SARS-CoV-2 RNA vaccine and application thereof

Abstract

The present disclosure relates to an SARS-CoV-2 RNA vaccine and a use thereof. The present disclosure also relates to conventional mRNA vaccines and self-replicating RNA vaccines for use in the treatment of SARS-CoV-2 infection or COVID-19.

Description

[0001] Related Application Data

[0002] This application claims priority to U.S. Patent Application No. 63 / 511,340, filed June 30, 2023, entitled “SARS-CoV-2 RNA Vaccines and Uses Thereof,” the entire contents of which are incorporated herein by reference.

[0003] SEQUENCE LISTING

[0004] The instant application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is incorporated by reference in its entirety. The Sequence Listing file contains 1 sequence (SEQ ID NO: 1) and is 1, 1, 1 bytes in size (zipped) and was created on June 30, 2023. TECHNICAL FIELD

[0005] The present disclosure relates to SARS-CoV-2 RNA vaccines and uses thereof. The present disclosure also relates to conventional mRNA vaccines and self-replicating RNA vaccines for the treatment of SARS-CoV-2 infection or COVID-19. BACKGROUND

[0006] Respiratory viral 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 cause global pandemics, killing millions of people worldwide. Recently, SARS-CoV-2 has caused the world pandemic of highly contagious coronavirus disease 2019 (COVID-19).

[0007] SARS-CoV-2, which belongs to the Coronaviridae family and the Orthocoronavirinae subfamily, is an enveloped, single-stranded positive-sense 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), the membrane protein (M protein), the envelope protein (E protein), and the nucleocapsid protein (N protein). The S protein is a transmembrane glycoprotein that forms prominent homotrimers on the virus surface, consisting of two functional subunits, S1 and S2, which have become the main targets for current genetically engineered vaccine development.

[0008] SARS-CoV-2 has a high mutation propensity, and now a large number of mutant strains have been identified globally. The major mutant strains include: Alpha (B.1.1.7) mutant, Beta (B.1.351) mutant, Gamma (P1) mutant, Epsilon (B.1.429) mutant, Delta (B.1.617.2) mutant, Kappa (B.1.617.1) mutant, and Omicron (B.1.1.529) mutant, which differ in terms of transmissibility, pathogenicity, and / or immune escape ability. Typically, these mutant strains are identified based on the number and location of mutations in the viral genome, particularly the genome encoding the S protein of the mutant SARS-CoV-2.

[0009] The Omicron strain contains up to 36 amino acid mutation sites, and this strain in particular shows improved affinity to the ACE2 target receptor, thereby enhancing its toxicity and infectivity and accelerating the escape of the virus. Currently, there are almost no vaccines available that target specific strains of SARS-CoV-2, such as the Omicron strain. The currently available vaccines (e.g. developed against the (original) strain) do not specifically target the evolving mutant strains, which are known to provide a reduced protective effect and / or immunogenicity.

[0010] Therefore, there is a need to develop new vaccines that can specifically target strains of SARS-CoV-2, such as the omicron strain of SARS-CoV-2. SUMMARY

[0011] The present disclosure is based on the inventors’ identification of RNAs comprising antigens from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus that are suitable for use in the treatment of SARS-Cov-2 infection or Coronavirus Disease 2019 (COVID-19). In particular, the inventors’ findings provide a basis for RNAs comprising antigens from SARS-CoV-2, such as the spike (S) protein from the omicron strain of SARS-CoV-2 (or variants thereof), that are suitable for use in the treatment of SARS-CoV-2 infection or COVID-19. Furthermore, the inventors’ findings provide a basis for methods of treating or preventing or delaying the progression of a disease or condition, such as SARS-CoV-2 infection or COVID-19 and its complications, including pneumonia, sepsis and acute respiratory distress syndrome (ARDS).

[0012] Accordingly, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding an antigen operably linked to a regulatory element, wherein the antigen is a spike (S) protein from the omicron strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0013] When discussing the “omicron strain” of SARS-CoV-2 herein, this term will be understood to include variants and sublineages derived from the omicron strain of SARS-CoV-2. For example, the omicron strain of SARS-CoV-2 includes the BA.1 strain, the BA.2 strain, the XB strain, the XBB strain, the JN.1 strain, the JN.2 strain, the JN.3 strain, the KP.1 strain, the KP.2 strain.

[0014] In one example, the polynucleotide comprises 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 set forth in SEQ ID NO: 17.

[0015] In one instance, the polynucleotide further comprises a nucleotide sequence encoding a second antigen operatively linked to a regulatory element. In one instance, the second antigen is a nucleocapsid (N) protein from SARS-CoV-2.

[0016] In one instance, the polynucleotide comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or the same nucleotide sequence as the sequence shown in SEQ ID NO:18.

[0017] In one instance, the polynucleotide comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or the same nucleotide sequence as the sequence shown in SEQ ID NO:19.

[0018] This disclosure also provides an RNA comprising a first nucleotide sequence encoding an antigen operatively linked to a regulatory element, wherein the antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0019] In one instance, the RNA also includes a nucleotide sequence encoding a second antigen that is operatively linked to a regulatory element. In one instance, the second antigen is a nucleocapsid (N) protein from SARS-CoV-2.

[0020] This disclosure also provides a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operatively linked to a regulatory element, wherein the antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0021] In one instance, 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 the sequence shown in SEQ ID NO:17.

[0022] In one instance, the self-replicating RNA further comprises a nucleotide sequence encoding a second antigen operatively linked to a regulatory element. In one instance, the second antigen is a nucleocapsid (N) protein from SARS-CoV-2.

[0023] In this example, the self-replicating RNA comprises, from 5' to 3', the following:

[0024] a) The nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2; and

[0025] b) The nucleotide sequence encoding the N protein antigen from SARS-CoV-2.

[0026] In another example, the self-replicating RNA comprises, from 5' to 3', the following:

[0027] a) The nucleotide sequence encoding the N protein antigen from SARS-CoV-2; and

[0028] b) The nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2.

[0029] In this example, the self-replicating RNA comprises, from 5' to 3', the following:

[0030] a) A nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2, operably linked to the SG promoter; and

[0031] b) A nucleotide sequence encoding the N protein antigen from SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES).

[0032] In another example, the self-replicating RNA comprises, from 5' to 3', the following:

[0033] a) A nucleotide sequence encoding the N protein antigen from SARS-CoV-2, operatively linked to the SG promoter; and

[0034] b) A nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2, which is operatively linked to a regulatory element selected from a group consisting of the SG promoter and the internal ribosome entry site (IRES).

[0035] In one instance, 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 the sequence shown in SEQ ID NO:18.

[0036] In one instance, 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 the sequence shown in SEQ ID NO:19.

[0037] This disclosure also provides a polynucleotide comprising a nucleotide sequence encoding an antigen operatively 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).

[0038] In one instance, the polynucleotide further comprises a nucleotide sequence encoding a second antigen operatively linked to a regulatory element. In one instance, the second antigen is the spike (S) protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0039] This disclosure also provides an RNA comprising a first nucleotide sequence encoding an antigen operatively 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).

[0040] In one instance, the RNA also includes a nucleotide sequence encoding a second antigen operatively linked to a regulatory element. In one instance, the second antigen is the spike (S) protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0041] This disclosure also provides a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operatively 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).

[0042] In one instance, the self-replicating RNA further comprises a nucleotide sequence encoding a second antigen operatively linked to a regulatory element. In one instance, the second antigen is the spike (S) protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0043] In this example, the self-replicating RNA comprises, from 5' to 3', the following:

[0044] a) The nucleotide sequence encoding the N protein antigen from the omicron strain of SARS-CoV-2; and

[0045] b) The nucleotide sequence encoding the S protein antigen from SARS-CoV-2.

[0046] In another example, the self-replicating RNA comprises, from 5' to 3', the following:

[0047] a) The nucleotide sequence encoding the S protein antigen from SARS-CoV-2; and

[0048] b) The nucleotide sequence encoding the N protein antigen from the omicron strain of SARS-CoV-2.

[0049] In this example, the self-replicating RNA comprises, from 5' to 3', the following:

[0050] a) A nucleotide sequence encoding the N protein antigen from the omicron strain of SARS-CoV-2, operably linked to the SG promoter; and

[0051] b) A nucleotide sequence encoding the S protein antigen from SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES).

[0052] In another example, the self-replicating RNA comprises, from 5' to 3', the following:

[0053] a) A nucleotide sequence encoding the S protein antigen from SARS-CoV-2, operably linked to the SG promoter; and

[0054] b) A nucleotide sequence encoding the N protein antigen from the omicron strain of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES).

[0055] In one instance, 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 the sequence shown in SEQ ID NO:18.

[0056] In one instance, 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 the sequence shown in SEQ ID NO:19.

[0057] In this example, the regulatory element is selected from: subgenomic (SG) promoters and internal ribosome entry sites (IRES), as well as Kozac concordant sequences or combinations thereof. In this example, the regulatory element is an SG promoter.

[0058] In this example, the nucleotide sequence encoding the second antigen is operatively linked to the same regulatory element as the nucleotide sequence encoding the first antigen.

[0059] This 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 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.

[0060] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first polypeptide of interest; and b) a second nucleotide sequence encoding a second polypeptide of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first polypeptide is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0061] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first polypeptide of interest; and b) a second nucleotide sequence encoding a second polypeptide of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first polypeptide is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0062] This 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, operatively 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.

[0063] In one instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0064] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0065] In one instance, the polynucleotide is RNA or DNA. In another instance, the RNA is messenger RNA (mRNA). In yet another instance, the mRNA is regular mRNA (cRNA) or self-replicating RNA.

[0066] Therefore, this 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, operatively 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.

[0067] In one instance, the RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0068] In one instance, the RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0069] This 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, operatively linked to a regulatory element selected from the group consisting of an SG promoter and 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.

[0070] In one instance, the cRNA contains, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0071] In one instance, the cRNA contains, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0072] This disclosure also 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, operatively linked to a regulatory element selected from the group consisting of an SG promoter and 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.

[0073] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0074] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0075] In one instance, a first nucleotide sequence encoding a first antigen of interest is operatively linked to a regulatory element. In another instance, the regulatory element is operatively linked to the 5' end of the first nucleotide sequence. In one instance, the regulatory element is selected from the group consisting of a Kozak concordant sequence, an IRES, an SG promoter, and combinations thereof. For example, the regulatory element is a Kozak concordant sequence. For example, the regulatory element is an IRES. In another instance, the regulatory element is an SG promoter.

[0076] In one instance, the Kozak public sequence comprises or consists of the sequence shown in SEQ ID NO:6 (GCCACC). In another instance, the Kozak public sequence consists of the sequence shown in NO:7 (ACCATGG).

[0077] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak common sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively 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.

[0078] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak common sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0079] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak common sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0080] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak concordant sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively 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.

[0081] In one instance, the RNA from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0082] In one instance, the RNA from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0083] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak concordant sequence, IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of an SG promoter and 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.

[0084] In one example, the cRNA from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0085] In one example, the cRNA from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0086] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak concordant sequence, IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of an SG promoter and 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.

[0087] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0088] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0089] In one instance, the first nucleotide sequence encoding the first antigen of interest is operatively linked to the Kozak shared sequence.

[0090] In one example, a first nucleotide sequence encoding a first antigen of interest is operatively linked to a Kozak concordant sequence and an SG promoter. For instance, the Kozak concordant sequence is operatively linked to the 5' end of an SG promoter, which is operatively linked to the 5' end of the first nucleotide sequence encoding the first antigen of interest.

[0091] In one instance, a first nucleotide sequence encoding a first antigen of interest is operatively linked to a Kozak concordant sequence and an IRES. For example, the Kozak concordant sequence is operatively linked to the 5' end of an IRES, which is operatively linked to the 5' end of the first nucleotide sequence encoding the first antigen of interest.

[0092] In one instance, the first nucleotide sequence encoding the first antigen of interest is operatively linked to the SG promoter.

[0093] In one instance, a first nucleotide sequence encoding the first antigen of interest is operatively ligated to the IRES.

[0094] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence; 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.

[0095] In one instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak common sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0096] In one instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak common sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0097] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; 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.

[0098] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0099] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0100] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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.

[0101] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0102] In one instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0103] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest operably linked to an SG promoter; 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.

[0104] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0105] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0106] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a first antigen of interest operably linked to an IRES; 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.

[0107] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, which is operatively linked to an IRES; and b) a second nucleotide sequence encoding a second antigen of interest, which is operatively 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0108] In one example, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0109] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence; 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 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 instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak common sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0111] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak common sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0112] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; 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.

[0113] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0114] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0115] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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.

[0116] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0117] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0118] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an SG promoter; 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.

[0119] In one instance, the RNA from 5' to 3' comprises: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0120] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0121] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an IRES; 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.

[0122] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0123] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0124] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence; 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.

[0125] In one instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak common sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0126] In one instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak common sequence; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0127] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and 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 instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0129] In one instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0130] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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.

[0131] In one instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0132] In one instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0133] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an SG promoter; 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 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 cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0135] In one example, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0136] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a first antigen of interest, operatively linked to an IRES; and b) a second nucleotide sequence encoding a second antigen of interest, operatively 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.

[0137] In one example, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0138] In one instance, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen of interest, operably linked to an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0139] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a first antigen operably linked to a Kozac concordance sequence; and b) a second nucleotide sequence encoding a second antigen operably linked 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.

[0140] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen operably linked to a Kozac common sequence; and b) a second nucleotide sequence encoding a second antigen operably linked to an IRES or SG promoter, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0141] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3',: a) a first nucleotide sequence encoding a first antigen operably linked to a Kozac common sequence; and b) a second nucleotide sequence encoding a second antigen operably linked 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).

[0142] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and 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.

[0143] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak common sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0144] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0145] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordance sequence and an IRES; 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.

[0146] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0147] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0148] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest operably linked to an SG promoter; 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.

[0149] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0150] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to the SG promoter; and b) a second nucleotide sequence encoding a second antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first antigen is a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0151] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest operably linked to an IRES; 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.

[0152] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to an IRES; 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 of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0153] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding an antigen of interest, operably linked to an IRES; 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 a nucleocapsid (N) protein from the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0154] In one instance, the polynucleotide is a bicistronic RNA. For example, the polynucleotide is a bicistronic cRNA. In another instance, the cRNA is a bicistronic cRNA. In yet another instance, the polynucleotide is a bicistronic self-replicating mRNA. For example, the self-replicating RNA is a bicistronic self-replicating RNA.

[0155] In one instance, a second nucleotide sequence encoding a second antigen is operatively linked to the IRES.

[0156] In one instance, a second nucleotide sequence encoding a second antigen is operatively linked to the SG promoter.

[0157] In one instance, the polynucleotide is a polycistronic RNA. For example, a polycistronic cRNA is a polycistronic cRNA. In another instance, the polynucleotide is a polycistronic self-replicating mRNA. For example, a self-replicating RNA is a polycistronic self-replicating mRNA.

[0158] This disclosure also provides a polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest; and b) a second nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of a subgenomic (SG) promoter and an internal ribosome entry site (IRES), wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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 polynucleotide comprises, from 5' to 3',: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0160] In one example, the polynucleotide comprises, from 5' to 3', a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2; 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 the SG promoter and IRES. In this 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] In one example, the polynucleotide is RNA or DNA. In another example, the RNA is messenger RNA (mRNA). In yet another example, the mRNA is regular mRNA (cRNA) or self-replicating RNA.

[0162] Therefore, this disclosure provides an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest; and b) a second nucleotide sequence encoding an antigen of interest, operatively 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 a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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.

[0163] In one example, the RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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] In one example, the RNA comprises, from 5' to 3',: 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 this 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] This disclosure also provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest; and b) a second nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of a subgenomic (SG) promoter and an internal ribosome entry site (IRES), wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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.

[0166] In one example, the cRNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 cRNA comprises, from 5' to 3', 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 this 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.

[0168] This disclosure also provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest; and b) a second nucleotide sequence encoding an antigen of interest, operatively 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 a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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.

[0169] In one example, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0170] In one example, the self-replicating RNA, from 5' to 3', contains, in sequence: 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0171] In one instance, the first nucleotide sequence is operatively linked to the regulatory element. For example, the regulatory element is operatively linked to the 5' end of the first nucleotide sequence. In one instance, the regulatory element is selected from the group consisting of a Kozak concordant sequence, an IRES, an SG promoter, and combinations thereof. For example, the regulatory element is a Kozak concordant sequence. For example, the regulatory element is an IRES. For example, the regulatory element is an SG promoter.

[0172] In one instance, the Kozak shared sequence includes or consists of the sequence shown in SEQ ID NO:6 (GCCACC). In another instance, the Kozak shared sequence consists of the sequence shown in SEQ ID NO:7 (ACCATGG).

[0173] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak common sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the antigen of interest, operatively 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2. In one example, the N protein is derived from the omicron strain of SARS-CoV-2. In another example, the N protein is derived from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0174] In one instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0175] In one example, the polynucleotide from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak common sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding an antigen of interest, operatively 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0177] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0178] In one example, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak common sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding an antigen of interest, operatively 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0180] In one instance, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0181] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of a Kozak common sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the antigen of interest, operatively 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0183] In one instance, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding the spike (S) protein of 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 concordant sequence, IRES, the SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0184] In one example, the self-replicating RNA comprises, from 5' to 3',: a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the Kozak concordant sequence, IRES, the SG promoter, and combinations thereof; 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 the SG promoter and IRES. In this 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 instance, the first nucleotide sequence is operatively linked to the Kozak common sequence.

[0186] In one instance, the first nucleotide sequence is operatively linked to a Kozak concordant sequence and an SG promoter. For example, the Kozak concordant sequence is operatively linked to the 5' end of an SG promoter, which is operatively linked to the 5' end of a first nucleotide sequence encoding the S protein.

[0187] In one instance, the first nucleotide sequence is operatively linked to a Kozak concordant sequence and an IRES. For example, the Kozak concordant sequence is operatively linked to the 5' end of an IRES, which is operatively linked to the 5' end of a first nucleotide sequence encoding the S protein.

[0188] In one instance, the first nucleotide sequence is operatively linked to the SG promoter.

[0189] In one instance, the first nucleotide sequence is operatively ligated to IRES.

[0190] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence; and b) a second nucleotide sequence encoding the antigen of interest, 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 a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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 polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the Kozak common sequence; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0192] In one example, the polynucleotide from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to a Kozak concordant sequence; 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 the SG promoter and IRES. In this 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 a polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding the antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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 polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the Kozak concordant sequence and the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0195] In one example, the polynucleotide from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to a Kozak concordant sequence and 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 a polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an IRES; and b) a second nucleotide sequence encoding the antigen of interest, 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 a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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 polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the Kozak concordance sequence and IRES; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0198] In one example, the polynucleotide from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 a polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest operably linked to an SG promoter; and b) a second nucleotide sequence encoding an antigen of interest 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 a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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.

[0200] In one instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0201] In one example, the polynucleotide from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 polynucleotide comprising: a) a first nucleotide sequence encoding an antigen of interest operably linked to an IRES; and b) a second nucleotide sequence encoding the antigen of interest 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 a nucleotide sequence encoding a spike (S) protein from the omicron strain of SARS-CoV-2 and a nucleotide sequence 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 instance, the polynucleotide from 5' to 3' comprises: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to IRES; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0204] In one example, the polynucleotide from 5' to 3' comprises, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and the IRES. In this 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 an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence; and b) a second nucleotide sequence encoding the antigen of interest, 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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 instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the Kozak common sequence; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0207] In one example, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding the antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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 instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the Kozak concordant sequence and the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0210] In one example, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an IRES; and b) a second nucleotide sequence encoding the antigen of interest, 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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 instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the Kozak concordance sequence and IRES; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0213] In one example, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to an SG promoter; and b) a second nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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 instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0216] In one example, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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 an RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to an IRES; and b) a second nucleotide sequence encoding the antigen of interest, operatively 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2. In an example, the N protein is from the omicron strain of SARS-CoV-2.

[0218] In one instance, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to IRES; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0219] In one example, the RNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and the IRES. In this 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] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence; and b) a second nucleotide sequence encoding an antigen of interest from SARS-CoV-2, 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0221] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the Kozak concordant sequence; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0222] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0223] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an SG promoter; and b) a second nucleotide sequence encoding the antigen of interest, operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0224] In one instance, the cRNA contains, from 5' to 3', the following: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the Kozak concordant sequence and the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0225] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0226] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operably linked to a Kozak concordant sequence and an IRES; and b) a second nucleotide sequence encoding the antigen of interest, 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0227] In one instance, the cRNA contains, from 5' to 3', the following: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the Kozak concordance sequence and IRES; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0228] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to the Kozak concordant 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 linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0229] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to an SG promoter; and b) a second nucleotide sequence encoding an antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0230] In one instance, the cRNA contains, from 5' to 3', the following: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0231] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES. In this 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.

[0232] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding an antigen of interest operably linked to an IRES; and b) a second nucleotide sequence encoding the antigen of interest 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 a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0233] In one instance, the cRNA contains, from 5' to 3', the following: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to IRES; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0234] In one example, the cRNA from 5' to 3' contains, in sequence: a) a first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a regulatory element selected from the group consisting of the SG promoter and the IRES. In this 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.

[0235] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding an antigen of interest, operatively linked to a subgenomic (SG) promoter; and b) a second nucleotide sequence encoding the antigen of interest, operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES, wherein the first and second nucleotide sequences are selected from the group consisting of a nucleotide sequence encoding the spike (S) protein of the omicron strain of SARS-CoV-2 and a nucleotide sequence encoding the nucleocapsid (N) protein of 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.

[0236] In one example, the self-replicating RNA of this disclosure comprises, from 5' to 3',: a) a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter; and b) a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to the IRES or SG promoter. In this example, the N protein is derived from the omicron strain of SARS-CoV-2. In another example, the N protein is derived from the delta, beta, alpha, gamma, or 2019-nCoV / USA-WA1 / 2020 strain of SARS-CoV-2.

[0237] In one example, the self-replicating RNA of this disclosure comprises, from 5' to 3', sequentially: a) a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operably linked to the 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 linked to the IRES or SG promoter. In this 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.

[0238] In one instance, the polynucleotide is a bicistronic RNA. For example, the polynucleotide is a bicistronic cRNA. In another instance, the cRNA is a bicistronic cRNA. In yet another instance, the polynucleotide is a bicistronic self-replicating mRNA. For example, the self-replicating RNA is a bicistronic self-replicating RNA.

[0239] In one instance, a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2 is operatively linked to IRES.

[0240] In one instance, a second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2 is operatively linked to the SG promoter.

[0241] In one instance, the polynucleotide is a polycistronic RNA. For example, the polynucleotide is a polycistronic cRNA. For example, the cRNA is a polycistronic cRNA. In another instance, the polynucleotide is a polycistronic self-replicating mRNA. In another instance, the self-replicating RNA is a monocistronic self-replicating mRNA. In another instance, the polynucleotide is a polycistronic self-replicating RNA. In another instance, the self-replicating RNA is a monocistronic self-replicating RNA.

[0242] In one instance, the SG promoter is a natural SG promoter. For example, a natural SG promoter is a natural promoter derived from and / or based on an RNA virus (e.g., alphavirus). In one instance, the natural SG promoter is the natural alphavirus SG promoter.

[0243] In one instance, the SG promoter is either the minimal SG promoter or the extended SG promoter.

[0244] In one instance, the SG promoter is the minimal SG promoter. In one instance, the natural SG promoter is the minimal SG promoter. For example, the minimal SG promoter is the minimum sequence required for transcription initiation. In one instance, the minimal natural SG promoter is 49 nucleotides in length. In one instance, the minimal SG promoter is 49 nucleotides in length. In one instance, the minimal natural SG promoter is encoded by a sequence comprising or consisting of the sequence shown in SEQ ID NO:1. In one instance, the minimal SG promoter is encoded by a sequence comprising or consisting of the sequence shown in SEQ ID NO:1.

[0245] In one instance, the SG promoter is an extended SG promoter. In another instance, the native SG promoter is an extended SG promoter. For example, the extended SG promoter is extended at the 5' end with nucleotides that appear in the sequence encoding a non-structural protein (e.g., NSP4) of an RNA virus (e.g., alphavirus). In one instance, the extended SG promoter is extended at the 5' end with nucleotides that appear in the sequence encoding alphavirus NSP4. Adding nucleotides to the 5' end of the SG promoter sequence does not interfere with the expression of non-structural proteins and viral replicases (e.g., alphavirus NSP4).

[0246] In one instance, the SG promoter extends at its 5' end by 51 or fewer nucleotides appearing in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In one instance, the extended SG promoter is a minimal SG promoter that extends at its 5' end by no more than 51 nucleotides appearing in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In one instance, the extended SG promoter is encoded by a sequence comprising or consisting of the sequence shown in SEQ ID NO:1, which extends at its 5' end by no more than 51 nucleotides appearing in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the length of the extended SG promoter is no more than 100 nucleotides. In one instance, the extended SG promoter is encoded by a sequence comprising or consisting of nucleotides 2 to 101 of SEQ ID NO:5.

[0247] In one example, the SG promoter extends at its 5' end by about 5 to about 20 nucleotides, for example, by about 5 nucleotides, or about 10 nucleotides, or about 12 nucleotides, or about 15 nucleotides, or about 20 nucleotides, in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the SG promoter extends at its 5' end by about 20 to about 35 nucleotides, for example, by about 25 nucleotides, or about 27 nucleotides, or about 30 nucleotides, or about 35 nucleotides, in a sequence encoding a non-structural protein (e.g., alphavirus NSP4).

[0248] In one example, the SG promoter extends at its 5' end by approximately 12 nucleotides that appear in 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 extends at its 5' end by 12 nucleotides that appear in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the length of the extended SG promoter does not exceed 61 nucleotides. In one example, the extended SG promoter is encoded by a sequence comprising or consisting of nucleotides 41 to 101 of SEQ ID NO:5. In yet another example, the extended SG promoter is encoded by a sequence comprising or consisting of the sequence shown in SEQ ID NO:2.

[0249] In one example, the SG promoter extends at its 5' end by approximately 31 nucleotides that appear in 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 extends at its 5' end by 31 nucleotides that appear in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the length of the extended SG promoter does not exceed 80 nucleotides. In one example, the extended SG promoter is encoded by a sequence comprising or consisting of nucleotides 22 to 101 of SEQ ID NO:5. In yet another example, the extended SG promoter is encoded by a sequence comprising or consisting of the sequence shown in SEQ ID NO:3.

[0250] In one example, the extended SG promoter contains repeating sequences corresponding to nucleotides 66 to 75 of SEQ ID NO:5. For example, the extended SG promoter is encoded by a sequence containing nucleotides 50 to 75 of SEQ ID NO:5 and nucleotides 66 to 101 of SEQ ID NO:5. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO:15.

[0251] In one instance, IRES is derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simultaneous immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-associated 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 protein (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0252] In one instance, the IRES is a wild-type IRES derived from encephalomyocarditis virus (EMCV). For example, wild-type EMCVIRES contains the sequence shown in SEQ ID NO:4.

[0253] In one instance, the first nucleotide sequence and / or the second nucleotide sequence and / or one or more additional nucleotide sequences are codon-optimized.

[0254] In one instance, the G / C content of the first nucleotide sequence and / or the second nucleotide sequence and / or one or more additional nucleotide sequences is modified.

[0255] In one instance, the G / C content of the first nucleotide sequence and / or the second nucleotide sequence and / or one or more additional nucleic acid sequences is increased by at least 5% compared to the G / C content of the unmodified sequence. For example, the G / C content of the first nucleotide sequence and / or the second nucleotide sequence and / or one or more additional nucleotide sequences is increased by at least 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40% compared to the G / C content of the unmodified sequence.

[0256] In one instance, a polynucleotide contains at least one chemically modified nucleotide.

[0257] In one example, the chemically modified nucleotide is selected from 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-methylpseudouridine (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, the chemically modified nucleotide is 2-thiouridine (s2U). For example, the chemically modified nucleotide is 5-methylcytidine (m5C). For example, the chemically modified nucleotide is N6-methyladenosine (m6a). For example, the chemically modified nucleotide is pseudouridine (ψ). For example, the chemically modified nucleotide is 1-methylpseudouridine (m1ψ).

[0258] In one instance, the first nucleotide sequence comprises the 5'-UTR of haptoglobin (HP), fibrinogen β chain (FGB), haptoglobin-associated protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen α chain (FGA), α6 collagen (Col6A), α-1-antitrypsin (SERPINA1), α-1-antichymotrypsin (SERPINA3), fragments thereof, and / or variants thereof.

[0259] In one instance, the 5'-UTR is the 5'-UTR of Venezuelan equine encephalitis virus (VEEV) or a modified form thereof. For example, the 5'-UTR contains the sequence shown in SEQ ID NO:13.

[0260] In one instance, the 5'-UTR, its fragments, and / or variants are 40 to 2000 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 40 to 100 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 100 to 250 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 250 to 500 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 500 to 750 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 750 to 1000 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 1000 to 1250 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 1250 to 1500 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 1500 to 1750 nucleotides in length. For example, the 5'-UTR, its fragments, and / or its variants are 1750 to 2000 nucleotides in length.

[0261] In one instance, the 5'-UTR, its fragments, and / or variants contain at least 90% identical nucleotide sequences to those shown in any of SEQ ID NO:9 to 12. For example, the 5'-UTR, its fragments, and / or variants contain 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical nucleotide sequences to those shown in any of SEQ ID NO:9 to 12.

[0262] In one instance, the polynucleotide comprises a combination of two or more 5'-UTRs, their fragments, and / or variants. In one instance, the two or more 5'-UTRs are identical. In one instance, the two or more 5'-UTRs are different.

[0263] In one example, the nucleotide sequence containing the 5'UTR includes at least one microRNA binding site, an AU enrichment element (ARE), a GC enrichment element, a stem-loop, or a combination thereof. In one example, the nucleotide sequence includes a microRNA binding site. In one example, the nucleotide sequence includes an AU enrichment element (ARE). In one example, the nucleotide sequence includes a GC enrichment element. In one example, the nucleotide sequence includes a stem-loop. For example, the stem-loop is a histone stem-loop.

[0264] In one instance, the polynucleotide also contains a nucleotide sequence comprising a 3'UTR. In one instance, the nucleotide sequence comprising a 3'UTR is located at the 3' of the second nucleotide sequence or one or more other nucleotide sequences. For example, the nucleotide sequence comprising a 3'UTR is located at the 3' of the second nucleotide sequence. In one instance, the 3'UTR comprises the 3'-UTR of arachidonic acid 5-lipoxygenase (ALOX5), αI collagen (COL1A1), tyrosine hydroxylase (TH) gene, split N-terminal enhancer (AES), human mitochondrial 12S rRNA (mtRNR1), fragments thereof, and / or variants thereof.

[0265] In one instance, the 3'UTR is the 3'UTR of Sindbis virus (SINV) or a modified form thereof. For example, the 3'UTR contains the sequence shown in SEQ ID NO:14 or 22.

[0266] In one instance, the 3'UTR, its fragments, and / or variants are 40 to 400 nucleotides in length. For example, the 3'-UTR is 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 nucleotides in length. For example, the 3'-UTR, its fragments, and / or variants are 40 to 50 nucleotides in length. For example, the 3'-UTR, its fragments, and / or variants are 50 to 60 nucleotides in length. For example, the length of the 3'-UTR, its fragments, and / or variants is 60 to 70 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 70 to 80 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 80 to 90 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 90 to 100 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 100 to 125 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 125 to 150 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 150 to 175 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 175 to 200 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 200 to 225 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 225 to 250 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 250 to 275 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 275 to 300 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 300 to 325 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 325 to 350 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 350 to 375 nucleotides. For example, the length of the 3'-UTR, its fragments, and / or variants is 375 to 400 nucleotides.

[0267] In one instance, the polynucleotide comprises a combination of two or more 3'-UTRs, their fragments, and / or variants. In one instance, the two or more 3'-UTRs are identical. In one instance, the two or more 3'-UTRs are different.

[0268] In one instance, the nucleotide sequence comprising the 3' UTR, fragments thereof, and / or variants thereof contains at least one microRNA binding site, an AU-enriched element (ARE), a GC-enriched element, a triple helix, a stem-loop, one or more stop codons, or combinations thereof. In one instance, the nucleotide sequence contains a microRNA binding site. In one instance, the nucleotide sequence contains an AU-enriched element (ARE). In one instance, the nucleotide sequence contains a GC-enriched element. In one instance, the nucleotide sequence contains a triple helix. In one instance, the nucleotide sequence contains a stem-loop. For example, the stem-loop is a histone stem-loop. In one instance, the nucleotide sequence contains one or more stop codons. For example, one or more stop codons are located at the 5' end of the 3'-UTR.

[0269] In one example, the polynucleotide comprises a nucleotide sequence including one or more 3' tail sequences located at the 3' end of a nucleotide sequence containing a 3' UTR. In one example, the one or more 3' tail sequences are selected from polyadenylated sequences, polyadenylated signals, G-quadruplexes, polycytidine sequences, stem-loops, and combinations thereof. For example, the 3' tail sequence comprises a polyadenylated sequence. In one example, the 3' tail sequence comprises a polyadenylated signal. In one example, the 3' tail sequence comprises a G-quadruplex. In one example, the 3' tail sequence comprises a polycytidine sequence. In one example, the 3' tail sequence comprises a stem-loop. For example, the stem-loop is a histone stem-loop. In one example, the 3' tail sequence comprises a polyadenylated sequence and a G-quadruplex. In one example, the 3' tail sequence comprises a stem-loop (e.g., a histone stem-loop) and a polyadenylated sequence.

[0270] In one example, one or more 3' tail sequences comprise one or more polyadenylated sequences, each polyadenylated sequence comprising 10 to 300 consecutive adenosine nucleotides. For example, each polyadenylated sequence comprises 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, each of one or more polyadenylated sequences comprises 10 to 20 consecutive adenosine nucleotides. For example, each of one or more polyadenylated sequences comprises 20 to 30 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 30 to 40 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 36 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 40 to 50 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 50 to 60 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 60 to 70 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 70 to 80 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 80 to 90 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 90 to 100 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 100 to 125 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 125 to 150 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 150 to 175 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 175 to 200 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 200 to 225 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 225 to 250 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 250 to 275 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 275 to 300 consecutive adenosine nucleotides.

[0271] In one example, one or more polyadenylated sequences each comprise 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 polyadenylated sequences each comprise 10 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each comprise 20 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each comprise 30 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each comprise 40 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each comprise 50 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each comprise 60 consecutive adenosine nucleotides. For example, one or more polyadenylated sequences each contain 70 consecutive adenosine monophosphate (AMP) nucleotides. For example, one or more polyadenylated sequences each contain 80 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 90 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 100 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 125 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 150 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 175 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 200 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 225 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 250 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 275 consecutive aMP nucleotides. For example, one or more polyadenylated sequences each contain 300 consecutive adenosine nucleotides.

[0272] In one example, the polyadenylated sequence comprises 36 consecutive adenosine nucleotides. For instance, the polyadenylated sequence comprises the sequence shown in SEQ ID NO:16.

[0273] In one instance, one or more polyadenylated sequences are separated by a break linker. For example, the 3' tail sequence from 5' to 3' consists of: a polyadenylated sequence containing consecutive adenosine nucleotides, a break linker, and a further polyadenylated sequence containing consecutive adenosine nucleotides.

[0274] In one instance, the length of the break linker is 10 to 50, 50 to 100, or 100 to 150 nucleotides. For example, the length of the break linker is 10 to 50 nucleotides. For example, the length of the break linker is 50 to 100 nucleotides. For example, the length of the break linker is 100 to 150 nucleotides.

[0275] In one instance, the length of the break linker can be 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, the break linker is 1 nucleotide long. For example, the break linker is 2 nucleotides long. For example, the break linker is 3 nucleotides long. For example, the length of the break linker is 4 nucleotides. For example, the length of the break linker is 5 nucleotides. For example, the length of the break linker is 6 nucleotides. For example, the length of the break linker is 7 nucleotides. For example, the length of the break linker is 8 nucleotides. For example, the length of the break linker is 9 nucleotides. For example, the length of the break linker is 10 nucleotides. For example, the length of the break linker is 11 nucleotides. For example, the length of the break linker is 12 nucleotides. For example, the length of the break linker is 13 nucleotides. For example, the length of the break linker is 14 nucleotides. For example, the length of the break linker is 15 nucleotides. For example, the length of the break linker is 16 nucleotides. For example, the length of the break linker is 17 nucleotides. For example, the length of the break linker is 18 nucleotides. For example, the length of the break linker is 19 nucleotides. For example, the length of the break linker is 20 nucleotides. For example, the length of the break linker is 25 nucleotides. For example, the length of the break linker is 30 nucleotides. For example, the length of the break linker is 35 nucleotides. For example, the length of the break linker is 40 nucleotides. For example, the length of the break linker is 45 nucleotides. For example, the length of the break linker is 50 nucleotides. For example, the length of the break linker is 55 nucleotides. For example, the length of the break linker is 60 nucleotides. For example, the length of the break linker is 65 nucleotides. For example, the length of the break linker is 70 nucleotides. For example, the length of the break linker is 75 nucleotides. For example, the length of the break linker is 80 nucleotides. For example, the length of the break linker is 85 nucleotides. For example, the length of the break linker is 90 nucleotides. For example, the length of the break linker is 95 nucleotides. For example, the length of the break linker is 100 nucleotides. For example, the length of the break linker is 110 nucleotides. For example, the length of the break linker is 120 nucleotides. For example, the length of the break linker is 130 nucleotides. For example, the length of the break linker is 140 nucleotides. For example, the length of the break linker is 150 nucleotides.

[0276] In one instance, the break linker is 10 nucleotides in length. In another instance, the break linker comprises or consists of the nucleotide sequence shown in SEQ ID NO:8. For example, the break linker comprises or consists of the nucleotide sequence GCAUAUGACU.

[0277] In one example, the 3' tail sequence from 5' to 3' consists of: a polyadenylated sequence containing 30 consecutive adenosine nucleotides, a 10-nucleotide break linker, and a further polyadenylated sequence containing 70 consecutive adenosine nucleotides.

[0278] In one example, the 3' tail sequence from 5' to 3' sequentially comprises: a polyadenylated sequence containing 30 consecutive adenosine nucleotides, a break linker comprising or consisting of the nucleotide sequence shown in SEQ ID NO:8, and a further polyadenylated sequence containing 70 consecutive adenosine nucleotides.

[0279] In one example, the polynucleotides consist of the following from 5' to 3':

[0280] a) 5'-UTR, its fragments and / or variants;

[0281] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0282] c) The first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);

[0283] d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0284] e) 3'-UTR, its fragments and / or variants; and

[0285] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0286] In one example, the polynucleotides consist of the following from 5' to 3':

[0287] a) 5'-UTR, its fragments and / or variants;

[0288] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0289] c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2;

[0290] d) The second nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0291] e) 3'-UTR, its fragments and / or variants; and

[0292] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0293] In one example, the RNA consists of the following from 5' to 3':

[0294] a) 5'-UTR, its fragments and / or variants;

[0295] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0296] c) The first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);

[0297] d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0298] e) 3'-UTR, its fragments and / or variants; and

[0299] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0300] In one example, the RNA consists of the following from 5' to 3':

[0301] a) 5'-UTR, its fragments and / or variants;

[0302] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0303] c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2;

[0304] d) The second nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0305] e) 3'-UTR, its fragments and / or variants; and

[0306] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0307] In one example, the cRNA consists of the following from 5' to 3':

[0308] a) 5'-UTR, its fragments and / or variants;

[0309] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0310] c) The first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);

[0311] d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0312] e) 3'-UTR, its fragments and / or variants; and

[0313] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0314] In one example, the cRNA consists of the following from 5' to 3':

[0315] a) 5'-UTR, its fragments and / or variants;

[0316] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0317] c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2;

[0318] d) The second nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0319] e) 3'-UTR, its fragments and / or variants; and

[0320] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0321] In one example, the self-replicating RNA contains, from 5' to 3', the following:

[0322] a) 5'-UTR, its fragments and / or variants;

[0323] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0324] c) The first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);

[0325] d) A second nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0326] e) 3'-UTR, its fragments and / or variants; and

[0327] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0328] In one example, the self-replicating RNA contains, from 5' to 3', the following:

[0329] a) 5'-UTR, its fragments and / or variants;

[0330] b) Select regulatory elements from groups consisting of Kozak common sequences, IRES, SG promoters and their combinations;

[0331] c) The first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2;

[0332] d) The second nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a regulatory element selected from the group consisting of the SG promoter and IRES;

[0333] e) 3'-UTR, its fragments and / or variants; and

[0334] f) Select one or more 3' tail sequences from the group consisting of polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops and combinations thereof.

[0335] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', the following:

[0336] a) A first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the minimal SG promoter; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to the minimal SG promoter; or

[0337] b) A first nucleotide sequence encoding the spike (S) protein of 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 the nucleocapsid (N) protein of SARS-CoV-2, operably linked to an extended SG promoter; or

[0338] c) A first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the minimal SG promoter; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to the wild-type EMCV IRES.

[0339] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', the following:

[0340] a) A first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operatively linked to a 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), operatively linked to a minimal SG promoter; or

[0341] b) A first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked to a 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 linked to an extended SG promoter; or

[0342] c) A first nucleotide sequence encoding the nucleocapsid (N) protein from SARS-CoV-2, operably linked 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 linked to the wild-type EMCV IRES.

[0343] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the minimal SG promoter; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to the minimal SG promoter.

[0344] In one instance, the self-replicating RNA of this disclosure comprises, 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 a minimal SG promoter.

[0345] For example, the self-replicating RNA of this disclosure comprises, from 5' to 3', the following: a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and operatively linked to a minimal SG promoter comprising the sequence shown in SEQ ID NO:1; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operatively linked to a minimal SG promoter comprising the sequence shown in SEQ ID NO:1.

[0346] For example, the self-replicating RNA of this disclosure comprises, 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.

[0347] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to a minimal SG promoter; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to an extended SG promoter.

[0348] In one instance, the self-replicating RNA of this disclosure comprises, 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.

[0349] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operatively linked to a minimal SG promoter encoded by the sequence shown in SEQ ID NO:1; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operatively linked to an extended SG promoter encoded by the sequence shown in SEQ ID NO:5.

[0350] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to an extended SG promoter encoded by the sequence shown in SEQ ID NO:5.

[0351] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is operatively linked to the minimal SG promoter; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, which is operatively linked to the wild-type EMCV IRES.

[0352] In one instance, the self-replicating RNA of this disclosure comprises, 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 wild-type EMCV IRES.

[0353] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the minimal SG promoter encoded by the sequence shown in SEQ ID NO:1; and a second nucleotide sequence encoding the nucleocapsid (N) protein of SARS-CoV-2, operably linked to the wild-type EMCV IRES encoded by the sequence shown in SEQ ID NO:4.

[0354] In one instance, the self-replicating RNA of this disclosure comprises, from 5' to 3', a first nucleotide sequence encoding a nucleocapsid (N) protein from SARS-CoV-2, operatively linked 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), operatively linked to a wild-type EMCV IRES encoded by the sequence shown in SEQ ID NO:4.

[0355] In one instance, the RNA also includes a 5' end cap structure.

[0356] In one instance, the 5' endocervical cap structure is an endogenous cap or the like. For example, the 5' endocervical cap structure is an endogenous cap. For example, the 5' endocervical cap structure is an analogue of an endogenous cap.

[0357] In one instance, the 5' cap structure contains guanine or a guanine analogue thereof. For example, the 5' cap structure contains guanine. For example, the 5' cap structure contains a guanine analogue of guanine.

[0358] In one example, the 5' cap structure is selected from anti-reverse cap analogs (ARCA), N7,2'-O-dimethylguanosine (mCAP), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazoguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, 2-azidoguanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), Cap1, and Cap2. For example, the 5' cap structure is an anti-reverse cap analog (ARCA). For example, the 5' cap structure is N7,2'-O-dimethylguanosine (mCAP). For example, the 5' cap structure is inosine. For example, the 5' cap structure is N1-methylguanosine. For example, the 5' cap structure is 2'-fluoroguanosine. For example, the 5' cap structure is 7-deazoguanosine. For example, the 5' cap structure is 8-oxoguanosine. For example, the 5' cap structure is 2-aminoguanosine. For example, the 5' cap structure is LNA-guanosine. For example, the 5' cap structure is 2-azidoguanosine. For example, the 5' cap structure is N6,2'-O-dimethyladenosine. For example, the 5' cap structure is 7-methylguanosine (m7G). For example, the 5' cap structure is Cap1. For example, the 5' cap structure is Cap2.

[0359] In one instance, the 5' cap structure is attached to the 5' end of the RNA via a 5'-5'-triphosphate linker or a 5'-5'-thiophosphate linker. For example, the 5' cap structure is attached to the 5' end of the RNA via a 5'-5'-triphosphate linker. For example, the 5' cap structure is attached to the 5' end of the RNA via a 5'-5'-thiophosphate linker.

[0360] In one instance, the antigens (i.e., the spike (S) protein from the omicron strain of SARS-CoV-2 or the nucleocapsid (N) protein from SARS-CoV-2) are expressed at substantially the same level. For example, the antigens have expression levels of about 10%, about 5%, or about 1% each. In another instance, the antigens are expressed at different levels. For example, the antigens have expression levels greater than about 10%, about 15%, or about 20% each. Methods for determining expression levels are known in the art and / or described herein.

[0361] In one instance, the self-replicating RNA originates from an alphavirus. For example, the alphavirus is selected from Semliki Forest Virus (SFV), Sindebeth Virus (SIN), and Venezuelan Equine Encephalitis Virus (VEE), and combinations thereof.

[0362] In one instance, the self-replicating RNA originated from the Semliki Forest Virus (SFV).

[0363] In one instance, the self-replicating RNA originated from the Sindbis virus (SIN).

[0364] In one instance, the self-replicating RNA originated from Venezuelan equine encephalitis virus (VEE).

[0365] In one instance, the S protein contains the mutant Q641R.

[0366] In one example, the antigen is derived 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 another example, the antigen is derived 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, BA.2.75, XB, XBB, JN.1, JN.2, JN.3, KP.1, and KP.2.

[0367] In one instance, the antigen is derived from the S protein of the omicron variant BA.1, and the S protein encoded by the polynucleotide contains one or more 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.

[0368] In one instance, the antigen is derived from the S protein of the omicron variant BA.2, and the S protein, encoded by a polynucleotide, contains one 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.

[0369] In one instance, the antigen is derived from the S protein of the omicron variant BA.4 or BA.5, and the S protein encoded by the polynucleotide contains 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.

[0370] In one instance, the antigen is derived from the S protein of the omicron variant of SARS-CoV-2, and the S protein contains one or all of the 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.

[0371] In one instance, the antigen is derived from the S protein of the omicron variant of SARS-CoV-2, and the S protein contains proteins selected from T19I, Δ24-26, A27S, V83A, Δ144, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, and D4. One or more of the mutations in the group consisting of 05N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K.

[0372] In one instance, the antigen is derived from the S protein of the omicron variant of SARS-CoV-2, and the S protein comprises the following proteins selected from 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, and R403K. One or all of the mutations in the group consisting of D405N, 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.

[0373] In one instance, the antigen is derived from the S protein of the 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.

[0374] In one instance, the antigen comes from one or more of the S protein containing mutants R346T, F456L, and T572I.

[0375] In the example, the nucleotide sequence encoding the antigen from the S protein contains a polynucleotide sequence that is 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% identical to SEQ ID NO:20.

[0376] In this example, the nucleotide sequence encoding the antigen from the S protein comprises the polynucleotide sequence shown in SEQ ID NO:20.

[0377] In the example, the S protein is encoded by 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% identical to SEQ ID NO:20.

[0378] In this example, the S protein is encoded by the polynucleotide sequence shown in SEQ ID NO:20.

[0379] In the example, the nucleotide sequence encoding the antigen from the N protein contains a polynucleotide sequence that is 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% identical to SEQ ID NO:21.

[0380] In this example, the nucleotide sequence encoding the antigen from the N protein comprises the polynucleotide sequence shown in SEQ ID NO:21.

[0381] In the example, the N protein is encoded by 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% identical to SEQ ID NO:21.

[0382] In this example, the N protein is encoded by the polynucleotide sequence shown in SEQ ID NO:21.

[0383] In one instance, the self-replicating RNA comprises or consists of the sequence according to SEQ ID NO:17. In another instance, the self-replicating RNA comprises or consists of the sequence according to SEQ ID NO:18. In yet another instance, the self-replicating RNA comprises or consists of the sequence according to SEQ ID NO:19.

[0384] This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure. This disclosure also 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, when administered, the compositions of this disclosure are capable of inducing an immune response in a subject. For example, administration of the composition induces a humoral and / or cell-mediated immune response. 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. In another example, the composition induces a cell-mediated immune response. For example, a cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells.

[0385] In one example, the immunogenic composition of this disclosure comprises a plurality of polynucleotides, wherein each polynucleotide encodes a different polypeptide antigen sequence. In another example, the immunogenic composition of this disclosure comprises a plurality of RNAs, wherein each RNA encodes a different polypeptide antigen sequence. In a further example, the immunogenic composition of this disclosure comprises a plurality of cRNAs, wherein each cRNA encodes a different polypeptide antigen sequence. In one example, the immunogenic composition comprises a plurality of polycistronic self-replicating RNAs, wherein each polycistronic self-replicating RNA encodes an antigenic polypeptide from a different SARS-CoV-2 strain (e.g., the S protein from the omicron strain of SARS-CoV-2 and the N protein from the delta strain of SARS-CoV-2). In another example, the different polypeptide antigen sequences originate from the same strain (e.g., both the S and N proteins originate from the omicron strain of SARS-CoV-2). In another example, the immunogenic composition of this disclosure comprises a plurality of self-replicating RNAs, wherein each self-replicating RNA encodes a different polypeptide antigen sequence. In yet another example, the immunogenic composition of this disclosure comprises a plurality of self-replicating RNAs, wherein each self-replicating RNA encodes the same polypeptide antigen sequence.

[0386] This disclosure also provides pharmaceutical compositions comprising the immunogenic compositions of this disclosure and a pharmaceutically acceptable carrier. Those skilled in the art will understand and / or as described herein, that a pharmaceutically acceptable carrier suitable for use in this disclosure is appropriate.

[0387] In one example, the pharmaceutical composition further comprises lipid nanoparticles (LNPs), polymeric microparticles, and an oil-in-water emulsion. For example, polynucleotides, RNA, cRNA, or self-replicating RNA are encapsulated in, bound to, or adsorbed onto the LNPs, polymeric microparticles, and oil-in-water emulsion. In one example, polynucleotides are encapsulated in, bound to, or adsorbed onto the LNPs, polymeric microparticles, and oil-in-water emulsion. In another example, RNA is encapsulated in, bound to, or adsorbed onto the LNPs, polymeric microparticles, and oil-in-water emulsion. For example, cRNA is encapsulated in, bound to, or adsorbed onto the LNPs, polymeric microparticles, and oil-in-water emulsion. For example, self-replicating RNA is encapsulated in, bound to, or adsorbed onto the LNPs, polymeric microparticles, and oil-in-water emulsion.

[0388] In one example, the pharmaceutical composition further comprises an LNP. For example, a polynucleotide is encapsulated in the LNP. In another example, RNA is encapsulated in the LNP. For example, cRNA is encapsulated in the LNP. For example, self-replicating RNA is encapsulated in the LNP. For example, a polynucleotide binds to the LNP. In another example, RNA binds to the LNP. For example, cRNA binds to the LNP. In another example, self-replicating RNA binds to the LNP. For example, a polynucleotide adsorbs onto the LNP. In another example, RNA adsorbs onto the LNP. For example, cRNA adsorbs onto the LNP. In a further example, self-replicating RNA adsorbs onto the LNP. In another example, each RNA is formulated together in the LNP. In another example, each RNA is formulated separately in the LNP.

[0389] In one instance, the LNP comprises PEG-lipids, structured lipids, and / or neutral lipids. For example, the LNP comprises PEG-lipids, structured lipids, and neutral lipids. In another instance, the LNP comprises PEG-lipids, structured lipids, or neutral lipids.

[0390] In one instance, the LNP also comprises cationic lipids. In another instance, the LNP does not contain cationic lipids.

[0391] In one example, the pharmaceutical composition further comprises polymeric microparticles. For example, the polynucleotide is encapsulated within polymeric microparticles. In another example, the RNA is encapsulated within polymeric microparticles. For example, the cRNA is encapsulated within polymeric microparticles. For example, the self-replicating RNA is encapsulated within polymeric microparticles. For example, the polynucleotide binds to the polymeric microparticles. In another example, the RNA binds to the polymeric microparticles. For example, the cRNA binds to the polymeric microparticles. In another example, the self-replicating RNA binds to the polymeric microparticles. For example, the polynucleotide is adsorbed onto the polymeric microparticles. In another example, the RNA is adsorbed onto the polymeric microparticles. For example, the cRNA is adsorbed onto the polymeric microparticles. In a further example, the self-replicating RNA is adsorbed onto the polymeric microparticles.

[0392] In one example, the pharmaceutical composition further comprises an oil-in-water emulsion. For example, the polynucleotide is encapsulated in an oil-in-water emulsion. In another example, the RNA is encapsulated in an oil-in-water emulsion. For example, the cRNA is encapsulated in an oil-in-water emulsion. For example, the self-replicating RNA is encapsulated in an oil-in-water emulsion. For example, the polynucleotide binds to the oil-in-water emulsion. In another example, the RNA binds to the oil-in-water emulsion. For example, the cRNA binds to the oil-in-water emulsion. In another example, the self-replicating RNA binds to the oil-in-water emulsion. In a further example, the self-replicating RNA is adsorbed onto the oil-in-water emulsion. In a further example, the self-replicating RNA is suspended in the oil-in-water emulsion.

[0393] This disclosure also provides immunogenic compositions or pharmaceutical compositions of this disclosure for use as vaccines.

[0394] In one instance, the polynucleotide is DNA. In one instance, this disclosure provides DNA encoding a cRNA vaccine of this disclosure. In one instance, this disclosure provides DNA encoding a self-replicating RNA vaccine of this disclosure.

[0395] In one instance, the DNA is a plasmid.

[0396] This disclosure provides methods for treating, preventing, or delaying the progression of a disease or condition in a subject, the methods comprising administering an immunogenic composition or pharmaceutical composition of the present disclosure to a subject in need. In one example, this disclosure provides a method for treating a disease or condition in a subject, the method comprising administering an immunogenic composition or pharmaceutical composition of the present disclosure to a subject in need. In another example, this disclosure provides a method for preventing a disease or condition in a subject, the method comprising administering an immunogenic composition or pharmaceutical composition of the present disclosure to a subject in need. In a further example, this disclosure provides a method for delaying the progression of a disease or condition in a subject, the method comprising administering an immunogenic composition or pharmaceutical composition of the present disclosure to a subject in need.

[0397] In one instance, this disclosure provides the use of the disclosed polynucleotides in the preparation of a medicament for treating, preventing, or delaying the progression of a disease or condition in a subject of need. For example, this disclosure provides the use of the disclosed polynucleotides in the preparation of a medicament for treating a disease or condition in a subject of need. In another instance, this disclosure provides the use of the disclosed polynucleotides in the preparation of a medicament for preventing a disease or condition in a subject of need. In a further instance, this disclosure provides the use of the disclosed polynucleotides in the preparation of a medicament for delaying the progression of a disease or condition in a subject of need.

[0398] In one instance, this disclosure provides the use of the disclosed RNA in the preparation of a medicament for treating, preventing, or delaying the progression of a disease or condition in a subject in need. For example, this disclosure provides the use of the disclosed RNA in the preparation of a medicament for treating a disease or condition in a subject in need. In another instance, this disclosure provides the use of the disclosed RNA in the preparation of a medicament for preventing a disease or condition in a subject in need. In a further instance, this disclosure provides the use of the disclosed RNA in the preparation of a medicament for delaying the progression of a disease or condition in a subject in need.

[0399] In one instance, this disclosure provides the use of the cRNA of this disclosure in the preparation of a medicament for treating, preventing, or delaying the progression of a disease or condition in a subject in need. For example, this disclosure provides the use of the cRNA of this disclosure in the preparation of a medicament for treating a disease or condition in a subject in need. In another instance, this disclosure provides the use of the cRNA of this disclosure in the preparation of a medicament for preventing a disease or condition in a subject in need. In a further instance, this disclosure provides the use of the cRNA of this disclosure in the preparation of a medicament for delaying the progression of a disease or condition in a subject in need.

[0400] In one instance, this disclosure provides the use of the self-replicating RNA of this disclosure in the preparation of a medicament for treating, preventing, or delaying the progression of a disease or condition in a subject in need. For example, this disclosure provides the use of the self-replicating RNA of this disclosure in the preparation of a medicament for treating a disease or condition in a subject in need. In another instance, this disclosure provides the use of the self-replicating RNA of this disclosure in the preparation of a medicament for preventing a disease or condition in a subject in need. In a further instance, this disclosure provides the use of the self-replicating RNA of this disclosure in the preparation of a medicament for delaying the progression of a disease or condition in a subject in need.

[0401] In one instance, the subject has an illness or condition. In one instance, the subject has been diagnosed with an illness or condition. In one instance, the subject is receiving treatment for an illness or condition.

[0402] In one example, this disclosure provides a method for treating or preventing or delaying the progression of SARS-CoV-2 infection in a subject in need, the method comprising administering to the subject an RNA disclosed herein (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0403] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for treating or preventing or delaying SARS-CoV-2 infection in a subject.

[0404] In examples, this disclosure provides RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for the treatment, prevention, or delay of SARS-CoV-2 infection.

[0405] In one instance, the subject with SARS-CoV-2 infection had at least one COVID-19 symptom. In another instance, such a COVID-19 symptom included runny nose, cough, sore throat, fever, headache, muscle aches, or fatigue. In yet another instance, at least one symptom was any of the mild COVID-19 symptoms described herein. In yet another instance, at least one symptom was any of the moderate to severe COVID-19 symptoms described herein.

[0406] In one instance, the participant was at risk of developing COVID-19 or SARS-CoV-2 infection. For example, the participant was at risk of developing COVID-19. In another instance, the participant was at risk of developing SARS-CoV-2 infection.

[0407] In one instance, the composition disclosed herein is applied in an amount sufficient to alleviate the severity of one or more symptoms of SARS-CoV-2 infection or COVID-19 or to prevent their occurrence. Those skilled in the art will understand and / or as described herein the symptoms of SARS-CoV-2 infection or COVID-19.

[0408] In one example, this disclosure provides a method for treating or preventing or delaying the progression of COVID-19 in a subject in need, the method comprising administering to the subject an RNA disclosed herein (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0409] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for treating or preventing or delaying the progression of COVID-19 in a subject.

[0410] In examples, this disclosure provides the RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for the treatment or prevention or delay of the progression of COVID-19 in subjects.

[0411] In one example, this disclosure provides a method for inducing an immune response in a subject, the method comprising administering to a subject in need an RNA disclosed herein (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0412] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for inducing an immune response in a subject in need.

[0413] In examples, this disclosure provides RNAs (e.g., self-replicating RNAs), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for inducing an immune response in subjects in need.

[0414] 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, such as the production of neutralizing antibodies. In another example, the composition induces a cell-mediated immune response. For example, a 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 a further example, the T cells are both CD4 and CD8 T cells.

[0415] In one instance, administration of the RNA disclosed herein (e.g., self-replicating RNA), the pharmaceutical composition disclosed herein, the immunogenic composition disclosed herein, or the vaccine disclosed herein induces a CD4 T cell-mediated immune response.

[0416] In one instance, administration of the RNA disclosed herein (e.g., self-replicating RNA), the pharmaceutical composition disclosed herein, the immunogenic composition disclosed herein, or the vaccine disclosed herein induces a CD8 T cell-mediated immune response.

[0417] In one instance, administration of the RNA disclosed herein (e.g., self-replicating RNA), the pharmaceutical composition disclosed herein, the immunogenic composition disclosed herein, or the vaccine disclosed herein induces a CD4 and CD8 T cell-mediated immune response.

[0418] In one instance, 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 instance, the CD4 T cell-mediated immune response is a Th1 response. In a further instance, the CD4 T cell-mediated immune response is a Th2 response. In one instance, the CD4 T cell-mediated immune response is a Th0 and Th1 response. In another instance, the CD4 T cell-mediated immune response is a Th0 and Th2 response. In a further instance, the CD4 T cell-mediated immune response is a Th1 and Th2 response. In yet another instance, the CD4 T cell-mediated immune response is a Th0, Th1, and Th2 response.

[0419] In one instance, the Th0-responsive cytokine expresses interleukin-2 (IL2+) and / or tumor necrosis factor-α (TNFα+); and / or is negative for interferon-γ (IFNg-), IL5-, and / or IL13-. For example, the cytokine is IL2+. In another instance, the cytokine is TNFα+. In one instance, the cytokine is IFNg-. In another instance, the cytokine is IL5-. In a further instance, the cytokine is IL13-.

[0420] In one instance, the Th1-responsive cytokine expresses interferon-γ (IFNg+) and / or is negative for IL5- and / or IL13-. For example, the cytokine is IFNg+. In another instance, the cytokine is IL5-. In a further instance, the cytokine is IL13-.

[0421] In one instance, the Th2-responsive cytokine expresses IL5+ and / or IL13+; and / or is negative for IFNg. For example, the cytokine is IL5+. In a further instance, the cytokine is IL13+. For example, the cytokine is IFNg-.

[0422] In one instance, the immune response is generated in response to the S protein from the omicron strain of SARS-CoV-2 and the N protein from SARS-CoV-2, optionally from the omicron strain of SARS-CoV-2. In another instance, 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 another instance, 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 another instance, the subject with SARS-CoV-2 infection has at least one symptom of COVID-19. In this instance, one such symptom of COVID-19 includes runny nose, cough, sore throat, fever, headache, muscle aches, or fatigue. In another instance, at least one symptom is any of the mild COVID-19 symptoms described herein. In another instance, at least one symptom is any of the moderate to severe COVID-19 symptoms described herein.

[0423] In one example, this disclosure provides a method for reducing the SARS-CoV-2 viral load in a subject suffering from COVID-19, comprising administering to the subject in need an RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein.

[0424] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for reducing the SARS-CoV-2 viral load in subjects with COVID-19.

[0425] In examples, this disclosure provides the RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for reducing SARS-CoV-2 viral load in subjects with COVID-19.

[0426] In one example, this disclosure provides a method for treating, preventing, or delaying the progression of pneumonia in a subject with COVID-19, comprising administering to the subject in need an RNA disclosed herein (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0427] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for treating, preventing, or delaying the progression of pneumonia in a subject with a confirmed case of COVID-19.

[0428] In examples, this disclosure provides the RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for the treatment, prevention, or delay of the progression of pneumonia in subjects with a confirmed COVID-19 diagnosis.

[0429] In one example, this disclosure provides a method for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject with COVID-19, comprising administering to the subject in need an RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein.

[0430] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject with COVID-19.

[0431] In examples, this disclosure provides the RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for the treatment, prevention, or delay of the progression of acute respiratory distress syndrome in subjects with COVID-19.

[0432] In one example, this disclosure provides a method for treating, preventing, or delaying the progression of sepsis in a subject with COVID-19, comprising administering to the subject in need an RNA disclosed herein (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0433] In examples, this disclosure provides the use of the RNA (e.g., self-replicating RNA), the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for treating, preventing, or delaying the progression of sepsis in a subject suffering from COVID-19.

[0434] In examples, this disclosure provides RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein for the treatment, prevention, or delay of the progression of sepsis in subjects with COVID-19.

[0435] In one example, this disclosure provides a method for preventing or reducing mortality in subjects suffering from COVID-19, comprising administering to a subject in need the disclosed RNA, the disclosed self-replicating RNA, the disclosed pharmaceutical composition, the disclosed immunogenic composition, or the disclosed vaccine.

[0436] In examples, this disclosure provides the use of the RNA disclosed herein, the self-replicating RNA disclosed herein, the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein in the preparation of a medicament for preventing or reducing mortality in subjects suffering from COVID-19.

[0437] In examples, this disclosure provides the RNA disclosed herein, the self-replicating RNA disclosed herein, the pharmaceutical compositions disclosed herein, the immunogenic compositions disclosed herein, or the vaccines disclosed herein for the prevention or reduction of mortality in subjects suffering from COVID-19.

[0438] In one instance, COVID-19 was caused by the omicron strain of SARS-CoV-2. In another instance, COVID-19 was caused by the delta, beta, alpha, or gamma strain 2019-nCoV / USA-WA1 / 2020 SARS-CoV-2.

[0439] In one instance, the subject was 18 years of age or older. In another instance, the subject was of any age, for example, from about 1 month to 100 years old, from about 2 months to about 80 years old, from about 6 months to about 3 years old, from about 3 years old to about 18 years old, from about 12 years old to about 18 years old, from about 18 years old to about 55 years old, from about 50 years old to about 75 years old, and from about 40 years old to about 65 years old. In another instance, the subject was 2 years or older. In another instance, the subject was 18 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older, 80 years or older, or about 90 years or older. In another instance, the subject was less than 2 years old, less than 18 months old, less than 12 months old, less than 6 months old, or less than 3 months old.

[0440] In one example, the composition or vaccine described herein is administered in a single-dose regimen. In another example, the composition is administered in a two-, three-, or four-dose regimen, with the doses administered approximately 1, 2, or 3 months apart.

[0441] This disclosure also provides a polynucleotide encoding the self-replicating RNA of this disclosure. For example, the polynucleotide is recombinant DNA.

[0442] In one instance, 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 the sequence shown in SEQ ID NO:17.

[0443] In one instance, 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 the sequence shown in SEQ ID NO:18.

[0444] In one instance, the polynucleotide comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or the same nucleotide sequence as the sequence shown in SEQ ID NO:19.

[0445] This disclosure also provides a kit containing at least one self-replicating RNA of this disclosure, optionally in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and packaged with instructions for treating or preventing or delaying the progression of a subject’s disease or condition (e.g., SARS-CoV-2 infection, COVID-19, and / or ARDS).

[0446] This disclosure also provides a kit containing at least one self-replicating RNA of this disclosure, optionally in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and packaged with instructions for administering the RNA to subjects who have or are at risk of having a disease or condition (e.g., SARS-CoV-2 infection, COVID-19, and / or ARDS).

[0447] 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.

[0448] Any discussion of documents, actions, materials, equipment, articles of manufacture, etc. included in this specification is not intended to acknowledge that any or all of these matters constitute part of the prior art or are common general knowledge in the relevant field prior to the priority date of each appended claim. Attached Figure Description

[0449] Figure 1-sa-mRNA construct design: Co96 comprises an alphavirus self-replicating RNA containing polynucleotides encoding the spike (S) protein antigen from the omicron strain of SARS-CoV-2. Co97 comprises an alphavirus self-replicating RNA containing polynucleotides encoding the S and N protein antigens from the omicron strain of SARS-CoV-2, driven by the synthetic genome promoter (SGP)v2. Co99 comprises an alphavirus self-replicating RNA containing polynucleotides encoding the N and S protein antigens from the omicron strain of SARS-CoV-2, driven by the synthetic genome promoter (SGP)v2. Co16 comprises an alphavirus self-replicating RNA containing polynucleotides encoding the S protein antigen from the original () strain of SARS-CoV-2. Co18 comprises an alphavirus self-replicating RNA containing polynucleotides encoding the S and N protein antigens from the original () strain of SARS-CoV-2, driven by the synthetic genome promoter (SGP)v2.

[0450] Figure 2 – In vitro detection of SARS-CoV-2N and S protein expression when expressed from sa-mRNA constructs Co96, Co97, Co99, Co18, Co16 and BHK-V.

[0451] Figure 3 - Measurement of the in vitro specific potency of sa-mRNA constructs Co96, Co97, Co99, Co18 and Co16, determined based on the RNA dose (ng) used in LNP formulation.

[0452] Figure 4 The in vitro effects of the –sa-mRNA constructs Co96, Co97, Co99, Co18 and Co16 on LV micro-neutralizing titers, PV micro-neutralizing titers and ACE-2 binding inhibition.

[0453] Figure 5 The in vitro effects of the -sa-mRNA constructs Co96, Co97, Co99, Co18, and Co16 on LV micro-neutralizing titers, PV micro-neutralizing titers, and ACE-2 binding inhibition of BA.1, BA.2, and BA.4 / 5 variants of the omicron strain of SARS-CoV-2.

[0454] Key information in the sequence list

[0455]

[0456] Detailed Explanation

[0457] generally

[0458] Throughout this specification, unless otherwise specifically stated or required by the context, references to a single step, composition of matter, group of steps, or group of composition of matter shall be understood to cover one or more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of composition of matter.

[0459] Those skilled in the art will recognize that, in addition to the variations and modifications specifically described herein, this disclosure is readily adaptable and modifiable. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations or any two or more of said steps or features.

[0460] The scope of this disclosure should not be limited to the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0461] Unless otherwise specified, any instance of this disclosure herein should be considered, with appropriate modifications, to be applicable to any other instance of this disclosure. In other words, any specific instance of this disclosure may be combined with any other specific instance of this disclosure (except where mutually exclusive).

[0462] Any instance of a specific feature or feature group or method or method step disclosed in this disclosure shall be deemed to provide explicit support for abandoning that specific feature or feature group or method or method step.

[0463] Unless otherwise specifically defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0464] Unless otherwise stated, the recombinant proteins, cell culture, and immunological techniques used in this disclosure are standard procedures and are well known to those skilled in the art. Such techniques are described and explained throughout the literature in the following sources, such as 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 (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); DM Glover and BD Hames (ed.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and FMAusubel et al. (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (ed.), Antibodies: A Laboratory Manual, Cold Spring Harbour. Laboratory, (1988), and JEColigan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0465] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be considered to provide explicit support for both meanings or either meaning.

[0466] Throughout this specification, the word “comprise” or its variations such as “comprises” or “comprising” will be understood to imply inclusion of the stated element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0467] As used herein, the term “derived from” should be considered to mean that a particular whole (integer) is available from a particular source, although not necessarily directly from that source. Similarly, the term “based on” should be considered to mean that a particular whole is developed or used from a particular source, although not necessarily directly from that source.

[0468] Selected definition

[0469] As used in this article, the term “monocistronic” refers to an RNA that encodes a single polypeptide, when referring to polynucleotides, RNA, cRNA, and / or self-replicating RNA.

[0470] As used herein, the term “multicistronic” (also known as “polycistronic”) referring to polynucleotides, RNA, cRNA, and / or self-replicating RNA refers to RNA that encodes two or more polypeptides. The term encompasses “bicistronic” (or “dicistronic”; i.e., encoding two polypeptides) and “tricistronic” (i.e., encoding three polypeptides) molecules. By “bicistronic”, it refers to a single nucleic acid capable of encoding two different polypeptides from different regions of the nucleic acid.

[0471] As used herein, the terms “regular mRNA” or “cRNA” or “non-amplified RNA” refer to constructs that allow the expression of heterologous RNA and proteins but whose RNA cannot be amplified in host cells.

[0472] As used herein, the term "self-replicating RNA" refers to an RNA virus-based construct that has been engineered to allow the expression of heterologous RNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can amplify in host cells, resulting in the expression of the desired gene product in the host cells.

[0473] As used herein, the term "naked" refers to nucleic acids that are essentially 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 onto, or bound to lipid nanoparticles (LNPs), liposomes, polymer microparticles, or oil-in-water emulsions.

[0474] As used herein, the terms “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of nucleotides (or bases) covalently linked to the phosphodiester backbone. By convention, unless otherwise stated, sequences are presented from the 5' end to the 3' end. For clarity of description of nucleic acids, specific sequence components are referred to, for example, as “first nucleotide sequence” and “second nucleotide sequence.” It should be understood that, unless otherwise stated, the first and second sequences may appear in any desired order or orientation, and the terms “first,” “second,” etc., do not imply any particular order or orientation.

[0475] As used herein, the term "antigen" refers to a molecule or structure containing one or more epitopes that induce, trigger, enhance, or promote cellular and / or humoral immune responses. Antigens may include proteins and peptides, for example, derived from pathogens (e.g., viruses, bacteria, fungi, protozoa, plants) or from tumors.

[0476] As used herein, the term "adjuvant" refers to a compound that, when used in combination with a specific immunogen (e.g., VLP) in a formulation, enhances or otherwise alters or modifies the resulting immune response. Modification of the immune response includes enhancing or amplifying the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean reducing or inhibiting certain antigen-specific immune responses.

[0477] As used herein, the term "operably linked" refers to a subgenomic promoter or regulatory element (e.g., an IRES) relative to a nucleic acid, such that the expression of the nucleic acid is controlled or regulated by that element. For example, a subgenomic promoter can be operably linked to many nucleic acids, for example, through another regulatory element, such as an internal ribosome entry site (IRES).

[0478] As used in this article, the term "subgenomic promoter" (also known as a "conjugation region" promoter) refers to a promoter that directs the expression of heterologous nucleotide sequences and regulates protein expression.

[0479] As used herein, the term "internal ribosome entry site" or "IRES" refers to a nucleotide sequence within mRNA to which a ribosome or its components (e.g., the 40S subunit of the ribosome) can bind. An IRES does not necessarily contain nucleic acids that induce mRNA translation (e.g., a start codon; AUG).

[0480] The terms "peptide" or "peptide chain" will be understood to refer to a continuous series of amino acids linked by peptide bonds. For example, a protein should be considered as comprising a single polypeptide chain, i.e., a continuous series of amino acids linked by peptide bonds, or a series of polypeptide chains covalently or nonvalently linked to each other (i.e., a polypeptide complex). A series of polypeptide chains can be covalently linked by the use of appropriate chemicals or disulfide bonds. Examples of nonvalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0481] The term "recombination" should be understood to refer to the product of artificial genetic recombination.

[0482] As used herein, unless the context implies otherwise, the term “substantially identical” when referring to expression levels means that the first and second antigens have expression levels of approximately 10% or less of each other.

[0483] As used herein, the terms “disease,” “symptom,” or “condition” refer to a disruption or interference with normal functioning, and are not limited to any particular condition, and will include diseases or symptoms.

[0484] As used herein, a subject “at risk” of having or developing SARS-CoV-2 infection may or may not have detectable SARS-CoV-2 infection or symptoms, and may or may not have shown detectable SARS-CoV-2 infection or symptoms prior to treatment according to this disclosure. “At risk” means that the subject has one or more risk factors that are measurable parameters associated with the development of SARS-CoV-2 infection, as known in the art and / or described herein.

[0485] As used herein, “treatment” or “treating” a subject includes the application or administration of the compounds or compositions of this disclosure to a subject (or the application or administration of the compounds of this disclosure to cells or tissues of a subject) for the purpose of delaying, slowing, stabilizing, curing, healing, reducing, alleviating, altering, remedying, reducing worsening, improving, modifying, or influencing a disease or condition, its symptoms, or its risk (or susceptibility) to the disease or condition. The term “treatment” means any indication of success in treating or improving an injury, pathology, or condition, including any objective or subjective parameter such as reduction; alleviation; a decrease in the rate of worsening; a decrease in the severity of the disease; stabilization, reduction of symptoms, or making the injury, pathology, or condition more tolerable for the subject; a decrease in the rate of degeneration or decline; or a lessening of the final point of degeneration.

[0486] As used herein, “preventing” or “prevention” means at least reducing the likelihood of acquiring a disease or condition (or susceptibility) (i.e., causing at least one clinical symptom of the disease to not develop in a patient who may be exposed to or susceptible to the disease but has not yet experienced or displayed symptoms of the disease). This article provides biological and physiological parameters for identifying such patients, and these are also well known to physicians. As used herein, the phrase “delaying the progression of…” includes reducing or slowing the progression of a disease or condition and / or the progression of at least one symptom of the disease or condition in an individual.

[0487] "Effective amount" refers to at least a certain amount, at the necessary dosage and for the necessary time period, that effectively achieves the desired result. For example, the desired result may be therapeutic or preventative. An effective amount may be provided by a single or multiple administrations. In some instances of this disclosure, the term "effective amount" refers to the amount necessary to effectively treat a disease or condition as described herein. In some instances of this disclosure, the term "effective amount" refers to the amount necessary to induce changes associated with a disease or condition as described herein. An effective amount can vary depending on the disease or condition to be treated or the factors to be changed, and also on factors related to the weight, age, ethnic background, sex, health status and / or physical condition of the mammal being treated, as well as other factors. Generally, an effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined by a medical practitioner through routine testing and experimentation. Therefore, this term should not be construed as limiting this disclosure to a specific amount, such as the weight or quantity of RNA. An effective amount may be administered in a single dose or in doses that are repeatedly administered once or several times during treatment.

[0488] A “therapeutic effective amount” is the minimum concentration required to induce at least a measurable improvement in a particular disease or condition. The therapeutic effective amount as described 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 in the individual to elicit the desired response. Therapeutic effective amount is also the amount in which any toxic or harmful effects of the RNA are offset by the beneficial therapeutic effect.

[0489] As used herein, the term “preventative effective amount” should be understood to mean the amount of RNA disclosed herein sufficient to prevent, suppress, or delay the onset of one or more detectable symptoms of the disease or condition described herein.

[0490] "Subject" can also be any animal susceptible to SARS-CoV-2. Subjects in this disclosure can be mammals, and in specific embodiments, humans, which can be infants, children, adults, or the elderly. "Subjects at risk of SARS-CoV-2 infection" or "subjects at risk of SARS-CoV-2 infection" refers to any subject who may have been exposed to SARS-CoV-2. Subjects can be primary contacts of individuals diagnosed with SARS-CoV-2 infection. "Subject" includes any human or non-human animal. Therefore, in addition to their use in human treatment, the compounds of this disclosure can also be used in veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to, dogs, cats, horses, cattle, sheep, and pigs.

[0491] As used herein, the term "lipid nanoparticle" or "LNP" should be understood to refer to lipid-based particles having at least one nanometer-sized (e.g., 1-1,000 nm) compound comprising any formula described herein. In embodiments, LNPs are formulated in compositions for delivering polynucleotides to desired targets (e.g., cells, tissues, organs, tumors, etc.). For example, lipid nanoparticles or LNPs are any lipid compositions, including, but not limited to, liposomes or vesicles, wherein the aqueous phase is encapsulated by an amphipathic lipid bilayer (e.g., monolayer; monolayer or multilayer; multilayer), micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles wherein the solid lipid nanoparticles lack a lipid bilayer.

[0492] Polynucleotides

[0493] As used herein, the term "polynucleotide" refers to a chain of nucleotide molecules chemically bonded between the phosphate group of a nucleotide and the hydroxyl group of a sugar in an adjacent nucleotide via a series of ester bonds. In one instance, a polynucleotide is DNA. In another instance, a polynucleotide is RNA, such as mRNA. For example, mRNA can be conventional mRNA (cRNA) or self-replicating RNA.

[0494] As used herein, the term “fragment” refers to a portion of the reference nucleotide sequence or polypeptide disclosed herein, which retains the defined activity of the full-length nucleotide sequence or polypeptide.

[0495] As a non-limiting example, the conventional mRNA or self-replicating RNA contains polynucleotides encoding more than one antigen from SARS-CoV-2, said antigens may be expressed by monocistronic polynucleotides, or each of said SARS-CoV-2 antigens may be expressed by polycistronic (or multicistronic) polynucleotides. For example, the S and N protein antigens may be expressed by monocistronic or polycistronic polynucleotides.

[0496] As used herein, the term "variant" refers to a nucleotide sequence that has one or more substitutions, insertions, deletions, and / or other modifications compared to an unmodified sequence. Those skilled in the art will appreciate that any variant described herein will have the same or similar expression of the encoded protein. For example, a variant is a functional variant. Exemplary modifications to nucleotide sequences and / or peptides will be apparent to those skilled in the art and / or described herein.

[0497] In one instance, modification is a chemical modification of one or more nucleotides in a nucleotide sequence. For example, at least one naturally occurring nucleotide of a polynucleotide is replaced by a chemically modified nucleotide (e.g., pseudouridine (ψ) and 1-methylpseudouridine (m1ψ)).

[0498] In one instance, the modification involves increasing the G / C content of the nucleotide sequence.

[0499] In one instance, the modification involves codon optimization of the nucleotide sequence.

[0500] In one instance, the substitution is a conservative substitution. Those skilled in the art will understand that a conservative substitution of a polypeptide involves replacing an amino acid in the polypeptide with a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, and size). In one instance, the substitution is a non-conservative substitution.

[0501] As used in this article, the terms “encode,” “encodes,” or “encoding” refer to a region of polynucleotides that can undergo translation into a polypeptide.

[0502] The polynucleotides disclosed herein include DNA and RNA (e.g., mRNA).

[0503] Deoxyribonucleic acid (DNA)

[0504] In one instance, a polynucleotide is DNA (e.g., a DNA vector).

[0505] It will be apparent to those skilled in the art that the DNA disclosed herein may also contain 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 the insertion of one or more nucleotide sequences (e.g., encoding an antigen of interest, a fragment thereof, and / or a variant thereof) without disrupting the rest of the DNA.

[0506] As used herein, the term "restriction endonuclease site" refers to the DNA sequence that binds to a restriction endonuclease. Typically, restriction endonuclease sites are short sequences (e.g., short sequences of about 4-8 base pairs) that are recognized and cleaved by restriction endonucleases.

[0507] As used herein, the term "restriction enzyme" or "restriction endonuclease" refers to a class of enzymes that are naturally found in bacteria and some viruses. Restriction endonucleases specifically bind to and cleave double-stranded DNA at specific sites within or near the restriction endonuclease site. Exemplary restriction 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.

[0508] In one instance, this 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, operatively linked to regulatory elements (e.g., SG promoters and IRES). For example, the polynucleotide is a DNA plasmid comprising the first and second nucleotide sequences.

[0509] In one example, the DNA comprises a nucleotide sequence containing a restriction endonuclease site at the 3' of the 3' UTR. The presence of the restriction endonuclease site at the 3' UTR allows for the production of linearized DNA. Linearization of the DNA ensures well-defined termination of in vitro transcribed DNA to produce mRNA.

[0510] Ribonucleic acid (RNA)

[0511] In one instance, the polynucleotide is mRNA containing a first nucleotide sequence encoding an antigen, the first nucleotide sequence being operatively linked to a promoter, wherein the antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0512] The mRNAs disclosed herein include non-replicating mRNAs (also known as conventional mRNA (cRNA) or non-amplified mRNA) and self-replicating RNAs (also known as self-amplified RNA or sa-mRNA).

[0513] Conventional (non-replicating) RNA

[0514] In one instance, the polynucleotide is cRNA containing a first nucleotide sequence encoding an antigen, the first nucleotide sequence being operatively linked to a promoter, wherein the antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0515] In an example, the cRNA of this disclosure comprises, from 5' to 3', the following sequence: a 5' cap structure, a 5'-UTR, a fragment thereof and / or a variant thereof, 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' tail sequence (e.g., a polyadenylation signal or one or more polyadenylated tails), wherein the first antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The cRNA of this disclosure may also include an internal ribosome entry site (e.g., a Kozak concordance sequence or IRES) operatively linked to the antigen of interest.

[0516] Self-replicating RNA

[0517] This disclosure provides a self-replicating RNA (also known as a replicon).

[0518] Those skilled in the art will understand that the self-replication of this disclosure is based on RNA-based genomic RNA from RNA viruses. The RNA should be positive (+)- strand, allowing it to be translated directly upon delivery to the cell without intermediate replication steps (e.g., reverse transcription). Translation of the RNA results in the production of non-structural proteins (NSPs), which bind to form a replicase complex (i.e., RNA-dependent RNA polymerase). This complex then amplifies the original RNA, producing antisense and sense transcripts, thereby generating multiple daughter RNAs that can be translated and transcribed to enhance overall protein expression.

[0519] In one instance, the self-replicating RNA disclosed herein comprises non-structural proteins of an RNA virus, 5' and 3' untranslated regions (UTRs), and a natural subgenomic promoter.

[0520] In one instance, the self-replicating RNA contains one or more non-structural proteins of an RNA virus. For example, the RNA contains at least one or more genes selected from: viral replicase (or viral polymerase), viral protease, viral helicase, and other non-structural viral proteins. For example, the self-replicating RNA contains viral replicase (or viral polymerase).

[0521] In another example, the self-replicating RNA contains the 5' and 3'-end UTRs of the RNA virus. Those skilled in the art will appreciate that the terms 5' and 3' UTR also include the terms 5' and 3' conserved sequence elements (CSEs). In one example, the self-replicating RNA contains 5'- and 3'-end CSEs.

[0522] The self-replicating RNA disclosed herein cannot induce the production of infectious viral particles. For example, the self-replicating RNA disclosed herein does not contain viral genes encoding structural proteins required for the production of viral particles.

[0523] In one instance, the self-replicating RNA is derived from or based on an alphavirus. Those skilled in the art will understand and / or describe suitable alphaviruses herein.

[0524] In another instance, the self-replicating RNA is derived from or based on a virus other than alphavirus, such as a positive-sense RNA virus. Those skilled in the art will recognize that the positive-sense RNA viruses used in this disclosure are suitable, and include, for example, picornaviruses, flaviviruses, rubella viruses, plague viruses, hepatoviruses, caliciviruses, or coronaviruses.

[0525] In one instance, 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 the sequence shown in SEQ ID NO:17.

[0526] In one instance, 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 the sequence shown in SEQ ID NO:18.

[0527] In one instance, 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 the sequence shown in SEQ ID NO:19.

[0528] A virus

[0529] In one instance, the self-replicating RNA disclosed herein is derived from (or based on) an alphavirus.

[0530] Alphaviruses are the only genus in the Bleoviridae 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 types of open reading frames (ORFs): non-structural and structural. The first ORF encodes four non-structural proteins (NSP1, NSP2, NSP3, and NSP4), which are essential for viral RNA transcription and replication. The second ORF encodes three structural proteins: the core nucleocapsid protein C and the envelope proteins P62 and E1, which associate as heterodimers. Viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells via membrane fusion.

[0531] In one instance, the self-replicating RNA disclosed herein contains a viral replicase (or viral polymerase). For example, the viral replicase is an alphavirus replicase, such as the alphavirus protein NSP4.

[0532] In one instance, the self-replicating RNA of this disclosure does not encode one or more alphavirus structural proteins (e.g., capsid and / or envelope glycoproteins). For example, the self-replicating RNA cannot produce RNA-containing alphavirus virions (i.e., infectious viral particles).

[0533] In one instance, the self-replicating RNA contains the natural alphavirus SG promoter. For example, the natural alphavirus SG promoter is a minimal SG promoter (i.e., the minimum sequence required to initiate transcription) and contains the sequence shown in SEQ ID NO:1.

[0534] Those skilled in the art will recognize the alphaviruses suitable for use in this disclosure. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEE; e.g., Trinidadian donkey, TC83CR), Semliki Forest Virus (SFV), Sindbius Virus (SIN), Ross River Virus, Western Equine Encephalitis Virus, Eastern Equine Encephalitis Virus, Chikungunya Virus, SA AR86 Virus, Everglades Virus, Mucambo Virus, Balma Forest Virus, Middelburg Virus, Pisuna Virus, Arlangi Virus, Geta Virus, Heron Mountain Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Wataroa Virus, Banbanki Virus, Ziragachi Virus, Highland J Virus, Morganburg Virus, Ndum Virus, and Baki Creek Virus. The term alphavirus may also include chimeric alphaviruses (e.g., described by Perri et al., (2003) J. Virol. 77(19): 10394-403) that contain genomic sequences from more than one alphavirus.

[0535] Control element

[0536] This disclosure provides a polynucleotide comprising a nucleotide sequence encoding an antigen operatively linked to a promoter wherein the antigen is the spike (S) protein of the omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0537] In this example, the regulatory element is selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES) and a Kozac concordant sequence, or a combination thereof. In this example, the regulatory element is the SG promoter.

[0538] Kozak public sequence

[0539] As used in this article, the term "Kozak concordance sequence" refers to a nucleotide sequence identified in a eukaryotic gene that facilitates gene translation by containing a start codon recognized by ribosomes (also known as a translation start codon).

[0540] Exemplary Kozak shared sequences are known in the art and / or described herein. In one instance, the Kozak shared sequence is shown as SEQ ID NO:6 (GCCACC). In another instance, the Kozak shared sequence is shown as SEQ ID NO:7 (ACCATGG).

[0541] subgenome promoters

[0542] Those skilled in the art will clearly understand and / or describe herein the SG promoter (also known as a 'junction region' promoter) suitable for use in this disclosure.

[0543] In one instance, the SG promoter is derived from or based on the alphavirus SG promoter. For example, the SG promoter is the natural alphavirus SG promoter. In one instance, the natural SG promoter is the minimal SG promoter. For example, the minimal SG promoter is the minimum sequence required for transcription initiation. In one instance, the natural SG promoter is the extended SG promoter. For example, the extended SG promoter is the minimal SG promoter extended at the 5' end with nucleotides that appear in the sequence encoding a non-structural protein (e.g., NSP4) of an RNA virus (e.g., alphavirus). In one instance, the extended SG promoter is the minimal SG promoter extended at the 5' end with nucleotides that appear in the sequence encoding alphavirus NSP4.

[0544] In one example, the SG promoter is extended at the 5' end by approximately 31 nucleotides that appear in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by a sequence as shown in SEQ ID NO:1, which is extended at the 5' end by 31 nucleotides that appear in a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the length of the extended SG promoter does not exceed 80 nucleotides. In one example, the extended SG promoter is encoded by a sequence comprising or consisting of nucleotides 22 to 101 of SEQ ID NO:5. In yet another example, the extended SG promoter is encoded by a sequence comprising or consisting of the sequence shown in SEQ ID NO:3.

[0545] In one example, the extended SG promoter contains repeating sequences corresponding to nucleotides 66 to 75 of SEQ ID NO:5. For example, the extended SG promoter is encoded by a sequence containing nucleotides 50 to 75 of SEQ ID NO:5 and nucleotides 66 to 101 of SEQ ID NO:5. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO:15.

[0546] In one instance, the polynucleotides of this disclosure contain the SG promoter from any alphavirus. For example, the RNA of this disclosure (e.g., cRNA or self-replicating RNA) contains the SG promoter from any alphavirus.

[0547] In one instance, the self-replicating RNA contains the SG promoter from any alphavirus.

[0548] The polynucleotides disclosed herein comprise two or more nucleotide sequences encoding two or more antigens of interest. In one instance, each of the two or more nucleotide sequences is operatively linked to an SG promoter. When 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 are derived from the same alphavirus. In another instance, the two or more SG promoters are derived from different alphaviruses.

[0549] When 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 be derived from the same alphavirus. In another instance, two or more SG promoters may be derived from different alphaviruses.

[0550] In another instance, the polynucleotide of this disclosure comprises two or more nucleotide sequences encoding two or more antigens of interest, the two or more nucleotide sequences being driven by the same promoter or by two or more promoters, the promoters themselves containing the same sequence or different sequences.

[0551] Internal ribosome entry site (IRES)

[0552] Those skilled in the art will clearly understand and / or see the IRES suitable for use in this disclosure as described herein.

[0553] In one instance, IRES is derived from encephalomyocarditis virus (EMCV). For example, IRES is a wild-type IRES derived from EMCV.

[0554] In one instance, IRES is derived from fibroblast growth factor 1A (FGF1A) IRES.

[0555] In addition, synthetic IRES elements have been described that can be designed to mimic the function of naturally occurring IRES elements according to methods known in the art (see Chappell, SA et al., Proc. Natl Acad. Sci. USA (2000) 97(4): 1536-41).

[0556] In one instance, IRES is derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simultaneous immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-associated 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 protein (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0557] In one instance, the IRES is a wild-type IRES derived from encephalomyocarditis virus (EMCV). For example, wild-type EMCVIRES contains the sequence shown in SEQ ID NO:4.

[0558] 5' - Untranslated region (5' - UTR)

[0559] In the example described herein, the polynucleotide contains a 5'-untranslated region (5'-UTR).

[0560] As used in this article, the term “5’-untranslated region” or “5’-UTR” refers to the non-coding region of mRNA located at the 5’ end of the translation initiation sequence (AUG).

[0561] Exemplary 5'-UTRs include, for example, the 5'-UTRs, fragments and / or variants of haptoglobin (HP), fibrinogen β chain (FGB), haptoglobin-associated protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen α chain (FGA), α6 collagen (Col6A), α-1-antitrypsin (SERPINA1), α-1-antiplasmin (SERPINA3).

[0562] In one instance, the 5'-UTR is the 5'-UTR of Venezuelan equine encephalitis virus (VEEV) or a modified form thereof. For example, the 5'-UTR contains the sequence shown in SEQ ID NO:13.

[0563] In one instance, the 5'-UTR contains at least one microRNA binding site, an AU enrichment element (ARE), a GC enrichment element, a stem-loop, or a combination thereof.

[0564] microRNA binding site

[0565] As used herein, the term “microRNA binding site” refers to a sequence within a polynucleotide (e.g., DNA or RNA transcript) that is sufficiently complementary to all or one region of a miRNA to interact, associate, or bind with the microRNA (miRNA).

[0566] As used herein, the term "microRNA" or "miRNA" refers to a 19-25 nucleotide-long non-coding RNA that binds to a multi-nucleotide 5'-UTR and downregulates gene expression (e.g., by inhibiting translation). The presence of microRNA binding sites in the 5'-UTR of this disclosure can inhibit translation via the 5'-UTR.

[0567] Those skilled in the art will clearly understand and / or see the microRNA binding sites suitable for use in this disclosure as described herein.

[0568] In one instance, the miRNA binding site contains the binding site for tissue-specific microRNAs or microRNAs that regulate biological processes. Examples include microRNAs from the liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid 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 (e.g., miR-132). Further examples of microRNA and microRNA binding sites are disclosed in U.S. Patent Application US14 / 043,927.

[0569] AU enriched element (ARE)

[0570] As used herein, the term “AU-enriched element (ARE)” or “AU-enriched elements (AREs)” refers to a region of a nucleotide sequence that contains adenosine (A) and uridine (U). Exemplary AREs include, for example, AREs derived from cytoplasmic myc (c-myc), myoblast-determining protein 1 (myoD), c-Jun, myoblastin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), or combinations thereof.

[0571] In one instance, the ARE contains a specific binding site for human antigen R, or “HuR” (also known as Elavl1). HuR is known to bind to the ARE to increase the stability of the mRNA.

[0572] GC enriched elements

[0573] As used herein, the term "GC-enriched element" refers to a nucleotide sequence that has a high concentration of guanine (G) and / or cytosine (C) compared to adenine (A) and thymine (T) / uracil (U). The presence of GC-enriched elements in polynucleotides (e.g., mRNA) can stabilize mRNA.

[0574] In one instance, the GC enrichment element comprises 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 in length.

[0575] In one example, the GC enrichment element contains 30% to 40%, or 40% to 50%, or 50% to 60%, or 60% to 70% cytosine. For example, the GC enrichment element contains 30% to 40% cytosine. For example, the GC enrichment element contains 40% to 50% cytosine. For example, the GC enrichment element contains 50% to 60% cytosine. For example, the GC enrichment element contains 60% to 70% cytosine.

[0576] In one example, the GC enrichment element contains 30%, 40%, 50%, 60%, or 70% cytosine. For example, the GC enrichment element contains 30% cytosine. For example, the GC enrichment element contains 40% cytosine. For example, the GC enrichment element contains 50% cytosine. For example, the GC enrichment element contains 60% cytosine. For example, the GC enrichment element contains 60% cytosine. For example, the GC enrichment element contains 70% cytosine.

[0577] In one instance, the GC-enriched element is at least 50% cytosine.

[0578] In one instance, the GC-enriched element was at least 60% cytosine.

[0579] In one instance, the GC-enriched element was at least 70% cytosine.

[0580] In one example, the GC enrichment element contains the nucleotide sequence CCCGGCGCC. In another example, the GC enrichment element contains the nucleotide sequence CCCCGGC. In a further example, the GC enrichment element contains the nucleotide sequence GCGCCCCGCGGCGCCCCGCG.

[0581] In one example, the GC enrichment element comprises the nucleotide sequence shown in SEQ ID NO:9 to 11. In another example, the GC enrichment element comprises the nucleotide sequence shown in SEQ ID NO:9. In yet another example, the GC enrichment element comprises the nucleotide sequence shown in SEQ ID NO:10. In a further example, the GC enrichment element comprises the nucleotide sequence shown in SEQ ID NO:11 (CCCCGGC).

[0582] Stem ring

[0583] As used herein, the term "stem-loop" refers to a nucleotide sequence comprising two adjacent, fully or partially anticomplementary sequences that pair together to form a stem-loop. Stem-loops can occur in single-stranded DNA, or more commonly in RNA. A stem-loop, also called a hairpin or hairpin loop, typically consists of a stem and a terminal loop within a continuous sequence, wherein the stem is formed by two adjacent, fully or partially anticomplementary sequences separated by a short sequence that constructs the loop within the stem-loop structure.

[0584] The stability of a paired stem-loop is determined by its length, the number of mismatches or protrusions it contains, and the nucleotide composition of the paired region.

[0585] In one instance, the length of the stem loop is 3 to 10 nucleotides. For example, the length of the stem loop is 3 to 8 nucleotides, or 3 to 7 nucleotides, or 3 to 6 nucleotides, or 4 to 5 nucleotides.

[0586] In one instance, the stem loop has a ring length of 4 nucleotides.

[0587] In one instance, the stem-loop is a histone stem-loop. For example, a histone stem-loop may contain or consist of the nucleotide sequence shown in SEQ ID NO:12.

[0588] 3'-Untranslated Region (3'-UTR)

[0589] In an example, the polynucleotide disclosed herein contains a 3'-untranslated region (3'-UTR).

[0590] As used in this article, the term "3'-UTR" refers to the region of mRNA located at the 3' end of the translation termination codon (i.e., the stop codon).

[0591] Exemplary 3'-UTRs include, for example, the 3'-UTR of arachidonic acid 5-lipoxygenase (ALOX5), αI collagen (COL1A1), tyrosine hydroxylase (TH) gene, split N-terminal enhancer (AES), human mitochondrial 12S rRNA (mtRNR1), fragments thereof, and / or variants thereof.

[0592] In one instance, the 3'-UTR is the 3'-UTR of Sindbis virus (SINV) or a modified form thereof. For example, the 3'-UTR contains the sequence shown in SEQ ID NO:14 or 22.

[0593] In one instance, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of the albumin gene. In another instance, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of the vertebrate α-globin gene. For example, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of the mammalian α-globin gene. For example, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of the human α-globin gene.

[0594] In one instance, the 3'-UTR of this disclosure also includes at least one microRNA binding site, an AU enrichment element (ARE), a GC enrichment element, a triple helix, a stem-loop, one or more stop codons, or a combination thereof.

[0595] stop codon

[0596] As used in this article, the term "stop codon" refers to a trinucleotide sequence within mRNA that signals the ribosomes to stop protein synthesis.

[0597] In one instance, the polynucleotide of this disclosure contains 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.

[0598] In one instance, the polynucleotide contains two consecutive stop codons, which contain the sequence UGAUGA.

[0599] In one instance, the polynucleotide contains two consecutive stop codons, which contain the sequence UAAUAG.

[0600] 3' tail sequence

[0601] In an example, the polynucleotide disclosed herein comprises one or more 3' tail sequences located at the 3' end of the 3'-UTR.

[0602] As described herein, the terms "3' tail sequence" or "multiple 3' tail sequences" refer to nucleotide sequences that induce the addition of non-coding nucleotides to the 3' end of mRNA (e.g., polyadenylation signals) or nucleotide sequences located at the 3' end of mRNA (e.g., polyadenylation sequences). Those skilled in the art will understand that the function of 3' tail sequences and / or the products of 3' tail sequences in mRNA is to stabilize mRNA and / or prevent mRNA degradation.

[0603] As used herein, when referring to the polyadenylated or polycytosine sequence of this disclosure, the term "break linker" refers to a single nucleotide or nucleotide sequence that links to and breaks a continuous adenosine or cytosine nucleotide segment in the polyadenylated or polycytosine sequence. For example, a break linker in a polyadenylated sequence is a single nucleotide or nucleotide sequence that comprises or is composed of nucleotides other than adenosine nucleotides. For example, a break linker in a polycytosine sequence is a single nucleotide or nucleotide sequence that comprises or is composed of nucleotides other than cytosine nucleotides.

[0604] In one instance, one or more 3' tail sequences are selected from polyadenylation sequences, polyadenylation signals, G-quadruplexes, polycytidine sequences, stem-loops, and combinations thereof.

[0605] Polyadenylation sequence

[0606] As used herein, the term "polyadenylated sequence" refers to the nucleotide sequence of adenine (A) located at the 3' end of mRNA. In the context of this disclosure, the polyadenylated sequence may be located within mRNA or DNA (e.g., a DNA plasmid used as a template for generating mRNA via a transcription vector).

[0607] Those skilled in the art will clearly understand and / or as described herein the suitable polyadenylated sequences used in this disclosure. In one example, the polyadenylated sequence comprises consecutive (i.e., one after another) adenosine nucleotides of any length (e.g., 10 to 300). In one example, the polyadenylated sequence comprises consecutive adenosine nucleotides separated by one or more break linkers. In one example, the polyadenylated sequence comprises consecutive adenosine nucleotides without break linkers.

[0608] Polyadenylation signaling

[0609] As used herein, the term "polyadenylation signal" refers to the nucleotide sequence that induces polyadenylation. Polyadenylation is generally understood as the addition of a polyadenylation sequence to RNA (e.g., adding a polyadenylation sequence to immature mRNA to produce mature mRNA). The polyadenylation signal may be located within the nucleotide sequence at the 3' end of the polynucleotide (e.g., mRNA) to be polyadenylated.

[0610] Those skilled in the art will clearly understand and / or as described herein the appropriate polyadenylation signal for use in this disclosure.

[0611] In one instance, the polyadenylation signal comprises a hexamer consisting of adenine and uracil / thymine nucleotides. In another instance, the hexamer sequence comprises or consists of AAUAAA.

[0612] In one instance, the 3' tail sequence contains a polyadenylation signal but not a polyadenylation sequence.

[0613] G-quadruplex

[0614] As used herein, the term “G-quadruplex” or “G4” refers to a nucleotide sequence rich in guanine residues that forms a four-strand secondary structure. For example, a G-quadruplex is a circular array of hydrogen-bonded guanine nucleotides formed by G-rich sequences in DNA and RNA.

[0615] In one example, the 3' tail sequence comprises a polyadenylated sequence and a G-tetramolecular. For instance, the 3' tail sequence comprises a polyadenylated sequence linked to a G-tetramolecular to produce a polyadenylated-guanine tetrad.

[0616] Polycytidine sequence

[0617] As used herein, the term "polycytosine sequence" refers to the nucleotide sequence of cytosine (C) located at the 3' end of mRNA. In the context of this disclosure, the polycytosine sequence may be located within mRNA or DNA (e.g., a DNA plasmid used as a template for generating mRNA via a transcription vector).

[0618] Those skilled in the art will clearly understand and / or as described herein the appropriate polycytosine sequence used in this disclosure.

[0619] In one instance, one or more 3' tail sequences comprise one or more polycytosine nucleotide sequences, each comprising 10 to 300 consecutive cytosine nucleotides. For example, one or more polycytosine nucleotide sequences each comprise 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 polycytosine 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.

[0620] In one instance, one or more polycytosine sequences are separated by a break linker. For example, a fourth nucleotide sequence containing one or more 3' tail sequences comprises, from 5' to 3', consecutive cytosine nucleotides, a break linker, and further consecutive cytosine nucleotides.

[0621] In one instance, the length of the break linker is 10 to 50, or 50 to 100, or 100 to 150 nucleotides. For example, the length of the break linker is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95, or 100, or 110, or 120, or 130, or 140, or 150 nucleotides.

[0622] 5' cap structure

[0623] In one instance, this disclosure provides an mRNA that includes a 5' end cap structure.

[0624] As used herein, the term "5' cap structure" refers to a structure located at the 5' end of mRNA that participates in nuclear export and binds to the mRNA cap-binding protein (CBP). The 5' cap structure is known to stabilize mRNA by associating the CBP with polyadenylate-binding proteins to form mature mRNA. Therefore, the presence of a 5' cap structure in the mRNA of this disclosure further increases mRNA stability compared to mRNA without a 5' cap.

[0625] Exemplary 5' cap structures include, for example, anti-reverse cap analogs (ARCA), N7,2'-O-dimethylguanosine (mCAP), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazonoguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, 2-azidoguanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), Cap1, and Cap2.

[0626] Typically, endogenous mRNA is 5'-capped with guanosine via a (5')ppp(5')-triphosphate bond to the 5' terminal nucleotide of the mRNA. The guanosine cap can then be methylated to 7-methylguanosine (m7G), resulting in 7mG(5')ppp(5')N,pN2p (Cap0 structure), where N represents the first and second 5' terminal nucleotides of the mRNA. The Cap0 structure can be further 2'-O-methylated to produce 7mG(5')ppp(5')NlmpNp (Cap1) and / or 7mG(5')-ppp(5')NlmpN2mp (Cap2).

[0627] In one instance, the polynucleotide disclosed herein comprises an endogenous cap.

[0628] As used herein, the term "endogenous cap" refers to a 5' cap synthesized in the cell. For example, an endogenous cap is a natural or wild-type 5' cap. For example, an endogenous cap is a Cap0, Cap1, or Cap2 structure.

[0629] In one instance, the polynucleotide disclosed herein comprises an analogue of an endogenous cap (also known as a cap analogue).

[0630] As used herein, the term "its analogue" in the context of endogenous caps or "cap analogues" refers to a synthetic 5' cap. Cap analogues can be used to generate 5'-capped mRNA in in vitro transcription reactions. Cap analogues can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked 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, m7G(5′)ppp(5′)G (New England BioLabs). In one example, the cap analogue is N7,3′-O-dimethylguanosine-5′-triphosphate-5′-guanosine (i.e., anti-reverse cap analogue (ARCA)).

[0631] In one instance, the 5' cap structure is a non-hydrolyzable cap structure. Non-hydrolyzable cap structures prevent mRNA from uncapping and increase the half-life of the mRNA.

[0632] In one example, the non-hydrolyzed cap structure comprises a modified nucleotide selected from α-thioguanosine nucleotides, α-methylphosphonates, selenophosphates, and combinations thereof. In one example, the modified nucleotide is linked to the 5' end of the mRNA via an α-thiophosphate bond. Those skilled in the art will understand methods for linking modified nucleotides to the 5' end of mRNA. For example, using a Vaccinia capping enzyme (New England BioLabs).

[0633] Modification

[0634] In one instance, the polynucleotide disclosed herein comprises one or more modifications. Typically, modifications are introduced into polynucleotides (e.g., mRNA) to increase their translation efficiency and / or stability. Suitable modifications to polynucleotides will be apparent to those skilled in the art and / or described herein.

[0635] In one instance, the first nucleotide sequence containing the 5'-UTR and / or a fragment thereof is modified. This 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 the fragment thereof.

[0636] In one instance, one or more nucleotide sequences of a polynucleotide are codon-optimized. Methods for codon optimization will be clear to those skilled in the art and / or described herein. For example, tools for codon optimization of polynucleotides include, for instance, GeneArt GeneOptimizer (Thermofisher). ® ) or GenSmart ® (GeneScript ® ).

[0637] In one instance, the polynucleotide is modified to increase the amount of guanine (G) and / or cytosine (C) in the polynucleotide. The amount of G / C in the polynucleotide (i.e., the G / C content) can affect the stability of the polynucleotide. Therefore, polynucleotides containing increased amounts of G / C nucleotides are functionally more stable than polynucleotides containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. The G / C content is increased by replacing A or T nucleotides with G or C nucleotides.

[0638] In one example, the G / C content in the first and / or second nucleotide sequences is increased, and the first and / or second nucleotide sequences encode a first and / or a second antigen of interest. For example, the G / C content in the first and / or second nucleotide sequences is increased, and the first and / or second nucleotide sequences encode S protein and / or N protein antigens. Modifications in the first and / or second and / or one or more nucleic acid sequences utilize the ability to replace codons containing fewer favorable nucleotide combinations (in terms of mRNA stability) with alternative codons (e.g., conserved amino acid substitutions), said alternative codons encoding the same amino acid or encoding an amino acid with similar chemical properties. For example, the G / C content is increased by replacing a codon containing an A or T nucleotide with a codon containing a G or C nucleotide encoding the same amino acid. For example, the G / C content is increased by replacing a codon containing an A or T nucleotide with a codon containing a G or C nucleotide encoding an amino acid with similar chemical properties.

[0639] In one instance, the G / C content is increased in one or more nucleotide sequences of a polynucleotide that does not encode the antigen of interest. For example, the G / C content is increased in the 5'-UTR, fragments, and / or variants thereof. For example, the G / C content is increased in the 3'-UTR, fragments, and / or variants thereof.

[0640] In one instance, a polynucleotide contains at least one chemically modified nucleotide.

[0641] As used herein, the term "chemically modified" or "chemically modified" in the context of nucleotides refers to a naturally occurring nucleotide (i.e., A, T, C, G, U) that has been modified compared to a naturally occurring nucleotide by substitution, insertion, or removal of individual or several atoms or groups of atoms. In one example, at least one naturally occurring nucleotide of a polynucleotide is replaced by a chemically modified nucleotide. In one example, at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100% of the naturally occurring nucleotide of a polynucleotide is replaced by a chemically modified nucleotide. Suitable chemically modified nucleotides for use in this disclosure will be apparent to those skilled in the art and / or described herein. Exemplary 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-methylpseudouridine (m1ψ).

[0642] SARS-CoV-2 antigen

[0643] The polynucleotides of this disclosure comprise a nucleic acid sequence encoding an antigen operatively linked to a promoter, wherein the antigen is the spike (S) protein of the omicron strain of SARS-CoV-2. In a further example, the polynucleotides of this disclosure comprise a nucleic acid sequence encoding a second antigen operatively linked to a promoter, wherein the antigen is the nucleocapsid (N) protein of SARS-CoV-2. In the context of this disclosure, the antigen is therefore a pathogen antigen. For example, antigens of interest are antigenic peptides, immunogenic fragments thereof, and / or variants that can induce an immune response in a subject.

[0644] The SARS-CoV-2 genome encodes at least four major structural proteins: spike (S), membrane (M), envelope (E), nucleocapsid (N) proteins, and other accessory proteins that contribute to the replication process and facilitate cell entry. The M protein is the most abundant component of the viral envelope and directs coronavirus assembly through interactions with all other structural proteins. The E protein is a small membrane protein, or viral pore protein, thought to promote viral particle budding by pinning the cell membrane surface. The S protein is a class I fusion protein that mediates the attachment of SARS-CoV-2 to the major cell surface receptor, human angiotensin-converting enzyme 2 (ACE2). Due to its exposed conformation on the viral surface, the S protein is highly immunogenic and is a major focus of current vaccine development. The N protein packages the RNA genome to form the nucleocapsid, although not necessarily required for envelope formation; it appears to play an important role in the assembly and stability of intact virions and in increasing VLP yield.

[0645] The S protein comprises three domains: (i) a large extracellular domain; (ii) a transmembrane domain (which passes through the viral envelope in a single pass); and (iii) a short intracellular tail. The extracellular domain consists of a trimer stem composed 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 the host and viral membranes, allowing the viral genome to enter the host cell. Receptor binding and membrane fusion are the initial and critical steps in the coronavirus infection cycle. Different CoVs target significantly different receptors.

[0646] The structure of the SARS-CoV-2 S protein is described, for example, by Cai et al. (Science (2020) 369:1586-1592), the full text of which is incorporated herein by reference. Each S1 subunit of the SARS-CoV-2 S protein contains an N-terminal domain (NTD), a receptor-binding domain (RBD), and two C-terminal domains (CTD). Prior to fusion with the host cell membrane, the S1 subunit of the SARS-CoV-2 S protein protects the S2 subunit. Upon binding to ACE2, the SARS-CoV-2 S protein refolds in a "folding knife" manner, forming a long central coiled helix, ultimately leading to membrane fusion and viral entry into the host cell.

[0647] In view of the tendency of RNA viruses such as SARS-CoV-2 to mutate, the inventors have provided a polynucleotide encoding an S protein sequence that may include mutations found in different strains of SARS-CoV-2, making the vaccine composition particularly useful in therapeutically targeted SARS-CoV-2 strains (e.g., the omicron strain of SARS-CoV-2).

[0648] In one instance, the polynucleotide of this disclosure encodes an S protein, said S protein comprising a mutation from the omicron strain of SARS-CoV-2. In one example, the omicron strain is variant BA.1, which encodes an S protein containing one or more 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.

[0649] In another example, where the omicron strain is variant BA.2, the polynucleotide encodes an S protein containing 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.

[0650] In another example, the omicron strain is variant BA.4 or BA.5, which encodes an S protein containing 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.

[0651] The mRNAs, conventional mRNAs, self-replicating RNAs, compositions, and vaccines disclosed herein can also be used to treat other variants of SARS-CoV-2. Specifically, the mRNAs, conventional mRNAs, self-replicating RNAs, compositions, and vaccines disclosed herein have been found to be useful in treating the B.1.1.7 SARS-CoV-2 strain (also known as 201 / 501Y.Vl, which was first detected in the UK and is now referred to as the Alpha variant); the B.1.351 SARS-CoV-2 strain (also known as 20H / 501.V2, which was first detected in South Africa and is now referred to as the Beta variant); the P1 SARS-CoV-2 strain (also known as 20J / 501Y.V3, which was first detected in Japan and Brazil and is now referred to as the Gamma variant); the B1.427 and B1.429 SARS-CoV-2 strains (which were first detected in California and are now referred to as the Epsilon variant); and / or the B.1.617.2 SARS-CoV-2 strain (which was first detected in India and is now referred to as the Delta variant). The mRNA, conventional mRNA, self-replicating RNA, compositions, and vaccines disclosed herein can also be used to treat the (original) strain of SARS-CoV-2.

[0652] According to the US CDC (SARS-CoV-2 Variant Classification and Definitions (cdc.govl)), the Alpha variant has been found to contain the following S protein mutations: 69 deletion, 70 deletion, 144 deletion, (E484K) (S494P) N501Y, A570D, D614G, P681H, T7161, S982A, D1118H and (K1191N) The key mutations are deletions of residues 69 / 70 and 144Y, and substitutions of N501Y, A570D, D614G, and P681H. Beta variants have been found to contain the following mutations: D80A, D215G, deletion of 241, deletion of 242, deletion of 243, K417N, E484K, N501Y, D614G, and A701V, with key mutations being substitutions of K417N, E484K, N501Y, and D614G. Gamma variants have been found to contain the following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T10271, with key mutations being E484K, K417N / T, N501Y, and D614G. Delta variants have been found to contain the following mutations: T19R, (G142D) The mutations found include 156 deletion, 157 deletion, R158G, L452R, T478K, D614G, P681R, and D950N, with key mutations being L452R, E484Q, and T478K. Epsilon variants have been found to contain the following mutations: S131, W152C, 30L452R, and D614G, with the key mutation being L452R. Therefore, this disclosure covers self-replicating RNAs containing polynucleotides encoding antigens derived from the S protein, which includes one or more of the mutations described above.

[0653] In the example, the nucleotide sequence encoding the antigen from the S protein comprises 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%, at least about 99% or the same as the S protein nucleotide sequence defined in SEQ ID NO:17.

[0654] In the example, the nucleotide sequence encoding the antigen from the S protein comprises 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%, at least about 99% or the same as the S protein nucleotide sequence defined in SEQ ID NO:18.

[0655] In the example, the nucleotide sequence encoding the antigen from the S protein comprises 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%, at least about 99% or the same as the S protein nucleotide sequence defined in SEQ ID NO:19.

[0656] In this example, the antigen from the S protein is encoded by a polynucleotide sequence comprising 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%, at least about 99%, or the same nucleotide sequence as defined in SEQ ID NO:17. In this example, the encoded sequence is self-replicating RNA.

[0657] In this example, the antigen from the S protein is encoded by a polynucleotide sequence comprising 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%, at least about 99%, or the same nucleotide sequence as defined in SEQ ID NO:18. In this example, the encoded sequence is self-replicating RNA.

[0658] In this example, the antigen from the S protein is encoded by a polynucleotide sequence comprising 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%, at least about 99%, or the same nucleotide sequence as defined in SEQ ID NO:19. In this example, the encoded sequence is self-replicating RNA.

[0659] In the example, the S protein is encoded by 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% of SEQ ID NO:20.

[0660] In this example, the S protein is encoded by the polynucleotide sequence shown in SEQ ID NO:20.

[0661] In one instance, the polynucleotide encodes an antigen from each of the S and N proteins of SARS-CoV-2. In another instance, the polynucleotide encodes an antigen from each of the S and N proteins of the omicron strain of SARS-CoV-2. In this instance, the omicron variant may be BA.1 or BA.2.

[0662] In one example, the polynucleotide encoding the second antigen may originate 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, the antigens do not originate 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 this example, both the S and N antigens originate from the omicron strain of SARS-CoV-2.

[0663] In one instance, the S protein from the omicron strain of SARS-CoV-2 originates from:

[0664] (a) The omicron variant BA.1 and the S protein contains one or more 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

[0665] (b) omicron variant BA.2 and the S protein 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

[0666] (c)omicron variant BA.4 or BA.5 and the S protein contains 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

[0667] (d) The 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; or

[0668] (e) The omicron variant of SARS-CoV-2 and whose S protein contains the following proteins selected from T19I, Δ24-26, A27S, V83A, Δ144, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, and R40. One or more of the mutations in the group consisting of 8S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, Q954H, and N969K; or

[0669] (f) The omicron variant of SARS-CoV-2, and the S protein contains selected from 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, R One or more of the mutations in the group consisting of 408S, 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; or

[0670] (g) The 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; or

[0671] (h) The omicron variant of SARS-CoV-2 and the S protein contains one or more of the mutations R346T, F456L and T572I.

[0672] In the example, the nucleotide sequence encoding the antigen from the N protein comprises 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%, at least about 99% or the same as the N protein nucleotide sequence defined in SEQ ID NO:18.

[0673] In the example, the nucleotide sequence encoding the antigen from the N protein comprises 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%, at least about 99% or the same as the N protein nucleotide sequence defined in SEQ ID NO:19.

[0674] In this example, the antigen from the N protein is encoded by a polynucleotide sequence comprising 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%, at least about 99%, or the same nucleotide sequence as defined in SEQ ID NO:18. In this example, the encoded sequence is a self-replicating RNA.

[0675] In this example, the antigen from the N protein is encoded by a polynucleotide sequence comprising 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%, at least about 99%, or the same nucleotide sequence as defined in SEQ ID NO:19. In this example, the encoded sequence is self-replicating RNA.

[0676] In the example, the N protein is encoded by 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% of SEQ ID NO:21.

[0677] In this example, the N protein is encoded by the polynucleotide sequence shown in SEQ ID NO:21.

[0678] In one instance, the polynucleotide encoding the second antigen from the N protein was derived from the omicron strain of SARS-CoV-2.

[0679] Production methods

[0680] Methods suitable for producing the polynucleotides, cRNAs and / or self-replicating RNAs disclosed herein will be clear to those skilled in the art and / or described herein.

[0681] In one instance, a polynucleotide is DNA. For example, a polynucleotide is plasmid DNA.

[0682] In one example, plasmid DNA is used to produce cRNA. In another example, plasmid DNA is used to produce self-replicating RNA. 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 disclosed herein. Single bacterial colonies are isolated and the resulting plasmid DNA is amplified in *E. coli* cultures.

[0683] In one instance, plasmid DNA is isolated after fermentation. For example, plasmid DNA is isolated using a commercially available kit (e.g., the Maxiprep DNA kit) or other conventional methods known to those skilled in the art. After isolation, the 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.

[0684] In one example, mRNA is prepared by in vitro transcription from a linearized DNA template using an RNA polymerase (e.g., T7 RNA polymerase). Following in vitro transcription, the DNA template is removed by DNase digestion. Those skilled in the art will understand that capping the synthetic mRNA corrects mRNA processing and contributes to mRNA stability. In one example, the mRNA is enzymatically 5'-capped. For example, the 5' cap is a cap0 structure or a cap1 structure. In one example, the 5' cap is a cap0 structure, such as a 5'-cap (i.e., cap0) consisting of a reverse 7-methylguanosine, which is linked to the rest of the mRNA via a 5'-5' triphosphate bridge. In one example, the 5' cap is a cap1 structure, such as a 5'-cap (i.e., cap1) consisting of an additional methylated cap0 with a 2'O position of the starting nucleotide.

[0685] In one example, mRNA is purified. Those skilled in the art will be familiar with various methods for purifying mRNA. 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 in, for example, nuclease-free water.

[0686] Composition

[0687] This disclosure provides immunogenic compositions comprising the polynucleotides disclosed herein.

[0688] This disclosure also provides immunogenic compositions comprising the cRNA of this disclosure.

[0689] This disclosure further provides immunogenic compositions comprising the self-replicating RNA of this disclosure.

[0690] This disclosure also provides pharmaceutical compositions comprising the immunogenic composition of this disclosure and a pharmaceutically acceptable carrier.

[0691] As will be clear to those skilled in the art and / or as described herein, 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 promote cell entry.

[0692] Delivery system

[0693] In one example, the pharmaceutical composition disclosed herein further comprises an LNP, polymeric microparticles, and an oil-in-water emulsion. For example, polynucleotides, cRNA, and / or self-replicating RNA are encapsulated in, bound to, or adsorbed onto the LNP, polymeric microparticles, and oil-in-water emulsion.

[0694] lipid nanoparticles

[0695] In one instance, the pharmaceutical composition disclosed herein also comprises LNP.

[0696] To clarify, the terms “lipid nanoparticles” or “LNP” refer to any lipid composition, including but not limited to liposomes or vesicles, in which water volumes are encapsulated by an amphiphilic lipid bilayer (e.g., monolayer; monolayer or multilayer; multilayer), micellar-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles in which solid lipid nanoparticles lack a lipid bilayer.

[0697] Those skilled in the art will understand and / or as described herein the lipid nanoparticles suitable for use in this disclosure. Lipids may have anionic, cationic, or zwitterionic hydrophilic head groups.

[0698] In one instance, the lipid nanoparticles comprise PEG-lipids, sterol-structured lipids, and / or neutral lipids. In one instance, the lipid nanoparticles also comprise cationic lipids. In one instance, the lipid nanoparticles do not contain cationic lipids.

[0699] In one instance, the LNP contains PEG-lipids. For example, the PEG-lipids are selected from PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0700] In one instance, LNP comprises structural lipids. For example, structural lipids are selected from cholesterol, coprosterol, sitosterol, campesterol, stigmasterol, ergosterol, tomatidine, tomatidine, ursolic acid, and α-tocopherol, and combinations thereof.

[0701] In one instance, the LNP comprises a neutral lipid. Exemplary phospholipids (anionic or zwitterionic) used in this disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. For example, neutral lipids are selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DOPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline. Phosphoric acid choline (DPPC), 1,2-eicosanoyl-sn-glycerol-choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidonicyl-sn-glycerol-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycerol-3-phosphocholine, 1,2-diphyranoyl-sn-glycerol-3-phosphoethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin and combinations thereof.

[0702] In one example, LNP comprises cationic lipids. Exemplary cationic lipids include, but are not limited to, dioleoyltrimethylammonium propane (DOTAP), 1,2-distearate-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleoyl-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleoyl-N,N-dimethyl-3-aminopropane (DLinDMA), and 2,5-bis((9z,12z)-octadec-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butyrate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadec-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butyrate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphatidylcholine. The lipids may be saturated or unsaturated.

[0703] Polymer microparticles

[0704] In one instance, the pharmaceutical composition disclosed herein also comprises polymeric microparticles.

[0705] Those skilled in the art will recognize that various polymers can form microparticles to encapsulate or adsorb the polynucleotides, cRNAs, and / or self-replicating RNAs disclosed herein. It is clear that the use of substantially non-toxic polymers implies that the particles are safe, and the use of biodegradable polymers implies that the particles can be metabolized post-delivery to avoid long-term persistence. Useful polymers are also sterilizable to facilitate the preparation of pharmaceutical-grade formulations.

[0706] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acid), polyhydroxybutyric acid, polylactone (including polycaprolactone), polydioxanone, polyvaline lactone, polyorthogonal ester, polyanhydride, polycyanoacrylate, tyrosine-derived polycarbonate, polyvinylpyrrolidone, or polyesteramide and combinations thereof.

[0707] oil-in-water cationic emulsion

[0708] In one instance, the pharmaceutical composition disclosed herein further comprises an oil-in-water cationic emulsion.

[0709] Those skilled in the art will understand and / or see the oils suitable for use in oil-in-water emulsions described herein. For example, the emulsion may contain one or more oils derived from, for example, animal (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 oil) include cod liver oil, shark liver oil, and whale oil. Exemplary plant 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.

[0710] In addition to oil, oil-in-water emulsions also include cationic lipids to promote emulsion formation and stability. Suitable cationic lipids will be apparent to those skilled in the art and / or described herein. Exemplary cationic lipids include, but are not limited to: 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyloctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl[C16:0]trimethylammonium propane (DPTAP), and distearyltrimethylammonium propane (DSTAP).

[0711] In some instances, the oil-in-water emulsion also includes nonionic surfactants and / or amphoteric surfactants. Those skilled in the art will recognize suitable surfactants for use in this disclosure. Exemplary surfactants include, but are not limited to, polyoxyethylene sorbitan ester surfactants (e.g., polysorbate 20 and polysorbate 80) and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butane oxide (BO).

[0712] Pharmaceutically acceptable carriers

[0713] Suitablely, in compositions or methods for administering cRNA and / or the self-replicating RNA of this disclosure to a subject, the cRNA and / or the self-replicating RNA is combined with a pharmaceutically acceptable vector as understood in the art. Thus, one example of this disclosure provides a composition (e.g., a pharmaceutical composition) comprising a combination of the self-replicating RNA of this disclosure (and any delivery system) with a pharmaceutically acceptable vector. Another example of this disclosure provides a composition (e.g., a pharmaceutical composition) comprising a combination of the cRNA of this disclosure (and any delivery system) with a pharmaceutically acceptable vector.

[0714] Generally, the term "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely applied to any subject (e.g., human). Depending on the specific route of administration, a variety of acceptable carriers known in the art may be used, such as those described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991).

[0715] The cRNA and / or self-replicating RNA disclosed herein can be used for prophylactic or therapeutic treatment via parenteral, topical, oral, local, intramuscular, aerosol, or transdermal administration. In one example, the self-replicating RNA is administered parenterally, such as intramuscularly, subcutaneously, or intravenously. For example, intramuscular administration of self-replicating RNA. In another example, cRNA is administered parenterally, such as intramuscularly, subcutaneously, or intravenously. For example, intramuscular administration of cRNA.

[0716] The formulation of the cRNA and / or self-replicating RNA to be administered will vary depending on the chosen route of administration and formulation (e.g., solution, emulsion, capsule). Suitable pharmaceutical compositions containing the cRNA and / or self-replicating RNA to be administered can be prepared using physiologically acceptable carriers. For solutions or emulsions, suitable carriers include, for example, aqueous solutions or alcohol / aqueous solutions, emulsions or suspensions, including saline and buffer media. Parenteral media may include sodium chloride solution, dextrose, glucose and sodium chloride, lactated Ringer's solution, or fixed oil. Various suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), glucose solutions, and glycine. Intravenous media may include various additives, preservatives or fluids, nutritional or electrolyte supplements (see generally Remington's Pharmaceutical Science, 16th edition, edited by Mack, 1980). The composition may optionally contain pharmaceutically acceptable excipients to meet the requirements of near-physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. cRNA and / or self-replicating RNA may be stored in a liquid stage, or may be freeze-dried for storage and reconstituted in a suitable carrier prior to use according to freeze-drying and reconstitution techniques known in the art.

[0717] The optimal concentration of the active ingredient in the selected medium can be determined based on procedural experience known to those skilled in the art, and will depend on the desired final pharmaceutical formulation.

[0718] After formulation, the disclosed compositions are administered in a dosage-compatible manner and at a therapeutically / prophylactically effective amount. The dosage range for administering the cRNA and / or self-replicating RNA of the present invention is sufficiently wide to produce the desired effect. For example, the composition contains an effective amount of self-replicating RNA. In one example, the composition contains a therapeutically effective amount of self-replicating RNA. In another example, the composition contains a prophylactically effective amount of self-replicating RNA. In one example, the composition contains an effective amount of cRNA. In one example, the composition contains a therapeutically effective amount of cRNA. In another example, the composition contains a prophylactically effective amount of cRNA.

[0719] The dosage should not be so high as to cause adverse side effects. Generally, the dosage will vary depending on the patient's age, condition, sex, and severity of the disease, and can be determined by a person skilled in the art. In the event of any complications, the dosage may be adjusted by an individual physician.

[0720] The dosage can vary from about 0.1 mg / kg to about 300 mg / kg, for example from about 0.2 mg / kg to about 200 mg / kg, for example from about 0.5 mg / kg to about 20 mg / kg, and may be administered once or more daily for one or more days.

[0721] In some instances, cRNA and / or self-replicating RNA are administered at an initial (or loading) dose higher than the subsequent dose (maintenance dose). For example, cRNA and / or self-replicating RNA are administered at an initial dose of about 10 mg / kg to about 30 mg / kg. Then, a maintenance dose of about 0.0001 mg / kg to about 10 mg / kg is administered. The maintenance dose may be administered every 7–35 days, for example, every 7, 14, or 28 days.

[0722] In some instances, a dose-escalation protocol is used, where cRNA and / or self-replicating RNA are initially administered at a lower dose than subsequently used. This dosage protocol is useful in cases where the subject initially experiences an adverse event.

[0723] In cases where the subject's response to treatment is insufficient, multiple doses may be administered over a week. Alternatively, or additionally, an increased dose may be administered.

[0724] Subjects can be retreated with the cRNA and / or self-replicating RNA disclosed herein. Subjects can be retreated with cRNA and / or self-replicating RNA by administering more than one exposure or dose set, such as at least about two exposures to the binding protein, for example from about 2 to 60 exposures, more specifically about 2 to 40 exposures, and most specifically about 2 to 20 exposures.

[0725] In one instance, any retreatment may be given when disease signs or symptoms recur.

[0726] In another instance, any retreatment may be administered at defined intervals. For example, subsequent exposure may be applied at various intervals, such as approximately 24–28 weeks, 48–56 weeks, or longer. For example, such exposure may be applied at intervals of approximately 24–26 weeks, approximately 38–42 weeks, or approximately 50–54 weeks.

[0727] In cases where the subject's response to treatment is insufficient, multiple doses may be administered over a week. Alternatively, or additionally, an increased dose may be administered.

[0728] In another instance, for subjects experiencing adverse reactions, the initial (or loading) dose may be split over multiple days of the week or over several consecutive days.

[0729] The administration of cRNA and / or self-replicating RNA according to the methods of this disclosure can be continuous or intermittent, depending on factors such as the recipient's physiological condition, whether the purpose of administration is therapeutic or prophylactic, and other factors known to a skilled practitioner. Administration of cRNA and / or self-replicating RNA can be substantially continuous over a pre-selected period of time, or can be a series of spaced-out doses, for example, during or after the development of the condition.

[0730] Screening assay

[0731] Methods suitable for selecting the cRNAs and / or self-replicating RNAs disclosed herein are available to those skilled in the art. Assays can be performed to assess the efficiency and potency of the RNA, including, for example, serological and immune responses.

[0732] antigen expression

[0733] In one instance, the expression of the polynucleotide of interest (PRNA) of self-replicating RNA (i.e., the PRNA encoding the S protein antigen from the omicron strain of SARS-CoV-2) was evaluated. In another instance, the expression of the PRNA of interest (i.e., the PRNA encoding the S protein antigen from the omicron strain of SARS-CoV-2) was evaluated.

[0734] For example, antibodies targeting the polynucleotide of interest are used to detect antigen expression. In one instance, the number of antigen-positive cells is measured, for example, by fluorescence-activated cell sorting (FACS). In another instance, mean fluorescence intensity (MFI) is determined using, for example, FACS. In a further instance, the specificity power or probability of successful transfection per unit mass of RNA is calculated.

[0735] Trace neutralization assay

[0736] In one instance, antibody responses to self-replicating RNA (naked and / or formulated) are evaluated. In another instance, antibody responses to cRNA (naked and / or formulated) are evaluated. For example, a micro-neutralization assay is used to evaluate cRNA and / or self-replicating RNA. Those skilled in the art will understand the methods for performing a micro-neutralization assay. In one instance, the micro-neutralization assay is a simplified assay. For one instance, a micro-neutralization assay based on viral fluorescent lesions is performed. In another instance, the micro-neutralization assay is a long assay.

[0737] Antigen-specific T cell response

[0738] In one instance, the ability of self-replicating RNA to induce an antigen-specific T cell response was evaluated. In another instance, the ability of cRNA to induce an antigen-specific T cell response was evaluated. Those skilled in the art will understand and / or see methods for evaluating the induction of antigen-specific T cell responses described herein.

[0739] For example, antigen-specific T cell detection can be performed on spleen cultures. In short, spleen cell cultures are established in T cell culture medium and the cell cultures are stimulated with or without antigenic peptides. In one instance, flow cytometry is used to determine antigen-specific T cell responses.

[0740] Neutralization determination

[0741] The self-replicating RNA disclosed herein can be screened in vitro for its ability to bind to the SARS-CoV-2 S protein and neutralize the binding of the S protein to ACE2. Those skilled in the art will recognize suitable assays, including, for example, the Vero micro-neutralization assay, the sVNT assay, or the pseudovirus neutralization assay (using, for example, HEK-293T cells or HeLa-ACE2 cells).

[0742] In one instance, the neutralization assay was a Vero micro-neutralization assay. In short, the SARS-CoV-2 wild-type virus was passaged in Vero cells (i.e., the Vero lineage isolated from kidney epithelial cells extracted from African green monkeys). Serial two-fold dilutions of the test protein were mixed with 100 TCID50. 50 Incubation with SARS-CoV-2 (i.e., median tissue culture infection dose) for 1 hour and assessment of residual viral infectivity in Vero cells; for example, viral cytopathic effects were read on day 5. Neutralizing antibody titers were calculated using the Reed / Muench method, as previously described (Houser et al., 2016; Subbarao et al., 2004).

[0743] In one example, the neutralization assay is an alternative neutralization test (sVNT). Briefly, the wells of a plate are coated with hACE2 protein in a carbonate-bicarbonate coating buffer (e.g., pH 9.6). HRP-conjugated SARS-CoV-2 pre-incubated with the test protein and HRP-conjugated SARS-CoV-2 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. A colorimetric signal is generated on the enzymatic reaction of HRP with a chromogenic substrate (e.g., 3,3',5,5'-tetramethylbenzidine (TMB)). In one example, absorbance readings are obtained at 450 nm and 570 nm.

[0744] In one instance, neutralization is a pseudovirus neutralization assay. Briefly, HIV reporter viruses pseudotyped with the SARS-CoV-2 S protein are generated by co-transfecting the SARS-2-CoV-2 spike plasmid with a viral backbone plasmid (e.g., pDR-NL Δenv FLUC) into, for example, HEK-293T cells. Pseudoviruses are harvested after transfection and clarified by filtration. Viral reservoir titers are calculated as a relative luciferase unit infection dose (RLU) by performing restricted dilution infection in Hela-hACE2 cells and measuring luciferase activity as a readout of viral infection.

[0745] Treatment or prevention methods

[0746] This disclosure provides, for example, methods for treating, preventing, or delaying the progression of COVID-19 caused by SARS-CoV-2. This disclosure also provides, for example, methods for treating, preventing, or delaying the progression of SARS-CoV-2 infection. In some instances of this disclosure, the subject has SARS-CoV-2 infection but no clinically diagnosed COVID-19. Thus, in one instance, the subject may exhibit one or more symptoms of SARS-CoV-2 infection, but COVID-19 has not yet been clinically detected.

[0747] Coronavirus disease 2019 (COVID-19)

[0748] This disclosure provides methods for treating, preventing, or delaying the progression of COVID-19 or SARS-CoV-2 infection in subjects.

[0749] 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. Although most cases result in mild symptoms, some cases develop ARDS or may lead to one or more complications, including pneumonia or sepsis. Therefore, this disclosure considers the treatment, prevention, or delay of the progression of mild or moderate to severe COVID-19, and includes the treatment, prevention, or delay of the progression of ARDS, pneumonia, or sepsis in subjects.

[0750] Mild COVID-19 can be considered to include the following symptoms: fever, cough, fatigue, shortness of breath, and loss of smell and taste. Specifically, mild COVID-19 is defined as a positive SARS-CoV-2 RT-PCR or molecular test result, and one of the following symptoms:

[0751] -fever;

[0752] - Sore throat;

[0753] -Headache;

[0754] - Muscle pain (myalgia);

[0755] - Gastrointestinal symptoms;

[0756] -cough;

[0757] - Chest tightness;

[0758] - Runny nose;

[0759] -respite;

[0760] -rash;

[0761] - Eye irritation or discharge;

[0762] - Feeling cold;

[0763] - New or altered olfactory or gustatory disorders;

[0764] - Feet or toes with a red or bruised appearance;

[0765] - Shivering, feeling cold or shivering;

[0766] - Discomfort (loss of appetite, general malaise, fatigue, weakness).

[0767] When a case meets the above case definition but does not meet the definition of moderate to severe / critical, the case is considered mild.

[0768] Moderate COVID-19 can be defined as: a positive SARS-CoV-2 RT-PCR or molecular test result, and any of the following new or worsening signs or symptoms:

[0769] -Respiratory rate ≥ 20 breaths / minute;

[0770] - Oxygen saturation (SpO2) in indoor air at sea level is abnormal but still ≥93%;

[0771] - Clinical or radiological evidence of pneumonia;

[0772] -Radiological evidence of DVT;

[0773] -Rapid breathing or difficulty breathing;

[0774] Or any two of the following new or worsening signs or symptoms:

[0775] -fever;

[0776] - Heart rate ≥ 90 beats / minute;

[0777] - Shivering, feeling cold or shivering;

[0778] - New or altered olfactory or gustatory disorders;

[0779] - Sore throat;

[0780] -Discomfort;

[0781] -Headache;

[0782] -cough;

[0783] - Muscle pain (myalgia);

[0784] - Gastrointestinal symptoms;

[0785] - Feet or toes with a red or bruised appearance.

[0786] Severe / critical COVID-19 is defined as: a positive SARS-CoV-2 RT-PCR or molecular test result; and any one or more of the following:

[0787] - Clinical signs of severe systemic disease at rest (respiratory rate ≥ 30 breaths / min, heart rate ≥ 125 beats / min, SpO2 ≤ 93% in indoor air at sea level, or PaO2 / FiO2 ≤ 300 mmHg);

[0788] - Respiratory failure (defined as requiring high-flow oxygen, non-invasive ventilation, mechanical ventilation, or ECMO [extracorporeal membrane oxygenation]);

[0789] - Evidence of shock (defined as systolic blood pressure ≤90 mmHg, diastolic blood pressure ≤60 mmHg or need for vasopressors);

[0790] - Significant acute renal, hepatic, or neurological dysfunction;

[0791] - Admitted to the ICU;

[0792] -die.

[0793] The time from exposure to symptom onset is typically about five days, but can range from two to fourteen 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).

[0794] Therefore, in some instances of this disclosure, the methods or uses of this disclosure can be used to treat, prevent, or delay the progression of ARDS in subjects with COVID-19. In one instance, the subject is at risk of contracting COVID-19 caused by SARS-CoV-2. In one instance, the methods of this disclosure can be used to treat ARDS in subjects with SARS-CoV-2 infection. In one instance, the methods of this disclosure can be used to prevent ARDS in subjects with SARS-CoV-2 infection. In one instance, the methods of this disclosure can be used to delay the progression of ARDS in subjects with SARS-CoV-2 infection.

[0795] Eligible participants may be individuals exhibiting symptoms of SARS-CoV-2 infection or who have been diagnosed with SARS-CoV-2 infection and / or COVID-19. Additionally, eligible participants may be those clinically or biochemically confirmed to be infected with SARS-CoV-2 or COVID-19. In one implementation, participants may be asymptomatic.

[0796] A reduction in SARS-CoV-2 infection can be determined using any method known in the art or the methods described herein, including measuring the viral load in a sample from a 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, such as the nose or pharynx (i.e., the throat). Alternatively, the response to treatment may result in a reduction in the severity of one or more of the symptoms described herein.

[0797] Acute respiratory distress syndrome (ARDS)

[0798] This disclosure provides methods for treating, preventing, or delaying the progression of ARDS in subjects.

[0799] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltration, severe hypoxemia, and disruption of the alveolar-capillary membrane barrier (i.e., pulmonary vascular leakage), leading to non-cardiac pulmonary edema. Currently, there is no effective pharmacological treatment.

[0800] Infectious causes, including influenza, are a major cause of ARDS. Therefore, in one instance of this disclosure, ARDS is associated with SARS-CoV-2 infection. For example, ARDS may be associated with SARS-CoV-2 or COVID-19.

[0801] ARDS is classified according to the Berlin Definition, which includes:

[0802] (1) It occurs within one week after the clinical injury or the onset of respiratory symptoms;

[0803] (2) Acute hypoxic respiratory failure, as determined by a PaO2 / FiO2 ratio of 300 mmHg or less associated with at least 5 cm continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP), where PaO2 is the partial pressure of oxygen in arterial blood and FiO2 is the fraction of oxygen inhaled.

[0804] (3) Bilateral opacity on lung radiographs cannot be fully explained by effusion, consolidation, or atelectasis; and

[0805] (4) Edema / respiratory failure cannot be fully explained by heart failure or fluid overload.

[0806] In one instance, the subject has or suffers from ARDS (i.e., the subject meets the Berlin definition of ARDS). For example, the subject requires treatment (i.e., there is a need for it).

[0807] In one instance, the subject has or suffers from symptoms associated with ARDS. Methods for identifying ARDS-related symptoms and identifying subjects at risk of developing ARDS will be clear to those skilled in the art and / or described herein. For example, the subject has one or more of the following symptoms:

[0808] a) A respiratory rate greater than 30 breaths per minute;

[0809] b) The oxygen saturation (SpO2) in indoor air is 93% or lower;

[0810] c) The ratio of arterial oxygen partial pressure to inhaled oxygen fraction (PaO2 / FiO2) is less than 300 mmHg;

[0811] d) The SpO2 / FiO2 ratio is less than 218; and

[0812] e) Radiographic lung infiltration of more than 50% of the body.

[0813] Currently, ARDS is classified as mild, moderate, or severe, with an associated increased mortality rate. The severity of ARDS can be classified according to the Berlin definition as follows:

[0814] (i) Mild ARDS: PaO2 / FiO2 associated with at least 5 cm CPAP or PEEP is 200-300 mmHg;

[0815] (ii) Moderate ARDS: PaO2 / FiO2 associated with at least 5 cm PEEP is 100-200 mmHg; and

[0816] (iii) Severe ARDS: PaO2 / FiO2 associated with at least 5 cm PEEP is less than or equal to 100 mmHg.

[0817] In one instance, ARDS is mild. In another instance, ARDS is moderate. In a further instance, ARDS is severe.

[0818] In addition to existing treatments for ARDS, the methods disclosed herein can also be used to prevent or delay the onset of ARDS. Therefore, in one instance, the subject did not have ARDS.

[0819] In one instance, the subject was at risk of developing one or more ARDS-related symptoms.

[0820] Reagent test kit

[0821] Another example of this disclosure provides a kit containing the self-replicating RNA of this disclosure, which is useful for treating or preventing diseases or conditions as described above.

[0822] Another example of this disclosure provides a kit containing cRNA of this disclosure that is useful for treating or preventing diseases or conditions as described above.

[0823] In one instance, the kit comprises (a) 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 condition in a subject, such as SARS-CoV-2 infection, COVID-19, or ARDS.

[0824] In one instance, the kit comprises (a) 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 condition in a subject, such as influenza, COVID-19, ARDS, or COVID-19-related sepsis or pneumonia.

[0825] According to this example of the disclosure, a packaging insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from various materials, such as glass or plastic. The container contains or holds a composition effective for the disease or condition of the disclosure and may have a sterile access port (e.g., the container may be an intravenous infusion bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the compositi...

Claims

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

2. The self-replicating RNA of claim 1, further comprising a nucleotide sequence encoding a second antigen operatively 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 derived 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 the SG promoter and IRES.

6. The self-replicating RNA according to claim 2, wherein the self-replicating RNA comprises, from 5' to 3', the following: a) The nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2; and b) The nucleotide sequence encoding the N protein antigen from SARS-CoV-2.

7. The self-replicating RNA according to claim 2, wherein the self-replicating RNA comprises, from 5' to 3', the following: a) The nucleotide sequence encoding the N protein antigen from SARS-CoV-2; and b) The nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2.

8. The self-replicating RNA according to claim 2, wherein the self-replicating RNA comprises, from 5' to 3', the following: a) A nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2, operably linked to the SG promoter; and b) A nucleotide sequence encoding the N protein antigen from SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES), optionally encoded by the sequence shown in SEQ ID NO:

18.

9. The self-replicating RNA according to claim 2, wherein the self-replicating RNA comprises, from 5' to 3', the following: a) A nucleotide sequence encoding the N protein antigen from SARS-CoV-2, operatively linked to the SG promoter; and b) A nucleotide sequence encoding the S protein antigen from the omicron strain of SARS-CoV-2, operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES), optionally encoded by the sequence shown in SEQ ID NO:

19.

10. The self-replicating RNA of claim 2, wherein the N protein is derived 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 according to claim 1.

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

15. The self-replicating RNA of claim 14, wherein the promoter is a subgenomic (SG) promoter.

16. The self-replicating RNA of claim 14, wherein the SG promoter is a minimal SG promoter or an extended SG promoter.

17. The self-replicating RNA of claim 16, wherein the extended SG promoter is extended at the 5' end with nucleotides appearing in the sequence encoding a non-structural protein of an RNA virus.

18. The self-replicating RNA of claim 16, wherein the minimal SG promoter is encoded by the sequence shown in SEQ ID NO:

1.

19. The self-replicating RNA of claim 16, wherein the extended SG promoter is encoded by the sequence shown in SEQ ID NO:

5.

20. The self-replicating RNA of claim 2, wherein the nucleotide sequence encoding the second antigen is operatively linked to IRES, optionally located at the 3' of the nucleotide sequence encoding the second antigen.

21. The self-replicating RNA of claim 20, wherein the IRES is derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simultaneous immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-associated 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 protein (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

22. The self-replicating RNA of claim 21, wherein the EMCV IRES is a wild-type IRES encoded by the sequence shown in SEQ ID NO:

4.

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

24. The self-replicating RNA of claim 1, wherein the antigen is derived 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. The self-replicating RNA according to claim 24, wherein: (a) The antigen is derived from the S protein of the omicron variant BA.1, and the S protein encoded by the polynucleotide comprises one or more 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 derived from the S protein of the 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 derived from the S protein of the omicron variant BA.4 or BA.5, and the S protein encoded by the polynucleotide contains 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.

26. The self-replicating RNA of 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% of SEQ ID NO:

20.

27. The self-replicating RNA of 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% of 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 of claim 28, wherein the alphavirus is selected from the group consisting of Semliki Forest Virus (SFV), Sindebeth Virus (SIN), and Venezuelan Equine Encephalitis Virus (VEE) and combinations thereof.

30. An immunogenic composition comprising the self-replicating RNA according to claim 1.

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

32. An immunogenic composition comprising a plurality of self-replicating RNAs according to 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 (LNP), polymer microparticles, or oil-in-water emulsion.

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

36. The pharmaceutical composition of claim 33, wherein each RNA is formulated together in an LNP.

37. The pharmaceutical composition of claim 33, wherein each RNA is separately formulated in an LNP.

38. The immunogenic composition according to claim 30, used as a vaccine.

39. The pharmaceutical composition according to claim 33, used as a vaccine.

40. A vaccine comprising the immunogenic composition according to claim 30.

41. A vaccine comprising the pharmaceutical composition according to claim 30.

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

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

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

45. A polynucleotide comprising: a) The first nucleotide sequence encoding the first antigen; and b) A second nucleotide sequence encoding the second antigen, operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES). The first antigen is the spike (S) protein of the omicron strain of SARS-CoV-2.

46. ​​The polynucleotide of claim 45, wherein the polynucleotide comprises, from 5' to 3', the following components in sequence: a) The first nucleotide sequence encoding the first antigen; and b) A second nucleotide sequence encoding a second antigen, operatively linked to an IRES or SG promoter, optionally comprising the sequence shown in SEQ ID NO:18 or 19.

47. A conventional mRNA (cRNA) comprising: a) The first nucleotide sequence encoding the first antigen; and b) A second nucleotide sequence encoding the second antigen, operatively linked to a regulatory element selected from the group consisting of the SG promoter and the internal ribosome entry site (IRES). The first antigen is the spike (S) protein of the omicron strain of SARS-CoV-2.

48. The cRNA of claim 47, wherein the cRNA comprises, from 5' to 3', the following components in sequence: a) The first nucleotide sequence encoding the first antigen; and b) A second nucleotide sequence encoding a second antigen, which is operatively linked to an IRES or SG promoter.

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

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

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

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

53. A method for treating or preventing or delaying the progression of SARS-CoV-2 infection in a subject in need, the method comprising administering the self-replicating RNA according to claim 1 to the subject.

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

55. The self-replicating RNA according to claim 1, used for treating or preventing or delaying the progression of SARS-CoV-2 infection in a subject.

56. A method for inducing an immune response in a subject, the method comprising administering the self-replicating RNA according to claim 1 to a subject in need of doing so.

57. Use of the self-replicating RNA according to claim 1 in the preparation of a medicament for inducing an immune response in a subject in need.

58. The self-replicating RNA according to claim 1, used to induce an immune response in a subject in need.

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

60. The method of 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 SARS-CoV-2 viral load in subjects suffering from COVID-19, comprising administering the self-replicating RNA according to claim 1 to the subject in need.

62. Use of the self-replicating RNA according to claim 1 in the preparation of a medicament for reducing the viral load of SARS-CoV-2 in subjects with COVID-19.

63. The self-replicating RNA according to claim 1, used to reduce the SARS-CoV-2 viral load in subjects suffering from COVID-19.

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

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

66. The self-replicating RNA according to claim 1, used for the treatment, prevention or delay of the progression of acute respiratory distress syndrome in subjects with COVID-19.

67. The method of claim 53, wherein the subject is 18 years of age or older.

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

69. The method of claim 53, wherein the self-replicating RNA, vaccine, or composition is administered in a two-, three-, or four-dose regimen, wherein the doses are administered at intervals of approximately 1, 2, or 3 months.

70. A reagent kit comprising: (a) The self-replicating RNA according to claim 1; (b) its instructions for use; and optionally (c) Pharmaceutically acceptable carriers, excipients or diluents.

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