] In one example, a first nucleotide sequence encoding the target first antigen is operably ligated to a regulatory element. In another example, the regulatory element is operably ligated to the 5' end of the first nucleotide sequence. In one example, the regulatory element is selected from the group consisting of Kozak consensus sequences, IRESs, SG promoters, and combinations thereof. For example, the regulatory element is a Kozak consensus sequence. For example, the regulatory element is an IRES. In another example, the regulatory element is an SG promoter.

[0058] For example, the Kozak consensus sequence contains or consists of the sequence shown in sequence number 6 (GCCACC). For example, the Kozak consensus sequence consists of the sequence shown in sequence number 7 (ACCATGG).

[0059] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen, operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target second antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0060] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0061] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0062] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0063] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0064] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0065] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0066] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0067] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0068] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0069] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0070] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0071] In one example, the first nucleotide sequence encoding the target first antigen is operably ligated to the Kozak consensus sequence.

[0072] In one example, the first nucleotide sequence encoding the target antigen is operably ligated to the Kozak consensus sequence and the SG promoter. For example, the Kozak consensus sequence is operably ligated to the 5' end of the SG promoter, and the SG promoter is operably ligated to the 5' end of the first nucleotide sequence encoding the target antigen.

[0073] In one example, the first nucleotide sequence encoding the target antigen is operably ligated to the Kozak consensus sequence and the IRES. For instance, the Kozak consensus sequence is operably ligated to the 5' end of the IRES, and the IRES is operably ligated to the 5' end of the first nucleotide sequence encoding the target antigen.

[0074] In one example, the first nucleotide sequence encoding the target antigen is operably ligated to the SG promoter.

[0075] In one example, the first nucleotide sequence encoding the target first antigen is operably ligated to the IRES.

[0076] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0077] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0078] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0079] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0080] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0081] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0082] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0083] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0084] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0085] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0086] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0087] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0088] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably linked to an IRES; and b) a second nucleotide sequence encoding a target second antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0089] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0090] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0091] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0092] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0093] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0094] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0095] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0096] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0097] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0098] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0099] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0100] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0101] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0102] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0103] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0104] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0105] In one example, the RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0106] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0107] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0108] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0109] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0110] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0111] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0112] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0113] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0114] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0115] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0116] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0117] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0118] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0119] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0120] In one example, the cRNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0121] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a first antigen operably ligated to a Kozac consensus sequence; and b) a second nucleotide sequence encoding a second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0122] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen operably ligated to a Kozac consensus sequence, and b) a second nucleotide sequence encoding a second antigen operably ligated to an IRES or SG promoter, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0123] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a first antigen operably ligated to a Kozac consensus sequence, and b) a second nucleotide sequence encoding a second antigen operably ligated to an IRES or SG promoter, wherein the first antigen is a nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0124] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0125] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0126] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0127] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0128] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0129] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target second antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0130] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0131] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0132] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0133] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first polypeptide is a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2.

[0134] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0135] In one example, the self-replicating RNA comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a target second antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first antigen is the nucleocapsid (N) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0136] In one example, a polynucleotide is a bicistronic RNA. For example, a polynucleotide is a bicistronic cRNA. In another example, a polynucleotide is a bicistronic self-replicating mRNA. For example, self-replicating RNA is a bicistronic self-replicating RNA.

[0137] In one example, a second nucleotide sequence encoding a second antigen is operably ligated to the IRES.

[0138] In one example, a second nucleotide sequence encoding a second antigen is operably ligated to the SG promoter.

[0139] For example, a polynucleotide is a multicistronic RNA. For example, a polynucleotide is a multicistronic cRNA. For example, cRNA is a multicistronic cRNA. In another example, a polynucleotide is a multicistronic self-replicating mRNA. For example, self-replicating RNA is a multicistronic self-replicating mRNA.

[0140] The disclosure also provides a polynucleotide comprising a) a first nucleotide sequence encoding a target antigen, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES), wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0141] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0142] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0143] For example, polynucleotides are RNA or DNA. For example, RNA is messenger RNA (mRNA). For example, mRNA is conventional mRNA (cRNA) or self-replicating RNA.

[0144] Accordingly, the present disclosure provides RNA comprising a) a first nucleotide sequence encoding a target antigen, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES), wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0145] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0146] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0147] The disclosure also provides a cRNA comprising a) a first nucleotide sequence encoding a target antigen, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES), wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0148] In one example, the cRNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0149] In one example, the cRNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0150] The disclosure further provides self-replicating RNA comprising: a) a first nucleotide sequence encoding a target antigen; and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES), wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0151] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0152] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0153] In one example, the first nucleotide sequence is operably ligated to a regulatory element. For example, the regulatory element is operably ligated to the 5' end of the first nucleotide sequence. In one example, the regulatory element is selected from the group consisting of Kozak consensus sequences, IRESs, SG promoters, and combinations thereof. For example, the regulatory element is a Kozak consensus sequence. For example, the regulatory element is an IRES. For example, the regulatory element is an SG promoter.

[0154] For example, the Kozak consensus sequence contains or consists of the sequence shown in sequence number 6 (GCCACC). For example, the Kozak consensus sequence consists of the sequence shown in sequence number 7 (ACCATGG).

[0155] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target antigen, operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0156] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0157] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0158] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0159] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0160] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0161] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0162] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0163] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0164] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0165] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0166] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0167] In one example, the first nucleotide sequence is operably ligated to the Kozak consensus sequence.

[0168] In one example, the first nucleotide sequence is operably ligated to the Kozak consensus sequence and the SG promoter. For instance, the Kozak consensus sequence is operably ligated to the 5' end of the SG promoter, and the SG promoter is operably ligated to the 5' end of the first nucleotide sequence encoding the S protein.

[0169] In one example, the first nucleotide sequence is operably ligated to the Kozak consensus sequence and the IRES. For instance, the Kozak consensus sequence is operably ligated to the 5' end of the IRES, which in turn is operably ligated to the 5' end of the first nucleotide sequence encoding the S protein.

[0170] In one example, the first nucleotide sequence is operably linked to the SG promoter.

[0171] In one example, the first nucleotide sequence is operably ligated to the IRES.

[0172] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0173] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0174] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0175] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0176] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoding a spike (S) protein, operably linked to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence from SARS-CoV-2 encoding a nucleocapsid (N) protein, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0177] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0178] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target antigen, operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0179] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0180] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0181] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0182] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0183] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0184] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0185] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an IRES, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0186] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to an IRES, and b) a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0187] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0188] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0189] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0190] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0191] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0192] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0193] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0194] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0195] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0196] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0197] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0198] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0199] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2.

[0200] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an IRES, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0201] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to an IRES, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0202] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a target antigen from SARS-CoV-2 operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0203] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0204] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0205] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0206] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0207] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0208] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0209] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0210] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0211] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0212] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0213] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0214] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0215] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an IRES, and b) a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0216] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to an IRES, and b) a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0217] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target antigen operably ligated to a subgenome (SG) promoter; and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, wherein the first and second nucleotide sequences are selected from the group consisting of nucleotide sequences encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and nucleotide sequences encoding a nucleocapsid (N) protein from SARS-CoV-2. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0218] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to an IRES or SG promoter. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0219] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an IRES or SG promoter. In one example, the N protein is from the Omicron strain of SARS-CoV-2. In another example, the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0220] In one example, a polynucleotide is a bicistronic RNA. For example, a polynucleotide is a bicistronic cRNA. In another example, a polynucleotide is a bicistronic self-replicating mRNA. For example, self-replicating RNA is a bicistronic self-replicating RNA.

[0221] In one example, the second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2 is operably ligated to the IRES.

[0222] In one example, the second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2 is operably ligated to the SG promoter.

[0223] In one example, a polynucleotide is a multicistronic RNA. For example, a polynucleotide is a multicistronic cRNA. For example, cRNA is a multicistronic cRNA. In another example, a polynucleotide is a multicistronic self-replicating mRNA. In another example, self-replicating RNA is a monocistronic self-replicating mRNA. In yet another example, a polynucleotide is a multicistronic self-replicating RNA. In yet another example, self-replicating RNA is a monocistronic self-replicating RNA.

[0224] For example, the SG promoter is a natural SG promoter. For instance, the natural SG promoter is a promoter that is natural to and / or based on RNA viruses (e.g., alphaviruses). For example, the natural SG promoter is a natural alphavirus SG promoter.

[0225] In one example, the SG promoter is either a minimum SG promoter or an extended SG promoter.

[0226] For example, the SG promoter is the minimal SG promoter. For example, the natural SG promoter is the minimal SG promoter. For example, the minimal SG promoter is the smallest sequence required to initiate transcription. For example, the minimal natural SG promoter is 49 nucleotides long. For example, the minimal SG promoter is 49 nucleotides long. For example, the minimal natural SG promoter is encoded by a sequence containing or consisting of the sequence shown in Sequence ID No. 1. For example, the minimal SG promoter is encoded by a sequence containing or consisting of the sequence shown in Sequence ID No. 1.

[0227] In one example, the SG promoter is an extended SG promoter. In another example, the natural SG promoter is an extended SG promoter. For example, the extended SG promoter is extended at its 5' end by nucleotides introduced into the sequence encoding a non-structural protein (e.g., NSP4) of an RNA virus (e.g., alphavirus). In one example, the extended SG promoter is extended at its 5' end by nucleotides introduced into the sequence encoding alphavirus NSP4. The addition of nucleotides to the 5' end of the SG promoter sequence did not interfere with the expression of non-structural proteins and viral replicases, such as alphavirus NSP4.

[0228] In one example, the SG promoter is extended at its 5' end by 51 or fewer nucleotides occurring within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is a minimal SG promoter extended at its 5' end by 51 or fewer nucleotides occurring within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 51 or fewer nucleotides occurring within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 100 or fewer. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 2-101 of SEQ ID NO: 5.

[0229] In one example, the SG promoter is extended at its 5' end by approximately 5 to 20 nucleotides, for example, about 5 nucleotides, or about 10 nucleotides, or about 12 nucleotides, or about 15 nucleotides, or about 20 nucleotides, occurring within the sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the SG promoter is extended at its 5' end by approximately 20 to 35 nucleotides, for example, about 25 nucleotides, or about 27 nucleotides, or about 30 nucleotides, or about 35 nucleotides, occurring within the sequence encoding a non-structural protein (e.g., alphavirus NSP4).

[0230] In one example, the SG promoter is extended at its 5' end by approximately 12 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 12 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 61 or less. In one example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 41-101 of SEQ ID NO: 5. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 2.

[0231] In one example, the SG promoter is extended at its 5' end by approximately 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 80 or less. In one example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 22-101 of SEQ ID NO: 5. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 3.

[0232] For example, the extended SG promoter contains a repeating sequence corresponding to nucleotides 66-75 of SEQ ID NO: 5. For instance, the extended SG promoter is encoded by a sequence containing nucleotides 50-75 and nucleotides 66-101 of SEQ ID NO: 5. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 15.

[0233] For example, IRESs are derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-related protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF-B), Antennapedia, X-linked apoptosis inhibitor (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0234] For example, IRES is a wild-type IRES derived from encephalomyocarditis virus (EMCV). For instance, wild-type EMCV IRES contains the sequence shown in SEQ ID NO: 4.

[0235] In one example, the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, are codon-optimized.

[0236] In one example, the G / C content of the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, is modified.

[0237] For example, the G / C content of the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, increases by at least 5% compared to the G / C content of the unmodified sequence. For instance, the G / C content of the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, increases by at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to the G / C content of the unmodified sequence.

[0238] For example, a polynucleotide contains at least one chemically modified nucleotide.

[0239] For example, the chemically modified nucleotides are selected from the group consisting of N6,2'-O-dimethyladenosine (m6Am), 5-methyluridine (m5U), N4-acetylcytidine (ac4C), 2-thiocytidine (s2C), 2-thiouridine (s2U), 5-methylcytidine (m5C), N6-methyladenosine (m6a), pseudouridine (ψ), 1-methylpseudridine (m1ψ), and combinations thereof. For example, the chemically modified nucleotide is N6,2'-O-dimethyladenosine (m6Am). For example, the chemically modified nucleotide is 5-methyluridine (m5U). For example, the chemically modified nucleotide is N4-acetylcytidine (ac4C). For example, the chemically modified nucleotide is 2-thiocytidine (s2C). For example, a chemically modified nucleotide is 2-thiouridine (s2U). For example, a chemically modified nucleotide is 5-methylcytidine (m5C). For example, a chemically modified nucleotide is N6-methyladenosine (m6a). For example, a chemically modified nucleotide is pseudouridine (ψ). For example, a chemically modified nucleotide is 1-methylpseudridine (m1ψ).

[0240] In one example, the first nucleotide sequence includes haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha-6 collagen (Col6A), alpha-1-antitrypsin (SERPINA1), and the 5'-UTR of alpha-1-antichymotrypsin (SERPINA3), or its fragments and / or variants.

[0241] In one example, the 5'UTR is the 5'UTR of Venezuelan encephalitis virus (VEEV) or a modified form thereof. For example, this 5'UTR contains the sequence shown in SEQ ID NO: 13.

[0242] For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 40 to 2000. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 40 to 100. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 100 to 250. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 250 to 500. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 500 to 750. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 750 to 1000. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 1000 to 1250. For example, a 5'-UTR, its fragments, and / or variants have a nucleotide length of 1250 to 1500. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 1500 to 1750. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 1750 to 2000.

[0243] For example, a 5'UTR, its fragments, and / or variants contain a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in any one of sequence numbers 9-12. For instance, a 5'-UTR, its fragments, and / or variants contain a nucleotide sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in any one of sequence numbers 9-12.

[0244] In one example, a polynucleotide contains two or more 5'-UTRs, their fragments, and / or variants. In one example, the two or more 5'-UTRs are the same. In another example, the two or more 5'-UTRs are different.

[0245] In one example, a nucleotide sequence containing the 5'UTR includes at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, and combinations thereof. In one example, the nucleotide sequence includes a microRNA binding site. In one example, the nucleotide sequence includes an AU-rich element (ARE). In one example, the nucleotide includes a GC-rich element. In one example, the nucleotide sequence includes a stem-loop. For example, the stem-loop is a histone stem-loop.

[0246] In one example, a polynucleotide further comprises a nucleotide sequence containing a 3'UTR. In one example, the nucleotide sequence containing the 3'UTR is located at 3' of a second or one or more additional nucleotide sequences. For example, the nucleotide sequence containing the 3'UTR is located at 3' of a second nucleotide sequence. In one example, the 3'UTR includes the arachidonic acid 5-lipoxygenase (ALOX5), alpha-I collagen (COL1A1), tyrosine hydroxylase (TH) gene, the amino-terminal enhancer (AES) of the split, the 3'-UTR of human mitochondrial 12S rRNA (mtRNR1), fragments thereof, and / or variants.

[0247] For example, the 3'UTR is the 3'UTR of Sindbisvirus (SINV) or a modified form thereof. For instance, the 3'UTR contains the sequence shown in SEQ ID NO: 14 or 22.

[0248] For example, a 3'UTR, its fragments, and / or variants have a nucleotide length of 40 to 400. For instance, a 3'-UTR has a nucleotide length of 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300, or 300 to 325, or 325 to 350, or 350 to 375, or 375 to 400. For example, a 3'-UTR, its fragments, and / or variants have a nucleotide length of 40 to 50. For example, a 3'-UTR, its fragments, and / or variants have a nucleotide length of 50 to 60. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 60-70. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 70-80. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 80-90. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 90-100. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 100-125. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 125-150. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 150-175. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 175-200. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 200-225. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 225-250. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 250-275. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 275-300. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 300-325.For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 325 to 350. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 350 to 375. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 375 to 400.

[0249] In one example, a polynucleotide contains two or more 3'-UTRs, their fragments, and / or variants. In one example, the two or more 3'-UTRs are the same. In another example, the two or more 3'-UTRs are different.

[0250] In one example, a nucleotide sequence containing the 3'UTR, fragments thereof, and / or variants includes at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a triple helix, a stem-loop, one or more stop codons, and combinations thereof. In one example, the nucleotide sequence includes a microRNA binding site. In one example, the nucleotide sequence includes an AU-rich element (ARE). In one example, the nucleotide sequence includes a GC-rich element. In one example, the nucleotide sequence includes a triple helix. In one example, the nucleotide sequence includes a stem-loop. For example, the stem-loop is a histone stem-loop. In one example, the nucleotide sequence includes one or more stop codons. For example, one or more stop codons are located at the 5' end of the 3'-UTR.

[0251] In one example, a polynucleotide comprises a nucleotide sequence containing one or more 3' tailing sequences located at the 3' end of a nucleotide sequence containing a 3' UTR. In one example, one or more 3' tailing sequences are selected from the group consisting of poly-A sequences, polyadenylation signals, G quadruplexes, poly-C sequences, stem-loops, and combinations thereof. For example, the 3' tailing sequence contains a poly-A sequence. In one example, the 3' tailing sequence contains a polyadenylation signal. In one example, the 3' tailing sequence contains a G quadruplex. In one example, the 3' tailing sequence contains a poly-C sequence. In one example, the 3' tailing sequence contains a stem-loop. For example, the stem-loop is a histone stem-loop. In one example, the 3' tailing sequence contains a poly-A sequence and a G quadruplex. In one example, the 3' tailing sequence contains a stem-loop (e.g., a histone stem-loop) and a poly-A sequence.

[0252] For example, one or more 3' tailing sequences contain one or more polyA sequences, each containing 10 to 300 consecutive adenosine nucleotides. For example, each polyA sequence contains 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 10 to 20 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 20 to 30 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 30 to 40 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 36 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 40 to 50 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 50 to 60 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 60 to 70 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 70 to 80 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 80 to 90 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 90 to 100 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 100 to 125 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 125 to 150 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 150 to 175 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 175 to 200 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 200 to 225 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 225 to 250 consecutive adenosine nucleotides.For example, one or more polyA sequences each contain 250 to 275 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 275 to 300 consecutive adenosine nucleotides.

[0253] For example, one or more polyA sequences each contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 10 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 20 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 30 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 40 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 50 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 60 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 70 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 80 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 90 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 100 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 125 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 150 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 175 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 200 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 225 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 250 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 275 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 300 consecutive adenosine nucleotides.

[0254] For example, a polyA sequence contains 36 consecutive adenosine nucleotides. For instance, a polyA sequence includes the sequence shown in Sequence ID No. 16.

[0255] In one example, one or more polyA sequences are separated by a break linker. For example, a 3' tailing sequence may contain, from 5' to 3', a polyA sequence containing consecutive adenosine nucleotides, a break linker, and a further polyA sequence containing consecutive adenosine nucleotides.

[0256] For example, a break linker has a nucleotide length of 10-50, 50-100, or 100-150.

[0257] For example, a break linker has a nucleotide length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides. For example, a break linker has a nucleotide length of 1. For example, a break linker has a nucleotide length of 2. For example, a interrupted linker has a length of 3 nucleotides. For example, a interrupted linker has a length of 4 nucleotides. For example, a interrupted linker has a length of 5 nucleotides. For example, a interrupted linker has a length of 6 nucleotides. For example, a interrupted linker has a length of 7 nucleotides. For example, a interrupted linker has a length of 8 nucleotides. For example, a interrupted linker has a length of 9 nucleotides. For example, a interrupted linker has a length of 10 nucleotides. For example, a interrupted linker has a length of 11 nucleotides. For example, a interrupted linker has a length of 12 nucleotides. For example, a interrupted linker has a length of 13 nucleotides. For example, a interrupted linker has a length of 14 nucleotides. For example, a interrupted linker has a length of 15 nucleotides. For example, a interrupted linker has a length of 16 nucleotides. For example, a interrupted linker has a length of 17 nucleotides. For example, a interrupted linker has a length of 18 nucleotides. For example, a interrupted linker has a length of 19 nucleotides. For example, a interrupted linker has a length of 20 nucleotides. For example, a break linker has a length of 25 nucleotides. For example, a break linker has a length of 30 nucleotides. For example, a break linker has a length of 35 nucleotides. For example, a break linker has a length of 40 nucleotides. For example, a break linker has a length of 45 nucleotides. For example, a break linker has a length of 50 nucleotides.For example, a break linker has a length of 55 nucleotides. For example, a break linker has a length of 60 nucleotides. For example, a break linker has a length of 65 nucleotides. For example, a break linker has a length of 70 nucleotides. For example, a break linker has a length of 75 nucleotides. For example, a break linker has a length of 80 nucleotides. For example, a break linker has a length of 85 nucleotides. For example, a break linker has a length of 90 nucleotides. For example, a break linker has a length of 95 nucleotides. For example, a break linker has a length of 100 nucleotides. For example, a break linker has a length of 110 nucleotides. For example, a break linker has a length of 120 nucleotides. For example, a break linker has a length of 130 nucleotides. For example, a break linker has a length of 140 nucleotides. For example, a break linker has a length of 150 nucleotides.

[0258] In one example, the interrupted linker is 10 nucleotides long. In another example, the interrupted linker contains or consists of the nucleotide sequence shown in Sequence ID No. 8. For example, the interrupted linker contains or consists of the nucleotide sequence GCAUAUGACU.

[0259] In one example, the 3' tailing sequence contains, in order from 5' to 3', a polyA sequence containing 30 consecutive adenosine nucleotides, a 10-nucleotide interruption linker, and a further polyA sequence containing 70 consecutive adenosine nucleotides.

[0260] In one example, the 3' tailing sequence includes, in order from 5' to 3', a polyA sequence containing 30 consecutive adenosine nucleotides, a interruption linker containing or consisting of the nucleotide sequence shown in SEQ ID NO: 8, and a further polyA sequence containing 70 consecutive adenosine nucleotides.

[0261] For example, polynucleotides are arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0262] For example, polynucleotides are arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, d) A second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0263] In one example, RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0264] In one example, RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, d) A second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0265] In one example, the cRNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0266] In one example, the cRNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, d) A second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0267] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0268] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, d) A second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) A 3' tailing sequence selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0269] For example, the self-replicating RNA of this disclosure is in the order from 5' to 3', a) A first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the minimal SG promoter, and a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the minimal SG promoter, or b) A first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to an extended SG promoter, or c) comprising a first nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a wild-type EMCV IRES.

[0270] For example, the self-replicating RNA of this disclosure is in the order from 5' to 3', a) A first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably ligated to the minimal SG promoter, and a second nucleotide sequence encoding the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the minimal SG promoter, or b) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2 operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a spike (S) protein from an Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to an extended SG promoter, or c) comprising a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2 operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a spike (S) protein from an Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to a wild-type EMCV IRES.

[0271] In one example, the self-replicating RNA of the present disclosure comprises, in order from 5' to 3', a first nucleotide sequence encoding a spike (S) protein from an Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2 operably linked to a minimal SG promoter.

[0272] In one example, the self-replicating RNA of the present disclosure comprises, in order from 5' to 3', a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2 operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a spike (S) protein from an Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to a minimal SG promoter.

[0273] For example, the self-replicating RNA of the present disclosure comprises, in order from 5' to 3', a first nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a minimal SG promoter comprising the sequence shown in SEQ ID NO: 1, and a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to a minimal SG promoter comprising the sequence shown in SEQ ID NO: 1.

[0274] For example, the self-replicating RNA of the present disclosure comprises, in order from 5' to 3', a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to a minimal SG promoter comprising the sequence shown in SEQ ID NO: 1, and a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a minimal SG promoter comprising the sequence shown in SEQ ID NO: 1.

[0275] In one example, the self-replicating RNA of the present disclosure comprises, in order from 5' to 3', a first nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to an extended SG promoter.

[0276] In one example, the self-replicating RNA of the present disclosure comprises, in order from 5' to 3', a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an extended SG promoter.

[0277] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 1, and a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to an extended SG promoter encoded by the sequence shown in SEQ ID NO: 5.

[0278] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 1, and a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an extended SG promoter encoded by the sequence shown in SEQ ID NO: 5.

[0279] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a wild-type EMCV IRES.

[0280] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a wild-type EMCV IRES.

[0281] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 1, and a second nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a wild-type EMCV IRES encoded by the sequence shown in SEQ ID NO: 4.

[0282] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably ligated to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 1, and a second nucleotide sequence encoding a spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a wild-type EMCV IRES encoded by the sequence shown in SEQ ID NO: 4.

[0283] In one example, RNA further includes a 5' end cap structure.

[0284] For example, the 5' end cap structure is an endogenous cap or an analogue of it.

[0285] For example, the 5'-terminal cap structure contains guanine or a guanine analog.

[0286] For example, the 5'-terminal cap structure is selected from the group consisting of anti-reverse cap analog (ARCA), N7,2'-O-dimethyl-guanosine (mCAP), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), cap 1, and cap 2. For example, the 5'-terminal cap structure is anti-reverse cap analog (ARCA). For example, the 5'-terminal cap structure is N7,2'-O-dimethyl-guanosine (mCAP). For example, the 5'-terminal cap structure is inosine. For example, the 5'-terminal cap structure is N1-methyl-guanosine. For example, the 5'-terminal cap structure is 2'-fluoro-guanosine. For example, the 5' end cap structure is 7-deaza-guanosine. For example, the 5' end cap structure is 8-oxo-guanosine. For example, the 5' end cap structure is 2-amino-guanosine. For example, the 5' end cap structure is LNA-guanosine. For example, the 5' end cap structure is 2-azido-guanosine. For example, the 5' end cap structure is N6,2'-O-dimethyladenosine. For example, the 5' end cap structure is 7-methylguanosine (m7G). For example, the 5' end cap structure is cap 1. For example, the 5' end cap structure is cap 2.

[0287] For example, the 5' end cap structure is ligated to the 5' end of the RNA by a 5'-5'-triphosphate ligation or a 5'-5'-phosphorothioate ligation.

[0288] In one example, antigens (i.e., the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or the nucleocapsid (N) protein from SARS-CoV-2) are expressed at substantially the same level. For example, the antigens may have expression levels within approximately 10%, 5%, or 1% of each other. In another example, the antigens are expressed at different levels. For example, the antigens may have expression levels exceeding approximately 10%, 15%, or 20% of each other. Methods for determining the levels of expression are known in the art and / or described herein.

[0289] In one example, the self-replicating RNA is derived from an alphavirus. For instance, the alphavirus is selected from a group consisting of Semliki Forest Virus (SFV), Sindobis Virus (SIN), Venezuelan Encephalitis Virus (VEE), and combinations thereof.

[0290] In one example, the self-replicating RNA originates from the Semliki Forest Virus (SFV).

[0291] In one example, the self-replicating RNA originates from the Sindbis virus (SIN).

[0292] In one example, the self-replicating RNA originates from the Venezuelan encephalitis virus (VEE).

[0293] In one example, the S protein contains the mutation Q641R.

[0294] In one example, the antigen is from the S protein of an Omicron variant selected from the group consisting of B.1.1.529, BA.1, BA.2, BA.4, BA.5, BA.2.12.1, and BA.2.75. In one example, the antigen is from the S protein of an Omicron variant selected from the group consisting of B.1.1.529, BA.1 strain, BA.2 strain, BA.4 strain, BA.5 strain, BA.2.12.1, BA.2.75, XB strain, XBB strain, JN.1 strain, JN.2 strain, JN.3 strain, KP.1 strain, and KP.2 strain.

[0295] In one example, the antigen is from the S protein of Omicron variant BA.1, and the S protein encoded by the polynucleotide comprises one or more or all of the mutations selected from the group consisting of A67V, T95I, Y145D, L212L, S371L, G446S, G496S, T547K, N856K, L981F, G142D, Q493R, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and del69-70.

[0296] In one example, the antigen is from the S protein of Omicron variant BA.2, and the S protein encoded by the polynucleotide comprises one or more or all of the mutations selected from the group consisting of G142D, Q493R, del24-26, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S.

[0297] In one example, the antigen is derived from the S protein of omicron variant BA.4 or BA.5, and the polynucleotide-encoded S protein contains one or more or all of the mutations selected from the group consisting of L452R, F486V, R493Q, del24-26, del69-70, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S.

[0298] In one example, the antigen is derived from the S protein of an omicron variant of SARS-CoV-2, and the S protein contains one or more mutations selected from the group consisting of T19I, Δ24-26, A27S, Δ144, G142D, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, F486P, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K.

[0299] In one example, the antigen is derived from the S protein of the omicron variant of SARS-CoV-2, and the S proteins are T19I, Δ24-26, A27S, V83A, Δ144, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N It contains one or more or all of the mutations selected from the group consisting of R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K.

[0300] In one example, the antigen is derived from the S protein of an omicron variant of SARS-CoV-2, and the S proteins are 16insMPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, Δ144, G142D, F157S, R158G, Δ211, L212I, V213G, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D40 It contains one or all of the mutations selected from the group consisting of 5N, R408S, K417N, N440K, V445P, G446S, N450D, L452W, N460K, S477N, T478K, N481K, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, Q954H, N969K, and P1143L.

[0301] In one example, the antigen is derived from the S protein of an omicron variant of SARS-CoV-2, and the S protein contains one or more mutations selected from the group consisting of T19I, Δ24-26, A27S, Δ144, G142D, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, L455S, N460K, S477N, T478K, F486P, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K.

[0302] In one example, the antigen is derived from an S protein containing one or more or all of the following mutations: R346T, F456L, and T572I.

[0303] For example, the nucleotide sequence encoding the antigen from the S protein includes a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% relative to SEQ ID NO: 20.

[0304] For example, the nucleotide sequence encoding the antigen from the S protein includes the polynucleotide sequence shown in SEQ ID NO: 20.

[0305] For example, the S protein is encoded by a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, and at least approximately 99% of SEQ ID NO: 20.

[0306] For example, the S protein is encoded by the polynucleotide sequence shown in SEQ ID NO: 20.

[0307] For example, the nucleotide sequence encoding the antigen from the N protein includes a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% relative to SEQ ID NO: 21.

[0308] For example, the nucleotide sequence encoding the antigen from the N protein includes the polynucleotide sequence shown in SEQ ID NO: 21.

[0309] For example, the N protein is encoded by a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, and at least approximately 99% of SEQ ID NO: 21.

[0310] For example, the N protein is encoded by the polynucleotide sequence shown in SEQ ID NO: 21.

[0311] In one example, the self-replicating RNA contains or consists of the sequence specified by SEQ ID NO: 17. In another example, the self-replicating RNA contains or consists of the sequence specified by SEQ ID NO: 18. In yet another example, the self-replicating RNA contains or consists of the sequence specified by SEQ ID NO: 19.

[0312] This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure. This disclosure further provides immunogenic compositions comprising the RNA of this disclosure. For example, this disclosure provides immunogenic compositions comprising the cRNA of this disclosure. This disclosure also provides immunogenic compositions comprising the self-replicating RNA of this disclosure. For example, compositions of this disclosure can induce an immune response in a subject when administered. For example, administration of a composition induces a humoral and / or cell-mediated immune response. In one example, a composition induces a humoral immune response in a subject. For example, a humoral immune response is an antibody-mediated immune response. In another example, a composition induces a cell-mediated immune response. For example, a cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells.

[0313] In one example, the immunogenic composition of this disclosure comprises multiple polynucleotides, each polynucleotide encoding a different polypeptide antigen sequence. In another example, the immunogenic composition of this disclosure comprises multiple RNAs, each RNA encoding a different polypeptide antigen sequence. In yet another example, the immunogenic composition of this disclosure comprises multiple cRNAs, each cRNA encoding a different polypeptide antigen sequence. In one example, the immunogenic composition comprises multiple multicistronic self-replicating RNAs, each multicistronic self-replicating RNA encoding antigen polypeptides from different SARS-CoV-2 strains (e.g., the S protein from the Omicron strain of SARS-CoV-2 and the N protein from the Delta strain). In yet another example, the different polypeptide antigen sequences are from the same strain (e.g., both the S and N proteins from the Omicron strain of SARS-CoV-2). In yet another example, the immunogenic composition of this disclosure comprises multiple self-replicating RNAs, each self-replicating RNA encoding a different polypeptide antigen sequence. In another example, the immunogenic composition of this disclosure comprises multiple self-replicating RNAs, each self-replicating RNA encoding the same polypeptide antigen sequence.

[0314] This disclosure also provides a pharmaceutical composition comprising the immunogenic composition of this disclosure and a pharmaceutically acceptable carrier. Pharmacochemically acceptable carriers suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0315] In one example, the pharmaceutical composition further comprises lipid nanoparticles (LNPs), polymer microparticles, and an oil-in-water emulsion. For example, polynucleotides, RNA, cRNA, or self-replicating RNA are encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. In one example, polynucleotides are encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. In another example, RNA is encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. For example, cRNA is encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. For example, self-replicating RNA is encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion.

[0316] In one example, the pharmaceutical composition further comprises LNPs. For example, polynucleotides are encapsulated within the LNPs. In another example, RNA is encapsulated within the LNPs. For example, cRNA is encapsulated within the LNPs. For example, self-replicating RNA is encapsulated within the LNPs. For example, polynucleotides are bound to the LNPs. In another example, RNA is bound to the LNPs. For example, cRNA is bound to the LNPs. In yet another example, self-replicating RNA is bound to the LNPs. For example, polynucleotides are adsorbed onto the LNPs. In yet another example, RNA is adsorbed onto the LNPs. For example, cRNA is adsorbed onto the LNPs. In yet another example, self-replicating RNA is adsorbed onto the LNPs. In yet another example, each RNA is formulated together within the LNPs. In yet another example, each RNA is formulated separately within the LNPs.

[0317] In one example, LNP includes PEG lipids, structural lipids, and / or neutral lipids. For example, LNP includes PEG lipids, structural lipids, and neutral lipids. In another example, LNP includes PEG lipids, structural lipids, or neutral lipids.

[0318] In one example, the LNP further contains cationic lipids. In another example, the LNP does not contain cationic lipids.

[0319] In one example, the pharmaceutical composition further comprises polymer microparticles. For example, polynucleotides are encapsulated within the polymer microparticles. In another example, RNA is encapsulated within the polymer microparticles. For example, cRNA is encapsulated within the polymer microparticles. For example, self-replicating RNA is encapsulated within the polymer microparticles. For example, polynucleotides are bound to polymer microparticles. In another example, RNA is bound to polymer microparticles. For example, cRNA is bound to polymer microparticles. In yet another example, self-replicating RNA is bound to polymer microparticles. For example, polynucleotides are adsorbed onto polymer microparticles. In yet another example, RNA is adsorbed onto polymer microparticles. For example, cRNA is adsorbed onto polymer microparticles. In yet another example, self-replicating RNA is adsorbed onto polymer microparticles.

[0320] In one example, the pharmaceutical composition further comprises an oil-in-water emulsion. For example, polynucleotides are encapsulated in the oil-in-water emulsion. In another example, RNA is encapsulated in the oil-in-water emulsion. For example, cRNA is encapsulated in the oil-in-water emulsion. For example, self-replicating RNA is encapsulated in the oil-in-water emulsion. For example, polynucleotides are bound to the oil-in-water emulsion. In another example, RNA is bound to the oil-in-water emulsion. For example, cRNA is bound to the oil-in-water emulsion. In yet another example, self-replicating RNA is bound to the oil-in-water emulsion. In a further example, self-replicating RNA is adsorbed onto the oil-in-water emulsion. In a further example, self-replicating RNA is resuspended in the oil-in-water emulsion.

[0321] This disclosure also provides immunogenic compositions or pharmaceutical compositions of this disclosure for use as vaccines.

[0322] For example, a polynucleotide is DNA. For example, the Disclosure provides DNA encoding the cRNA vaccine of the Disclosure. For example, the Disclosure provides DNA encoding the self-replicating RNA vaccine of the Disclosure.

[0323] For example, DNA is a plasmid.

[0324] This disclosure provides a method for treating, preventing, or slowing the progression of a disease or condition in a subject, wherein the method comprises administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need. In one example, this disclosure provides a method for treating a disease or condition in a subject, wherein the method comprises administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need. In another example, this disclosure provides a method for preventing a disease or condition in a subject, wherein the method comprises administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need. In yet another example, this disclosure provides a method for slowing the progression of a disease or condition in a subject, wherein the method comprises administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need.

[0325] In one example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of a disease or condition in a subject that requires treatment or prevention of such disease or condition. For example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment of such disease or condition. In another example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of such disease or condition. In yet another example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for delaying the progression of a disease or condition in a subject that requires delaying the progression of such disease or condition.

[0326] In one example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for treating, preventing, or slowing the progression of a disease or condition in a subject that requires treatment or prevention of such disease or condition. For example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment of such disease or condition. In another example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of such disease or condition. In yet another example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for slowing the progression of a disease or condition in a subject that requires slowing the progression of such disease or condition.

[0327] In one example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for treating, preventing, or slowing the progression of a disease or condition in a subject that requires treatment or prevention or delay of its progression. For example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment or prevention or delay of its progression. In another example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of a disease or condition. In yet another example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for slowing the progression of a disease or condition in a subject that requires delay of its progression.

[0328] In one example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for treating, preventing, or slowing the progression of a disease or condition in a subject that requires treatment or prevention or delay of its progression. For example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment or prevention of a disease or condition. In another example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of a disease or condition. In yet another example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for slowing the progression of a disease or condition in a subject that requires delay of its progression.

[0329] In one example, the subject suffers from a disease or condition. In another example, the subject has been diagnosed with a disease or condition. In yet another example, the subject is receiving treatment for a disease or condition.

[0330] For example, the present disclosure provides a method for treating, preventing, or delaying the progression of SARS-CoV-2 infection in a subject requiring treatment, prevention, or delay of its progression, the method comprising administering RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to the subject.

[0331] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of SARS-CoV-2 infection in a subject.

[0332] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in the treatment, prevention, or delay of the progression of SARS-CoV-2 infection.

[0333] In one example, a subject with SARS-CoV-2 infection has at least one symptom of COVID-19. In one example, one such symptom of COVID-19 may include runny nose, cough, sore throat, fever, headache, muscle aches, or fatigue. In another example, at least one symptom is any one of the mild COVID-19 symptoms described herein. In yet another example, at least one symptom is any one of the moderate to severe COVID-19 symptoms described herein.

[0334] In one example, the subject is at risk of developing COVID-19 or SARS-CoV-2 infection. For example, the subject is at risk of developing COVID-19. In another example, the subject is at risk of developing SARS-CoV-2 infection.

[0335] In one example, the compositions of this disclosure are administered in an amount sufficient to reduce the severity of or prevent the onset of one or more symptoms of SARS-CoV-2 infection or COVID-19. The symptoms of SARS-CoV-2 infection or COVID-19 are obvious to those skilled in the art and / or described herein.

[0336] For example, the present disclosure provides a method for treating, preventing, or delaying the progression of COVID-19 in a subject requiring treatment, prevention, or delay of its progression, the method comprising administering RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to the subject.

[0337] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a medicine for treating, preventing, or delaying the progression of COVID-19 in a subject.

[0338] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in the treatment, prevention, or delay of the progression of COVID-19 in a subject.

[0339] For example, the present disclosure provides a method for inducing an immune response in a subject, comprising administering to a subject in need of such response RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0340] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a pharmaceutical product for inducing an immune response in subjects requiring induction of an immune response.

[0341] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in inducing an immune response in subjects requiring the induction of an immune response.

[0342] In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. For example, the production of neutralizing antibodies. In another example, the composition induces a cell-mediated immune response. For example, the cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells. For example, the T cells are CD4 T cells and / or CD8 T cells. In one example, the T cells are CD4 T cells. In another example, the T cells are CD8 T cells. In yet another example, the T cells are CD4 and CD8 T cells.

[0343] For example, administration of RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD4 T cell-mediated immune response.

[0344] For example, administration of RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein induces a CD8 T cell-mediated immune response.

[0345] For example, administration of RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD4 and CD8 T cell-mediated immune response.

[0346] In one example, the CD4 T cell-mediated immune response is a Th0, Th1, and / or Th2 response. For example, the CD4 T cell-mediated immune response is a Th0 response. In another example, the CD4 T cell-mediated immune response is a Th1 response. In yet another example, the CD4 T cell-mediated immune response is a Th2 response. In one example, the CD4 T cell-mediated immune response is a Th0 and Th1 response. In another example, the CD4 T cell-mediated immune response is a Th0 and Th2 response. In yet another example, the CD4 T cell-mediated immune response is a Th1 and Th2 response. In yet another example, the CD4 T cell-mediated immune response is a Th0, Th1, and Th2 response.

[0347] In one example, the Th0 response cytokine expresses interleukin-2 (IL2+) and / or tumor necrosis factor alpha (TNFa+), and / or is negative for interferon-gamma (IFNg-), IL5-, and / or IL13-. For example, the cytokine is IL2+. In another example, the cytokine is TNFa+. In one example, the cytokine is IFNg-. In yet another example, the cytokine is IL5-. In yet another example, the cytokine is IL13-.

[0348] In one example, the Th1 response cytokine expresses interferon-gamma (IFNg+) and / or is negative for IL5- and / or IL13-. For example, the cytokine is IFNg+. In another example, the cytokine is IL5-. In yet another example, the cytokine is IL13-.

[0349] In one example, the Th2-responsive cytokine expresses IL5+ and / or IL13+ and / or is negative for IFNg. For example, the cytokine is IL5+. In another example, the cytokine is IL13+. For example, the cytokine is IFNg-.

[0350] In one example, the immune response is elevated in response to the S protein from the Omicron strain of SARS-CoV-2 and the N protein from SARS-CoV-2, optionally the Omicron strain of SARS-CoV-2. In another example, the immune response is sufficient to treat, prevent, or delay the progression of at least one symptom of SARS-CoV-2 infection caused by the Omicron strain of SARS-CoV-2. In yet another example, the immune response is sufficient to treat, prevent, or delay the progression of at least one symptom of SARS-CoV-2 infection caused by the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2. In yet another example, a subject with SARS-CoV-2 infection has at least one symptom of COVID-19. In one example, one such symptom of COVID-19 may include runny nose, cough, sore throat, fever, headache, muscle aches, or fatigue. In another example, at least one symptom is one of the mild COVID-19 symptoms described herein. In yet another example, at least one symptom is one of the moderate to severe COVID-19 symptoms described herein.

[0351] For example, the present disclosure provides a method for reducing the SARS-CoV-2 viral load in a subject having COVID-19, comprising administering RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need thereof.

[0352] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a drug for reducing the SARS-CoV-2 viral load in a subject having COVID-19.

[0353] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in reducing the SARS-CoV-2 viral load in subjects having COVID-19.

[0354] For example, the present disclosure provides a method for treating, preventing, or delaying the progression of pneumonia in a subject having COVID-19, comprising administering RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need thereof.

[0355] For example, the present disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical product for treating, preventing, or delaying the progression of pneumonia in a subject having confirmed COVID-19.

[0356] For example, the present disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in treating, preventing, or delaying the progression of pneumonia in subjects having confirmed COVID-19.

[0357] For example, the present disclosure provides a method for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject having COVID-19, comprising administering RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, or a vaccine or immunogenic composition disclosed herein to a subject in need thereof.

[0358] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical product for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject having COVID-19.

[0359] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in treating, preventing, or delaying the progression of acute respiratory distress syndrome in subjects having COVID-19.

[0360] For example, the present disclosure provides a method for treating, preventing, or delaying the progression of sepsis in a subject having COVID-19, comprising administering RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need thereof.

[0361] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein in the preparation of pharmaceuticals for treating, preventing, or delaying the progression of sepsis in subjects having COVID-19.

[0362] For example, the present disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in treating, preventing, or delaying the progression of sepsis in subjects having COVID-19.

[0363] For example, the present disclosure provides a method for preventing or reducing death in a subject having COVID-19, comprising administering the RNA disclosed herein, the self-replicating RNA disclosed herein, the pharmaceutical composition disclosed herein, the immunogenic composition disclosed herein, or the vaccine disclosed herein to a subject in need thereof.

[0364] For example, this disclosure provides the use of RNA, self-replicating RNA, pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein in the preparation of pharmaceuticals for preventing or reducing death in subjects having COVID-19.

[0365] For example, this disclosure provides RNA, self-replicating RNA, pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for use in preventing or reducing death in subjects having COVID-19.

[0366] In one example, COVID-19 is caused by the Omicron strain of SARS-CoV-2. In another example, COVID-19 is caused by the delta, beta, alpha, or gamma strains of 2019-nCoV / USA-WA1 / 2020 SARS-CoV-2.

[0367] In one example, the subjects are people aged 18 and over. In another example, the subjects are people of any age, for example, from about 1 month to 100 years old, for example, from about 2 months to about 80 years old, from about 6 months to about 3 years old, from about 3 years to about 18 years old, from about 12 years to about 18 years old, from about 18 years to about 55 years old, from about 50 years to about 75 years old, and from about 40 years to about 65 years old. In yet another example, the subjects are people from 2 years old. In yet another example, the subjects are people from 18 years old, from 30 years old, from 40 years old, from 50 years old, from 60 years old, from 70 years old, from 80 years old, or from about 90 years old. In yet another example, the subjects are people under 2 years old, under 18 months old, under 12 months old, under 6 months old, or under 3 months old.

[0368] In one example, the compositions or vaccines described herein are administered in a single-dose regimen. In another example, the compositions are administered in two, three, or four-dose regimens, with doses administered at intervals of approximately one, two, or three months.

[0369] This disclosure also provides polynucleotides encoding the self-replicating RNA of this disclosure. For example, the polynucleotides are recombinant DNA.

[0370] For example, the polynucleotide contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in SEQ ID NO: 17.

[0371] For example, the polynucleotide contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in SEQ ID NO: 18.

[0372] For example, the polynucleotide contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in SEQ ID NO: 19.

[0373] The Disclosure also provides a kit comprising at least one self-replicating RNA of the Disclosure in an optional delivery system and / or a pharmaceutically acceptable carrier or diluent, packaged with instructions for use in treating, preventing or delaying the progression of a disease or disorder (e.g., SARS-CoV-2 infection, COVID-19, and / or ARDS) in a subject.

[0374] The Disclosure also provides a kit comprising at least one self-replicating RNA of the Disclosure in an optional delivery system and / or a pharmaceutically acceptable carrier or diluent, packaged with instructions for administering the RNA to subjects suffering from or at risk of suffering from a disease or disorder (e.g., SARS-CoV-2 infection, COVID-19, and / or ARDS).

[0375] In one example, the self-replicating RNA, RNA, immunogenic composition, or pharmaceutical composition of this disclosure is supplied in a vial. In another example, the RNA, self-replicating RNA, immunogenic composition, or pharmaceutical composition of this disclosure is supplied in a syringe.

[0376] Nothing included herein with respect to any documents, actions, materials, devices, articles, etc., should be construed as an admission that any or all of these matters constitute part of the basis of the prior art or were common knowledge in the art relating to this disclosure, because they existed prior to the priority date of each of the attached claims. [Brief explanation of the drawing]

[0377] [Figure 1]Design of sa-mRNA constructs: Co96 contains alphaviral self-replicating RNA containing polynucleotides encoding the spike (S) protein antigen from the Omicron strain of SARS-CoV-2. Co97 contains alphaviral self-replicating RNA containing polynucleotides encoding the S protein antigen and N protein antigen from the Omicron strain of SARS-CoV-2, driven by the synthetic genome promoter (SGP)v2. Co99 contains alphaviral self-replicating RNA containing polynucleotides encoding the N protein antigen and S protein antigen from the Omicron strain of SARS-CoV-2, driven by the synthetic genome promoter (SGP)v2. Co16 contains alphaviral self-replicating RNA containing polynucleotides encoding the S protein antigen from the original (Wuhan) strain of SARS-CoV-2. Co18 contains alphaviral self-replicating RNA containing polynucleotides encoding the S protein antigen and N protein antigen from the original (Wuhan) strain of SARS-CoV-2, driven by the synthetic genome promoter (SGP)v2. [Figure 2] Detection of SARS-CoV-2 N and S protein expression in vitro when expressed from sa-mRNA constructs Co96, Co97, Co99, Co18, Co16, and BHK-V. [Figure 3] Measurement of the specific potency of sa-mRNA constructs Co96, Co97, Co99, Co18, and Co16 in vitro, determined according to the dose of RNA (ng) when formulated into LNPs. [Figure 4] Effects of sa-mRNA constructs Co96, Co97, Co99, Co18, and Co16 on LV microneutralization titer, PV microneutralization titer, and in vitro ACE-2 binding inhibition. [Figure 5] Effects of sa-mRNA constructs Co96, Co97, Co99, Co18, and Co16 on LV microneutralization titer, PV microneutralization titer, and ACE-2 binding inhibition in vitro of BA.1, BA.2, and BA.4 / 5 variants of the Omicron strain of SARS-CoV-2.

[0378] [Table 1] [Modes for carrying out the invention]

[0379] overview Throughout this specification, unless otherwise specifically stated or the context requires, any reference to a single step, composition, group of steps, or group of compositions shall be construed as encompassing one or more (i.e., one or more) of those steps, compositions, groups of steps, or groups of compositions.

[0380] Those skilled in the art will understand that this disclosure is subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations or any two or more of such steps or features.

[0381] This disclosure is intended for illustrative purposes only and should not be limited in scope by the specific examples described herein. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0382] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specifically stated. In other words, any specific example in this disclosure may be combined with any other specific example in this disclosure (unless they are mutually exclusive).

[0383] Any example in this disclosure disclosing specific features or sets of features or methods or method steps would be construed as providing express support for rejecting those specific features or sets of features or methods or method steps.

[0384] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0385] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. This is described and explained through the literature provided by sources such as al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0386] The terms "and / or," for example, "X and / or Y," are understood to mean "X and Y" or "X or Y," and are considered to provide explicit support for both meanings or either of them.

[0387] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to mean including the element, element, or step, or group of elements, elements, or steps, that is described, but not to mean excluding any other element, element, or step, or group of elements, elements, or steps.

[0388] Where used herein, the term “derived from” shall be interpreted as indicating that a particular element may be derived from a particular source, but not necessarily directly from that source. Similarly, the term “based on” shall be interpreted as indicating that a particular element may be developed or used from a particular source, but not necessarily directly from that source.

[0389] Selected definition As used herein, the term “monocistronic” refers to an RNA encoding a single polypeptide, with respect to polynucleotides, RNA, cRNA, and / or self-replicating RNA.

[0390] As used herein, the term “multicistronic” (also known as “polycistronic”) refers to RNA encoding two or more polypeptides with respect to polynucleotides, RNA, cRNA, and / or self-replicating RNA. The term encompasses “bisistric” (or “disistric,” i.e., encoding two polypeptides) and “tricistric” (i.e., encoding three polypeptides) molecules. “Bicistronic” means a single nucleic acid capable of encoding two different polypeptides from different regions of the nucleic acid.

[0391] As used herein, the terms “conventional mRNA,” “cRNA,” or “non-amplified RNA” refer to RNA that is a construct enabling the expression of heterologous RNA and proteins, but which cannot be amplified within a host cell.

[0392] As used herein, the term “self-replicating RNA” refers to constructs based on RNA viruses that have been engineered to enable the expression of heterologous mRNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified in host cells, resulting in the expression of desired gene products in those host cells.

[0393] As used herein, the term “naked” refers to nucleic acids that are substantially free of other macromolecules, such as lipids, polymers, and proteins. “Naked” nucleic acids, such as self-replicating RNA, are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated in, adsorbed to, or bound to lipid nanoparticles (LNPs), liposomes, macromolecular microparticles, or oil-in-water emulsions.

[0394] As used herein, the terms “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of consecutive nucleotides (or bases) covalently linked to a phosphodiester backbone. Traditionally, sequences are presented from the 5' end to the 3' end unless otherwise specified. To facilitate a clear description of nucleic acids, specific sequence components are referred to, for example, “first nucleotide sequence” and “second nucleotide sequence.” The first and second sequences may appear in any desired order or orientation unless otherwise specified, and it should be understood that no particular order or orientation is intended by the terms “first,” “second,” etc.

[0395] As used herein, the term “antigen” refers to a molecule or structure containing one or more epitopes that induce, trigger, enhance, or boost a cellular and / or humoral immune response. Antigens may include proteins and peptides derived from pathogens such as viruses, bacteria, fungi, protozoa, plants, or tumors.

[0396] As used herein, the term “adjuvant” refers to a compound that, when used in combination with a specific immunogen (e.g., a VLP) in a formulation, enhances, or otherwise alters or modifies the resulting immune response. Modification of the immune response includes enhancing or expanding the specificity of either or both the antibody and / or cellular immune responses. Modification of the immune response can also mean reducing or suppressing a particular antigen-specific immune response.

[0397] As used herein, the term “operably linked to” means positioning a subgenome promoter or regulatory element (e.g., IRES) relative to a nucleic acid such that nucleic acid expression is controlled or regulated by the element. For example, a subgenome promoter can be operably linked to a number of nucleic acids via another regulatory element, such as an internal ribosome entry site (IRES).

[0398] As used herein, the term “subgenome promoter” (also known as “junction region” promoter) refers to a promoter that directs the expression of heterogeneous nucleotide sequences and regulates protein expression.

[0399] As used herein, the terms “internal ribosome entry site” or “IRES” refer to a sequence of nucleotides within mRNA to which a ribosome or its components, such as the 40S subunit of a ribosome, can bind. An IRES does not necessarily have to contain the nucleic acid that induces translation of the mRNA (e.g., the start codon; AUG).

[0400] The terms “polypeptide” or “polypeptide chain” will be understood to mean a sequence of amino acids linked by peptide bonds. For example, a protein will be interpreted as containing a single polypeptide chain, i.e., a sequence of amino acids linked by peptide bonds, or a sequence of polypeptide chains (i.e., polypeptide complexes) covalently or non-covalently linked to one another. A sequence of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0401] The term "recombinant" shall be understood to mean a product of artificial genetic modification.

[0402] As used herein, the term “substantially identical” with respect to levels of expression means, unless the context indicates otherwise, that the first and second antigens have (at least) levels of expression of each other that are approximately 10% or less of each other.

[0403] As used herein, the terms “disease,” “disorder,” or “condition” refer to the disruption or interference of normal function and include, but are not limited to, any specific condition.

[0404] As used herein, a subject who has developed or is “at risk” of developing SARS-CoV-2 infection may or may not have a detectable disease or symptoms of SARS-CoV-2 infection, and may or may not have shown a detectable disease or symptoms of SARS-CoV-2 infection prior to treatment according to this disclosure. “At risk” means that the subject has one or more risk factors which are measurable parameters known in the art and / or described herein that correlate with the development of SARS-CoV-2 infection.

[0405] As used herein, the terms “treatment” or “to treat” a subject include the application or administration of the compounds or compositions of the Disclosure to a subject (or the application or administration of the compounds of the Disclosure to cells or tissues from a subject) for the purpose of delaying, slowing, stabilizing, healing, curing, reducing, releasing, modifying, restoring, reducing, mitigating, improving, or affecting a disease or condition, the symptoms of a disease or condition, or the risk (or susceptibility to such risk) of a disease or condition. The terms “to treat” include any indication of successful treatment or mitigation of an injury, pathology or condition, including any objective or subjective parameters such as reduction, remission, a decrease in the rate of exacerbation, a decrease in the severity of the disease, stabilization, a reduction in symptoms, or making the injury, pathology or condition more tolerable to the subject, slowing the rate of degeneration or decline, or preventing the final stage of degeneration from becoming more debilitating.

[0406] As used herein, “prevention” or “prevention” means at least reducing the likelihood of acquiring a disease or disability (or susceptibility to it) (i.e., preventing the development of at least one of the clinical symptoms of the disease in a patient who may be exposed to the disease or susceptible to the disease but has not yet experienced or shown any symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known to physicians.

[0407] As used herein, the phrase "delay the progression of ~" includes reducing or delaying the progression of a disease or condition and / or at least one symptom of a disease or condition in an individual.

[0408] "Effective dose" refers to the minimum effective amount in the required dosage and duration to achieve the desired outcome. For example, the desired outcome may be a therapeutic or prophylactic outcome. An effective dose may be provided in one or more doses. In some examples of this disclosure, the term "effective dose" means the amount required to treat a disease or condition previously described herein. In some examples of this disclosure, the term "effective dose" means the amount required to make a change associated with a disease or condition previously described herein. The effective dose may vary depending on the disease or condition being treated or the factor being modified, and also depending on body weight, age, racial background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, the effective dose will fall within a relatively broad range (e.g., "dosage" range) that can be determined by healthcare professionals through routine testing and experimentation. Therefore, this term should not be construed as limiting this disclosure to a specific amount of RNA, e.g., weight or number. The effective dose may be administered in a single dose or in doses repeated once or several times over a period of treatment.

[0409] The "therapeutic dose" is the minimum concentration required to produce at least a measurable improvement in a particular disease or condition. The therapeutic dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the RNA disclosed herein to induce a desired response in the individual. The therapeutic dose is also defined as the therapeutically beneficial effect outweighing any toxic or adverse effects of the RNA.

[0410] As used herein, the term “preventive effective dose” shall be interpreted as meaning an amount of RNA of the Disclosure sufficient to prevent, inhibit, or delay the onset of one or more detectable symptoms of any disease or disorder described herein.

[0411] "Subjects" can also be any animal susceptible to infection with SARS-CoV-2. Subjects of the Disclosure may be mammals, and in certain embodiments may be humans, which may be infants, children, adults, or the elderly. "Subjects at risk of infection with SARS-CoV-2" or "Subjects at risk of SARS-CoV-2 infection" is any subject that has been or has been exposed to SARS-CoV-2. Subjects may also be primary contacts of individuals diagnosed with SARS-CoV-2 infection. "Subjects" include any human or non-human animals. Thus, in addition to their usefulness in human treatment, the compounds of the Disclosure may also be useful in the veterinary treatment of mammals, including companion animals and farm animals, not limited to dogs, cats, horses, cattle, sheep, and pigs.

[0412] As used herein, the terms “lipid nanoparticles” or “LNPs” are understood to mean lipid-based particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) and containing a compound of any of the formulas described herein. In embodiments, LNPs are formulated in compositions for the delivery of polynucleotides to desired targets, e.g., cells, tissues, organs, tumors, etc. For example, lipid nanoparticles or LNPs refer to any lipid composition and may be selected from, for example, liposomes or vesicles in which the aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; monolayer, or multiple; multilayer), micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles lacking a lipid bilayer.

[0413] Polynucleotides As used herein, the term “polynucleotide” refers to a molecular chain of nucleotides chemically linked by a series of esterifications (linkages) between the phosphoryl group of one nucleotide and the hydroxyl group of a sugar in an adjacent nucleotide. For example, a polynucleotide is DNA. For example, a polynucleotide is RNA, such as mRNA. For example, mRNA is conventional mRNA (cRNA) or self-replicating RNA.

[0414] As used herein, the term “fragment” refers to a portion of a nucleotide sequence or polypeptide of a reference nucleotide sequence or polypeptide disclosed herein that maintains the defined activity of the full-length nucleotide sequence or polypeptide.

[0415] As a non-limiting example, if conventional mRNA or self-replicating RNA contains polynucleotides encoding two or more antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), then these two or more antigens may be expressed by monocistronic polynucleotides, or each of the SARS-CoV-2 antigens may be expressed by polycistronic (or multicistronic) polynucleotides. For example, S and N protein antigens may be expressed by monocistronic polynucleotides or by polycistronic polynucleotides.

[0416] As used herein, the term “variant” refers to a nucleotide sequence having one or more substitutions, insertions, deletions, and / or other modifications compared to an unmodified sequence. It will be apparent to those skilled in the art that any variant described herein has the same or similar expression of the encoded protein. For example, a variant is a functional variant. Exemplary modifications to nucleotide sequences and / or polypeptides are apparent to those skilled in the art and / or are described herein.

[0417] In one example, modification is a chemical modification of one or more nucleotides in a nucleotide sequence. For instance, at least one naturally occurring nucleotide in a polynucleotide is replaced by a chemically modified nucleotide (e.g., pseudouridine (ψ) and 1-methylpseudridine (m1ψ)).

[0418] In one example, the modification involves increasing the G / C content of the nucleotide sequence.

[0419] In one example, the modification involves codon optimization of the nucleotide sequence.

[0420] In one example, the substitution is a conserved substitution. Those skilled in the art will understand that a conserved substitution in a polypeptide involves replacing an amino acid in the polypeptide with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). In another example, the substitution is a non-conservative substitution.

[0421] As used herein, the terms “encode,” “encodes,” or “the act of coding” refer to a region of polynucleotide that can be translated into a polypeptide.

[0422] The polynucleotides of this disclosure include DNA and RNA (e.g., mRNA).

[0423] Deoxyribonucleic acid (DNA) One example of a polynucleotide is DNA (for example, a DNA vector).

[0424] It will be apparent to those skilled in the art that the DNA of this disclosure further comprises an endonuclease restriction site at the 3' end of the 3' UTR. Those skilled in the art will understand that the endonuclease restriction site allows for the insertion of one or more nucleotide sequences (e.g., encoding the antigen of interest, a fragment thereof, and / or variants) without disrupting the rest of the DNA.

[0425] As used herein, the term “restriction endonuclease site” refers to a sequence of DNA that binds to a restriction endonuclease. Typically, a restriction endonuclease site is a short sequence (e.g., approximately 4–8 base pairs) that is recognized and cleaved by a restriction endonuclease.

[0426] As used herein, the terms “restriction enzyme” or “restriction endonuclease” refer to a class of enzymes naturally present in bacteria and some viruses. Restriction endonucleases specifically bind to double-stranded DNA at or adjacent to a restriction endonuclease site and cleave the double-stranded DNA. Examples of restrictive endonucleases include, for example, BciVI (Bful), Bcul (Spel), EcoRI, Aatll, AgeI (BshTI), Apal, BamHI, BglII, Blpl (Bpu1102I), BsrGI (Bsp1407), Clal (Bsu15I), EcoRI, EcoRV (Eco32I), Eam1104I (EarI), Hindlll, Kpnl, Mlul, Ncol, Ndel, Nhel, Notl, Nsil, Mph1103I), Pstl, Pvul, Pvull, SacI, SalI, ScaI, SpeI, Xbal, Xhol, Sacll (Cfr42I), and Xbal.

[0427] In one example, the present disclosure provides a transcribed polynucleotide comprising a first nucleotide sequence encoding a first antigen of interest, and / or a second nucleotide sequence encoding a second antigen of interest, operably ligated to a regulatory element such as an SG promoter and an IRES. For example, the polynucleotide is a DNA plasmid comprising the first and second nucleotide sequences.

[0428] In one example, the DNA contains a nucleotide sequence that includes a restriction endonuclease site located at 3' of the 3' UTR. The presence of the restriction endonuclease site at 3' of the 3' UTR enables the production of linearized DNA. Linearization of the DNA ensures the defined termination of the in vitro transcription DNA for mRNA production.

[0429] Ribonucleic acid (RNA) In one example, the polynucleotide is an mRNA containing a first nucleotide sequence encoding an antigen, operably linked to a promoter, and the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0430] The mRNAs in this disclosure include non-replicating mRNAs (conventionally also referred to as mRNA (cRNA) or non-amplified mRNAs) in addition to self-replicating RNAs (also known as self-amplified RNAs or sa-mRNAs).

[0431] Conventional (non-replicating) RNA In one example, the polynucleotide is a cRNA containing a first nucleotide sequence encoding an antigen, operably linked to a promoter, and the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0432] In one example, the cRNA of the Disclosure comprises, in 5' to 3' order, a 5' cap structure, a 5'-UTR, a fragment thereof and / or a variant, a first nucleotide sequence encoding a first antigen of interest, a second nucleotide sequence encoding a second antigen of interest, a 3'-UTR, and a 3' tailing sequence (e.g., a polyadenylation signal or one or more poly-A tails), where the first antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The cRNA of the Disclosure may further comprise an intratranslational ribosome entry site (e.g., a Kozak consensus sequence or IRES) operably linked to the antigen of interest.

[0433] self-replicating RNA This disclosure provides self-replicating RNA (also known as a replicon).

[0434] Those skilled in the art will understand that the self-replicating RNA of this disclosure is based on the genomic RNA of an RNA virus. The RNA should be a positive (+) strand and therefore can be directly translated after delivery to cells without requiring an intervening replication step (e.g., reverse transcription). Translation of the RNA results in the production of non-structural proteins (NSPs) that combine to form a replicase complex (i.e., RNA-dependent RNA polymerase). The complex then amplifies the original RNA, resulting in the production of multiple daughter RNAs that can be translated and transcribed, thereby enhancing overall protein expression.

[0435] For example, the self-replicating RNA of this disclosure includes non-structural proteins of an RNA virus, 5' and 3' untranslated regions (UTRs), and a natural subgenome promoter.

[0436] For example, self-replicating RNA contains one or more non-structural proteins of an RNA virus. For instance, RNA contains at least one gene selected from the group consisting of viral replicases (or viral polymerases), viral proteases, viral helicases, and other non-structural viral proteins. For example, self-replicating RNA contains viral replicases (or viral polymerases).

[0437] In another example, self-replicating RNA includes the 5' and 3' UTRs of an RNA virus. Those skilled in the art will see that the terms 5' and 3' UTR also encompass the terms 5' and 3' conserved sequence elements (CSEs). In one example, self-replicating RNA includes the 5' and 3' CSEs.

[0438] The self-replicating RNAs of this disclosure cannot induce the production of infectious viral particles. For example, the self-replicating RNAs of this disclosure do not contain viral genes that encode structural proteins necessary for the production of viral particles.

[0439] In one example, the self-replicating RNA is derived from or based on an alphavirus. Suitable alphaviruses will be obvious to those skilled in the art and / or are described herein.

[0440] In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus, such as a positive-strand RNA virus. Positive-strand RNA viruses suitable for use in this disclosure will be apparent to those skilled in the art and include, for example, picornaviruses, flaviviruses, rubiviruses, pestiviruses, hepaciviruses, caliciviruses, or coronaviruses.

[0441] In one example, the self-replicating RNA is encoded by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in Sequence ID No. 17.

[0442] In one example, the self-replicating RNA encodes at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in Sequence ID No. 18.

[0443] For example, the self-replicating RNA is encoded by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the same nucleotide sequence as shown in Sequence ID No. 19.

[0444] Alphavirus For example, the self-replicating RNA of this disclosure is derived from (or based on) an alphavirus.

[0445] Alphaviruses are the only genus in the Togaviridae family and are enveloped viruses with a positive sense single-stranded RNA genome. Those skilled in the art will understand that the alphavirus genome contains two open reading frames (ORFs), one non-structural and one structural. The first ORF encodes four non-structural proteins (NSP1, NSP2, NSP3, and NSP4) necessary for the transcription and replication of viral RNA. The second encodes three structural proteins that associate as heterodimers: the coanucleocapsid protein C, as well as envelope proteins P62 and E1. Viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells via membrane fusion.

[0446] In one example, the self-replicating RNA of this disclosure includes a viral replicase (or viral polymerase). For example, the viral replicase is an alphaviral replicase, such as the alphaviral protein NSP4.

[0447] For example, the self-replicating RNA of this disclosure does not encode one or more alphaviral structural proteins (e.g., capsid and / or envelope glycoproteins). For instance, the self-replicating RNA cannot produce RNA-containing alphaviral virions (i.e., infectious viral particles).

[0448] For example, self-replicating RNA contains a natural alphavirus SG promoter. For instance, the natural alphavirus SG promoter is a minimal SG promoter (i.e., the minimum sequence required for transcription initiation) and contains the sequence shown in Sequence ID No. 1.

[0449] Those skilled in the art will recognize alphaviruses suitable for use in this disclosure. Exemplary alphaviruses include, but are not limited to, Venezuelan horse encephalitis virus (VEE, e.g., Trinidadorova, TC83CR), Semlik Forest virus (SFV), Sindobis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mukambo virus, Burma Forest virus, Middelberg virus, Pixna virus, Onyonnyon virus, Geta virus, Sagiyama virus, Beval virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Bangbanki virus, Kyzilagachi virus, Highland J virus, Fort Morgan virus, Ndum virus, and Baggy Creek virus. The term alphavirus may also include chimeric alphaviruses that contain genome sequences from two or more alphaviruses (as described, for example, by Perri et al, (2003) J. Virol. 77(19):10394-403).

[0450] Adjustment element This disclosure relates to a polynucleotide comprising a nucleotide sequence encoding an antigen operably ligated to a promoter, wherein the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0451] In one example, the regulatory element is selected from a group consisting of a subgenome (SG) promoter, an internal ribosome entry site (IRES), and a Kozac consensus sequence, or a combination thereof. In one example, the regulatory element is the SG promoter.

[0452] Kozak Consensus Array As used herein, the term "Kozak consensus sequence" refers to a nucleotide sequence identified in a eukaryotic gene that facilitates gene translation by containing a start codon (also known as a translation start codon) that is recognized by a ribosome.

[0453] Exemplary Kozak consensus sequences are known in the art and / or described herein. In one example, the Kozak consensus sequence is shown in SEQ ID NO: 6 (GCCACC). In another example, the Kozak consensus sequence is shown in SEQ ID NO: 7 (ACCATGG).

[0454] Subgenome promoters Suitable SG promoters for use in this disclosure (also known as “junction area” promoters) will be obvious to those skilled in the art and / or are described herein.

[0455] For example, the SG promoter is derived from or based on the alphavirus SG promoter. For instance, the SG promoter is the natural alphavirus SG promoter. For example, the natural SG promoter is the minimal SG promoter. For instance, the minimal SG promoter is the minimum sequence required for transcription initiation. For example, the natural SG promoter is the extended SG promoter. For instance, the extended SG promoter is the minimal SG promoter extended at the 5' end by nucleotides generated in the sequence encoding a non-structural protein (e.g., NSP4) of an RNA virus (e.g., alphavirus). For example, the extended SG promoter is the minimal SG promoter extended at the 5' end by nucleotides generated in the sequence encoding alphavirus NSP4.

[0456] In one example, the SG promoter is extended at its 5' end by approximately 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 80 or less. In one example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 22-101 of SEQ ID NO: 5. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 3.

[0457] For example, the extended SG promoter contains a repeating sequence corresponding to nucleotides 66-75 of SEQ ID NO: 5. For instance, the extended SG promoter is encoded by a sequence containing nucleotides 50-75 and nucleotides 66-101 of SEQ ID NO: 5. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 15.

[0458] For example, the polynucleotides of this disclosure contain an SG promoter from any alphavirus. For example, the RNA (e.g., cRNA or self-replicating RNA) of this disclosure contains an SG promoter from any alphavirus.

[0459] In one example, the self-replicating RNA contains an SG promoter from any alphavirus.

[0460] The polynucleotides of this disclosure comprise two or more nucleotide sequences encoding two or more antigens of interest. In one example, each of the two or more nucleotide sequences is operably ligated to an SG promoter. If two or more SG promoters are present in the RNA of this disclosure, the promoters may be the same or different. For example, the two or more SG promoters may originate from the same alphavirus. In another example, the two or more SG promoters may originate from different alphaviruses.

[0461] If two or more SG promoters are present in the self-replicating RNA of this disclosure, the promoters may be the same or different. For example, two or more SG promoters may originate from the same alphavirus. In another example, two or more SG promoters may originate from different alphaviruses.

[0462] In another example, if the polynucleotide of the present disclosure comprises two or more nucleotide sequences encoding two or more antigens of interest, the two or more nucleotide sequences may be driven by the same promoter, or by two or more promoters which themselves may contain the same or different sequences.

[0463] Internal ribosome entry site (IRES) The IRESs suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0464] In one example, IRES originates from encephalomyocarditis virus (EMCV). For instance, IRES is a wild-type IRES derived from EMCV.

[0465] In one example, IRES is derived from fibroblast growth factor 1A (FGF1A)IRES.

[0466] In addition, synthetic IRES elements that can be designed according to methods known in the art to mimic the functions of naturally occurring IRES elements are described (see Chappell, SA et al. Proc. Natl Acad. Sci. USA (2000) 97(4):1536-41).

[0467] For example, IRESs are derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-related protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF-B), Antennapedia, X-linked apoptosis inhibitor (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0468] For example, IRES is a wild-type IRES derived from encephalomyocarditis virus (EMCV). For instance, wild-type EMCV IRES contains the sequence shown in SEQ ID NO: 4.

[0469] 5' Untranslated region (5'-UTR) For example, the polynucleotides described herein include a 5' untranslated region (5'-UTR).

[0470] As used herein, the terms “5'-untranslated region” or “5'-UTR” refer to the non-coding region of mRNA located at the 5' end of the translation initiation sequence (AUG).

[0471] Examples of 5'-UTRs include, for example, the 5'-UTRs of haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha-6 collagen (Col6A), alpha-1-antitrypsin (SERPINA1), alpha-1-antichymotrypsin (SERPINA3), and their fragments and / or variants.

[0472] In one example, the 5'UTR is the 5'UTR of Venezuelan encephalitis virus (VEEV) or a modified form thereof. For example, this 5'UTR contains the sequence shown in SEQ ID NO: 13.

[0473] In one example, the 5'UTR includes at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, and combinations thereof.

[0474] microRNA binding site As used herein, the term “microRNA binding site” refers to a sequence within a polyn reoside (e.g., within a DNA or RNA transcript) that is sufficiently complementary to all or one region of a microRNA (miRNA) that interacts with, associates with, or binds to the miRNA.

[0475] As used herein, the terms “microRNA” or “miRNA” refer to a 19-25 nucleotide-length non-coding RNA that binds to the 5'UTR of a polynucleotide and downregulates gene expression (e.g., by inhibiting translation). The presence of microRNA binding sites(s) in the 5'UTR of this disclosure can function to inhibit translation of the 5'UTR.

[0476] Suitable miRNA binding sites for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0477] For example, miRNA binding sites include binding sites for tissue-specific microRNAs or microRNAs that regulate biological processes. These include miRNAs from liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). For example, microRNAs that regulate biological processes such as angiogenesis (miR-132). Further exemplary miRNAs and miRNA-binding sites are disclosed in U.S. Patent Application No. 14 / 043,927.

[0478] AU Rich Element (ARE) As used herein, the terms “AU-rich element (ARE)” or “AU-rich element (ARE)” refer to a region of nucleotide sequence containing stretches of adeonysin (A) and uridine (U). Exemplary AREs include, for example, AREs from cytoplasmic myc (c-myc), myoblast-determining protein 1 (myoD), c-Jun, myogenin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α), or combinations thereof.

[0479] For example, ARE contains a human antigen R (HuR, also known as Elavl1) specific binding site. HuR is known to bind to ARE and enhance mRNA stability.

[0480] GC Rich Element As used herein, the term “GC-rich element” refers to a nucleotide sequence having a larger amount of guanine (G) and / or cytosine (C) compared to adenine (A) and thymine (T) / uracil (U). The presence of GC-rich elements in polynucleotides (e.g., mRNA) can stabilize the mRNA.

[0481] For example, a GC-rich element contains a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0482] For example, a GC-rich element contains 30%-40%, 40%-50%, 50%-60%, or 60%-70% cytosine. For example, a GC-rich element contains 30%-40% cytosine. For example, a GC-rich element contains 40%-50% cytosine. For example, a GC-rich element contains 50%-60% cytosine. For example, a GC-rich element contains 60%-70% cytosine.

[0483] For example, a GC-rich element contains 30%, 40%, 50%, 60%, or 70% cytosine. For example, a GC-rich element contains 30% cytosine. For example, a GC-rich element contains 40% cytosine. For example, a GC-rich element contains 50% cytosine. For example, a GC-rich element contains 60% cytosine. For example, a GC-rich element contains 60% cytosine. For example, a GC-rich element contains 70% cytosine.

[0484] For example, a GC-rich element is at least 50% cytosine.

[0485] For example, a GC-rich element is at least 60% cytosine.

[0486] For example, a GC-rich element is at least 70% cytosine.

[0487] In one example, the GC-rich element contains the nucleotide sequence CCCCGGCGCC. In another example, the GC-rich element contains the nucleotide sequence CCCCGGC. In yet another example, the GC-rich element contains the nucleotide sequence GGCCCCGCGGCGCCCCGCG.

[0488] In one example, the GC-rich element contains the nucleotide sequence shown in SEQ ID NOs: 9-11. In another example, the GC-rich element contains the nucleotide sequence shown in SEQ ID NO: 9. In yet another example, the GC-rich element contains the nucleotide sequence shown in SEQ ID NO: 10. In yet another example, the GC-rich element contains the nucleotide sequence (CCCCGGC) shown in SEQ ID NO: 11.

[0489] stem loop As used herein, the term “stem-loop” refers to a nucleotide sequence containing an intramolecular base pairing of two adjacent complete or partial reverse-complementary sequences that form a stem-loop. Stem-loops can occur in single-stranded DNA or, more commonly, in RNA. Stem-loops can also be referred to as hairpins or hairpin loops, which typically consist of a stem and a terminal loop in a contiguous sequence, with the stem being formed by two adjacent complete or partial reverse-complementary sequences separated by a shorter sequence that constructs the loop into a stem-loop structure.

[0490] The stability of a paired stem-loop is determined by its length, the number of mismatches or bulges it contains, and the nucleotide composition of the paired region.

[0491] For example, the loop in a stem-loop has a length of 3 to 10 nucleotides. For instance, the loop in a stem-loop has a length of 3 to 8, or 3 to 7, or 3 to 6, or 4 to 5 nucleotides.

[0492] In one example, the loop in a stem-loop has a length of 4 nucleotides.

[0493] For example, a stem-loop is a histone stem-loop. For instance, a histone stem-loop contains or consists of the nucleotide sequence shown in Sequence ID No. 12.

[0494] 3' Untranslated region (3'-UTR) For example, the polynucleotides of this disclosure include a 3'-untranslated region (3'-UTR).

[0495] As used herein, the term "3'-UTR" refers to the region of mRNA located 3' to the translation termination codon (i.e., stop codon).

[0496] Examples of 3'-UTRs include, for example, the 3'-UTRs of arachidonic acid 5-lipoxygenase (ALOX5), alpha-I collagen (COL1A1), tyrosine hydroxylase (TH) genes, split amino-terminal enhancers (AES), human mitochondrial 12S rRNA (mtRNR1), fragments thereof, and / or variants.

[0497] For example, the 3'UTR is the 3'UTR of Sindbisvirus (SINV) or a modified form thereof. For instance, the 3'UTR contains the sequence shown in SEQ ID NO: 14 or 22.

[0498] For example, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the albumin gene. For example, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the vertebrate α-globin gene. For instance, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the mammalian α-globin gene. For instance, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the human α-globin gene.

[0499] In one example, the 3'-UTR of this disclosure further comprises at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a triple helix, a stem-loop, one or more stop codons, or a combination thereof.

[0500] Stop codon As used herein, the term “stop codon” refers to a trinucleotide sequence within mRNA that signals the termination of protein synthesis by ribosomes.

[0501] For example, the polynucleotides of this disclosure include at least one stop codon at the 5' end of the 3'-UTR. For example, the stop codon is selected from UAG, UAA, and UGA.

[0502] For example, a polynucleotide contains two consecutive stop codons with the sequence UGAUGA.

[0503] For example, a polynucleotide contains two consecutive stop codons with the sequence UAAUAG.

[0504] 3' Tailing arrangement For example, the polynucleotides of this disclosure include one or more 3' tailing sequences located on the 3' side of the 3' UTR.

[0505] As used herein, the terms “3' tailing sequence” or “3' tailing sequence(plural)” refer to a nucleotide sequence that induces the addition of a non-coding nucleotide to the 3' end of mRNA (e.g., a polyadenylation signal), or a nucleotide sequence located at the 3' end of mRNA (e.g., a poly-A sequence). Those skilled in the art will understand that 3' tailing sequences and / or products of 3' tailing sequences in mRNA function to stabilize mRNA and / or prevent mRNA degradation.

[0506] As used herein, the term “interrupting linker” in relation to polyA or polyC sequences in this disclosure refers to a single nucleotide or nucleotide sequence that links to and interrupts a stretch of consecutive adenosine or cytosine nucleotides in a polyA or polyC sequence. For example, an interrupting linker in a polyA sequence is a single nucleotide or nucleotide sequence consisting of or containing a nucleotide other than an adenosine nucleotide. For example, an interrupting linker in a polyC sequence is a single nucleotide or nucleotide sequence consisting of or containing a nucleotide other than a cytosine nucleotide.

[0507] In one example, one or more 3' tailing sequences are selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0508] Poly-A array As used herein, the term “polyA sequence” refers to the adenine (A) nucleotide sequence located at the 3' end of mRNA. In the context of this disclosure, the polyA sequence may be located within mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating mRNA by transcription of a vector).

[0509] Suitable polyA sequences for use in this disclosure will be obvious to those skilled in the art and / or are described herein. In one example, the polyA sequence comprises a sequence of adenosine nucleotides of any length (e.g., 10 to 300). In another example, the polyA sequence comprises a sequence of adenosine nucleotides separated by one or more interrupting linkers. In yet another example, the polyA sequence comprises a sequence of adenosine nucleotides without interrupting linkers.

[0510] Polyadenylation signal As used herein, the term “polyadenylation signal” refers to a nucleotide sequence that induces polyadenylation. Polyadenylation is typically understood as the addition of a polyA sequence to RNA (e.g., to immature mRNA to produce mature mRNA). The polyadenylation signal may be located within the nucleotide sequence at the 3' end of the polynucleotide being polyadenylated (e.g., mRNA).

[0511] Suitable polyadenylation signals for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0512] In one example, the polyadenylation signal contains a hexamer composed of adenine and uracil / thymidine nucleotides. In another example, the hexamer sequence contains or consists of AAUAAA.

[0513] In one example, the 3' tailing sequence contains a polyadenylation signal but does not contain a poly(A) sequence.

[0514] G-quadruplex As used herein, the terms “G quadruple” or “G4” refer to nucleotide sequences rich in guanine residues that form a quadruple-stranded secondary structure. For example, a G quadruple is a cyclic hydrogen-bonded array of four guanine nucleotides formed by a G-rich sequence in both DNA and RNA.

[0515] In one example, the 3' tailing sequence contains a poly(A) sequence and a G quadruplex. For instance, the 3' tailing sequence contains a poly(A) sequence linked to a G quadruplex to produce a poly(AG) quartet.

[0516] PolyC sequence As used herein, the term “polyC sequence” refers to the cytosine (C) nucleotide sequence located at the 3' end of mRNA. In the context of this disclosure, the polyC sequence may be located within mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating mRNA by vector transcription).

[0517] Suitable polyC sequences for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0518] For example, one or more 3' tailing sequences contain one or more polyC sequences, each containing 10 to 300 consecutive cytosine nucleotides. For example, one or more polyC sequences each contain 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300 consecutive cytosine nucleotides. For example, one or more polyC sequences each contain 10, or 20, or 30, or 40, or 50, or 60, or 70, or 80, or 90, or 100, or 125, or 150, or 175, or 200, or 225, or 250, or 275, or 300 consecutive cytosine nucleotides.

[0519] In one example, one or more polyC sequences are separated by interrupting linkers. For instance, a fourth nucleotide sequence containing one or more 3' tailing sequences may contain, from 5' to 3', consecutive cytosine nucleotides, interrupting linkers, and further consecutive cytosine nucleotides.

[0520] In one example, the interrupted linker has a nucleotide length of 10–50, 50–100, or 100–150. For example, a break linker has a nucleotide length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides.

[0521] 5' cap structure In one example, this disclosure provides mRNA containing a 5' terminal cap structure.

[0522] As used herein, the term “5' cap structure” refers to a structure at the 5' end of mRNA involved in nuclear export that binds to mRNA cap-binding protein (CBP). The 5' cap structure is known to stabilize mRNA through association with CBP and poly(A)-binding protein, thereby forming mature mRNA. Therefore, the presence of a 5' cap structure in the mRNA of this disclosure can further increase the stability of the mRNA compared to mRNA without a 5' cap.

[0523] Examples of 5' cap structures include, for example, anti-reverse cap analogues (ARCA), N7,2'-O-dimethyl-guanosine (mCAP), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), cap 1, and cap 2.

[0524] Typically, endogenous mRNA is 5'-capped with guanosine via a (5)'-ppp-(5)'-triphosphate ligation attached to the 5' terminal nucleotide of the mRNA. The guanosine cap can then be methylated to 7-methylguanosine (m7G) to produce 7mG(5')ppp(5')N,pN2p (cap 0 structure), where N represents the first and second 5' terminal nucleotides of the mRNA. The cap 0 structure can be further 2'-O-methylated to produce 7mG(5')ppp(5')NlmpNp (cap 1) and / or 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[0525] In one example, the polynucleotides of this disclosure include an endogenous cap.

[0526] As used herein, the term “endogenous cap” refers to a 5' cap synthesized in a cell. For example, the endogenous cap is a natural 5' cap or a wild-type 5' cap. For example, the endogenous cap is a cap 0, cap 1, or cap 2 structure.

[0527] For example, the polynucleotides of this disclosure include analogues of endogenous caps (also referred to as cap analogues).

[0528] As used herein, the terms "endogenous cap" or "its analogue" in the context of "cap analogue" refer to a synthetic 5' cap. Cap analogues can be used to produce 5'-capped mRNA in in vitro transcription reactions. Cap analogues can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or ligated to nucleotides (e.g., the 5' terminal nucleotide of mRNA). Exemplary cap analogues are commercially available and include, for example, 3″-O-Me-m7G(5′)ppp(5′)G, G(5′)ppp(5′)A, G(5′)ppp(5′)G, m7G(5′)ppp(5′)A, and m7G(5′)ppp(5′)G (New England BioLabs). One example of a cap analogue is N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (i.e., anti-reverse cap analogue (ARCA)).

[0529] In one example, a 5' cap structure is a non-hydrolyzable cap structure. A non-hydrolyzable cap structure can prevent mRNA decapping and increase the mRNA half-life.

[0530] In one example, the non-hydrolyzable cap structure contains a modified nucleotide selected from the group consisting of α-thio-guanosine nucleotides, α-methyl-phosphonates, seleno-phosphates, and combinations thereof. In one example, the modified nucleotide is ligated to the 5' end of the mRNA via an α-phosphorothieate linkage. The method of ligating the modified nucleotide to the 5' end of the mRNA will be obvious to those skilled in the art. For example, a Vaccina Capping Enzyme (New England Biolabs) is used.

[0531] qualification In one example, the polynucleotides of this disclosure include one or more modifications. Typically, the modifications are introduced into a polynucleotide (e.g., mRNA) to increase the translation efficiency and / or stability of the polynucleotide. Suitable modifications to polynucleotides are obvious to those skilled in the art and / or are described herein.

[0532] In one example, a first nucleotide sequence containing the 5'-UTR and / or a fragment thereof is modified. The modification of the first nucleotide sequence containing the 5'-UTR and / or a fragment thereof results in a variant of the 5'-UTR and / or a fragment thereof.

[0533] In one example, one or more nucleotide sequences of a polynucleotide are codon-optimized. Methods of codon optimization are apparent to those skilled in the art and / or are described herein. For example, tools for codon optimization of polynucleotides include, for example, GeneArt GeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).

[0534] In one example, a polynucleotide is modified to increase the amount of guanine (G) and / or cytosine (C) in the polynucleotide. The amount of G / C in a polynucleotide (i.e., the G / C content) can affect the stability of the polynucleotide. Therefore, a polynucleotide containing an increased amount of G / C nucleotides can be functionally more stable than a polynucleotide containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. The G / C content is increased by substituting A or T nucleotides with G or C nucleotides.

[0535] For example, the G / C content increases in the first and / or second nucleotide sequences encoding the first and / or second antigen of interest. For instance, the G / C content increases in the first and / or second nucleotide sequences encoding the S protein and / or N protein antigen. Modifications in the first and / or second nucleotide sequences, and / or one or more nucleotide sequences, utilize the ability to substitute codons containing less desirable combinations of nucleotides (from the standpoint of mRNA stability) with alternative codons encoding the same amino acid or amino acid(s) with similar chemistry (e.g., conserved amino acid substitutions). For example, the G / C content increases by substituting codons containing A or T nucleotides with codons containing G or C nucleotides encoding the same amino acid.

[0536] For example, the G / C content increases in one or more nucleotide sequences of polynucleotides that do not code for the target antigen. For instance, the G / C content increases in the 5'-UTR, its fragments, and / or variants. For instance, the G / C content increases in the 3'-UTR, its fragments, and / or variants.

[0537] For example, a polynucleotide contains at least one chemically modified nucleotide.

[0538] As used herein, the terms “chemically modified” or “chemically modified” in the context of nucleotides refer to naturally occurring nucleotides (i.e., A, T, C, G, U) that are modified by the substitution, insertion, or removal of individual or several atoms or atomic groups compared to naturally occurring nucleotides. For example, at least one naturally occurring nucleotide in a polynucleotide is replaced by a chemically modified nucleotide. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the naturally occurring nucleotides in a polynucleotide are replaced by chemically modified nucleotides. Chemically modified nucleotides suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein. Examples of chemically modified nucleotides include, for example, N6,2'-O-dimethyladenosine (m6Am), 5-methyluridine (m5U), N4-acetylcytidine (ac4C), 2-thiocytidine (s2C), 2-thiouridine (s2U), 5-methylcytidine (m5C), N6-methyladenosine (m6a), pseudouridine (ψ), and 1-methylpseudridine (m1ψ).

[0539] SARS-CoV-2 antigen The polynucleotides of this disclosure comprise a nucleotide sequence encoding an antigen, operably linked to a promoter, wherein the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a further example, the polynucleotides of this disclosure comprise a nucleotide sequence encoding a second antigen, operably linked to a promoter, wherein the antigen is the nucleocapsid (N) protein from SARS-CoV-2. Thus, in the context of this disclosure, the antigen is a pathogenic antigen. For example, the antigen of interest is an antigen polypeptide, its immunogenic fragment, and / or variant that can induce an immune response in a subject.

[0540] The SARS-CoV-2 genome encodes at least four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, as well as other accessory proteins that assist the replication process and facilitate entry into cells. The M protein is the most abundant component of the viral envelope and directs the assembly of the coronavirus through interaction with all other structural proteins. The E protein is a small membrane protein or biloporin that is thought to facilitate the budding of viral particles by pinching the cell membrane surface. The S protein is the class I fusion protein of SARS-CoV-2 that mediates the attachment of the virus to the major cell surface receptor, human angiotensin-converting enzyme 2 (ACE2). Due to its conformation exposed to the surface of the virus, the S protein is highly immunogenic and is the main focus of current vaccine development. The N protein packages the RNA genome to form the nucleocapsid and, although not necessarily required for envelope formation, appears to play a crucial role in the assembly and stability of the complete virion, as well as in enhancing VLP yield.

[0541] The S protein contains three domains: (i) a large ectodomain, (ii) a transmembrane domain (which traverses the viral envelope in a single path), and (iii) a short intracellular tail. The ectodomain consists of a trimer stem made up of three receptor-binding subunits (3×S1) and three membrane-fusion subunits (3×S2). Thus, the SARS-CoV-2 S protein is a homotrimer. During viral entry, S1 binds to receptors on the host cell surface for viral attachment, and S2 fuses with the host and viral membranes, allowing the viral genome to enter the host cell. Receptor binding and membrane fusion are the first and crucial steps in the coronavirus infection cycle. There is significant dissociation in receptors targeted by different CoVs.

[0542] The structure of the SARS-CoV-2S protein is described, for example, in Cai et al. (Science (2020) 369:1586-1592), which is incorporated herein by reference in its entirety. Each S1 subunit of the SARS-CoV-2S protein contains an N-terminal domain (NTD), a receptor-binding domain (RBD), and two C-terminal domains (CTD). Before fusion with the host cell membrane, the S1 subunit of the SARS-CoV-2S protein protects the S2 subunit. Upon binding to ACE2, the SARS-CoV-2S protein refolds in a "jackknife" fashion, forming a long, coiled coil at its center, ultimately leading to membrane fusion and viral entry into the host cell.

[0543] Considering the mutational tendencies of RNA viruses such as SARS-CoV-2, the present inventors provide a polynucleotide encoding an S protein that may contain mutations found in different strains of SARS-CoV-2, and as a result, the vaccine composition finds particular utility in the treatment of target strains of SARS-CoV-2, such as the Omicron strain of SARS-CoV-2.

[0544] For example, the polynucleotides of this disclosure encode an S protein containing a mutation from the Omicron strain of SARS-CoV-2. For example, if the Omicron strain is variant BA.1, the polynucleotide encodes an S protein containing one or more or all of the mutations selected from the group consisting of A67V, T95I, Y145D, L212L, S371L, G446S, G496S, T547K, N856K, L981F, G142D, Q493R, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and del69-70.

[0545] In another example, if the Omicron strain is variant BA.2, the polynucleotide encodes an S protein containing one or more or all of the mutations selected from the group consisting of G142D, Q493R, del24-26, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S.

[0546] In another example, if the Omicron strain is variant BA.4 or BA.5, the polynucleotide encodes an S protein containing one or more or all of the mutations selected from the group consisting of L452R, F486V, R493Q, del24-26, del69-70, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S.

[0547] The mRNA, conventional mRNA, self-replicating RNA, compositions, and vaccines disclosed herein may also be useful for treating other variants of SARS-CoV-2. In particular, the mRNA, conventional mRNA, self-replicating RNA, compositions, and vaccines of this disclosure find useful in the treatment of the B.1.1.7 SARS-CoV-2 strain (also known as 201 / 501Y.Vl, first detected in the United Kingdom, and now known as the alpha variant), the B.1.351 SARS-CoV-2 strain (also known as 20H / 501.V2, first detected in South Africa, and now known as the beta variant), the Pl SARS-CoV-2 strain (also known as 20J / 501Y.V3, first detected in Japan and Brazil, and now known as the gamma variant), the Bl.427 and Bl.429 SARS-CoV-2 strains (first detected in California, and now known as the epsilon variant), and / or the B.1.617.2 SARS-CoV-2 strain (first detected in India, and now known as the delta variant). The mRNA, conventional mRNA, self-replicating RNA, compositions, and vaccines disclosed herein may also be useful in treating the Wuhan (original) strain of SARS-CoV-2.

[0548] According to the CDC (SARS-CoV-2 Variant Classification and Definition (cdc.govl)), the alpha variant includes the following mutations in the S protein: 69 deletion, 70 deletion, 144 deletion, (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T7161, S982A, D1118H, and (K1191N*), and is the main variant. Key mutations found are deletions of residues 69 / 70 and 144Y, as well as substitutions of N501Y, A570D, D614G, and P681H. Beta variants include the following mutations: D80A, D215G, 241 deletion, 242 deletion, 243 deletion, K417N, E484K, N501Y, D614G, and A701V, with the major mutation being K417N. The E484K, N501Y, and D614G substitutions have been found. The gamma variants include the following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T10271, with the major mutations being E484K, K417N / T, N501Y, and D614G. The delta variants include the following mutations: T19R, (G142D*), 156 deletion, 157 deletion, R158G, L452R, T478K, D614G, P681R, and D950N, with the major mutations being L452R, E484Q, and T478K. The epsilon variants include the following mutations: S131, W152C, 30 L452R, and D614G, with the major mutation being L452R. Accordingly, this disclosure encompasses self-replicating RNA containing polynucleotides encoding antigens from the S protein, which include one or more of the above mutations.

[0549] For example, the nucleotide sequence encoding the antigen from the S protein includes a polynucleotide sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the S protein nucleotide sequence defined in SEQ ID NO: 17, or is identical to it.

[0550] For example, the nucleotide sequence encoding the antigen from the S protein includes a polynucleotide sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the S protein nucleotide sequence defined in SEQ ID NO: 18, or is identical to it.

[0551] For example, the nucleotide sequence encoding the antigen from the S protein includes a polynucleotide sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the S protein nucleotide sequence defined in SEQ ID NO: 19, or is identical to it.

[0552] In one example, the antigen from the S protein is encoded by a polynucleotide sequence that contains or is identical to at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of the S protein nucleotide sequence defined in SEQ ID NO: 17. In this example, the encoded sequence is self-replicating RNA.

[0553] In one example, the antigen from the S protein is encoded by a polynucleotide sequence that contains or is identical to at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of the S protein nucleotide sequence defined in SEQ ID NO: 18. In this example, the encoded sequence is self-replicating RNA.

[0554] In one example, the antigen from the S protein is encoded by a polynucleotide sequence that contains or is identical to at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of the S protein nucleotide sequence defined in SEQ ID NO: 19. In this example, the encoded sequence is self-replicating RNA.

[0555] For example, the S protein is encoded by a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, and at least approximately 99% of SEQ ID NO: 20.

[0556] For example, the S protein is encoded by the polynucleotide sequence shown in SEQ ID NO: 20.

[0557] In one example, the polynucleotide encodes an antigen from the S and N proteins of SARS-CoV-2, respectively. In another example, the polynucleotide encodes an antigen from the S and N proteins of the Omicron strain of SARS-CoV-2, respectively. In yet another example, the Omicron variant may be BA.1 or BA.2.

[0558] In one example, the polynucleotide encoding the second antigen could be from the delta, beta, alpha, gamma, or other strains of SARS-CoV-2, or from the SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020. In another example, none of the antigens are from the delta, beta, alpha, or gamma strains of SARS-CoV-2, or from the SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020. Therefore, in one example, each of the S and N antigens is from the SARS-CoV-2 Omicron strain.

[0559] For example, the S protein from the Omicron strain of SARS-CoV-2 is derived from the following: (a) The omicron variant BA.1 and S protein contain one or more of the mutations selected from the group consisting of A67V, T95I, Y145D, L212L, S371L, G446S, G496S, T547K, N856K, L981F, G142D, Q493R, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and del69-70, or (b) The omicron variant BA.2 and S protein contain one or more of the mutations selected from the group consisting of G142D, Q493R, del24-26, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S, or (c) The omicron variant BA.4 or BA.5 and S protein contain one or more mutations selected from the group consisting of L452R, F486V, R493Q, del24-26, del69-70, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S, or (d) The omicron variant and S protein of SARS-CoV-2 contain one or more mutations selected from the group consisting of T19I, Δ24-26, A27S, Δ144, G142D, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, F486P, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K, or (e) SARS-CoV-2 omicron variants and S proteins include T19I, Δ24-26, A27S, V83A, Δ144, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, It contains one or all of the mutations selected from the group consisting of N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K, or (f) SARS-CoV-2 omicron variants and S proteins are 16insMPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, Δ144, G142D, F157S, R158G, Δ211, L212I, V213G, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417 It contains one or all of the mutations selected from the group consisting of N, N440K, V445P, G446S, N450D, L452W, N460K, S477N, T478K, N481K, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, Q954H, N969K, and P1143L, or (g) The omicron variant and S protein of SARS-CoV-2 contain one or more mutations selected from the group consisting of T19I, Δ24-26, A27S, Δ144, G142D, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, L455S, N460K, S477N, T478K, F486P, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K, or (h) The omicron variant and S protein of SARS-CoV-2 contain one or more of the R346T, F456L, and T572I mutations.

[0560] For example, the nucleotide sequence encoding an antigen from the N protein includes a polynucleotide sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the N protein nucleotide sequence defined in SEQ ID NO: 18, or is identical to it.

[0561] For example, the nucleotide sequence encoding an antigen from the N protein includes a polynucleotide sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the N protein nucleotide sequence defined in SEQ ID NO: 19, or is identical to it.

[0562] In one example, the antigen from the N protein is encoded by a polynucleotide sequence that contains or is identical to at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of the N protein nucleotide sequence defined in SEQ ID NO: 18. In this example, the encoded sequence is self-replicating RNA.

[0563] In one example, the antigen from the N protein is encoded by a polynucleotide sequence that contains or is identical to at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of the N protein nucleotide sequence defined in SEQ ID NO: 19. In this example, the encoded sequence is self-replicating RNA.

[0564] For example, the N protein is encoded by a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, and at least approximately 99% of SEQ ID NO: 21.

[0565] For example, the N protein is encoded by the polynucleotide sequence shown in SEQ ID NO: 21.

[0566] In one example, the polynucleotide encoding the second antigen from the N protein is from the Omicron strain of SARS-CoV-2.

[0567] Method of production Preferred methods for producing the polynucleotides, cRNAs, and / or self-replicating RNAs of this disclosure will be obvious to those skilled in the art and / or are described herein.

[0568] For example, polynucleotides are DNA. For instance, a polynucleotide is plasmid DNA.

[0569] In one example, cRNA is produced using plasmid DNA. In another example, self-replicating RNA is produced using plasmid DNA. Those skilled in the art will understand that plasmid DNA is relatively stable. Briefly, competent bacterial cells (e.g., Escherichia coli) are transformed with a DNA plasmid encoding the self-replicating RNA of this disclosure. Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in an E. coli culture.

[0570] In one example, plasmid DNA is isolated after fermentation. For example, plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other routine methods known to those skilled in the art. After isolation, plasmid DNA is linearized by restriction digestion (i.e., using restriction enzymes). The restriction enzymes are removed using methods known in the art, including, for example, phenol / chloroform extraction and ethanol precipitation.

[0571] In one example, mRNA is prepared by in vitro transcription from a linearized DNA template using RNA polymerase (e.g., T7 RNA polymerase). After in vitro transcription, the DNA template is removed by DNase digestion. Those skilled in the art will understand that synthetic mRNA capping is performed to correct mRNA processing and contribute to mRNA stabilization. In one example, mRNA is enzymatically 5' capped. For example, the 5' cap is either a cap 0 structure or a cap 1 structure. In one example, the 5' cap is a cap 0 structure, for example, the 5'-cap (i.e., cap 0) consists of inverted 7-methylguanosine attached to the remainder of the mRNA via a 5'-5' triphosphate crosslink. In another example, the 5' cap is a cap 1 structure, for example, the 5'-cap (i.e., cap 1) consists of cap 0 having an additional methylation at the 2'O position of the start nucleotide.

[0572] In one example, mRNA is purified. Various methods for purifying mRNA will be apparent to those skilled in the art. For example, mRNA is purified using lithium chloride (LiCl) precipitation. In another example, mRNA is purified using tangential flow filtration (TFF). After purification, the mRNA is resuspended, for example, in nuclease-free water.

[0573] composition This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure.

[0574] This disclosure also provides immunogenic compositions comprising the cRNA of this disclosure.

[0575] This disclosure further provides immunogenic compositions comprising the self-replicating RNA of this disclosure.

[0576] This disclosure also provides a pharmaceutical composition comprising the immunogenic composition of this disclosure and a pharmaceutically acceptable carrier.

[0577] It will be apparent to those skilled in the art, and / or as described herein, that the polynucleotides, cRNAs, and / or self-replicating RNAs of this disclosure may exist as naked RNA or in combination with lipids, polymers, or other delivery systems that facilitate entry into cells.

[0578] Delivery system For example, the pharmaceutical composition of the present disclosure further comprises LNPs, polymer microparticles, and an oil-in-water emulsion. For instance, polynucleotides, cRNAs, and / or self-replicating RNAs are encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, or the oil-in-water emulsion.

[0579] Lipid nanoparticles In one example, the pharmaceutical composition of this disclosure further comprises LNP.

[0580] The terms “lipid nanoparticles” or “LNPs” refer to any lipid composition, and it will be clear that they include, for example, liposomes or vesicles in which the aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; monolayer, or multiple; multilayer), micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles lacking a lipid bilayer.

[0581] Lipid nanoparticles suitable for use in this disclosure will be obvious to those skilled in the art and / or described herein. Lipids may have anionic, cationic, or zwitterionic hydrophilic head groups.

[0582] In one example, the lipid nanoparticles include PEG lipids, sterol-structured lipids, and / or neutral lipids. In another example, the lipid nanoparticles further include cationic lipids. In yet another example, the lipid nanoparticles do not include cationic lipids.

[0583] For example, LNPs include PEG lipids. For instance, PEG lipids are selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0584] For example, LNPs include structural lipids. Structural lipids are selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol, as well as combinations thereof.

[0585] For example, LNPs include neutral lipids. Examples of phospholipids (anionic or zwitterionic) for use in this disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. For example, neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Choline (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanol-sn-glycero-3-phosphoethanolamine (ME 16.0 Selected from the group consisting of PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin, as well as combinations thereof.

[0586] For example, LNPs include cationic lipids. Examples of cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), and 2,5-bis((9z,12z)-octadeca-9,12,diene-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). One example of a phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,diene-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphocholine. Lipids can be saturated or unsaturated.

[0587] Polymer microparticles In one example, the pharmaceutical composition of this disclosure further comprises polymer microparticles.

[0588] Those skilled in the art will recognize that various polymers can form microparticles for encapsulating or adsorbing the polynucleotides, cRNAs, and / or self-replicating RNAs of this disclosure. The use of substantially non-toxic polymers will obviously mean that the particles are safe, and the use of biodegradable polymers will obviously mean that the particles can be metabolized after delivery to avoid long-term persistence. Useful polymers are also sterilizable to aid in the preparation of pharmaceutical-grade formulations.

[0589] Examples of non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxybutyrate, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinylpyrrolidinone or polyesteramides, and combinations thereof.

[0590] Oil-in-water cationic emulsion For example, the pharmaceutical composition of this disclosure further comprises an oil-in-water cationic emulsion.

[0591] Suitable oils for use in oil-in-water emulsions will be obvious to those skilled in the art and / or described herein. For example, an emulsion may contain one or more oils derived from, for example, animals (e.g., fish) or plant sources (e.g., nuts, seeds, grains). Those skilled in the art will recognize that biocompatible and biodegradable oils are preferred. Exemplary animal oils (i.e., fish oils) include cod liver oil, shark liver oil, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, and corn oil.

[0592] In addition to oils, oil-in-water emulsions also contain cationic lipids that promote emulsion formation and stabilization. Suitable cationic lipids are obvious to those skilled in the art and / or are described herein. Exemplary cationic lipids include, but are not limited to, 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl[C16:0]trimethylammoniumpropane (DPTAP), and distearoyltrimethylammoniumpropane (DSTAP).

[0593] In some examples, oil-in-water emulsions also include nonionic surfactants and / or zwitterionic surfactants. Those skilled in the art will recognize surfactants suitable for use in this disclosure. Exemplary surfactants include, but are not limited to, polyoxyethylene sorbitan surfactants (e.g., polysorbate 20 and polysorbate 80), as well as copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0594] Pharmacologically acceptable carriers Preferably, in a composition or method for administering the cRNA and / or self-replicating RNA of the present disclosure, the cRNA and / or self-replicating RNA is combined with a pharmaceutically acceptable carrier as understood in the art. Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the self-replicating RNA of the present disclosure (and an optional delivery system) combined with a pharmaceutically acceptable carrier. Another example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the cRNA of the present disclosure (and an optional delivery system) combined with a pharmaceutically acceptable carrier.

[0595] Generally speaking, "carrier" means any solid or liquid filler, binder, diluent, encapsulant, emulsifier, wetting agent, solvent, suspension agent, coating, or lubricant that can be safely administered to any target, such as a human. Depending on the specific route of administration, a variety of acceptable carriers known in the art may be used, as described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0596] The cRNAs and / or self-replicating RNAs of this disclosure are useful for prophylactic or therapeutic treatment by parenteral, topical, oral, or local, intramuscular, aerosol, or transdermal administration. In one example, the self-replicating RNA is administered parenterally, such as intramuscular, subcutaneous, or intravenous. For example, the self-replicating RNA is administered intramuscularly. In another example, the cRNA is administered parenterally, such as intramuscular, subcutaneous, or intravenous. For example, the cRNA is administered intramuscularly.

[0597] The formulation of the administered cRNA and / or self-replicating RNA will vary depending on the chosen route of administration and formulation (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition containing the administered cRNA and / or self-replicating RNA can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, for example, those containing saline and a buffer medium. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, Ringer's lactate solution, or fixative oil. Various suitable aqueous carriers, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine, are known to those skilled in the art. Intravenous vehicles may contain additives, preservatives, or fluids, nutrients, or electrolyte replacements (see, in general, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition may optionally contain pH adjusters and buffers, as well as pharmaceutically acceptable auxiliary substances necessary to approximate the physiological state, such as toxicity adjusters, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. cRNA and / or self-replicating RNA can be stored in liquid form or lyophilized for storage and reconstituted in a suitable carrier before use according to lyophilization and reconstitution techniques known in the art.

[0598] The optimal concentration of the active ingredient(s) in the selected culture medium can be determined empirically according to procedures known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0599] When formulated, the compositions of this disclosure will be administered in a manner compatible with the administered formulation and in a therapeutically / prophylactically effective amount. The dosage range for the administration of the cRNA and / or self-replicating RNA of this disclosure is large enough to produce the desired effect. For example, a composition contains an effective amount of self-replicating RNA. In one example, a composition contains a therapeutically effective amount of self-replicating RNA. In another example, a composition contains a prophylactically effective amount of self-replicating RNA. In one example, a composition contains an effective amount of cRNA. In one example, a composition contains a therapeutically effective amount of cRNA. In another example, a composition contains a prophylactically effective amount of cRNA.

[0600] The dosage should not be so high as to cause adverse side effects. Generally, the dosage varies depending on the patient's age, condition, sex, and the severity of the disease, and can be determined by those skilled in the art. The dosage may be adjusted by the individual physician if complications occur.

[0601] The dosage can vary over a day or several days, administered at least once a day, from approximately 0.1 mg / kg to approximately 300 mg / kg, for example, from approximately 0.2 mg / kg to approximately 200 mg / kg, for example, from approximately 0.5 mg / kg to approximately 20 mg / kg.

[0602] In some cases, cRNA and / or self-replicating RNA are administered at an initial (or loading) dose higher than the subsequent (maintenance) dose. For example, cRNA and / or self-replicating RNA are administered at an initial dose of approximately 10 mg / kg to approximately 30 mg / kg. Then, cRNA and / or self-replicating RNA are administered at a maintenance dose of approximately 0.0001 mg / kg to approximately 10 mg / kg. The maintenance dose may be administered every 7 to 35 days, for example, every 7, 14, or 28 days.

[0603] In some cases, a dose-escalation regime is used, in which cRNA and / or self-replicating RNA are initially administered at a lower dose than that used in subsequent doses. This dose regime is useful when subjects experience adverse events early on.

[0604] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, increased doses may be administered.

[0605] The subjects may be re-treated with the cRNA and / or self-replicating RNA of this disclosure. The subjects may be re-treated with the cRNA and / or self-replicating RNA by being given two or more sets of exposures or doses, such as at least about two exposures of the binding protein, e.g., about 2 to 60 exposures, more specifically about 2 to 40 exposures, most specifically about 2 to 20 exposures.

[0606] For example, optional retreatment may be given when signs or symptoms of the disease return.

[0607] In another example, any retreatment may be given at defined intervals. For instance, subsequent exposures may be administered at various intervals, such as approximately 24–28 weeks, 48–56 weeks, or longer. For example, such exposures may be administered at intervals of approximately 24–26 weeks, 38–42 weeks, or 50–54 weeks, respectively.

[0608] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, increased doses may be administered.

[0609] In another example, for subjects experiencing adverse reactions, the initial (or loading) dose may be divided over several days of the week or over a number of consecutive days.

[0610] The administration of cRNA and / or self-replicating RNA by the method of this disclosure may be continuous or intermittent, depending, for example, the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. The administration of cRNA and / or self-replicating RNA may be essentially continuous over a pre-selected period, or it may be a series of intervald doses, for example, either during or after the onset of a condition.

[0611] Screening assay Preferred methods for selecting cRNA and / or self-replicating RNA of this disclosure are available to those skilled in the art. Assays may be performed to evaluate the efficiency and effectiveness of RNA, including, for example, serological and immune responses.

[0612] antigen expression In one example, self-replicating RNA is evaluated for the expression of a target polynucleotide(s) (i.e., a polynucleotide encoding the S protein antigen from the SARS-CoV-2 Omicron strain). In another example, cRNA is evaluated for the expression of a target polynucleotide(s) (i.e., a polynucleotide encoding the S protein antigen from the SARS-CoV-2 Omicron strain).

[0613] For example, antigen expression is detected using antibodies against the target polynucleotide(s). In one example, the number of cells positive for antigen expression is measured, for example, by fluorescence-activated cell sorting (FACS). In another example, mean fluorescence intensity (MFI) is determined, for example, using FACS. In yet another example, a specific potency value or transfection success rate per unit mass of RNA is calculated.

[0614] Microneutralization assay In one example, self-replicating RNA (naked and / or formulated) is evaluated for antibody response. In another example, cRNA (naked and / or formulated) is evaluated for antibody response. For example, cRNA and / or self-replicating RNA are evaluated using a microneutralization assay. The method for performing a microneutralization assay will be obvious to those skilled in the art. In one example, the microneutralization assay is a shortened assay. In one example, a viral fluorescence focus-based microneutralization assay is performed. In another example, the microneutralization assay is a long-term assay.

[0615] Antigen-specific T cell response In one example, self-replicating RNA is evaluated for its ability to induce an antigen-specific T cell response. In another example, cRNA is evaluated for its ability to induce an antigen-specific T cell response. Methods for evaluating the induction of an antigen-specific T cell response are apparent to those skilled in the art and / or are described herein.

[0616] For example, antigen-specific T cell detection is performed in spleen cultures. Briefly, the spleen cell culture is established in T cell medium, and the cell culture is either stimulated with the antigen peptide or not. In one example, the antigen-specific T cell response is determined using flow cytometry.

[0617] Neutralization assay The self-replicating RNAs of this disclosure can be screened in vitro for their ability to bind to the SARS-CoV-2 S protein and neutralize the binding of the S protein to ACE2. Suitable assays will be apparent to those skilled in the art and include, for example, the Vero microneutralization assay, the sVNT assay, or the pseudoviral neutralization assay (e.g., using HEK-293T cells or HeLa-ACE2 cells).

[0618] In one example, the neutralization assay is the Vero microneutralization assay. Briefly, SARS-CoV-2 wild-type virus is passaged in Vero cells (i.e., a Vero strain isolated from kidney epithelial cells extracted from African green monkeys). Two-fold serial dilutions of the test protein are passed over 1 hour at 100 TCID. 50 SARS-CoV-2 (i.e., the median infectious dose for tissue culture) is incubated with Vero cells, and residual viral infectivity is evaluated in Vero cells, for example, on day 5, to determine the viral cytotoxic effect. Neutralizing antibody titers are calculated using the Reed / Muench method as described above (Houser et al., 2016, Subbarao et al. 2004).

[0619] In one example, the neutralization assay is a surrogate neutralization test (sVNT). Briefly, plate wells are coated with hACE2 protein in a carbonate-bicarbonate coated buffer (e.g., pH 9.6). HRP-conjugated SARS-CoV-2 and HRP-conjugated SARS-CoV-2 pre-incubated with the test protein are added to hACE2 at different concentrations and incubated, for example, at room temperature for 1 hour. Unbound HRP-conjugated antigen is removed by washing. The colorimetric signal is developed by an enzymatic reaction of HRP with a chromogenic substrate, e.g., 3,3',5,5'-tetramethylbenzidine (TMB). In one example, absorbance readings at 450 nm and 570 nm are obtained.

[0620] In one example, neutralization is a pseudoviral neutralization assay. Briefly, an HIV reporter virus pseudotyped with the SARS-CoV-2 S protein is produced by co-transfection of the SARS-2-CoV-2 spike plasmid, together with a viral backbone plasmid (e.g., pDR-NL Δenv FLUC), into, for example, HEK-293T cells. The pseudovirus is collected after transfection and clarified by filtration. The viral stock titer, reported as the relative luciferase unit infectious dose (RLU), is calculated by limiting dilution infection in Hela-hACE2 cells, measuring luciferase activity as a readout of viral infection.

[0621] Methods of treatment or prevention This disclosure provides methods for treating, preventing, or delaying the progression of, for example, COVID-19, caused by SARS-CoV-2. This disclosure also provides methods for treating, preventing, or delaying the progression of, for example, SARS-CoV-2 infection. In some examples of this disclosure, subjects have SARS-CoV-2 infection but do not have clinically diagnosed COVID-19. Therefore, in one example, a subject may exhibit one or more symptoms of SARS-CoV-2 infection, but COVID-19 is not yet clinically detectable.

[0622] Coronavirus disease 2019 (COVID-19) This disclosure provides methods for treating, preventing, or delaying the progression of COVID-19 or SARS-CoV-2 infection in subjects.

[0623] COVID-19 is an infectious disease caused by SARS-CoV-2. Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. The majority of cases present with mild symptoms, but some may develop ARDS or one or more complications, including pneumonia or sepsis. Therefore, this disclosure is intended to treat, prevent or delay the progression of mild or moderate to severe COVID-19, and includes treating, preventing or delaying the progression of ARDS, pneumonia, or sepsis in subjects.

[0624] Mild COVID-19 may include symptoms such as fever, cough, fatigue, shortness of breath, and loss of smell and taste. Specifically, mild COVID-19 is defined as a SARS-CoV-2 positive RT-PCR or molecular test result and one of the following symptoms: - Fever, - Sore throat, -headache, - Muscle pain (muscle soreness), -Gastrointestinal symptoms, -cough, - A tightness in the chest, -snot, -wheezing, -Skin rash, - Eye irritation or discharge, -cold, - New or altered olfactory or gustatory disorders, - Redness or bruising of the feet or toes, - Chills or shivers, - Fatigue (loss of appetite, generally feeling unwell, tired, decreased physical strength).

[0625] The case meets the above definition of a case, but is moderate to If the definition of severe / critical is not met, it is considered mild.

[0626] Moderate COVID-19 may be defined as a SARS-CoV-2 positive RT-PCR or molecular test result and any one of the following new or worsening signs or symptoms: -20 breaths / minute or more, -The oxygen saturation (SpO2) in the indoor air at sea level is abnormal, but it is still over 93%. - Clinical or radiological evidence of pneumonia, -Radiological evidence of DVT, -Shortness of breath or difficulty breathing, Or any two of the following new or worsening signs or symptoms: - Fever, -Heart rate of 90 beats / min or more, - Chills or shivers, - New or altered olfactory or gustatory disorders, - Sore throat, - Fatigue, -headache, -cough, - Muscle pain (muscle soreness), -Gastrointestinal symptoms, - Redness or bruising on the feet or toes.

[0627] Severe / critical COVID-19 is defined as a SARS-CoV-2 positive RT-PCR or molecular test result, and one or more of the following: - Resting clinical signs indicating severe systemic disease (respiratory rate 2: 30 breaths / min, heart rate 2 > 125 beats / min, SpO2 < 93% of room air at sea level, or PaO2 / FiO2 < 300 mmHg), - Respiratory failure (defined as requiring high-flow oxygen, non-invasive ventilation, mechanical ventilation, or ECMO [extracorporeal membrane oxygenation]), - Evidence of shock (defined as systolic blood pressure below 90 mmHg, diastolic blood pressure below 60 mmHg, or requiring vascular pressurizers), - Marked acute renal dysfunction, hepatic dysfunction, or neurological dysfunction, - Admission to the ICU, -death.

[0628] The time from exposure to symptom onset is typically about 5 days, but can range from 2 to 14 days. Complications of SARS-CoV-2 infection can include viral pneumonia, secondary bacterial pneumonia, sinus infections, and exacerbation of pre-existing health conditions such as asthma or heart failure. Viral pneumonia can also lead to acute respiratory distress syndrome (ARDS).

[0629] Therefore, in some examples of this disclosure, the methods or uses of the disclosure can be used to treat, prevent or delay the progression of ARDS in subjects infected with COVID-19. In one example, subjects are at risk of having COVID-19 caused by SARS-CoV-2. In one example, the methods of the disclosure can be used to treat ARDS in subjects infected with SARS-CoV-2. In one example, the methods of the disclosure can be used to prevent ARDS in subjects infected with SARS-CoV-2. In one example, the methods of the disclosure can be used to delay the progression of ARDS in subjects infected with SARS-CoV-2.

[0630] The subjects requiring this may be individuals exhibiting symptoms of SARS-CoV-2 infection, or diagnosed with SARS-CoV-2 infection, and / or having COVID-19. Furthermore, the subjects requiring this may be those clinically or biochemically determined to be infected with SARS-CoV-2 infection or COVID-19. In one embodiment, the subjects may be asymptomatic.

[0631] Reduction in SARS-CoV-2 infection can be determined using any method known in the art or described herein, including measuring the viral load in a sample from the subject after treatment and comparing it to the viral load in a sample from the same subject before treatment. Preferably, the sample is taken from the respiratory tract, preferably the upper respiratory tract, e.g., the nose or pharynx (i.e., throat). Alternatively, responsiveness to treatment may result in a reduction in the severity of one or more of the symptoms described herein.

[0632] Acute respiratory distress syndrome (ARDS) This disclosure provides a method for treating, preventing, or delaying the progression of ARDS in subjects.

[0633] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltration, severe hypoxemia, and disruption of the alveolar-capillary barrier (i.e., pulmonary vascular leakage), leading to non-cardiogenic pulmonary edema. Currently, there is no effective pharmacological treatment.

[0634] Infectious pathogens, including influenza, are the primary causes of ARDS. Therefore, in one example of this disclosure, ARDS is associated with SARS-CoV-2 infection. For example, ARDS is associated with SARS-CoV-2 and / or COVID-19.

[0635] ARDS are classified according to the Berlin definition, including the following: (1) Presentation of a clinical attack of respiratory symptoms or within one week of onset, (2) Acute hypoxemic respiratory failure, as determined by a PaO2 / FiO2 ratio of 300 mmHg or less at at least 5 cm of continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP) (PaO2 is the partial pressure of oxygen in arterial blood, and FiO2 is the fraction of inspired oxygen), (3) Bilateral shadows on chest radiographs that are not fully explained by exudation, alveolar parenchymaling, or atelectasis, and (4) Edema / respiratory failure not fully explained by heart failure or fluid overload.

[0636] For example, the subject has or is suffering from ARDS (i.e., the subject meets the Berlin definition of ARDS). For instance, the subject requires treatment (i.e., needs it).

[0637] For example, a subject may have or be suffering from symptoms associated with ARDS. The symptoms associated with ARDS, and methods for identifying subjects at risk of developing ARDS, are evident to those skilled in the art and / or are described herein. For example, a subject may have one or more or all of the following symptoms: a) A respiratory rate exceeding 30 breaths per minute, b) Oxygen saturation (SpO2) of 93% or less in indoor air, c) Ratio of arterial oxygen partial pressure to the fraction of inhaled oxygen below 300 mmHg (PaO2 / FiO2), d) SpO2 / FiO2 ratio less than 218, and e) More than 50% of the lungs covered by radiographs.

[0638] Currently, ARDS is classified as mild, moderate, or severe based on the associated increased mortality rate. The severity of ARDS can be classified according to the Berlin definition as follows: (i) Mild ARDS: PaO2 / FiO2 of 200-300 mmHg on CPAP or PEEP at least 5 cm, (ii) Moderate ARDS: PaO2 / FiO2 of 100-200 mmHg on at least 5 cm PEEP, (iii) Severe ARDS: PaO2 / FiO2 less than 100 mmHg on at least 5 cm PEEP.

[0639] In one example, ARDS is mild ARDS. In another example, ARDS is moderate ARDS. In yet another example, ARDS is severe ARDS.

[0640] The method described herein can be used to prevent or delay the onset of ARDS in addition to existing treatments for ARDS. Therefore, in one example, the subject does not have ARDS.

[0641] In one example, the subject is at risk of developing one or more symptoms associated with ARDS.

[0642] kit Another example of the present disclosure provides a kit containing the self-replicating RNA of the present disclosure that is useful for treating or preventing the diseases or disorders described above.

[0643] Another example of the present disclosure provides a kit containing the cRNA of the present disclosure that is useful for treating or preventing the diseases or disorders described above.

[0644] For example, the kit includes (a) optionally a container containing self-replicating RNA in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and (b) a package insert with instructions for treating or preventing a disease or disorder in the subject (e.g., SARS-CoV-2 infection, COVID-19, or ARDS).

[0645] For example, the kit includes (a) optionally a container containing cRNA in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and (b) a package insert with instructions for treating or preventing a disease or disorder in the subject (e.g., influenza, COVID-19, ARDS, or COVID-19-related sepsis or pneumonia).

[0646] In this example of the Disclosure, the accompanying information is on or associated with the container. Suitable containers include, for example, bottles, vials, and syringes. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold or contain the composition effective for the disease or disorder of the Disclosure and may have a sterile access port (for example, the container may be a vial with a stopper that can be pierced by an intravenous solution bag or a subcutaneous injection needle). At least one activator in the composition is self-replicating RNA and / or cRNA. The label or accompanying information indicates that the composition is to be used to treat subjects suitable for treatment, e.g., subjects having or susceptible to developing SARS-CoV-2 infection, COVID-19, ARDS, or COVID-19-related sepsis or pneumonia, and provides specific guidance on dosage and treatment intervals, as well as any other medicinal products. The kit may further include additional containers containing pharmaceutically acceptable diluent buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0647] This disclosure includes the following non-limiting embodiments. [Examples]

[0648] Example 1: Method Generation of self-replicating RNA The DNA templates encoding the self-replicating RNA described herein were produced in competent Escherichia coli cells transformed with DNA plasmids. Individual bacterial colonies were isolated, and the resulting plasmid DNA was amplified in E. coli cultures. After fermentation, the plasmid DNA was isolated using the Maxiprep DNA kit and linearized by restriction digestion. Restriction enzymes were removed using phenol / chloroform extraction and ethanol precipitation.

[0649] mRNA was prepared by in vitro transcription from a linearized DNA template using T7 RNA polymerase. The DNA template was then removed by DNase digestion. Enzymatic capping was performed using cap 0 to provide functional mRNA. The obtained mRNA was then purified and resuspended in nuclease-free water.

[0650] The self-replicating RNAs used in the experiment are outlined in Figure 1 and include the following: 1. Co96 containing alphaviral self-replicating RNA, including polynucleotides encoding the spike (S) protein antigen from the Omicron strain of SARS-CoV-2. 2. Co97 containing alphaviral self-replicating RNA, which includes polynucleotides encoding S protein antigen and N protein antigen from the Omicron strain of SARS-CoV-2, driven by a synthetic genome promoter (SGP)v2. 3. Co99 containing alphaviral self-replicating RNA, including polynucleotides encoding N protein antigen and S protein antigen from the Omicron strain of SARS-CoV-2...

Claims

1. A self-replicating RNA comprising a nucleotide sequence encoding an antigen, operably ligated to a regulatory element, wherein the antigen is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

2. The self-replicating RNA according to claim 1, further comprising a nucleotide sequence encoding a second antigen, operably linked to a regulatory element.

3. The self-replicating RNA according to claim 2, wherein the second antigen is a nucleocapsid (N) protein from SARS-CoV-2.

4. The self-replicating RNA according to claim 3, wherein the N protein is from the Omicron strain of SARS-CoV-2.

5. The self-replicating RNA according to claim 1, wherein the regulatory element is selected from the group consisting of SG promoter and IRES.

6. The self-replicating RNA is in the order from 5' to 3', a) The nucleotide sequence encoding the S protein antigen from the Omicron strain of SARS-CoV-2, b) The self-replicating RNA according to claim 2, comprising a nucleotide sequence encoding an N protein antigen from SARS-CoV-2.

7. The self-replicating RNA is in the order from 5' to 3', a) The nucleotide sequence encoding the N protein antigen from SARS-CoV-2, b) The self-replicating RNA according to claim 2, comprising a nucleotide sequence encoding the S protein antigen from the Omicron strain of SARS-CoV-2.

8. The self-replicating RNA is in the order from 5' to 3', a) A nucleotide sequence encoding the S protein antigen from the Omicron strain of SARS-CoV-2, operably linked to the SG promoter, b) The self-replicating RNA according to claim 2, comprising a nucleotide sequence encoding the N protein antigen from SARS-CoV-2, which is optionally encoded by the sequence shown in Sequence ID No. 18 and operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

9. The self-replicating RNA is in the order from 5' to 3', a) A nucleotide sequence encoding the N protein antigen from SARS-CoV-2, operably linked to the SG promoter, b) The self-replicating RNA according to claim 2, comprising a nucleotide sequence encoding the S protein antigen from the Omicron strain of SARS-CoV-2, which is optionally operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), encoded by the sequence shown in Sequence ID No.

19.

10. The self-replicating RNA according to claim 2, wherein the N protein is from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

11. The self-replicating RNA according to claim 1, wherein the self-replicating RNA is a monocistronic self-replicating RNA.

12. The self-replicating RNA according to claim 2, wherein the self-replicating RNA is a polycistronic self-replicating RNA.

13. The self-replicating RNA according to claim 2, wherein the regulatory element is the same as the regulatory element described in claim 1.

14. The self-replicating RNA according to claim 1, wherein the regulatory element is a promoter, an internal ribosome entry site (IRES), a Kozak consensus sequence, or a combination thereof.

15. The self-replicating RNA according to claim 14, wherein the promoter is a subgenome (SG) promoter.

16. The self-replicating RNA according to claim 14, wherein the SG promoter is a minimum SG promoter or an extended SG promoter.

17. The self-replicating RNA according to claim 16, wherein the extended SG promoter is extended at its 5' end by a nucleotide generated in a sequence encoding a non-structural protein of the RNA virus.

18. The self-replicating RNA according to claim 16, wherein the minimum SG promoter is encoded by the sequence shown in Sequence ID No.

1.

19. The self-replicating RNA according to claim 16, wherein the extended SG promoter is encoded by the sequence shown in Sequence ID No.

5.

20. The self-replicating RNA according to claim 2, wherein the nucleotide sequence encoding the second antigen is operably ligated to an IRES at an optionally selectable 3' position relative to the nucleotide sequence encoding the second antigen.

21. The self-replicating RNA according to claim 20, wherein the IRES is an IRES derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-related protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF-B), Antennapedia, X-linked apoptosis inhibitor (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or a combination thereof.

22. The self-replicating RNA according to claim 21, wherein the EMCV IRES is a wild-type IRES encoded by the sequence shown in Sequence ID No.

4.

23. The self-replicating RNA according to claim 2, wherein the antigen is expressed at substantially the same level.

24. The self-replicating RNA according to claim 1, wherein the antigen is from the S protein of an omicron variant selected from the group consisting of B. 1.1.529, BA. 1, BA. 2, BA. 4, BA. 5, BA. 2.12.1, and BA. 2.

75.

25. (a) The antigen is from the S protein of omicron variant BA. 1, and the polynucleotide-encoded S protein contains one or more or all of the mutations selected from the group consisting of A67V, T95I, Y145D, L212L, S371L, G446S, G496S, T547K, N856K, L981F, G142D, Q493R, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and del69-70, or (b) The antigen is from the S protein of omicron variant BA. 2, and the S protein encoded by the polynucleotide contains one or more mutations selected from the group consisting of G142D, Q493R, del24-26, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S, or (c) The antigen is an omicron variant BA.4 or BA. The self-replicating RNA according to claim 24, wherein the S protein encoded by the polynucleotide comprises one or more or all of the mutations selected from the group consisting of L452R, F486V, R493Q, del24-26, del69-70, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, T19I, A27S, G142D, V213G, S371F, T376A, D405N, and R408S.

26. The self-replicating RNA according to claim 1, wherein the S protein is encoded by a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% relative to SEQ ID NO:

20.

27. The self-replicating RNA according to claim 1, wherein the N protein is encoded by a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% relative to SEQ ID NO:

21.

28. The self-replicating RNA according to claim 1, wherein the self-replicating RNA is derived from an alphavirus.

29. The self-replicating RNA according to claim 28, wherein the alphavirus is selected from the group consisting of Semliki Forest Virus (SFV), Sindobis Virus (SIN), Venezuelan Encephalitis Virus (VEE), and combinations thereof.

30. An immunogenic composition comprising the self-replicating RNA described in claim 1.

31. An immunogenic composition comprising a plurality of self-replicating RNAs as described in claim 1, wherein each self-replicating RNA encodes a different polypeptide antigen sequence.

32. An immunogenic composition comprising a plurality of self-replicating RNAs as described in claim 1, wherein each self-replicating RNA encodes the same polypeptide antigen sequence.

33. A pharmaceutical composition comprising the immunogenic composition according to claim 30 and a pharmaceutically acceptable carrier.

34. The pharmaceutical composition according to claim 33, further comprising lipid nanoparticles (LNPs), polymer microparticles, or an oil-in-water emulsion.

35. The pharmaceutical composition according to claim 33, wherein the self-replicating RNA is encapsulated in, bound to, or adsorbed to LNPs, polymer microparticles, or an oil-in-water emulsion.

36. The pharmaceutical composition according to claim 33, wherein each RNA is formulated together in the LNP.

37. The pharmaceutical composition according to claim 33, wherein each RNA is formulated separately in the LNP.

38. The immunogenic composition according to claim 30 for use as a vaccine.

39. The pharmaceutical composition according to claim 33 for use as a vaccine.

40. A vaccine comprising the immunogenic composition described in claim 30.

41. A vaccine comprising the pharmaceutical composition described in claim 30.

42. A polynucleotide encoding the self-replicating RNA according to claim 1, comprising optionally the sequence shown in SEQ ID NO: 17, 18, or 19.

43. The polynucleotide according to claim 42, wherein the polynucleotide is recombinant DNA.

44. The polynucleotide according to claim 43, wherein the recombinant DNA is a plasmid.

45. a) A first nucleotide sequence encoding the first antigen, b) A polynucleotide comprising a second nucleotide sequence encoding a second antigen, which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), The first antigen is a polynucleotide, which is the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

46. The polynucleotides are arranged in the order from 5' to 3', a) The first nucleotide sequence encoding the first antigen, b) The polynucleotide according to claim 45, comprising a second nucleotide sequence encoding a second antigen, operably linked to an IRES or SG promoter, optionally including the sequence shown in SEQ ID NO: 18 or 19.

47. a) A first nucleotide sequence encoding the first antigen, b) A conventional mRNA (cRNA) comprising a second nucleotide sequence encoding a second antigen, which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), The first antigen is conventional mRNA (cRNA) of the spike (S) protein from the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

48. The cRNA is arranged in the order from 5' to 3', a) The first nucleotide sequence encoding the first antigen, b) The cRNA according to claim 47, comprising the second nucleotide sequence encoding a second antigen, which is operably linked to an IRES or SG promoter.

49. The polynucleotide according to claim 45, wherein the first nucleotide sequence is operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, an SG promoter, and combinations thereof.

50. The cRNA according to claim 47, wherein the first nucleotide sequence is operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, an SG promoter, and combinations thereof.

51. The polynucleotide according to claim 45, wherein the second nucleotide sequence optionally encodes a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2, from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

52. The cRNA according to claim 47, wherein the second nucleotide sequence optionally encodes a nucleocapsid (N) protein from the Omicron strain of SARS-CoV-2, from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

53. A method for treating, preventing, or delaying the progression of SARS-CoV-2 infection in a subject requiring treatment, prevention, or delay of its progression, wherein the method comprises administering the self-replicating RNA described in claim 1 to the subject.

54. Use of the self-replicating RNA according to claim 1 in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of SARS-CoV-2 infection in a subject.

55. The self-replicating RNA according to claim 1, for use in the treatment or prevention of SARS-CoV-2 infection in a subject, or in delaying its progression.

56. A method for inducing an immune response in a subject, wherein the method comprises administering the self-replicating RNA described in claim 1 to a subject in need of such response.

57. The use of the self-replicating RNA according to claim 1 in the manufacture of a pharmaceutical product for inducing an immune response in a subject requiring induction of an immune response.

58. The self-replicating RNA according to claim 1, for use in inducing an immune response in subjects requiring induction of an immune response.

59. The method according to claim 56, wherein the immune response is a humoral and / or cell-mediated immune response.

60. The method according to claim 59, wherein the immune response is induced in response to at least one antigen from the Omicron strain of SARS-CoV-2.

61. A method for reducing the SARS-CoV-2 viral load in a subject having COVID-19, comprising administering the self-replicating RNA described in claim 1 to the subject in need of such reduction.

62. Use of self-replicating RNA according to claim 1 in the preparation of a pharmaceutical product for reducing the SARS-CoV-2 virus load in a subject having COVID-19.

63. The self-replicating RNA according to claim 1, for use in reducing the SARS-CoV-2 virus load in subjects having COVID-19.

64. A method for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject having COVID-19, comprising administering the self-replicating RNA described in claim 1 to a subject in need thereof.

65. Use of the self-replicating RNA according to claim 1 in the preparation of a pharmaceutical product for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject having COVID-19.

66. The self-replicating RNA according to claim 1, for use in treating, preventing, or delaying the progression of acute respiratory distress syndrome in subjects having COVID-19.

67. The method according to claim 53, wherein the subject is a human being 18 years of age or older.

68. The method according to claim 53, wherein the self-replicating RNA, vaccine, or composition is administered in a single-dose regimen.

69. The method according to claim 53, wherein the self-replicating RNA, vaccine, or composition is administered in two, three, or four dose regimens, with the doses administered at intervals of approximately one, two, or three months.

70. It's a kit, (a) Self-replicating RNA as described in claim 1, (b) The instruction manual, and optionally, (c) A kit comprising a pharmaceutically acceptable carrier, excipient, or diluent.

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