Combination RNA vaccine

Abstract

This disclosure relates to combination RNA vaccines and their use. This disclosure also relates to conventional mRNA vaccines and self-replicating RNA vaccines for the treatment of diseases or conditions including respiratory syncytial virus (RSV).

Description

Technical Field

[0001] Related Application Data This application claims priority from U.S. Patent Application No. 63 / 511,332, filed June 30, 2023, entitled "Combination RNA Vaccine," the entire content of which is incorporated herein by reference.

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

[0003] This disclosure relates to combination RNA vaccines and their use. This disclosure also relates to conventional mRNA vaccines and self-replicating RNA vaccines for the treatment of diseases or conditions including respiratory syncytial virus (RSV).

Background Art

[0004] Respiratory virus infections are a major threat to human health. Infections such as those caused by influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV) are known to have caused worldwide pandemics and led to the deaths of millions of people around the world. More recently, SARS-CoV-2 has been the cause of the ongoing worldwide pandemic of severe infectious coronavirus disease 2019 (COVID-19). Furthermore, respiratory syncytial virus (RSV) is the most common single cause of hospitalization due to respiratory disease in infants, and reinfection remains common throughout later life. Several vaccines are available for viral infections such as SARS-CoV-2 and RSV, such as mRNA vaccines developed to treat or prevent SARS-CoV-2 infection, but further improvements can be made to increase their effectiveness and / or improve treatment strategies.

[0005] Currently, egg-based manufacturing processes are the most common method of vaccine production. This process requires considerable time to optimize viral replication within the eggs and necessitates resources (i.e., eggs) to produce sufficient quantities of vaccine, especially during a pandemic. Furthermore, given the long development time required, vaccine strain selection takes place before the vaccine becomes available, making it difficult to adapt to changes in the virus. Vaccines are also produced using cell-based manufacturing processes involving cultured mammalian cells (e.g., Madin Darby canine kidney, or MDCK cells) instead of eggs, and virus-based platforms involving recombinant viruses (e.g., baculoviruses encoding influenza antigens) are also being utilized.

[0006] There is still a need for the development of specific and efficient viral vaccines that can be produced more rapidly and with broader utility than current egg-based techniques, for the treatment or prevention of respiratory viral infections such as RSV and SARS-CoV-2. Nucleic acid-based vaccines offer clear advantages over current egg-based manufacturing platforms, but several challenges remain. For example, due to the inherently unstable nature of mRNA, most RNA-based vaccines consequently have a limited ability to deliver antigens at the doses and durations required to produce a potent and durable immune response.

[0007] Therefore, it will be clear to those skilled in the art that there is a need in the art for compositions having broader utility and / or improved efficacy that are suitable for use as vaccines. [Overview of the project]

[0008] This disclosure is based on the inventors' identification of RNAs, including antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, antigens from influenza, and antigens from respiratory syncytial virus (RSV) which are suitable as vaccines for the treatment of SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), influenza, and / or RSV. Furthermore, the inventors' research results provide a basis for methods to treat, prevent, or delay the progression of diseases or disorders, including SARS-CoV-2 infection or COVID-19, influenza, and / or RSV, and their complications, including acute respiratory distress syndrome (ARDS), in subjects.

[0009] Therefore, this disclosure is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) A polynucleotide comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), The present invention provides a polynucleotide in which each nucleotide sequence is operably linked to a regulatory element.

[0010] For example, polynucleotides are arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding an antigen from influenza, (c) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and the following:

[0011] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) A nucleotide sequence encoding an antigen from influenza, and

[0012] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) Nucleotide sequences encoding antigens from respiratory syncytial virus (RSV), (c) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the following:

[0013] For example, polynucleotides are RNA or DNA. For example, RNA is messenger RNA (mRNA). For example, mRNA is conventional mRNA (cRNA) or self-replicating RNA.

[0014] Therefore, this disclosure also, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) RNA comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), The present invention provides RNA in which each nucleotide sequence is operably linked to a regulatory element.

[0015] In one example, RNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding an antigen from influenza, (c) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and the following:

[0016] In another example, the RNA comprises, in order from 5' to 3', (a) a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and (b) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and (c) a nucleotide sequence encoding an antigen from influenza.

[0017] In another example, the RNA comprises, in order from 5' to 3', (a) a nucleotide sequence encoding an antigen from influenza, and (b) a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and (c) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0018] The present disclosure also provides (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and (b) a nucleotide sequence encoding a second antigen from influenza, and (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), wherein the crRNA comprises each nucleotide sequence operably linked to a regulatory element.

[0019] In one example, the crRNA comprises, in order from 5' to 3', (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and (b) a nucleotide sequence encoding an antigen from influenza, and (c) a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV).

[0020] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) A nucleotide sequence encoding an antigen from influenza, and

[0021] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) Nucleotide sequences encoding antigens from respiratory syncytial virus (RSV), (c) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the following:

[0022] This disclosure also states, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) Self-replicating RNA comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), The present invention provides a self-replicating RNA in which each nucleotide sequence is operably linked to a regulatory element.

[0023] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding an antigen from influenza, (c) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and the following:

[0024] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) A nucleotide sequence encoding an antigen from influenza, and

[0025] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) Nucleotide sequences encoding antigens from respiratory syncytial virus (RSV), (c) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the following:

[0026] In one example, the regulatory element is operably ligated to the 5' end of the first nucleotide sequence defined in (a). In one example, the regulatory element is selected from the group consisting of Kozak consensus sequences, IRESs, promoters, and combinations thereof. For example, the regulatory element is a Kozak consensus sequence. In another example, the regulatory element is an IRES. In yet another example, the promoter is an SG promoter.

[0027] In one example, the nucleotide sequences are operably linked to the same regulatory element (e.g., the same SG promoter). In another example, the nucleotide sequences are operably linked to different regulatory elements (e.g., different SG promoters).

[0028] For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0029] For example, polynucleotides are arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0030] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0031] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0032] For example, RNA is (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0033] In one example, RNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0034] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0035] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0036] In one example, cRNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0037] In one example, the cRNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0038] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0039] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0040] In one example, self-replicating RNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0041] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0042] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0043] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0044] For example, polynucleotides are (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0045] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0046] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0047] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0048] For example, RNA is (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0049] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0050] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0051] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0052] In one example, cRNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0053] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0054] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0055] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0056] In one example, self-replicating RNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0057] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0058] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

[0059] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

[0060] For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0061] For example, polynucleotides are arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0062] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0063] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0064] For example, RNA is (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0065] In one example, RNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0066] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0067] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0068] In one example, cRNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0069] In one example, the cRNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0070] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0071] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0072] In one example, self-replicating RNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0073] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0074] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0075] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0076] For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0077] For example, polynucleotides are arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0078] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0079] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0080] For example, RNA is (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0081] In one example, RNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0082] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0083] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0084] In one example, cRNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0085] In one example, the cRNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0086] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0087] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0088] In one example, self-replicating RNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0089] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0090] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0091] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0092] For example, polynucleotides are (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0093] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0094] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0095] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0096] For example, RNA is (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0097] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0098] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0099] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0100] In one example, cRNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0101] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0102] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0103] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0104] In one example, self-replicating RNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0105] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0106] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0107] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0108] For example, polynucleotides are (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0109] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0110] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0111] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0112] For example, RNA is (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0113] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0114] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0115] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0116] In one example, cRNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0117] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0118] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0119] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0120] In one example, self-replicating RNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0121] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0122] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0123] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the IRES, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0124] For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0125] For example, polynucleotides are arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0126] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0127] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0128] For example, RNA is (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0129] In one example, RNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0130] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0131] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0132] In one example, cRNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0133] In one example, the cRNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0134] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0135] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0136] In one example, self-replicating RNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0137] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0138] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0139] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0140] For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0141] For example, polynucleotides are arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0142] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0143] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0144] For example, RNA is (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0145] In one example, RNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0146] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0147] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0148] In one example, cRNA is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0149] In one example, the cRNA is arranged in the order from 5' to 3'. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0150] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0151] In another example, the cRNA, in the order from 5' to 3', (a) a nucleotide sequence encoding an antigen from influenza, and (b) a nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter, and (c) a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an SG promoter.

[0152] In one example, the self-replicating RNA (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and (b) a nucleotide sequence encoding a second antigen from influenza, operably linked to an SG promoter, and (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0153] In one example, the self-replicating RNA, in the order from 5' to 3', (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and (b) a nucleotide sequence encoding a second antigen from influenza, operably linked to an SG promoter, and (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0154] In one example, the self-replicating RNA, in the order from 5' to 3', (a) a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and (b) a nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an SG promoter, (c) a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0155] In another example, the self-replicating RNA, in the order from 5' to 3', (a) a nucleotide sequence encoding an antigen from influenza, (b) a nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0156] In one example, the polynucleotide (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) a nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0157] In one example, the polynucleotide, in the order from 5' to 3', (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) a nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0158] In one example, the polynucleotide, in the order from 5' to 3', (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0159] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0160] For example, RNA is (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0161] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV) operably linked to an IRES.

[0162] > In one example, the RNA is, in the 5' to 3' order, (a) a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV) operably linked to an SG promoter, (b) a nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to an SG promoter, (c) a nucleotide sequence encoding a third antigen from influenza operably linked to an IRES.

[0163] In another example, the RNA is, in the 5' to 3' order, (a) a nucleotide sequence encoding an antigen from influenza operably linked to an SG promoter, (b) a nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV) operably linked to an SG promoter, (c) a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to an IRES.

[0164] In one example, the cRNA (a) a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably linked to an SG promoter, (b) a nucleotide sequence encoding a second antigen from influenza operably linked to an SG promoter, (c) a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV) operably linked to an IRES.

[0165] In one example, the cRNA is, in the 5' to 3' order, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0166] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0167] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0168] In one example, self-replicating RNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0169] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to the IRES.

[0170] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the IRES.

[0171] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the IRES.

[0172] For example, polynucleotides are (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0173] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0174] For example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0175] In another example, polynucleotides are arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0176] For example, RNA is (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0177] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0178] In one example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0179] In another example, RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0180] In one example, cRNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0181] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0182] In one example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0183] In another example, the cRNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0184] In one example, self-replicating RNA is, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0185] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an SG promoter.

[0186] In one example, self-replicating RNA is structured from 5' to 3' in that order. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to the SG promoter.

[0187] In another example, self-replicating RNA is in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (c) comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter.

[0188] In one example, the polynucleotide, RNA, cRNA, or self-replicating RNA of this disclosure includes a Kozak consensus sequence. In one example, the Kozak consensus sequence includes or consists of the sequence shown in SEQ ID NO: 6 (GCCACC). In another example, the Kozak consensus sequence consists of the sequence shown in SEQ ID NO: 7 (ACCATGG).

[0189] In one example, a polynucleotide is a multicistronic RNA. For example, a polynucleotide is a multicistronic cRNA. For example, cRNA is a multicistronic cRNA. In another example, a polynucleotide is a multicistronic self-replicating mRNA. In another example, self-replicating RNA is a monocistronic self-replicating mRNA. In yet another example, a polynucleotide is a multicistronic self-replicating RNA. In yet another example, self-replicating RNA is a monocistronic self-replicating RNA.

[0190] For example, the SG promoter is a natural SG promoter. For instance, the natural SG promoter is a promoter that is natural to and / or based on RNA viruses (e.g., alphaviruses). For example, the natural SG promoter is a natural alphavirus SG promoter.

[0191] In one example, the SG promoter is either a minimum SG promoter or an extended SG promoter.

[0192] For example, the SG promoter is the minimal SG promoter. For example, the natural SG promoter is the minimal SG promoter. For example, the minimal SG promoter is the smallest sequence required to initiate transcription. For example, the minimal natural SG promoter is 49 nucleotides long. For example, the minimal SG promoter is 49 nucleotides long. For example, the minimal natural SG promoter is encoded by a sequence containing or consisting of the sequence shown in Sequence ID No. 1. For example, the minimal SG promoter is encoded by a sequence containing or consisting of the sequence shown in Sequence ID No. 1.

[0193] In one example, the SG promoter is an extended SG promoter. In another example, the natural SG promoter is an extended SG promoter. For example, the extended SG promoter is extended at its 5' end by nucleotides introduced into the sequence encoding a non-structural protein (e.g., NSP4) of an RNA virus (e.g., alphavirus). In one example, the extended SG promoter is extended at its 5' end by nucleotides introduced into the sequence encoding alphavirus NSP4. The addition of nucleotides to the 5' end of the SG promoter sequence did not interfere with the expression of non-structural proteins and viral replicases, such as alphavirus NSP4.

[0194] In one example, the SG promoter is extended at its 5' end by 51 or fewer nucleotides occurring within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is a minimal SG promoter extended at its 5' end by 51 or fewer nucleotides occurring within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 51 or fewer nucleotides occurring within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 100 or fewer. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 2-101 of SEQ ID NO: 5.

[0195] In one example, the SG promoter is extended at its 5' end by approximately 5 to 20 nucleotides, for example, about 5 nucleotides, or about 10 nucleotides, or about 12 nucleotides, or about 15 nucleotides, or about 20 nucleotides, occurring within the sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the SG promoter is extended at its 5' end by approximately 20 to 35 nucleotides, for example, about 25 nucleotides, or about 27 nucleotides, or about 30 nucleotides, or about 35 nucleotides, occurring within the sequence encoding a non-structural protein (e.g., alphavirus NSP4).

[0196] In one example, the SG promoter is extended at its 5' end by approximately 12 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 12 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 61 or less. In one example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 41-101 of SEQ ID NO: 5. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 2.

[0197] In one example, the SG promoter is extended at its 5' end by approximately 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 80 or less. In one example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 22-101 of SEQ ID NO: 5. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 3.

[0198] For example, the extended SG promoter contains a repeating sequence corresponding to nucleotides 66-75 of SEQ ID NO: 5. For instance, the extended SG promoter is encoded by a sequence containing nucleotides 50-75 and nucleotides 66-101 of SEQ ID NO: 5. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 15.

[0199] For example, IRESs are derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-related protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF-B), Antennapedia, X-linked apoptosis inhibitor (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0200] For example, IRES is a wild-type IRES derived from encephalomyocarditis virus (EMCV). For instance, wild-type EMCV IRES contains the sequence shown in SEQ ID NO: 4.

[0201] In one example, the first and / or second and / or third nucleotide sequences are codon-optimized.

[0202] In one example, the G / C content of the first and / or second and / or third nucleotide sequence is modified.

[0203] For example, the G / C content of the first and / or second and / or third nucleotide sequence increases by at least 5% compared to the G / C content of the unmodified sequence. For instance, the G / C content of the first and / or second and / or third nucleotide sequence increases by at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to the G / C content of the unmodified sequence.

[0204] For example, a polynucleotide contains at least one chemically modified nucleotide.

[0205] For example, the chemically modified nucleotides are selected from the group consisting of N6,2'-O-dimethyladenosine (m6Am), 5-methyluridine (m5U), N4-acetylcytidine (ac4C), 2-thiocytidine (s2C), 2-thiouridine (s2U), 5-methylcytidine (m5C), N6-methyladenosine (m6a), pseudouridine (ψ), 1-methylpseudridine (m1ψ), and combinations thereof. For example, the chemically modified nucleotide is N6,2'-O-dimethyladenosine (m6Am). For example, the chemically modified nucleotide is 5-methyluridine (m5U). For example, the chemically modified nucleotide is N4-acetylcytidine (ac4C). For example, the chemically modified nucleotide is 2-thiocytidine (s2C). For example, a chemically modified nucleotide is 2-thiouridine (s2U). For example, a chemically modified nucleotide is 5-methylcytidine (m5C). For example, a chemically modified nucleotide is N6-methyladenosine (m6a). For example, a chemically modified nucleotide is pseudouridine (ψ). For example, a chemically modified nucleotide is 1-methylpseudridine (m1ψ).

[0206] In one example, the first nucleotide sequence includes haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha-6 collagen (Col6A), alpha-1-antitrypsin (SERPINA1), alpha-1-antichymotrypsin (SERPINA3), its 5'-UTR, fragments, and / or variants.

[0207] In one example, the 5'UTR is the 5'UTR of Venezuelan encephalitis virus (VEEV) or a modified form thereof. For example, the 5'UTR contains the sequence shown in SEQ ID NO: 13.

[0208] For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 40 to 2000. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 40 to 100. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 100 to 250. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 250 to 500. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 500 to 750. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 750 to 1000. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 1000 to 1250. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 1250 to 1500. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 1500 to 1750. For example, 5'-UTR, its fragments, and / or variants have a nucleotide length of 1750 to 2000.

[0209] For example, a 5'UTR, its fragments, and / or variants contain a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in any one of sequence numbers 9-12.

[0210] In one example, a polynucleotide contains a combination of two or more 5'-UTRs, their fragments, and / or variants. In one example, two or more 5'-UTRs are the same. In another example, two or more 5'-UTRs are different.

[0211] In one example, a nucleotide sequence containing the 5'UTR includes at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, and combinations thereof. In one example, the nucleotide sequence includes a microRNA binding site. In one example, the nucleotide sequence includes an AU-rich element (ARE). In one example, the nucleotide includes a GC-rich element. In one example, the nucleotide sequence includes a stem-loop. For example, the stem-loop is a histone stem-loop.

[0212] In one example, a polynucleotide further comprises a nucleotide sequence containing a 3'UTR. In one example, the nucleotide sequence containing the 3'UTR is located at 3' of a second or one or more additional nucleotide sequences. For example, the nucleotide sequence containing the 3'UTR is located at 3' of a second nucleotide sequence. In one example, the 3'UTR includes the arachidonic acid 5-lipoxygenase (ALOX5), alpha-I collagen (COL1A1), tyrosine hydroxylase (TH) gene, the amino-terminal enhancer (AES) of a split, the 3'-UTR of human mitochondrial 12S rRNA (mtRNR1), fragments thereof, and / or variants.

[0213] For example, the 3'UTR is the 3'UTR of Sindbisvirus (SINV) or a modified form thereof. For instance, the 3'UTR contains the sequence shown in SEQ ID NO: 14.

[0214] For example, a 3'UTR, its fragments, and / or variants have a nucleotide length of 40 to 400. For instance, a 3'-UTR has a nucleotide length of 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300, or 300 to 325, or 325 to 350, or 350 to 375, or 375 to 400. For example, a 3'-UTR, its fragments, and / or variants have a nucleotide length of 40 to 50. For example, a 3'-UTR, its fragments, and / or variants have a nucleotide length of 50 to 60. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 60-70. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 70-80. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 80-90. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 90-100. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 100-125. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 125-150. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 150-175. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 175-200. For example, 3'-UTR, its fragments and / or variants have a nucleotide length of 200-225. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 225-250. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 250-275. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 275-300. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 300-325. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 325-350.For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 350 to 375. For example, 3'-UTR, its fragments, and / or variants have a nucleotide length of 375 to 400.

[0215] In one example, a polynucleotide contains a combination of two or more 3'-UTRs, their fragments, and / or variants. In one example, the two or more 3'-UTRs are identical. In another example, the two or more 3'-UTRs are different.

[0216] In one example, a nucleotide sequence comprising the 3'UTR, its fragments, and / or variants includes at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a triple helix, a stem-loop, one or more stop codons, and combinations thereof. In one example, the nucleotide sequence includes a microRNA binding site. In one example, the nucleotide sequence includes an AU-rich element (ARE). In one example, the nucleotide sequence includes a GC-rich element. In one example, the nucleotide sequence includes a triple helix. In one example, the nucleotide sequence includes a stem-loop. For example, the stem-loop is a histone stem-loop. In one example, the nucleotide sequence includes one or more stop codons. For example, one or more stop codons are located at the 5' end of the 3'-UTR.

[0217] In one example, a polynucleotide comprises a nucleotide sequence containing one or more 3' tailing sequences located at the 3' end of a nucleotide sequence containing a 3' UTR. In one example, one or more 3' tailing sequences are selected from the group consisting of poly-A sequences, polyadenylation signals, G quadruplexes, poly-C sequences, stem-loops, and combinations thereof. For example, the 3' tailing sequence contains a poly-A sequence. In one example, the 3' tailing sequence contains a polyadenylation signal. In one example, the 3' tailing sequence contains a G quadruplex. In one example, the 3' tailing sequence contains a poly-C sequence. In one example, the 3' tailing sequence contains a stem-loop. For example, the stem-loop is a histone stem-loop. In one example, the 3' tailing sequence contains a poly-A sequence and a G quadruplex. In one example, the 3' tailing sequence contains a stem-loop (e.g., a histone stem-loop) and a poly-A sequence.

[0218] For example, one or more 3' tailing sequences contain one or more polyA sequences, each containing 10 to 300 consecutive adenosine nucleotides. For example, each polyA sequence contains 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 10 to 20 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 20 to 30 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 30 to 40 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 36 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 40 to 50 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 50 to 60 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 60 to 70 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 70 to 80 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 80 to 90 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 90 to 100 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 100 to 125 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 125 to 150 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 150 to 175 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 175 to 200 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 200 to 225 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 225 to 250 consecutive adenosine nucleotides.For example, one or more polyA sequences each contain 250 to 275 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 275 to 300 consecutive adenosine nucleotides.

[0219] For example, one or more polyA sequences each contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 10 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 20 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 30 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 40 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 50 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 60 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 70 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 80 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 90 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 100 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 125 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 150 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 175 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 200 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 225 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 250 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 275 consecutive adenosine nucleotides. For example, one or more polyA sequences each contain 300 consecutive adenosine nucleotides.

[0220] For example, a polyA sequence contains 36 consecutive adenosine nucleotides. For instance, a polyA sequence includes the sequence shown in Sequence ID No. 16.

[0221] In one example, one or more polyA sequences are separated by a break linker. For example, a 3' tailing sequence may contain, from 5' to 3', a polyA sequence containing consecutive adenosine nucleotides, a break linker, and a further polyA sequence containing consecutive adenosine nucleotides.

[0222] For example, a break linker has a nucleotide length of 10-50, 50-100, or 100-150.

[0223] For example, a break linker has a nucleotide length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides. For example, a break linker has a nucleotide length of 1. For example, a break linker has a nucleotide length of 2. For example, a interrupted linker has a length of 3 nucleotides. For example, a interrupted linker has a length of 4 nucleotides. For example, a interrupted linker has a length of 5 nucleotides. For example, a interrupted linker has a length of 6 nucleotides. For example, a interrupted linker has a length of 7 nucleotides. For example, a interrupted linker has a length of 8 nucleotides. For example, a interrupted linker has a length of 9 nucleotides. For example, a interrupted linker has a length of 10 nucleotides. For example, a interrupted linker has a length of 11 nucleotides. For example, a interrupted linker has a length of 12 nucleotides. For example, a interrupted linker has a length of 13 nucleotides. For example, a interrupted linker has a length of 14 nucleotides. For example, a interrupted linker has a length of 15 nucleotides. For example, a interrupted linker has a length of 16 nucleotides. For example, a interrupted linker has a length of 17 nucleotides. For example, a interrupted linker has a length of 18 nucleotides. For example, a interrupted linker has a length of 19 nucleotides. For example, a interrupted linker has a length of 20 nucleotides. For example, a break linker has a length of 25 nucleotides. For example, a break linker has a length of 30 nucleotides. For example, a break linker has a length of 35 nucleotides. For example, a break linker has a length of 40 nucleotides. For example, a break linker has a length of 45 nucleotides. For example, a break linker has a length of 50 nucleotides.For example, a break linker has a length of 55 nucleotides. For example, a break linker has a length of 60 nucleotides. For example, a break linker has a length of 65 nucleotides. For example, a break linker has a length of 70 nucleotides. For example, a break linker has a length of 75 nucleotides. For example, a break linker has a length of 80 nucleotides. For example, a break linker has a length of 85 nucleotides. For example, a break linker has a length of 90 nucleotides. For example, a break linker has a length of 95 nucleotides. For example, a break linker has a length of 100 nucleotides. For example, a break linker has a length of 110 nucleotides. For example, a break linker has a length of 120 nucleotides. For example, a break linker has a length of 130 nucleotides. For example, a break linker has a length of 140 nucleotides. For example, a break linker has a length of 150 nucleotides.

[0224] In one example, the interrupted linker is 10 nucleotides long. In another example, the interrupted linker contains or consists of the nucleotide sequence shown in Sequence ID No. 8. For example, the interrupted linker contains or consists of the nucleotide sequence GCAUAUGACU.

[0225] In one example, the 3' tailing sequence contains, in order from 5' to 3', a polyA sequence containing 30 consecutive adenosine nucleotides, a 10-nucleotide interruption linker, and a further polyA sequence containing 70 consecutive adenosine nucleotides.

[0226] In one example, the 3' tailing sequence includes, in order from 5' to 3', a polyA sequence containing 30 consecutive adenosine nucleotides, a interruption linker containing or consisting of the nucleotide sequence shown in SEQ ID NO: 8, and a further polyA sequence containing 70 consecutive adenosine nucleotides.

[0227] In one example, with RNA, and by arbitrary selection, self-replicating RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments, and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) Nucleotide sequence encoding the antigen from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), d) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, e) A nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter. f) 3'-UTR, its fragments, and / or variants, and g) comprising one or more 3' tailing sequences selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0228] In one example, with RNA, and by arbitrary selection, self-replicating RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments, and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) Nucleotide sequences encoding antigens from respiratory syncytial virus (RSV), d) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter. e) A nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter, f) 3'-UTR, its fragments, and / or variants, and g) comprising one or more 3' tailing sequences selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0229] In one example, with RNA, and by arbitrary selection, self-replicating RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of Kozak consensus sequences, IRES, SG promoters, and combinations thereof. c) Nucleotide sequence encoding an antigen from influenza, d) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter. e) A nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, f) 3'-UTR, its fragments, and / or variants, and g) comprising one or more 3' tailing sequences selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0230] For example, the RNA disclosed herein, and optionally, the self-replicating RNA, are arranged in the order from 5' to 3'. a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operatively ligated to the minimal SG promoter, a nucleotide sequence encoding an antigen from influenza, and a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), or b) A nucleotide sequence antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to the minimal SG promoter, a nucleotide sequence encoding an antigen from influenza operably ligated to the minimal SG promoter, and a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV) operably ligated to the extended SG promoter, or c) comprising a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a minimal SG promoter; a nucleotide sequence encoding an antigen from influenza, operably ligated to a minimal SG promoter; and a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably ligated to a wild-type EMCV IRES.

[0231] In another example, the RNA in this disclosure, of optional choice, self-replicating RNA is in the order from 5' to 3'. a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operatively ligated to the minimal SG promoter, a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a nucleotide sequence encoding a third antigen from influenza, operatively ligated to the minimal SG promoter, or b) A nucleotide sequence antigen from respiratory syncytial virus (RSV) operably ligated to a minimal SG promoter, a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to a minimal SG promoter, and a nucleotide sequence encoding a third antigen from influenza operably ligated to an extended SG promoter, or c) comprising a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV) operably ligated to a minimal SG promoter, a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to a minimal SG promoter, and a nucleotide sequence encoding a third antigen from influenza operably ligated to a wild-type EMCV IRES.

[0232] In another example, the RNA in this disclosure, of optional choice, self-replicating RNA is in the order from 5' to 3'. a) A nucleotide sequence encoding an antigen from influenza, operably ligated to the minimal SG promoter, a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), and a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to the minimal SG promoter, or b) A nucleotide sequence antigen from influenza operably ligated to the minimal SG promoter, a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV) operably ligated to the minimal SG promoter, and a nucleotide sequence encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to the extended SG promoter, or It includes a nucleotide sequence encoding an antigen from influenza, operably ligated to a minimal SG promoter; a nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably ligated to a minimal SG promoter; and a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to a wild-type EMCV IRES.

[0233] In one example, the RNA of the present disclosure, optionally, a self-replicating RNA, comprises, in 5' to 3' order, a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1, a nucleotide sequence encoding an antigen from influenza operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1, and a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV) operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1.

[0234] In one example, the RNA of the present disclosure, optionally, a self-replicating RNA, comprises, in 5' to 3' order, a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1, a nucleotide sequence encoding an antigen from influenza operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1, and a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV) operably ligated to an extended SG promoter containing the sequence shown in SEQ ID NO: 5.

[0235] In one example, the RNA of the present disclosure, optionally, a self-replicating RNA, comprises, in 5' to 3' order, a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1, a nucleotide sequence encoding an antigen from influenza operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 1, and a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV) operably ligated to a wild-type EMCV IRES containing the sequence shown in SEQ ID NO: 4.

[0236] In one example, RNA further includes a 5' end cap structure.

[0237] For example, the 5' end cap structure is an endogenous cap or an analogue of it.

[0238] For example, the 5'-terminal cap structure contains guanine or a guanine analog.

[0239] For example, the 5'-terminal cap structure is selected from the group consisting of anti-reverse cap analog (ARCA), N7,2'-O-dimethyl-guanosine (mCAP), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), cap 1, and cap 2. For example, the 5'-terminal cap structure is anti-reverse cap analog (ARCA). For example, the 5'-terminal cap structure is N7,2'-O-dimethyl-guanosine (mCAP). For example, the 5'-terminal cap structure is inosine. For example, the 5'-terminal cap structure is N1-methyl-guanosine. For example, the 5'-terminal cap structure is 2'-fluoro-guanosine. For example, the 5' end cap structure is 7-deaza-guanosine. For example, the 5' end cap structure is 8-oxo-guanosine. For example, the 5' end cap structure is 2-amino-guanosine. For example, the 5' end cap structure is LNA-guanosine. For example, the 5' end cap structure is 2-azido-guanosine. For example, the 5' end cap structure is N6,2'-O-dimethyladenosine. For example, the 5' end cap structure is 7-methylguanosine (m7G). For example, the 5' end cap structure is cap 1. For example, the 5' end cap structure is cap 2.

[0240] For example, the 5' end cap structure is ligated to the 5' end of the RNA by a 5'-5'-triphosphate ligation or a 5'-5'-phosphorothioate ligation.

[0241] In one example, antigens (i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), or influenza antigen) are expressed at substantially the same level. For example, the antigens are expressed at about 10%, 5%, or 1% of each other's levels of expression. In another example, the antigens are expressed at different levels. For example, the antigens are expressed at about 10%, 15%, or more than 20% of each other's levels of expression. Methods for determining the levels of expression are known in the art and / or described herein.

[0242] In one example, the self-replicating RNA is derived from an alphavirus. For instance, the alphavirus is selected from a group consisting of Semliki Forest Virus (SFV), Sindobis Virus (SIN), Venezuelan Encephalitis Virus (VEE), and combinations thereof.

[0243] In one example, the self-replicating RNA originates from the Semliki Forest Virus (SFV).

[0244] In one example, the self-replicating RNA originates from the Sindbis virus (SIN).

[0245] In one example, the self-replicating RNA originates from the Venezuelan encephalitis virus (VEE).

[0246] In one example, the antigen is selected from a group consisting of respiratory syncytial virus (RSV), influenza virus, and / or SARS-CoV-2.

[0247] In one example, the antigen is derived from respiratory syncytial virus (RSV).

[0248] In one example, the antigen is derived from influenza.

[0249] In one example, the antigen is derived from SARS-CoV-2.

[0250] For example, the antigen may be from a single strain (i.e., monovalent) of influenza virus, or from multiple strains (i.e., polyvalent). For instance, an immunogenic composition may contain antigens from one or more (e.g., one, two, or three) influenza virus strains.

[0251] In one example, the antigen is derived from an influenza A virus strain. For instance, the antigen may be an influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), non-structural (NS) protein, or an immunogenic fragment or variant thereof. In one example, the antigen may be an influenza A hemagglutinin (HA) subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, and / or an influenza A neuraminidase (NA) subtype N1, N2, N3, N4, N5, N6, N7, N8, or N9, and / or an influenza A matrix (M) protein subtype M1 or M2, and / or an influenza A non-structural (NS) protein subtype NS1 or NS2.

[0252] Those skilled in the art will recognize that pandemic strains of influenza viruses are generally H1, H2, H3, H5, H6, H7, or H9 subtype influenza A virus strains. For example, H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 strains.

[0253] In one example, the antigen is from an H1, H2, H3, H5, H6, H7, or H9 subtype influenza A virus strain. For example, the antigen could be H1 hemagglutinin, or H2 hemagglutinin, or H3 hemagglutinin, or H5 hemagglutinin, or H6 hemagglutinin, or H7 hemagglutinin, or H9 hemagglutinin. In another example, the antigen is an H5 subtype influenza A virus strain (i.e., H5 hemagglutinin). In one example, the H5 hemagglutinin is from the A / turkey / Turkey / 1 / 2005 virus strain. In another example, the H3 hemagglutinin is from the A / Delaware / 39 / 2019 virus strain.

[0254] In one example, the antigen is an N1, N2, N3, N7, or N9 subtype influenza A virus strain. For example, the antigen is an N1 neuraminidase, or an N2 neuraminidase, or an N3 neuraminidase, or an N7 neuraminidase, or an N9 neuraminidase. For example, the antigen is an N1 neuraminidase subtype influenza A virus strain. In one example, the N1 neuraminidase is the A / turkey / turkey / 1 / 2005 strain. In another example, the N2 neuraminidase is the A / Delaware / 39 / 2019 virus strain.

[0255] In one example, the antigen is H5 hemagglutinin protein and / or N1 neuraminidase protein. In another example, the antigen is an H5 hemagglutinin subtype influenza A virus strain. In yet another example, the antigen is an N1 neuraminidase subtype influenza A virus strain.

[0256] For example, an antigen from an influenza protein contains a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 19.

[0257] For example, antigens from influenza proteins are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 19.

[0258] For example, an antigen from an influenza protein contains a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 20.

[0259] For example, antigens from influenza proteins are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 20.

[0260] In one example, the antigen is an influenza B virus strain. Those skilled in the art will recognize that influenza B viruses are not divided into subtypes, but rather into two lineages, namely B / Yamagata and B / Victoria.

[0261] In one example, the antigen is the B / Yamagata influenza B virus strain. For example, the influenza B virus strain is the B / Singapore / INFTT 16 0610 / 16(By) virus strain. In another example, the antigen is the B / Victoria influenza B virus strain.

[0262] In one example, the antigen is the influenza B virus Hyam protein and / or Nyam protein. For example, the antigen is the influenza B virus Hyam protein. In another example, the antigen is the influenza B virus Nyam protein. In yet another example, the antigen is the influenza B virus Hyam and Nyam proteins. In one example, the antigen is the Hyam subtype influenza B virus strain. In yet another example, the antigen is the Nyam subtype influenza B virus strain.

[0263] In one example, the antigen is derived from influenza C.

[0264] In one example, the antigens are from SARS-CoV-2 strains alpha (B.1.1.7), beta (B.1.351), gamma (P1), epsilon (B.1.429), delta (B.1.617.2) variant, kappa (B.1.617.1) variant, Wuhan (original) strain, and / or omicron (B.1.1.529).

[0265] In one example, the antigen is the SARS-CoV-2 spike (S) protein or nucleocapsid (N) protein. For example, the antigen is the SARS-CoV-2 N protein or S protein from the alpha (B.1.1.7), beta (B.1.351), gamma (P1), epsilon (B.1.429), delta (B.1.617.2) variant, kappa (B.1.617.1) variant, Wuhan (original) strain, and / or omicron (B.1.1.529) strain. In one example, the antigen is the SARS-CoV-2 N protein or S protein from the Wuhan (original) SARS-CoV-2 strain. In another example, the antigen is the SARS-CoV-2 N protein or S protein from the delta SARS-CoV-2 strain. In one example, the antigen is the SARS-CoV-2 N protein or S protein from the delta-SARS-CoV-2 omicron strain.

[0266] For example, antigens from the SARS-CoV-2 S protein contain polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 21.

[0267] For example, antigens from the SARS-CoV-2 S protein are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 21.

[0268] For example, the antigen from the SARS-CoV-2 S protein contains a polynucleotide sequence that has at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 22.

[0269] For example, antigens from the SARS-CoV-2 S protein are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 22.

[0270] In one example, the antigen is the SARS-CoV-2 nucleocapsid (N) protein. In another example, the antigen is the SARS-CoV-2 envelope (E) protein. In yet another example, the antigen is the SARS-CoV-2 matrix (M) protein.

[0271] In another example, the S protein is a mutant S protein.

[0272] For example, mutant S proteins contain mutations in the receptor-binding domain. Examples of mutations include: S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, The group is selected from P479L, P479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, and Y505H, Y508H. In one example, the mutant S protein contains a mutation in the receptor-binding domain selected from the group consisting of N439K, N439L, L452R, S477N, T478I, V483A, and E484D.

[0273] For example, mutant S proteins contain mutations in the receptor-binding domain. Examples of mutations include: R346K, K417N, K417T, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, The group is selected from T478I, T478K, P479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, and Y508H. For example, the mutant S protein contains a mutation in the receptor-binding domain selected from the group consisting of R346K, K417N, K417T, N439K, N439L, L452R, S477N, T478I, V483A, E484D, E484K, and N501Y.

[0274] For example, mutant S proteins include P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V395I, F400C, R403K, R403S, and D4 This includes mutations selected from the group consisting of 05V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417R, K417N, I418V, Y421S, Y423C, Y423F, Y423S, D427Y, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, and D614G.

[0275] For example, mutant S proteins include L18F, D80A, T95I, Y144S, Y145N, D215G, P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V39 5I, F400C, R403K, R403S, D405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417N, K417T, K417R, I418V, Y421S, Y423C , Y423F, Y423S, D427Y, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K 458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, T478K, P4 79L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490 This includes mutations selected from the group consisting of L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, Y508H, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, A570D, D614G, P680H, P681H, A701V, T716I, and D950N.

[0276] In one example, the antigen from RSV is an RSV surface glycoprotein selected from the fusion (F), glycoprotein (G), small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P and L, and non-structural proteins NS1 and NS2. In a specific example, the antigen is the RSV-F antigen.

[0277] For example, the antigen from RSV contains a polynucleotide sequence that has at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 17.

[0278] In one example, antigens from RSV are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 17.

[0279] For example, the antigen from RSV contains a polynucleotide sequence that has at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with SEQ ID NO: 18.

[0280] For example, antigens from RSV are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 18.

[0281] In one example, the composition further comprises one or more additional antigens. Thus, in some examples, the composition comprises a fourth and / or fifth antigen. In one example, one or more additional antigens are viral antigens. In one example, the viral antigens are from respiratory viruses. In one example, the respiratory viruses are selected from the group consisting of influenza viruses, respiratory syncytial viruses, parainfluenza viruses, metapneumonia viruses, rhinoviruses, coronaviruses, adenoviruses, and bocaviruses.

[0282] In one example, one or more additional viral antigens are from the influenza virus.

[0283] In one example, one or more additional viral antigens are from respiratory syncytial virus.

[0284] In one example, one or more additional viral antigens are from the parainfluenza virus.

[0285] In one example, one or more additional viral antigens are from metapneumonia virus.

[0286] In one example, one or more additional viral antigens are derived from rhinovirus.

[0287] In one example, one or more additional viral antigens are derived from coronaviruses.

[0288] In one example, one or more additional viral antigens are derived from adenovirus.

[0289] In one example, one or more additional viral antigens are derived from the bocavirus.

[0290] In one example, one or more additional antigens are viral antigens from influenza viruses or coronaviruses.

[0291] This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure. This disclosure further provides immunogenic compositions comprising the RNA of this disclosure. For example, this disclosure provides immunogenic compositions comprising the cRNA of this disclosure. This disclosure also provides immunogenic compositions comprising the self-replicating RNA of this disclosure. In one example, a composition of this disclosure can induce an immune response in a subject when administered. 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, a 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.

[0292] In one example, the immunogenic composition of this disclosure comprises multiple polynucleotides, each polynucleotide encoding a different polypeptide antigen sequence. In another example, the immunogenic composition of this disclosure comprises multiple RNAs, each RNA encoding a different polypeptide antigen sequence. Therefore, this disclosure is, (a) RNA containing a nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) RNA containing a nucleotide sequence encoding a second antigen from influenza, (c) an immunogenic composition comprising RNA containing a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), The present invention provides an immunogenic composition in which each nucleotide sequence is operably linked to a regulatory element. Each RNA may be, for example, cRNA, mRNA, or self-replicating RNA.

[0293] In a further example, the immunogenic composition of the present disclosure comprises multiple cRNAs, each encoding a different polypeptide antigen sequence. In another example, the immunogenic composition of the present disclosure comprises multiple self-replicating RNAs, each encoding a different polypeptide antigen sequence.

[0294] This disclosure also provides a pharmaceutical composition comprising the immunogenic composition of this disclosure and a pharmaceutically acceptable carrier. Pharmacochemically acceptable carriers suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0295] In one example, the pharmaceutical composition further comprises lipid nanoparticles (LNPs), polymer microparticles, and an oil-in-water emulsion. For example, polynucleotides, RNA, cRNA, or self-replicating RNA are encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. In one example, polynucleotides are encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. In another example, RNA is encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. For example, cRNA is encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion. For example, self-replicating RNA is encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, and the oil-in-water emulsion.

[0296] In one example, the pharmaceutical composition further comprises LNPs. For example, polynucleotides are encapsulated within the LNPs. In another example, RNA is encapsulated within the LNPs. For example, cRNA is encapsulated within the LNPs. For example, self-replicating RNA is encapsulated within the LNPs. For example, polynucleotides are bound to the LNPs. In another example, RNA is bound to the LNPs. For example, cRNA is bound to the LNPs. In yet another example, self-replicating RNA is bound to the LNPs. For example, polynucleotides are adsorbed onto the LNPs. In yet another example, RNA is adsorbed onto the LNPs. For example, cRNA is adsorbed onto the LNPs. In yet another example, self-replicating RNA is adsorbed onto the LNPs. In yet another example, each RNA is formulated together within the LNPs. In yet another example, each RNA is formulated separately within the LNPs.

[0297] In one example, LNP includes PEG lipids, structural lipids, and / or neutral lipids. For example, LNP includes PEG lipids, structural lipids, and neutral lipids. In another example, LNP includes PEG lipids, structural lipids, or neutral lipids.

[0298] In one example, the LNP further contains cationic lipids. In another example, the LNP does not contain cationic lipids.

[0299] In one example, the pharmaceutical composition further comprises polymer microparticles. For example, polynucleotides are encapsulated within the polymer microparticles. In another example, RNA is encapsulated within the polymer microparticles. For example, cRNA is encapsulated within the polymer microparticles. For example, self-replicating RNA is encapsulated within the polymer microparticles. For example, polynucleotides are bound to polymer microparticles. In another example, RNA is bound to polymer microparticles. For example, cRNA is bound to polymer microparticles. In yet another example, self-replicating RNA is bound to polymer microparticles. For example, polynucleotides are adsorbed onto polymer microparticles. In yet another example, RNA is adsorbed onto polymer microparticles. For example, cRNA is adsorbed onto polymer microparticles. In yet another example, self-replicating RNA is adsorbed onto polymer microparticles.

[0300] In one example, the pharmaceutical composition further comprises an oil-in-water emulsion. For example, polynucleotides are encapsulated in the oil-in-water emulsion. In another example, RNA is encapsulated in the oil-in-water emulsion. For example, cRNA is encapsulated in the oil-in-water emulsion. For example, self-replicating RNA is encapsulated in the oil-in-water emulsion. For example, polynucleotides are bound to the oil-in-water emulsion. In yet another example, RNA is bound to the oil-in-water emulsion. For example, cRNA is bound to the oil-in-water emulsion. In yet another example, self-replicating RNA is bound to the oil-in-water emulsion. In a further example, self-replicating RNA is adsorbed onto the oil-in-water emulsion. In a further example, self-replicating RNA is resuspended in the oil-in-water emulsion.

[0301] This disclosure also provides immunogenic compositions or pharmaceutical compositions of this disclosure for use as vaccines.

[0302] For example, a polynucleotide is DNA. For example, the Disclosure provides DNA encoding the cRNA vaccine of the Disclosure. For example, the Disclosure provides DNA encoding the self-replicating RNA vaccine of the Disclosure.

[0303] For example, DNA is a plasmid.

[0304] This disclosure provides a method for treating, preventing, or slowing the progression of a disease or condition in a subject, wherein the method comprises administering an immunogenic composition, pharmaceutical composition, or vaccine of this disclosure to a subject in need. In one example, this disclosure provides a method for treating a disease or condition in a subject, wherein the method comprises administering an immunogenic composition, pharmaceutical composition, or vaccine of this disclosure to a subject in need. In another example, this disclosure provides a method for preventing a disease or condition in a subject, wherein the method comprises administering an immunogenic composition, pharmaceutical composition, or vaccine of this disclosure to a subject in need. In yet another example, this disclosure provides a method for slowing the progression of a disease or condition in a subject, wherein the method comprises administering an immunogenic composition, pharmaceutical composition, or vaccine of this disclosure to a subject in need.

[0305] In one example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of a disease or condition in a subject that requires treatment or prevention of such disease or condition. For example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment of such disease or condition. In another example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of such disease or condition. In yet another example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for delaying the progression of a disease or condition in a subject that requires delaying the progression of such disease or condition.

[0306] In one example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for treating, preventing, or slowing the progression of a disease or condition in a subject that requires treatment or prevention of such disease or condition. For example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment of such disease or condition. In another example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of such disease or condition. In yet another example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for slowing the progression of a disease or condition in a subject that requires slowing the progression of such disease or condition.

[0307] In one example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for treating, preventing, or slowing the progression of a disease or condition in a subject that requires treatment or prevention of such disease or condition. For example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment of such disease or condition. In another example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of such disease or condition. In yet another example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for slowing the progression of a disease or condition in a subject that requires slowing the progression of such disease or condition.

[0308] In one example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for treating, preventing, or slowing the progression of a disease or condition in a subject that requires treatment or prevention of such disease or condition. For example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for treating a disease or condition in a subject that requires treatment of such disease or condition. In another example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for preventing a disease or condition in a subject that requires prevention of such disease or condition. In yet another example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for slowing the progression of a disease or condition in a subject that requires slowing the progression of such disease or condition.

[0309] In another example, the Disclosure provides the use of the immunogenic compositions, pharmaceutical compositions, or vaccines of the Disclosure in the preparation of pharmaceuticals for treating, preventing, or delaying the progression of a disease or condition in subjects who require treatment or prevention of a disease or condition or delaying its progression. For example, the Disclosure provides the use of the immunogenic compositions, pharmaceutical compositions, or vaccines of the Disclosure in the manufacture of pharmaceuticals for treating a disease or condition in subjects who require treatment of a disease or condition. In another example, the Disclosure provides the use of the immunogenic compositions, pharmaceutical compositions, or vaccines of the Disclosure in the manufacture of pharmaceuticals for preventing a disease or condition in subjects who require prevention of a disease or condition. In yet another example, the Disclosure provides the use of the immunogenic compositions, pharmaceutical compositions, or vaccines of the Disclosure in the manufacture of pharmaceuticals for delaying the progression of a disease or condition in subjects who require delaying the progression of a disease or condition.

[0310] In one example, the Disclosure provides polynucleotides for use in treating, preventing, or delaying the progression of a disease or condition in subjects requiring treatment or prevention of such disease or condition or delaying its progression. In another example, the Disclosure provides RNA for use in treating, preventing, or delaying the progression of a disease or condition in subjects requiring treatment or prevention of such disease or condition or delaying its progression. In yet another example, the Disclosure provides cRNA for use in treating, preventing, or delaying the progression of a disease or condition in subjects requiring treatment or prevention of such disease or condition or delaying its progression. In yet another example, the Disclosure provides self-replicating RNA for use in treating, preventing, or delaying the progression of a disease or condition in subjects requiring treatment or prevention of such disease or condition or delaying its progression. In yet another example, the Disclosure provides immunogenic compositions or vaccines disclosed herein for use in treating, preventing, or delaying the progression of a disease or condition in subjects requiring treatment or prevention of such disease or condition or delaying its progression.

[0311] In one example, the subject suffers from a disease or condition. In another example, the subject has been diagnosed with a disease or condition. In yet another example, the subject is receiving treatment for a disease or condition.

[0312] In one example, the disease or condition is selected from a group consisting of influenza, COVID-19, SARS-CoV-2 infection, RSV, and acute respiratory distress syndrome (ARDS), or a combination thereof. In another example, the disease or condition is influenza, SARS-CoV-2 infection, and RSV. In yet another example, the disease or condition is influenza and COVID-19. In yet another example, the disease or condition is SARS-CoV-2 and influenza. In yet another example, the disease or condition is RSV and influenza. In yet another example, the disease or condition is SARS-CoV-2 and RSV. In yet another example, the disease or condition is RSV and COVID-19. In yet another example, the disease or condition is SARS-CoV-2. In yet another example, the disease or condition is RSV. In yet another example, the disease or condition is influenza. In yet another example, the disease or condition is COVID-19. In yet another example, the disease or condition is ARDS.

[0313] In one example, a composition or vaccine of the present disclosure is administered in an amount sufficient to reduce the severity of or prevent the onset of one or more of the following conditions: SARS-CoV-2 infection, COVID-19, influenza, RSV, and / or ARDS. These conditions are obvious to those skilled in the art and / or are described herein.

[0314] For example, the present disclosure provides a method for inducing an immune response in a subject, the method comprising administering a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need thereof.

[0315] For example, this disclosure provides the use of a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a pharmaceutical product for inducing an immune response in a subject requiring induction of an immune response.

[0316] For example, this disclosure provides a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in inducing an immune response in subjects requiring the induction of an immune response.

[0317] In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. For example, the production of neutralizing antibodies. In another example, the composition induces a cell-mediated immune response. For example, the cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells. For example, the T cells are CD4 T cells and / or CD8 T cells. In one example, the T cells are CD4 T cells. In another example, the T cells are CD8 T cells. In yet another example, the T cells are CD4 and CD8 T cells.

[0318] For example, administration of a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD4 T cell-mediated immune response.

[0319] For example, administration of a pharmaceutical composition, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD8 T cell-mediated immune response.

[0320] For example, administration of a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD4 and CD8 T cell-mediated immune response.

[0321] In one example, the CD4 T cell-mediated immune response is a Th0, Th1, and / or Th2 response. For example, the CD4 T cell-mediated immune response is a Th0 response. In another example, the CD4 T cell-mediated immune response is a Th1 response. In yet another example, the CD4 T cell-mediated immune response is a Th2 response. In one example, the CD4 T cell-mediated immune response is a Th0 and Th1 response. In another example, the CD4 T cell-mediated immune response is a Th0 and Th2 response. In yet another example, the CD4 T cell-mediated immune response is a Th1 and Th2 response. In yet another example, the CD4 T cell-mediated immune response is a Th0, Th1, and Th2 response.

[0322] In one example, the Th0 response cytokine expresses interleukin-2 (IL2+) and / or tumor necrosis factor alpha (TNFa+), and / or is negative for interferon-gamma (IFNg-), IL5-, and / or IL13-. For example, the cytokine is IL2+. In another example, the cytokine is TNFa+. In one example, the cytokine is IFNg-. In yet another example, the cytokine is IL5-. In yet another example, the cytokine is IL13-.

[0323] In one example, the Th1 response cytokine expresses interferon-gamma (IFNg+) and / or is negative for IL5- and / or IL13-. For example, the cytokine is IFNg+. In another example, the cytokine is IL5-. In yet another example, the cytokine is IL13-.

[0324] In one example, the Th2-responsive cytokine expresses IL5+ and / or IL13+ and / or is negative for IFNg. For example, the cytokine is IL5+. In another example, the cytokine is IL13+. For example, the cytokine is IFNg-.

[0325] For example, the present disclosure provides a method for reducing the viral load in a subject having a viral infection, the method comprising administering to the subject having a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein.

[0326] For example, this disclosure provides the use of a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical for reducing the viral load in a subject having a viral infection.

[0327] For example, this disclosure provides a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in reducing the viral load in a subject having a viral infection.

[0328] In one example, the subjects are people aged 18 and over. In another example, the subjects are people of any age, for example, from about 1 month to 100 years old, for example, from about 2 months to about 80 years old, from about 6 months to about 3 years old, from about 3 years to about 18 years old, from about 12 years to about 18 years old, from about 18 years to about 55 years old, from about 50 years to about 75 years old, and from about 40 years to about 65 years old. In yet another example, the subjects are people from 2 years old. In yet another example, the subjects are people from 18 years old, from 30 years old, from 40 years old, from 50 years old, from 60 years old, from 70 years old, from 80 years old, or from about 90 years old. In yet another example, the subjects are people under 2 years old, under 18 months old, under 12 months old, under 6 months old, or under 3 months old.

[0329] In one example, the immunogenic compositions described herein are administered to an infant. In one example, the infant has or is suspected of having RSV, influenza, and / or SARS-CoV-2 infection.

[0330] In one example, the infants are approximately 1 to 12 months old, or 2 to 11 months old, or 3 to 10 months old, or 4 to 9 months old, or 5 to 8 months old, or 6 to 7 months old. In another example, the infants are approximately 1 to 11 months old, 1 to 10 months old, 1 to 9 months old, 1 to 8 months old, 1 to 7 months old, 1 to 6 months old, 1 to 5 months old, 1 to 4 months old, 1 to 3 months old, or 1 to 2 months old. In yet another example, the infants are approximately 1 month old, 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, or 12 months old.

[0331] Therefore, one example provided is a method for treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in an infant, comprising administering an immunogenic composition, pharmaceutical composition, or vaccine disclosed herein to the infant.

[0332] Another example provided is the use of immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein in the preparation of pharmaceuticals for treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in infants.

[0333] In another example, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein are provided for use in treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in infants.

[0334] Another example provided is a method for inducing an immune response to respiratory syncytial virus (RSV) in an infant, comprising administering an immunogenic composition, pharmaceutical composition, or vaccine disclosed herein to the infant.

[0335] Another example provided is the use of immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein in the preparation of pharmaceuticals for inducing an immune response to respiratory syncytial virus (RSV) in infants.

[0336] In another example, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein are provided for use in inducing an immune response to respiratory syncytial virus (RSV) in infants.

[0337] When the treatment, prevention, or delay of the progression of RSV is sought in infants, those skilled in the art will recognize the preferred immunogenic compositions described herein for administration to infants.

[0338] In one example, the compositions or vaccines described herein are administered in a single-dose regimen. In another example, the compositions are administered in two, three, or four-dose regimens, with doses administered at intervals of approximately one, two, or three months.

[0339] The Disclosure also provides a kit, packaged with instructions for use, for use in treating, preventing, or delaying the progression of a disease or disorder in a subject (e.g., influenza virus infection, RSV infection, SARS-CoV-2 infection, COVID-19, and / or ARDS), comprising at least one RNA of the Disclosure (e.g., self-replicating RNA) in an optional delivery system and / or a pharmaceutically acceptable carrier or diluent.

[0340] In one example, the RNA, immunogenic composition, or pharmaceutical composition of this disclosure is supplied in a vial. In another example, the RNA, immunogenic composition, or pharmaceutical composition of this disclosure is supplied in a syringe.

[0341] Any considerations relating to the documents, actions, materials, devices, articles, etc. included herein, even if they existed prior to the priority date of each of the attached claims, should not be construed as acknowledging that any or all of these matters constitute part of the basis of the prior art or were common knowledge in the art relating to this disclosure.

[0342] Key for sequence listing [Table 1] [Modes for carrying out the invention]

[0343] overview Throughout this specification, unless otherwise specifically stated or the context requires, any reference to a single step, composition, group of steps, or group of compositions shall be construed as encompassing one or more (i.e., one or more) of those steps, compositions, groups of steps, or groups of compositions.

[0344] Those skilled in the art will understand that this disclosure is subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations or any two or more of such steps or features.

[0345] This disclosure is intended for illustrative purposes only and should not be limited in scope by the specific examples described herein. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0346] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specifically stated. In other words, any specific example in this disclosure may be combined with any other specific example in this disclosure (unless they are mutually exclusive).

[0347] Any example in this disclosure disclosing specific features or sets of features or methods or method steps would be construed as providing express support for rejecting those specific features or sets of features or methods or method steps.

[0348] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0349] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. This is described and explained through the literature provided by sources such as al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0350] The terms "and / or," for example, "X and / or Y," are understood to mean "X and Y" or "X or Y," and are considered to provide explicit support for both meanings or either of them.

[0351] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to mean including the element, element, or step, or group of elements, elements, or steps, that is described, but not to mean excluding any other element, element, or step, or group of elements, elements, or steps.

[0352] Where used herein, the term “derived from” shall be interpreted as indicating that a particular element may be derived from a particular source, but not necessarily directly from that source. Similarly, the term “based on” shall be interpreted as indicating that a particular element may be developed or used from a particular source, but not necessarily directly from that source.

[0353] Selected definition As used herein, the term “monocistronic” refers to an RNA encoding a single polypeptide, with respect to polynucleotides, RNA, cRNA, and / or self-replicating RNA.

[0354] As used herein, the term “multicistronic” (also known as “polycistronic”) refers to RNA encoding two or more polypeptides with respect to polynucleotides, RNA, cRNA, and / or self-replicating RNA. The term encompasses “bisistric” (or “disistric,” i.e., encoding two polypeptides) and “tricistric” (i.e., encoding three polypeptides) molecules. “Bicistronic” means a single nucleic acid capable of encoding two different polypeptides from different regions of the nucleic acid.

[0355] As used herein, the terms “conventional mRNA,” “cRNA,” or “non-amplified RNA” refer to constructs that enable the expression of heterologous RNA and proteins, but whose RNA cannot be amplified within a host cell.

[0356] As used herein, the term “self-replicating RNA” refers to constructs based on RNA viruses that have been engineered to enable the expression of heterologous mRNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified in host cells, resulting in the expression of desired gene products in those host cells.

[0357] As used herein, the term “naked” refers to nucleic acids that are substantially free of other macromolecules, such as lipids, polymers, and proteins. “Naked” nucleic acids, such as self-replicating RNA, are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated in, adsorbed to, or bound to lipid nanoparticles (LNPs), liposomes, macromolecular microparticles, or oil-in-water emulsions.

[0358] As used herein, the terms “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of consecutive nucleotides (or bases) covalently linked to a phosphodiester backbone. Traditionally, sequences are presented from the 5' end to the 3' end unless otherwise specified. To facilitate a clear description of nucleic acids, specific sequence components are referred to, for example, “first nucleotide sequence” and “second nucleotide sequence.” The first and second sequences may appear in any desired order or orientation unless otherwise specified, and it should be understood that no particular order or orientation is intended by the terms “first,” “second,” etc.

[0359] As used herein, the term “antigen” refers to a molecule or structure containing one or more epitopes that induce, trigger, enhance, or boost a cellular and / or humoral immune response. Antigens may include proteins and peptides derived from pathogens such as viruses, bacteria, fungi, protozoa, plants, or tumors.

[0360] As used herein, the term “adjuvant” refers to a compound that, when used in combination with a specific immunogen (e.g., a VLP) in a formulation, enhances, or otherwise alters or modifies the resulting immune response. Modification of the immune response includes enhancing or expanding the specificity of either or both the antibody and / or cellular immune responses. Modification of the immune response can also mean reducing or suppressing a particular antigen-specific immune response.

[0361] As used herein, the term “operably linked to” means positioning a subgenome promoter or regulatory element (e.g., IRES) relative to a nucleic acid such that nucleic acid expression is controlled or regulated by the element. For example, a subgenome promoter can be operably linked to a number of nucleic acids via another regulatory element, such as an internal ribosome entry site (IRES).

[0362] As used herein, the term “subgenome promoter” (also known as “junction region” promoter) refers to a promoter that directs the expression of heterogeneous nucleotide sequences and regulates protein expression.

[0363] As used herein, the terms “internal ribosome entry site” or “IRES” refer to a sequence of nucleotides within mRNA to which a ribosome or its components, such as the 40S subunit of a ribosome, can bind. An IRES does not necessarily have to contain the nucleic acid that induces translation of the mRNA (e.g., the start codon; AUG).

[0364] The terms “polypeptide” or “polypeptide chain” will be understood to mean a sequence of amino acids linked by peptide bonds. For example, a protein will be interpreted as containing a single polypeptide chain, i.e., a sequence of amino acids linked by peptide bonds, or a sequence of polypeptide chains (i.e., polypeptide complexes) covalently or non-covalently linked to one another. A sequence of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0365] The term "recombinant" shall be understood to mean a product of artificial genetic modification.

[0366] As used herein, the term “substantially identical” with respect to levels of expression means, unless the context indicates otherwise, that the first and second antigens have (at least) levels of expression of each other that are approximately 10% or less of each other.

[0367] As used herein, the terms “disease,” “disorder,” or “condition” refer to the disruption or interference of normal function and include, but are not limited to, any specific condition.

[0368] As used herein, a subject who has developed or is “at risk” of developing a SARS-CoV-2 infection may or may not have a detectable disease or symptoms of SARS-CoV-2 infection, and may or may not have exhibited a detectable disease or symptoms of SARS-CoV-2 infection prior to treatment according to this disclosure. “At risk” means that the subject has one or more risk factors which are measurable parameters known in the art and / or described herein that correlate with the development of a SARS-CoV-2 infection.

[0369] As used herein, the terms “treatment” or “to treat” a subject include the application or administration of the compounds or compositions of the Disclosure to a subject (or the application or administration of the compounds of the Disclosure to cells or tissues from a subject) for the purpose of delaying, slowing, stabilizing, healing, curing, reducing, releasing, modifying, restoring, reducing, mitigating, improving, or affecting a disease or condition, symptoms of a disease or condition, or the risk (or susceptibility to such risk) of a disease or condition. The terms “to treat” include any indication of successful treatment or mitigation of an injury, pathology or condition, including any objective or subjective parameters such as reduction, remission, a decrease in the rate of exacerbation, a decrease in the severity of the disease, stabilization, a reduction in symptoms, or making the injury, pathology or condition more tolerable to the subject, slowing the rate of degeneration or decline, or preventing the final stage of degeneration from becoming more debilitating.

[0370] As used herein, “prevention” or “prevention” means at least reducing the likelihood of acquiring a disease or disability (or susceptibility to it) (i.e., preventing the development of at least one of the clinical symptoms of the disease in a patient who may be exposed to the disease or susceptible to the disease but has not yet experienced or shown any symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are well known to physicians.

[0371] As used herein, the phrase “delaying the progression of” includes reducing or delaying the progression of a disease or condition and / or at least one symptom of a disease or condition in an individual.

[0372] "Effective dose" refers to the minimum effective amount in the required dosage and duration to achieve the desired outcome. For example, the desired outcome may be a therapeutic or prophylactic outcome. An effective dose may be provided in one or more doses. In some examples of this disclosure, the term "effective dose" means the amount required to treat a disease or condition previously described herein. In some examples of this disclosure, the term "effective dose" means the amount required to make a change associated with a disease or condition previously described herein. The effective dose may vary depending on the disease or condition being treated or the factor being modified, and also depending on body weight, age, racial background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, the effective dose will fall within a relatively broad range (e.g., "dosage" range) that can be determined by healthcare professionals through routine testing and experimentation. Therefore, this term should not be construed as limiting this disclosure to a specific amount of RNA, e.g., weight or number. The effective dose may be administered in a single dose or in doses repeated once or several times over a period of treatment.

[0373] The "therapeutic dose" is the minimum concentration required to produce at least a measurable improvement in a particular disease or condition. The therapeutic dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the RNA disclosed herein to induce a desired response in the individual. The therapeutic dose is also defined as the therapeutically beneficial effect outweighing any toxic or adverse effects of the RNA.

[0374] As used herein, the term “preventive effective dose” shall be construed to mean an amount of RNA of the Disclosure sufficient to prevent, inhibit, or delay the onset of one or more detectable symptoms of any disease or disorder described herein.

[0375] "Subjects" can also be any animals susceptible to infection by, for example, SARS-CoV-2, RSV, and / or influenza. Subjects of the Disclosure may be mammals, and in certain embodiments may be humans, which may be infants, children, adults, or the elderly. "Subjects at risk of infection" is any subject that could or is exposed to infection. Subjects may also be primary contacts of individuals diagnosed with an infection. "Subjects" include any human or non-human animals. Thus, in addition to their usefulness in human treatment, the compounds of the Disclosure may also be useful in veterinary treatment of mammals, including companion animals and farm animals, not limited to dogs, cats, horses, cattle, sheep, and pigs.

[0376] As used herein, the terms “lipid nanoparticles” or “LNPs” are understood to mean lipid-based particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) and containing a compound of any formula described herein. In embodiments, LNPs are formulated in compositions for the delivery of polynucleotides to desired targets, e.g., cells, tissues, organs, tumors, etc. For example, lipid nanoparticles or LNPs refer to any lipid composition, including, but not limited to, liposomes or vesicles in which aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; monolayer, or multiple; multilayer), micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles lacking a lipid bilayer.

[0377] antigen The antigens suitable for use in the compositions described herein will be obvious to those skilled in the art.

[0378] For example, the immunogenic compositions described herein include (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), Each nucleotide sequence is operablely linked to a regulatory element.

[0379] influenza Influenza, also known as "the flu," is an infectious disease caused by the influenza virus. It will be apparent to those skilled in the art that there are currently four types of influenza viruses: A, B, C, and D. Influenza A virus is the most common influenza virus, infecting humans, animals, and birds, while influenza B virus infections primarily occur in humans. Influenza C virus infections do not cause any severe symptoms in humans or mammals, and influenza D has so far infected only pigs and cattle.

[0380] In one example, the antigen is derived from an influenza A virus strain. For instance, the antigen may be an influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), non-structural (NS) protein, or an immunogenic fragment or variant thereof. In one example, the antigen may be an influenza A hemagglutinin (HA) subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, and / or an influenza A neuraminidase (NA) subtype N1, N2, N3, N4, N5, N6, N7, N8, or N9, and / or an influenza A matrix (M) protein subtype M1 or M2, and / or an influenza A non-structural (NS) protein subtype NS1 or NS2.

[0381] For example, influenza virus antigens are derived from different subtypes of the influenza virus. For instance, different hemagglutinin subtypes and / or different neuraminidase subtypes and / or matrix protein subtypes and / or nucleoprotein subtypes and / or non-structural protein subtypes.

[0382] Those skilled in the art will recognize that pandemic strains of influenza viruses are generally H1, H2, H3, H5, H6, H7, or H9 subtype influenza A virus strains. For example, H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 strains.

[0383] In one example, the antigen is an H1, H2, H3, H5, H6, H7, or H9 subtype influenza A virus strain. For example, the antigen is an H1 hemagglutinin, or an H2 hemagglutinin, or an H3 hemagglutinin, or an H5 hemagglutinin, or an H6 hemagglutinin, or an H7 hemagglutinin, or an H9 hemagglutinin. For example, the antigen is an H5 subtype influenza A virus strain (i.e., H5 hemagglutinin). In one example, the H5 hemagglutinin is the A / turkey / turkey / 1 / 2005 virus strain. In another example, the H3 hemagglutinin is the A / Delaware / 39 / 2019 virus strain.

[0384] In one example, the antigen is an N1, N2, N3, N7, or N9 subtype influenza A virus strain. For example, the antigen is an N1 neuraminidase, or an N2 neuraminidase, or an N3 neuraminidase, or an N7 neuraminidase, or an N9 neuraminidase. For example, the antigen is an N1 neuraminidase subtype influenza A virus strain. In one example, the N1 neuraminidase is the A / turkey / turkey / 1 / 2005 strain. In another example, the N2 neuraminidase is the A / Delaware / 39 / 2019 virus strain.

[0385] In one example, the antigen is H5 hemagglutinin protein and / or N1 neuraminidase protein. For example, the antigen is an H5 hemagglutinin subtype influenza A virus strain. In another example, the second antigen is an N1 neuraminidase subtype influenza A virus strain.

[0386] In one example, the antigen is an influenza B virus strain. Those skilled in the art will recognize that influenza B viruses are not divided into subtypes, but rather into two lineages, namely B / Yamagata and B / Victoria.

[0387] In one example, the antigen is the B / Yamagata influenza B virus strain. For example, the influenza B virus strain is the B / Singapore / INFTT 16 0610 / 16(By) virus strain. In another example, the antigen is the B / Victoria influenza B virus strain.

[0388] In one example, the antigen is the influenza B virus Hyam protein and / or Nyam protein. For example, the antigen is the influenza B virus Hyam protein. In another example, the antigen is the influenza B virus Nyam protein. In yet another example, the antigen is the influenza B virus Hyam and Nyam proteins.

[0389] For example, an antigen from an influenza protein contains a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 19.

[0390] For example, antigens from influenza proteins are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 19.

[0391] For example, an antigen from an influenza protein contains a polynucleotide sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 20.

[0392] For example, antigens from influenza proteins are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 20.

[0393] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) COVID-19 is an infectious disease caused by SARS-CoV-2. It was first identified in Wuhan, Hubei Province, China in December 2019, and is currently causing an ongoing pandemic.

[0394] In one example, the antigen is from a single strain (i.e., monovalent) of SARS-CoV-2. In another example, the antigen is from the alpha (B.1.1.7), beta (B.1.351), gamma (P1), epsilon (B.1.429), delta (B.1.617.2) variant, kappa (B.1.617.1) variant, Wuhan (original) strain, or omicron (B.1.1.529) strain of SARS-CoV-2.

[0395] In one example, the antigen is the SARS-CoV-2 spike (S) protein or nucleocapsid (N) protein. For example, the antigen is the SARS-CoV-2 N protein or S protein from the alpha (B.1.1.7), beta (B.1.351), gamma (P1), epsilon (B.1.429), delta (B.1.617.2) variant, kappa (B.1.617.1) variant, Wuhan (original) strain, or omicron (B.1.1.529) strain. In one example, the antigen is the SARS-CoV-2 N protein or S protein from the Wuhan (original) SARS-CoV-2 strain. In another example, the antigen is the SARS-CoV-2 N protein or S protein from the delta SARS-CoV-2 strain. In one example, the antigen is the SARS-CoV-2 N protein or S protein from the delta-SARS-CoV-2 omicron strain.

[0396] For example, antigens from the SARS-CoV-2 S protein contain polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 21.

[0397] For example, antigens from the SARS-CoV-2 S protein are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 21.

[0398] For example, the antigen from the SARS-CoV-2 S protein contains a polynucleotide sequence that has at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 22.

[0399] For example, antigens from the SARS-CoV-2 S protein are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 22.

[0400] Respiratory syncytial virus (RSV) RSV is an enclosed, non-segmented, negative-strand RNA virus belonging to the Paramyxoviridae family of the Pneumovirus genus. To infect host cells, paramyxoviruses, including RSV, require fusion of the viral membrane with the host cell membrane, similar to other enveloped viruses such as influenza viruses. In the case of RSV, a conserved fusion protein (RSV-F glycoprotein) fuses the viral membrane and cell membrane by coupling irreversible protein refolding with membrane juxtaposition. Based on paramyxovirus research, the RSV-F protein initially folds into a metastable pre-fusion conformation. During cell entry, the pre-fusion conformation undergoes refolding and conformational changes to its stable post-fusion conformation.

[0401] In one example, the antigen is from an RSV surface glycoprotein selected from the fusion (F), glycoprotein (G), small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P and L, and non-structural proteins NS1 and NS2. In a specific example, the antigen is the RSV-F antigen.

[0402] The RSV F glycoprotein is a type I single-pass endogenous membrane protein with four common domains: an N-terminal ER translocation signal sequence (SS), an extracellular domain (ED), a transmembrane domain (TM), and a cytoplasmic tail (CT). The CT contains a single palmitoylated cysteine ​​residue. The F protein sequence is highly conserved among RSV isolates but evolves over time. Unlike most paramyxoviruses, the F protein in RSV can mediate entry and syncytium formation independently of other viral proteins (other paramyxoviruses typically require an HN protein in addition to the F protein).

[0403] The RSV-F glycoprotein is translated from mRNA into a protein of approximately 574 amino acids, denoted as F0. Post-translational processing of F0 involves the removal of the N-terminal signal peptide by signal peptidases in the endoplasmic reticulum. F0 is also cleaved at two sites (approximately 109 / 110 and approximately 136 / 137) by cellular proteases (particularly furin) in the trans-Golgi. This cleavage results in the removal of a short intervening sequence, generating two subunits denoted as F1 (approximately 50 kDa; C-terminus; approximately residues 137-574) and F2 (approximately 20 kDa; N-terminus; approximately residues 1-109), which remain associated with each other. F1 contains a hydrophobic fusion peptide at its N-terminus and also contains two amphiphilic 7-residue repeat regions (heptad-repeat regions) (HRA and HRB). The HRA is located near the fusion peptide, and the HRB is located near the transmembrane domain. The three F1-F2 heterodimers are organized as F1-F2 homotrimers in the virion.

[0404] Suitable RSV-F antigens for inclusion in the immunogenic compositions described herein include RSV-F glycoproteins and RSV-F glycoprotein variants. Suitable RSV-F glycoprotein variants include, for example, cleavage variants of the full-length F protein and soluble extracellular domain, each optionally containing one or more mutations such as furin cleavage mutations, trypsin cleavage mutations, fusion peptide mutations (e.g., whole or partial deletions), mutations that stabilize the HRB trimer, and mutations that destabilize the HRA trimer.

[0405] Full-length and truncated RSV-F glycoproteins, including those having one or more such mutations in various combinations, are well known in the art and are disclosed, for example, in WO2011 / 008974, which is incorporated herein by reference in its entirety.

[0406] In one example, the antigen is from the Pre-F or F protein of RSV. In another example, the antigen is from the G protein of RSV. In another example, the composition described herein includes an additional antigen. In another example, the additional antigen is a virus, bacterium, fungus, or protozoan.

[0407] For example, the antigen from RSV contains a polynucleotide sequence that has at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with respect to SEQ ID NO: 17.

[0408] In one example, antigens from RSV are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 17.

[0409] For example, the antigen from RSV contains a polynucleotide sequence that has at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity with SEQ ID NO: 18.

[0410] For example, antigens from RSV are encoded by polynucleotide sequences that have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity to SEQ ID NO: 18.

[0411] Viral antigens In one example, the polynucleotides of this disclosure further comprise additional viral antigens.

[0412] Additional viral antigens that can be encoded by RNA according to this disclosure will be apparent to those skilled in the art, and include, for example, orthomyxoviruses (e.g., influenza A, B, and C), paramyxoviridae viruses (pneumonia viruses (e.g., respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, and turkey rhinotracheitis virus), paramyxovirus types 1-4 (PIV), mumps, Sendai virus, Simian virus 5), bovine parainfluenza virus, nipah virus, henipavirus, and (and Newcastle disease virus), poxviridae (e.g., including but not limited to varicella and varicella major, varicella minor, varicella metapneumonia viruses, e.g., human varicella metapneumonia virus (hMPV) and trimeta pneumonia virus (aMPV)), measles virus genus (e.g., measles), picornaviruses (e.g., enteroviruses, rhinoviruses, heparnaviruses, parechoviruses, cardioviruses, and aftoviruses), enteroviruses (e.g., poliovirus type 1, 2, or 3, coxavirus) Coxsackie A viruses types 1-22 and 24, Coxsackie B viruses types 1-6, Echovirus (ECHO) viruses types 1-9, 11-27, and 29-34, as well as Enteroviruses 68-71), Banyavirus (e.g., California Encephalitis Virus), Phlebovirus (e.g., Rift Valley Fever Virus), Nairovirus (e.g., Crimean-Congo Hemorrhagic Fever Virus), Heparnavirus (e.g., Hepatitis A Virus (HAV)), Togavirus (e.g., Rubivirus, Alphavirus, or Alterivirus), Flavy Viruses (e.g., tick-borne encephalitis (TBE) virus, dengue fever (type 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus), pestiviruses (e.g., bovine viral diarrhea (BVDV), classical swine fever (CSFV), or border disease (BDV)), hepadnaviruses (e.g., hepatitis B virus, hepatitis C virus), rhabdoviruses (e.g., lyssavirus (rabies virus),and becyclovirus (VSV), caliciviridae (e.g., Norwalk virus and Norwalk-like viruses (e.g., Hawaii virus and Snow Mountain virus)), coronaviruses (e.g., Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV), SARS coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine infectious gastroenteritis virus (TGEV)), retroviruses (e.g., oncovirus, lentivirus, or spumavirus), reoviruses (e.g., orthoreovirus, rotau) This includes proteins and peptides from viruses (such as orbiviruses or cortiviruses), parvoviruses (e.g., parvovirus B19), hepatitis delta virus (HDV), hepatitis E virus (HEV), human herpesviruses (e.g., herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8)), papovaviruses (e.g., papillomaviruses and polyomaviruses), adenoviruses, and arenaviruses.

[0413] In one example, the additional viral antigens are derived from the parainfluenza virus.

[0414] In one example, the additional viral antigen comes from the metapneumonia virus.

[0415] In one example, the additional viral antigens are derived from rhinovirus.

[0416] In one example, the additional viral antigen is from a coronavirus. In this example, the coronavirus may be SARS-CoV-2, and the antigen is RNA encoding SARS-CoV-2 spike glycoprotein peptide or a fragment thereof, RNA encoding SARS-CoV-2 nucleocapsidrin protein peptide or a fragment thereof, RNA encoding SARS-CoV-2 membrane glycoprotein peptide or a fragment thereof, RNA encoding SARS-CoV-2 orf3a or a fragment thereof, RNA encoding SARS-CoV-2 orflab or a fragment thereof, or a variant of the above.

[0417] In one example, the additional viral antigen comes from an adenovirus.

[0418] In one example, the additional viral antigens are derived from the bocavirus.

[0419] In one example, the additional antigen may come from a single strain (i.e., monovalent) of the influenza virus, or from multiple strains (i.e., polyvalent).

[0420] In one example, the additional antigens are from influenza A, B, and / or C virus strains.

[0421] In one example, the additional antigens are derived from influenza A virus strains. For instance, the antigens may be influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), non-structural (NS) protein, or immunogenic fragments or variants thereof. In one example, the additional antigens may be influenza A hemagglutinin (HA) subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, and / or influenza A neuraminidase (NA) subtypes N1, N2, N3, N4, N5, N6, N7, N8, or N9, and / or influenza A matrix (M) protein subtype M1 or M2, and / or influenza A non-structural (NS) protein subtype NS1 or NS2.

[0422] Those skilled in the art will recognize that pandemic strains of influenza viruses are generally H1, H2, H3, H5, H6, H7, or H9 subtype influenza A virus strains. For example, H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 strains.

[0423] In one example, the antigen is an H1, H2, H3, H5, H6, H7, or H9 subtype influenza A virus strain. For example, the antigen is an H1 hemagglutinin, or an H2 hemagglutinin, or an H3 hemagglutinin, or an H5 hemagglutinin, or an H6 hemagglutinin, or an H7 hemagglutinin, or an H9 hemagglutinin. For example, the antigen is an H5 subtype influenza A virus strain (i.e., H5 hemagglutinin). In one example, the H5 hemagglutinin is the A / turkey / turkey / 1 / 2005 virus strain. In another example, the H3 hemagglutinin is the A / Delaware / 39 / 2019 virus strain.

[0424] In one example, the antigen is an N1, N2, N3, N7, or N9 subtype influenza A virus strain. For example, the antigen is an N1 neuraminidase, or an N2 neuraminidase, or an N3 neuraminidase, or an N7 neuraminidase, or an N9 neuraminidase. For example, the antigen is an N1 neuraminidase subtype influenza A virus strain. In one example, the N1 neuraminidase is the A / turkey / turkey / 1 / 2005 strain. In another example, the N2 neuraminidase is the A / Delaware / 39 / 2019 virus strain.

[0425] In one example, the antigen is either an H5 hemagglutinin protein or an N1 neuraminidase protein. For instance, the antigen is either an H5 hemagglutinin subtype influenza A virus strain or an N1 neuraminidase subtype influenza A virus strain.

[0426] Infections such as influenza and coronavirus infections are major causes of ARDS. Therefore, in one example of this disclosure, ARDS is associated with influenza, RSV, or SARS-CoV-2 infection. In one example, ARDS is associated with SARS-CoV-2 infection. Therefore, those skilled in the art will understand that antigens targeting SARS-CoV-2 infection or influenza, including those listed above, may be suitable antigens for the treatment of ARDS.

[0427] bacterial antigen In one example, the additional antigens of this disclosure are bacterial antigens.

[0428] In the case of free RNA, the scaffolds were introduced It's just like Neisseria meningitides Streptococcus pneumoniae Streptococcus pyogenes Moraxella catarrhalis Bordetella pertussis Burkholderia sp cepacia) 、Staphylococcus aureus 、Haemophilus influenzae 、Clostridium tetani(vaccine) 、Clostridium perfringens 、Clostridium botulinums 、Cornynebacterium diphtheriae(diphtheria) Pseudomonas aeruginosa、Legionella pneumophila、Coxiella burnetii、Brucella sp.(B.abortus、B.canis、B.melitensis、B.neotomae、B.ovis、B.suis、およびB.pinnipediae)、Francisella sp.(F. novicida, F. philomiragia, F. tularensis), Streptococcus agalactiae, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum(New), Haemophilus ducreyi、Enterococcus faecalis、Enterococcus faecium、Helicobacter pylori、Staphylococcus saprophyticus、Yersinia enterocolitica、E.Contains proteins and peptides from coli, Bacillus anthracis (anthrax), Yersinia pestis (plague), Mycobacterium tuberculosis, Rickettsia, Listeria, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi (typhoid fever), Borrelia burgdorfer, Porphyromonas sp., and Klebsiella sp.

[0429] fungal antigen In one example, the additional antigens of this disclosure are fungal antigens.

[0430] The fungal antigens that can be encoded by RNA or provided in polypeptide form according to this disclosure will be apparent to those skilled in the art, for example, Dermatophytes (Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T verrucosum var. album, var. discoides, var.ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme), Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces This includes proteins and peptides from *Dermatidis*, *Cryptococcus neoformans*, *Geotrichum clavatum*, *Histoplasma capsulatum*, *Klebsiella pneumoniae*, *Microsporidia*, *Encephalitozoon* spp., *Septata intestinalis*, and *Enterocytozoon bieneusi*.

[0431] Protazoan Antigen In one example, the additional antigen of this disclosure is a protazoan antigen.

[0432] The protazoane antigens that can be encoded by RNA or provided in polypeptide form according to this disclosure will be apparent to those skilled in the art and include, for example, proteins and peptides from Entamoeba histolytica, Giardia lambli, Cryptosporidium parvum, Cyclospora cayatanensis, and Toxoplasma.

[0433] Polynucleotides As used herein, the term “polynucleotide” refers to a molecular chain of nucleotides chemically linked by a series of esterifications (linkages) between the phosphoryl group of one nucleotide and the hydroxyl group of a sugar in an adjacent nucleotide. For example, a polynucleotide is DNA. For example, a polynucleotide is RNA, such as mRNA. For example, mRNA is conventional mRNA (cRNA) or self-replicating RNA.

[0434] As used herein, the term “fragment” refers to a portion of a nucleotide sequence or polypeptide of a reference nucleotide sequence or polypeptide disclosed herein that maintains the defined activity of the full-length nucleotide sequence or polypeptide.

[0435] As a non-limiting example, if conventional mRNA or self-replicating RNA contains polynucleotides encoding two or more antigens, these two or more antigens may be expressed by monocistronic polynucleotides, or each of these antigens may be expressed by polycistronic (or multicistronic) polynucleotides. For example, an antigen may be expressed by monocistronic polynucleotides or by polycistronic polynucleotides.

[0436] As used herein, the term “variant” refers to a nucleotide sequence having one or more substitutions, insertions, deletions, and / or other modifications compared to an unmodified sequence. It will be apparent to those skilled in the art that any variant described herein has the same or similar expression of the encoded protein. For example, a variant is a functional variant. Exemplary modifications to nucleotide sequences and / or polypeptides are apparent to those skilled in the art and / or are described herein.

[0437] In one example, modification is a chemical modification of one or more nucleotides in a nucleotide sequence. For instance, at least one naturally occurring nucleotide in a polynucleotide is replaced by a chemically modified nucleotide (e.g., pseudouridine (ψ) and 1-methylpseudridine (m1ψ)).

[0438] In one example, the modification involves increasing the G / C content of the nucleotide sequence.

[0439] In one example, the modification involves codon optimization of the nucleotide sequence.

[0440] In one example, the substitution is a conserved substitution. Those skilled in the art will understand that a conserved substitution in a polypeptide involves replacing an amino acid in the polypeptide with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). In another example, the substitution is a non-conservative substitution.

[0441] As used herein, the terms “encode,” “encodes,” or “the act of coding” refer to a region of polynucleotide that can be translated into a polypeptide.

[0442] The polynucleotides of this disclosure include DNA and RNA (e.g., mRNA).

[0443] Deoxyribonucleic acid (DNA) One example of a polynucleotide is DNA (for example, a DNA vector).

[0444] It will be apparent to those skilled in the art that the DNA of this disclosure further comprises an endonuclease restriction site at the 3' end of the 3' UTR. Those skilled in the art will understand that the endonuclease restriction site allows for the insertion of one or more nucleotide sequences (e.g., encoding the antigen of interest, a fragment thereof, and / or a variant) without disrupting the rest of the DNA.

[0445] As used herein, the term “restriction endonuclease site” refers to a sequence of DNA that binds to a restriction endonuclease. Typically, a restriction endonuclease site is a short sequence (e.g., approximately 4–8 base pairs) that is recognized and cleaved by a restriction endonuclease.

[0446] As used herein, the terms “restriction enzyme” or “restriction endonuclease” refer to a class of enzymes naturally present in bacteria and some viruses. Restriction endonucleases specifically bind to double-stranded DNA at or adjacent to a restriction endonuclease site and cleave the double-stranded DNA. Examples of restrictive endonucleases include, for example, BciVI (Bful), Bcul (Spel), EcoRI, Aatll, AgeI (BshTI), Apal, BamHI, BglII, Blpl (Bpu1102I), BsrGI (Bsp1407), Clal (Bsu15I), EcoRI, EcoRV (Eco32I), Eam1104I (EarI), Hindlll, Kpnl, Mlul, Ncol, Ndel, Nhel, Notl, Nsil, Mph1103I), Pstl, Pvul, Pvull, SacI, SalI, ScaI, SpeI, Xbal, Xhol, Sacll (Cfr42I), and Xbal.

[0447] In one example, the present disclosure provides a transcribed polynucleotide comprising a nucleotide sequence encoding a target antigen operably linked to a regulatory element such as an SG promoter and an IRES. For example, the polynucleotide is a DNA plasmid comprising a first and a second nucleotide sequence.

[0448] In one example, the DNA contains a nucleotide sequence that includes a restriction endonuclease site located at 3' of the 3' UTR. The presence of the restriction endonuclease site at 3' of the 3' UTR enables the production of linearized DNA. Linearization of the DNA ensures the defined termination of the in vitro transcription DNA for mRNA production.

[0449] Ribonucleic acid (RNA) As used herein, the term “RNA (ribonucleic acid)” refers to a single-stranded molecular chain of nucleotides chemically linked by a series of esterifications (linkages) between the phosphoryl group of one nucleotide and the hydroxyl group of a sugar in an adjacent nucleotide. Preferred forms of RNA will be apparent to those skilled in the art. In one example, RNA is messenger RNA (mRNA). In one example, mRNA encoding a chemoattractant and / or antigen is monocistronic mRNA. For example, monocistronic mRNA is conventional mRNA (cRNA) or self-replicating RNA. In another example, mRNA encoding a chemoattractant and / or antigen is multicistronic mRNA. For example, multicistronic mRNA is conventional mRNA (cRNA) or self-replicating RNA.

[0450] For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) mRNA comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), Each nucleotide sequence is operablely linked to a regulatory element.

[0451] The mRNAs in this disclosure include not only self-replicating mRNA (also known as self-amplified RNA or sa-mRNA) but also non-replicating mRNA (conventionally referred to as mRNA (cRNA) or non-amplified mRNA).

[0452] Conventional (non-replicating) RNA For example, polynucleotides are (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) A cRNA comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), Each nucleotide sequence is operablely linked to a regulatory element.

[0453] In one example, the cRNA of the Disclosure comprises, in 5' to 3' order, a 5' cap structure, a 5'-UTR, a fragment and / or variant thereof, a first nucleotide sequence encoding a first antigen of interest, a second nucleotide sequence encoding a second antigen of interest, a third nucleotide sequence encoding a second antigen of interest, a 3'-UTR, and a 3' tailing sequence (e.g., a polyadenylation signal or one or more poly-A tails). The cRNA of the Disclosure may further comprise an intratranslational ribosome entry site (e.g., a Kozak consensus sequence or IRES) operably linked to the antigen of interest.

[0454] self-replicating RNA This disclosure is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) A self-replicating RNA (also known as a replicon) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), The present invention provides a self-replicating RNA in which each nucleotide sequence is operably linked to a regulatory element.

[0455] Those skilled in the art will understand that the self-replicating RNA of this disclosure is based on the genomic RNA of an RNA virus. The RNA should be a positive (+) strand and therefore can be directly translated after delivery to cells without requiring an intervening replication step (e.g., reverse transcription). Translation of the RNA results in the production of non-structural proteins (NSPs) that combine to form a replicase complex (i.e., RNA-dependent RNA polymerase). The complex then amplifies the original RNA, resulting in the production of multiple daughter RNAs that can be translated and transcribed, thereby enhancing overall protein expression.

[0456] For example, the self-replicating RNA of this disclosure includes non-structural proteins of an RNA virus, 5' and 3' untranslated regions (UTRs), and a natural subgenome promoter.

[0457] For example, self-replicating RNA contains one or more non-structural proteins of an RNA virus. For instance, RNA contains at least one gene selected from the group consisting of viral replicases (or viral polymerases), viral proteases, viral helicases, and other non-structural viral proteins. For example, self-replicating RNA contains viral replicases (or viral polymerases).

[0458] In another example, self-replicating RNA includes the 5' and 3' UTRs of an RNA virus. Those skilled in the art will see that the terms 5' and 3' UTR also encompass the terms 5' and 3' conserved sequence elements (CSEs). In one example, self-replicating RNA includes the 5' and 3' CSEs.

[0459] The self-replicating RNAs of this disclosure cannot induce the production of infectious viral particles. For example, the self-replicating RNAs of this disclosure do not contain viral genes that encode structural proteins necessary for the production of viral particles.

[0460] In one example, the self-replicating RNA is derived from or based on an alphavirus. Suitable alphaviruses will be obvious to those skilled in the art and / or are described herein.

[0461] In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus, such as a positive-strand RNA virus. Positive-strand RNA viruses suitable for use in this disclosure will be apparent to those skilled in the art and include, for example, picornaviruses, flaviviruses, rubiviruses, pestiviruses, hepaciviruses, caliciviruses, or coronaviruses.

[0462] Alphavirus For example, the self-replicating RNA of this disclosure is derived from (or based on) an alphavirus.

[0463] Alphaviruses are the only genus in the Togaviridae family and are enveloped viruses with a positive sense single-stranded RNA genome. Those skilled in the art will understand that the alphavirus genome contains two open reading frames (ORFs), one non-structural and one structural. The first ORF encodes four non-structural proteins (NSP1, NSP2, NSP3, and NSP4) necessary for the transcription and replication of viral RNA. The second encodes three structural proteins that associate as heterodimers: the coanucleocapsid protein C, as well as envelope proteins P62 and E1. Viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells via membrane fusion.

[0464] In one example, the self-replicating RNA of this disclosure includes a viral replicase (or viral polymerase). For example, the viral replicase is an alphaviral replicase, such as the alphaviral protein NSP4.

[0465] For example, the self-replicating RNA of this disclosure does not encode one or more alphaviral structural proteins (e.g., capsid and / or envelope glycoproteins). For instance, the self-replicating RNA cannot produce RNA-containing alphaviral virions (i.e., infectious viral particles).

[0466] For example, self-replicating RNA contains a natural alphavirus SG promoter. For instance, the natural alphavirus SG promoter is a minimal SG promoter (i.e., the minimum sequence required for transcription initiation) and contains the sequence shown in Sequence ID No. 1.

[0467] Those skilled in the art will recognize alphaviruses suitable for use in this disclosure. Exemplary alphaviruses include, but are not limited to, Venezuelan horse encephalitis virus (VEE, e.g., Trinidadorova, TC83CR), Semlik Forest virus (SFV), Sindobis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mukambo virus, Burma Forest virus, Middelberg virus, Pixna virus, Onyonnyon virus, Geta virus, Sagiyama virus, Beval virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Bangbanki virus, Kyzilagachi virus, Highland J virus, Fort Morgan virus, Ndum virus, and Baggy Creek virus. The term alphavirus may also include chimeric alphaviruses that contain genome sequences from two or more alphaviruses (as described, for example, by Perri et al, (2003) J. Virol. 77(19):10394-403).

[0468] Adjustment element This disclosure is, (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) A polynucleotide comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), The present invention provides a polynucleotide in which each nucleotide sequence is operably linked to a regulatory element.

[0469] In one example, the regulatory element is selected from a group consisting of a subgenome (SG) promoter, an internal ribosome entry site (IRES), and a Kozak consensus sequence, or a combination thereof. In one example, the regulatory element is the SG promoter.

[0470] Kozak Consensus Array As used herein, the term "Kozak consensus sequence" refers to a nucleotide sequence identified in a eukaryotic gene that facilitates gene translation by containing a start codon (also known as a translation start codon) that is recognized by a ribosome.

[0471] Exemplary Kozak consensus sequences are known in the art and / or described herein. In one example, the Kozak consensus sequence is shown in SEQ ID NO: 6 (GCCACC). In another example, the Kozak consensus sequence is shown in SEQ ID NO: 7 (ACCATGG).

[0472] Subgenome promoters Suitable SG promoters for use in this disclosure (also known as “junction area” promoters) will be obvious to those skilled in the art and / or are described herein.

[0473] For example, the SG promoter is derived from or based on the alphavirus SG promoter. For instance, the SG promoter is the natural alphavirus SG promoter. For example, the natural SG promoter is the minimal SG promoter. For instance, the minimal SG promoter is the minimum sequence required for transcription initiation. For example, the natural SG promoter is the extended SG promoter. For instance, the extended SG promoter is the minimal SG promoter extended at the 5' end by nucleotides generated in the sequence encoding a non-structural protein (e.g., NSP4) of an RNA virus (e.g., alphavirus). For example, the extended SG promoter is the minimal SG promoter extended at the 5' end by nucleotides generated in the sequence encoding alphavirus NSP4.

[0474] In one example, the SG promoter is extended at its 5' end by approximately 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). In another example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 1, which is extended at its 5' end by 31 nucleotides that occur within a sequence encoding a non-structural protein (e.g., alphavirus NSP4). For example, the extended SG promoter has a nucleotide length of 80 or less. In one example, the extended SG promoter is encoded by a sequence containing or consisting of nucleotides 22-101 of SEQ ID NO: 5. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 3.

[0475] For example, the extended SG promoter contains a repeating sequence corresponding to nucleotides 66-75 of SEQ ID NO: 5. For instance, the extended SG promoter is encoded by a sequence containing nucleotides 50-75 and nucleotides 66-101 of SEQ ID NO: 5. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 15.

[0476] For example, the polynucleotides of this disclosure contain an SG promoter from any alphavirus. For example, the RNA (e.g., cRNA or self-replicating RNA) of this disclosure contains an SG promoter from any alphavirus.

[0477] In one example, the self-replicating RNA contains an SG promoter from any alphavirus.

[0478] The polynucleotides of this disclosure comprise three nucleotide sequences encoding three antigens of interest. In one example, each of the three nucleotide sequences is operably ligated to an SG promoter. When the three nucleotide sequences are present in the RNA of this disclosure, the operably ligated promoters may be the same or different. For example, the three SG promoters may originate from the same alphavirus. In another example, the three SG promoters may originate from different alphaviruses.

[0479] Internal ribosome entry site (IRES) The IRESs suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0480] In one example, IRES originates from encephalomyocarditis virus (EMCV). For instance, IRES is a wild-type IRES derived from EMCV.

[0481] In one example, IRES is derived from fibroblast growth factor 1A (FGF1A)IRES.

[0482] In addition, synthetic IRES elements that can be designed according to methods known in the art to mimic the functions of naturally occurring IRES elements are described (see Chappell, SA et al. Proc. Natl Acad. Sci. USA (2000) 97(4):1536-41).

[0483] For example, IRESs are derived from encephalomyocarditis virus (EMCV), poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-related protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF-B), Antennapedia, X-linked apoptosis inhibitor (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0484] For example, IRES is a wild-type IRES derived from encephalomyocarditis virus (EMCV). For instance, wild-type EMCV IRES contains the sequence shown in SEQ ID NO: 4.

[0485] 5' Untranslated region (5'-UTR) For example, the polynucleotides described herein include a 5' untranslated region (5'-UTR).

[0486] As used herein, the terms “5'-untranslated region” or “5'-UTR” refer to the non-coding region of mRNA located at the 5' end of the translation initiation sequence (AUG).

[0487] Examples of 5'-UTRs include, for example, the 5'-UTRs of haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha-6 collagen (Col6A), alpha-1-antitrypsin (SERPINA1), alpha-1-antichymotrypsin (SERPINA3), their fragments, and / or variants.

[0488] In one example, the 5'UTR is the 5'UTR of Venezuelan encephalitis virus (VEEV) or a modified form thereof. For example, the 5'UTR contains the sequence shown in SEQ ID NO: 13.

[0489] In one example, the 5'UTR includes at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, and combinations thereof.

[0490] microRNA binding site As used herein, the term “microRNA binding site” refers to a sequence within a polyn reoside (e.g., within a DNA or RNA transcript) that is sufficiently complementary to all or one region of a microRNA (miRNA) to interact with, associate with, or bind to the miRNA.

[0491] As used herein, the terms “microRNA” or “miRNA” refer to a 19-25 nucleotide-length non-coding RNA that binds to the 5'UTR of a polynucleotide and downregulates gene expression (e.g., by inhibiting translation). The presence of microRNA binding sites(s) in the 5'UTR of this disclosure can function to inhibit translation of the 5'UTR.

[0492] Suitable miRNA binding sites for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0493] For example, miRNA binding sites include binding sites for tissue-specific microRNAs or those that regulate biological processes. These include miRNAs from liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). Other examples include microRNAs that regulate biological processes such as angiogenesis (miR-132). Further exemplary miRNAs and miRNA-binding sites are disclosed in U.S. Patent Application No. 14 / 043,927.

[0494] AU Rich Element (ARE) As used herein, the terms “AU-rich element (ARE)” or “AU-rich element (ARE)” refer to a region of nucleotide sequence containing stretches of adeonysin (A) and uridine (U). Exemplary AREs include, for example, AREs from cytoplasmic myc (c-myc), myoblast-determining protein 1 (myoD), c-Jun, myogenin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α), or combinations thereof.

[0495] For example, AREs contain a human antigen R or "HuR" (also known as Elavl1) specific binding site. HuR is known to bind to AREs, increasing mRNA stability.

[0496] GC Rich Element As used herein, the term “GC-rich element” refers to a nucleotide sequence having a larger amount of guanine (G) and / or cytosine (C) compared to adenine (A) and thymine (T) / uracil (U). The presence of GC-rich elements in polynucleotides (e.g., mRNA) can stabilize the mRNA.

[0497] For example, a GC-rich element contains a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0498] For example, a GC-rich element contains 30%-40%, 40%-50%, 50%-60%, or 60%-70% cytosine. For example, a GC-rich element contains 30%-40% cytosine. For example, a GC-rich element contains 40%-50% cytosine. For example, a GC-rich element contains 50%-60% cytosine. For example, a GC-rich element contains 60%-70% cytosine.

[0499] For example, a GC-rich element contains 30%, 40%, 50%, 60%, or 70% cytosine. For example, a GC-rich element contains 30% cytosine. For example, a GC-rich element contains 40% cytosine. For example, a GC-rich element contains 50% cytosine. For example, a GC-rich element contains 60% cytosine. For example, a GC-rich element contains 60% cytosine. For example, a GC-rich element contains 70% cytosine.

[0500] For example, a GC-rich element is at least 50% cytosine.

[0501] For example, a GC-rich element is at least 60% cytosine.

[0502] For example, a GC-rich element is at least 70% cytosine.

[0503] In one example, the GC-rich element contains a nucleotide sequence following SEQ ID NO: 9. In another example, the GC-rich element contains the nucleotide sequence CCCCGGCGCC (SEQ ID NO: 10). In yet another example, the GC-rich element contains the nucleotide sequence CCCCGGC (SEQ ID NO: 11).

[0504] stem loop As used herein, the term “stem-loop” refers to a nucleotide sequence containing an intramolecular base pairing of two adjacent complete or partial reverse-complementary sequences that form a stem-loop. Stem-loops can occur in single-stranded DNA or, more commonly, in RNA. Stem-loops can also be referred to as hairpins or hairpin loops, which typically consist of a stem and a terminal loop in a contiguous sequence, with the stem being formed by two adjacent complete or partial reverse-complementary sequences separated by a shorter sequence that constructs the loop into a stem-loop structure.

[0505] The stability of a paired stem-loop is determined by its length, the number of mismatches or bulges it contains, and the nucleotide composition of the paired region.

[0506] For example, the loop in a stem-loop has a length of 3 to 10 nucleotides. For instance, the loop in a stem-loop has a length of 3 to 8, or 3 to 7, or 3 to 6, or 4 to 5 nucleotides.

[0507] In one example, the loop in a stem-loop has a length of 4 nucleotides.

[0508] For example, a stem-loop is a histone stem-loop. For instance, a histone stem-loop contains or consists of the nucleotide sequence shown in Sequence ID No. 12.

[0509] 3' Untranslated region (3'-UTR) For example, the polynucleotides of this disclosure include a 3'-untranslated region (3'-UTR).

[0510] As used herein, the term "3'-UTR" refers to the region of mRNA located at the 3' end of a translation termination codon (i.e., a stop codon).

[0511] Examples of 3'-UTRs include, for example, the 3'-UTRs of arachidonic acid 5-lipoxygenase (ALOX5), alpha-I collagen (COL1A1), tyrosine hydroxylase (TH) genes, split amino-terminal enhancers (AES), human mitochondrial 12S rRNA (mtRNR1), fragments thereof, and / or variants.

[0512] For example, the 3'UTR is the 3'UTR of Sindbisvirus (SINV) or a modified form thereof. For instance, the 3'UTR contains the sequence shown in SEQ ID NO: 14.

[0513] For example, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the albumin gene. For example, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the vertebrate α-globin gene. For instance, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the mammalian α-globin gene. For instance, the 3'-UTR contains or consists of a nucleotide sequence derived from the 3'-UTR of the human α-globin gene.

[0514] In one example, the 3'-UTR of this disclosure further comprises at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a triple helix, a stem-loop, one or more stop codons, or a combination thereof.

[0515] Stop codon As used herein, the term “stop codon” refers to a trinucleotide sequence within mRNA that signals the termination of protein synthesis by ribosomes.

[0516] For example, the polynucleotides of this disclosure include at least one stop codon at the 5' end of the 3'-UTR. For example, the stop codon is selected from UAG, UAA, and UGA.

[0517] For example, a polynucleotide contains two consecutive stop codons with the sequence UGAUGA.

[0518] For example, a polynucleotide contains two consecutive stop codons with the sequence UAAUAG.

[0519] 3' Tailing arrangement For example, the polynucleotides of this disclosure include one or more 3' tailing sequences located at the 3' end of the 3' UTR.

[0520] As described herein, the terms “3' tailing sequence” or “3' tailing sequence” refer to a nucleotide sequence that induces the addition of a non-coding nucleotide to the 3' end of mRNA (e.g., a polyadenylation signal), or a nucleotide sequence located at the 3' end of mRNA (e.g., a poly-A sequence). Those skilled in the art will understand that 3' tailing sequences and / or products of 3' tailing sequences in mRNA function to stabilize mRNA and / or prevent mRNA degradation.

[0521] As used herein, the term “interrupting linker” in relation to polyA or polyC sequences in this disclosure refers to a single nucleotide or nucleotide sequence that ligates to and interrupts a stretch of consecutive adenosine or cytosine nucleotides in a polyA or polyC sequence. For example, an interrupting linker in a polyA sequence is a single nucleotide or nucleotide sequence consisting of or containing a nucleotide other than an adenosine nucleotide. For example, an interrupting linker in a polyC sequence is a single nucleotide or nucleotide sequence consisting of or containing a nucleotide other than a cytosine nucleotide.

[0522] In one example, one or more 3' tailing sequences are selected from the group consisting of poly(A) sequences, polyadenylation signals, G quadruplexes, poly(C) sequences, stem-loops, and combinations thereof.

[0523] Poly-A sequence As used herein, the term “polyA sequence” refers to the adenine (A) nucleotide sequence located at the 3' end of mRNA. In the context of this disclosure, the polyA sequence may be located within mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating mRNA by transcription of a vector).

[0524] Suitable polyA sequences for use in this disclosure will be obvious to those skilled in the art and / or are described herein. In one example, the polyA sequence comprises a sequence of adenosine nucleotides of any length (e.g., 10 to 300). In another example, the polyA sequence comprises a sequence of adenosine nucleotides separated by one or more interrupting linkers. In yet another example, the polyA sequence comprises a sequence of adenosine nucleotides without interrupting linkers.

[0525] Polyadenylation signal As used herein, the term “polyadenylation signal” refers to a nucleotide sequence that induces polyadenylation. Polyadenylation is typically understood as the addition of a polyA sequence to RNA (e.g., to immature mRNA to produce mature mRNA). The polyadenylation signal may be located within the nucleotide sequence at the 3' end of the polynucleotide being polyadenylated (e.g., mRNA).

[0526] Suitable polyadenylation signals for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0527] In one example, the polyadenylation signal contains a hexamer composed of adenine and uracil / thymidine nucleotides. In another example, the hexamer sequence contains or consists of AAUAAA.

[0528] In one example, the 3' tailing sequence contains a polyadenylation signal but does not contain a poly(A) sequence.

[0529] G-quadruplex As used herein, the terms “G quadruple” or “G4” refer to a nucleotide sequence rich in guanine residues that forms a quadruple-stranded secondary structure. For example, a G quadruple is a cyclic hydrogen-bonded array of four guanine nucleotides formed by a G-rich sequence in both DNA and RNA.

[0530] In one example, the 3' tailing sequence contains a poly(A) sequence and a G quadruplex. For instance, the 3' tailing sequence contains a poly(A) sequence linked to a G quadruplex to produce a poly(AG) quartet.

[0531] PolyC sequence As used herein, the term “polyC sequence” refers to the cytosine (C) nucleotide sequence located at the 3' end of mRNA. In the context of this disclosure, the polyC sequence may be located within mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating mRNA by vector transcription).

[0532] Suitable polyC sequences for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0533] For example, one or more 3' tailing sequences contain one or more polyC sequences, each containing 10 to 300 consecutive cytosine nucleotides. For example, one or more polyC sequences each contain 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300 consecutive cytosine nucleotides. For example, one or more polyC sequences each contain 10, or 20, or 30, or 40, or 50, or 60, or 70, or 80, or 90, or 100, or 125, or 150, or 175, or 200, or 225, or 250, or 275, or 300 consecutive cytosine nucleotides.

[0534] In one example, one or more polyC sequences are separated by interrupting linkers. For instance, a fourth nucleotide sequence containing one or more 3' tailing sequences may contain, from 5' to 3', consecutive cytosine nucleotides, interrupting linkers, and further consecutive cytosine nucleotides.

[0535] In one example, the interrupted linker has a nucleotide length of 10–50, 50–100, or 100–150. For example, a break linker has a nucleotide length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides.

[0536] 5' cap structure In one example, this disclosure provides mRNA containing a 5' terminal cap structure.

[0537] As used herein, the term “5' cap structure” refers to a structure at the 5' end of mRNA involved in nuclear export that binds to mRNA cap-binding protein (CBP). The 5' cap structure is known to stabilize mRNA through association with CBP and poly(A)-binding protein, thereby forming mature mRNA. Therefore, the presence of a 5' cap structure in the mRNA of this disclosure can further increase the stability of the mRNA compared to mRNA without a 5' cap.

[0538] Examples of 5' cap structures include, for example, anti-reverse cap analogues (ARCA), N7,2'-O-dimethyl-guanosine (mCAP), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), cap 1, and cap 2.

[0539] Typically, endogenous mRNA is 5'-capped with guanosine via a (5)'-ppp-(5)'-triphosphate ligation attached to the 5' terminal nucleotide of the mRNA. The guanosine cap can then be methylated to 7-methylguanosine (m7G) to produce 7mG(5')ppp(5')N,pN2p (cap 0 structure), where N represents the first and second 5' terminal nucleotides of the mRNA. The cap 0 structure can be further 2'-O-methylated to produce 7mG(5')ppp(5')NlmpNp (cap 1) and / or 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[0540] In one example, the polynucleotides of this disclosure include an endogenous cap.

[0541] As used herein, the term “endogenous cap” refers to a 5' cap synthesized in a cell. For example, the endogenous cap is a natural 5' cap or a wild-type 5' cap. For example, the endogenous cap is a cap 0, cap 1, or cap 2 structure.

[0542] For example, the polynucleotides of this disclosure include analogues of endogenous caps (also referred to as cap analogues).

[0543] As used herein, the terms "endogenous cap" or "its analogue" in the context of "cap analogue" refer to a synthetic 5' cap. Cap analogues can be used to produce 5'-capped mRNA in in vitro transcription reactions. Cap analogues can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or ligated to nucleotides (e.g., the 5' terminal nucleotide of mRNA). Exemplary cap analogues are commercially available and include, for example, 3”-O-Me-m7G(5')ppp(5')G, G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, and m7G(5')ppp(5')G (New England BioLabs). One example of a cap analogue is N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (i.e., anti-reverse cap analogue (ARCA)).

[0544] In one example, a 5' cap structure is a non-hydrolyzable cap structure. A non-hydrolyzable cap structure can prevent mRNA decapping and increase the mRNA half-life.

[0545] In one example, the non-hydrolyzable cap structure contains a modified nucleotide selected from the group consisting of α-thio-guanosine nucleotides, α-methyl-phosphonates, seleno-phosphates, and combinations thereof. In one example, the modified nucleotide is ligated to the 5' end of the mRNA via an α-phosphorothieate linkage. The method of ligating the modified nucleotide to the 5' end of the mRNA will be obvious to those skilled in the art. For example, a Vaccina Capping Enzyme (New England Biolabs) is used.

[0546] qualification In one example, the polynucleotides of this disclosure include one or more modifications. Typically, the modifications are introduced into a polynucleotide (e.g., mRNA) to increase the translation efficiency and / or stability of the polynucleotide. Suitable modifications to polynucleotides are obvious to those skilled in the art and / or are described herein.

[0547] For example, a nucleotide sequence containing the 5'-UTR and / or a fragment thereof is modified. Modification of a nucleotide sequence containing the 5'-UTR and / or a fragment thereof results in a variant of the 5'-UTR and / or a fragment thereof.

[0548] In one example, one or more nucleotide sequences of a polynucleotide are codon-optimized. Methods of codon optimization are apparent to those skilled in the art and / or are described herein. For example, tools for codon optimization of polynucleotides include, for example, GeneArt GeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).

[0549] In one example, a polynucleotide is modified to increase the amount of guanine (G) and / or cytosine (C) in the polynucleotide. The amount of G / C in a polynucleotide (i.e., the G / C content) can affect the stability of the polynucleotide. Therefore, a polynucleotide containing an increased amount of G / C nucleotides can be functionally more stable than a polynucleotide containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. The G / C content is increased by substituting A or T nucleotides with G or C nucleotides.

[0550] For example, the G / C content increases in the nucleotide sequence encoding the target antigen. Modifications in the nucleotide sequence utilize the ability (e.g., conserved amino acid substitutions) to replace codons containing less desirable combinations of nucleotides (from the standpoint of mRNA stability) with alternative codons encoding the same amino acid or amino acid(s) with similar chemistry. For example, the G / C content increases by replacing codons containing A or T nucleotides with codons containing G or C nucleotides that encode the same amino acid.

[0551] For example, the G / C content increases in nucleotide sequences of polynucleotides that do not encode the target antigen. For instance, the G / C content increases in the 5'-UTR, its fragments, and / or variants. For instance, the G / C content increases in the 3'-UTR, its fragments, and / or variants.

[0552] For example, a polynucleotide contains at least one chemically modified nucleotide.

[0553] As used herein, the terms “chemically modified” or “chemically modified” in the context of nucleotides refer to naturally occurring nucleotides (i.e., A, T, C, G, U) that are modified by the substitution, insertion, or removal of individual or several atoms or atomic groups compared to naturally occurring nucleotides. For example, at least one naturally occurring nucleotide in a polynucleotide is replaced by a chemically modified nucleotide. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the naturally occurring nucleotides in a polynucleotide are replaced by chemically modified nucleotides. Chemically modified nucleotides suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein. Examples of chemically modified nucleotides include, for example, N6,2'-O-dimethyladenosine (m6Am), 5-methyluridine (m5U), N4-acetylcytidine (ac4C), 2-thiocytidine (s2C), 2-thiouridine (s2U), 5-methylcytidine (m5C), N6-methyladenosine (m6a), pseudouridine (ψ), and 1-methylpseudridine (m1ψ).

[0554] Method of production Preferred methods for producing the polynucleotides, cRNAs, and / or self-replicating RNAs of this disclosure will be obvious to those skilled in the art and / or are described herein.

[0555] For example, polynucleotides are DNA. For instance, a polynucleotide is plasmid DNA.

[0556] In one example, cRNA is produced using plasmid DNA. In another example, self-replicating RNA is produced using plasmid DNA. Those skilled in the art will understand that plasmid DNA is relatively stable. Briefly, competent bacterial cells (e.g., Escherichia coli) are transformed with a DNA plasmid encoding the self-replicating RNA of this disclosure. Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in an E. coli culture.

[0557] In one example, plasmid DNA is isolated after fermentation. For example, plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other routine methods known to those skilled in the art. After isolation, plasmid DNA is linearized by restriction digestion (i.e., using restriction enzymes). The restriction enzymes are removed using methods known in the art, including, for example, phenol / chloroform extraction and ethanol precipitation.

[0558] In one example, mRNA is prepared by in vitro transcription from a linearized DNA template using RNA polymerase (e.g., T7 RNA polymerase). After in vitro transcription, the DNA template is removed by DNase digestion. Those skilled in the art will understand that synthetic mRNA capping is performed to correct mRNA processing and contribute to mRNA stabilization. In one example, mRNA is enzymatically 5' capped. For example, the 5' cap is either a cap 0 structure or a cap 1 structure. In one example, the 5' cap is a cap 0 structure, for example, the 5'-cap (i.e., cap 0) consists of inverted 7-methylguanosine attached to the remainder of the mRNA via a 5'-5' triphosphate crosslink. In another example, the 5' cap is a cap 1 structure, for example, the 5'-cap (i.e., cap 1) consists of cap 0 with additional methylation at the 2'O position of the start nucleotide.

[0559] In one example, mRNA is purified. Various methods for purifying mRNA will be apparent to those skilled in the art. For example, mRNA is purified using lithium chloride (LiCl) precipitation. In another example, mRNA is purified using tangential flow filtration (TFF). After purification, the mRNA is resuspended, for example, in nuclease-free water.

[0560] composition This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure.

[0561] This disclosure also provides immunogenic compositions comprising the RNA of this disclosure.

[0562] This disclosure also provides immunogenic compositions comprising the cRNA of this disclosure.

[0563] This disclosure further provides immunogenic compositions comprising the self-replicating RNA of this disclosure.

[0564] This disclosure also provides a pharmaceutical composition comprising the immunogenic composition of this disclosure and a pharmaceutically acceptable carrier.

[0565] It will be apparent to those skilled in the art, and / or as described herein, that the polynucleotides, RNAs, cRNAs, and / or self-replicating RNAs of this disclosure may exist as naked RNA or in combination with lipids, polymers, or other delivery systems that facilitate entry into cells.

[0566] Delivery system For example, the pharmaceutical composition of the present disclosure further comprises LNPs, polymer microparticles, and an oil-in-water emulsion. For instance, polynucleotides, cRNAs, and / or self-replicating RNAs are encapsulated, bound to, or adsorbed within the LNPs, polymer microparticles, or the oil-in-water emulsion.

[0567] Lipid nanoparticles In one example, the pharmaceutical composition of this disclosure further comprises LNP.

[0568] The terms “lipid nanoparticles” or “LNPs” refer to any lipid composition, including, but not limited to, liposomes or vesicles in which aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; monolayer, or multiple; multilayer), micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles lacking a lipid bilayer.

[0569] Lipid nanoparticles suitable for use in this disclosure will be obvious to those skilled in the art and / or described herein. Lipids may have anionic, cationic, or zwitterionic hydrophilic head groups.

[0570] In one example, the lipid nanoparticles include PEG lipids, sterol-structured lipids, and / or neutral lipids. In another example, the lipid nanoparticles further include cationic lipids. In yet another example, the lipid nanoparticles do not include cationic lipids.

[0571] For example, LNPs include PEG lipids. For instance, PEG lipids are selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0572] For example, LNPs include structural lipids. Structural lipids are selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol, as well as combinations thereof.

[0573] For example, LNPs include neutral lipids. Examples of phospholipids (anionic or zwitterionic) for use in this disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. For example, neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Choline (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanol-sn-glycero-3-phosphoethanolamine (ME 16.0 Selected from the group consisting of PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin, as well as combinations thereof.

[0574] For example, LNPs include cationic lipids. Examples of cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), and 2,5-bis((9z,12z)-octadeca-9,12,diene-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). One example of a phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,diene-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphocholine. Lipids can be saturated or unsaturated.

[0575] Polymer microparticles In one example, the pharmaceutical composition of this disclosure further comprises polymer microparticles.

[0576] Those skilled in the art will recognize that various polymers can form microparticles for encapsulating or adsorbing the polynucleotides, RNA, cRNA, and / or self-replicating RNA of this disclosure. The use of substantially non-toxic polymers will obviously mean that the particles are safe, and the use of biodegradable polymers will mean that the particles will not remain in the body long-term as they are metabolized after delivery. Useful polymers are also sterilizable for use in the preparation of pharmaceutical-grade formulations.

[0577] Examples of non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxybutyrate, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinylpyrrolidinone or polyesteramides, and combinations thereof.

[0578] Oil-in-water cationic emulsion For example, the pharmaceutical composition of this disclosure further comprises an oil-in-water cationic emulsion.

[0579] Suitable oils for use in oil-in-water emulsions will be obvious to those skilled in the art and / or described herein. For example, an emulsion may contain one or more oils derived from, for example, animals (e.g., fish) or plant sources (e.g., nuts, seeds, grains). Those skilled in the art will recognize that biocompatible and biodegradable oils are preferred. Exemplary animal oils (i.e., fish oils) include cod liver oil, shark liver oil, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, and corn oil.

[0580] In addition to oils, oil-in-water emulsions also contain cationic lipids that promote emulsion formation and stabilization. Suitable cationic lipids are obvious to those skilled in the art and / or are described herein. Exemplary cationic lipids include, but are not limited to, 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl[C16:0]trimethylammoniumpropane (DPTAP), and distearoyltrimethylammoniumpropane (DSTAP).

[0581] In some examples, oil-in-water emulsions also include nonionic surfactants and / or zwitterionic surfactants. Those skilled in the art will recognize surfactants suitable for use in this disclosure. Exemplary surfactants include, but are not limited to, polyoxyethylene sorbitan surfactants (e.g., polysorbate 20 and polysorbate 80), as well as copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0582] Pharmacologically acceptable carriers Preferably, in a composition or method for administering the polynucleotide, RNA, cRNA, and / or self-replicating RNA of the present disclosure, the polynucleotide, RNA, cRNA, and / or self-replicating RNA are combined with a pharmaceutically acceptable carrier as understood in the art. Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the RNA of the present disclosure (e.g., self-replicating RNA) (and an optional delivery system) combined with a pharmaceutically acceptable carrier. Another example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the cRNA of the present disclosure (and an optional delivery system) combined with a pharmaceutically acceptable carrier.

[0583] Generally speaking, "carrier" means any solid or liquid filler, binder, diluent, encapsulant, emulsifier, wetting agent, solvent, suspension agent, coating, or lubricant that can be safely administered to any target, such as a human. Depending on the specific route of administration, a variety of acceptable carriers known in the art may be used, as described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0584] The polynucleotides, RNAs, cRNAs, and / or self-replicating RNAs of this disclosure are useful for prophylactic or therapeutic treatment by parenteral, topical, oral, or topical administration, intramuscular, aerosol, or transdermal administration. In one example, the polynucleotides, RNAs, cRNAs, or self-replicating RNAs are administered parenterally, such as intramuscular, subcutaneous, or intravenous. For example, the polynucleotides, RNAs, cRNAs, or self-replicating RNAs are administered intramuscularly. In another example, the cRNAs are administered parenterally, such as intramuscular, subcutaneous, or intravenous. For example, the cRNAs are administered intramuscularly.

[0585] The formulation of the administered polynucleotide, RNA, cRNA, or self-replicating RNA will vary depending on the chosen route of administration and formulation (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition containing the administered polynucleotide, RNA, cRNA, or self-replicating RNA can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic solutions / aqueous solutions, emulsions, or suspensions containing saline and a buffer medium. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, Ringer's lactate solution, or fixative oil. Various suitable aqueous carriers, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine, are known to those skilled in the art. Intravenous vehicles may contain additives, preservatives, or fluids, nutritional supplements, or electrolyte supplements (see, for general, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition may optionally contain pH adjusters and buffers, as well as pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as toxicity adjusters, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. Polynucleotides, RNA, cRNA, and / or self-replicating RNA can be stored in liquid form or lyophilized for storage and reconstituted in a suitable carrier before use according to lyophilization and reconstitution techniques known in the art.

[0586] The optimal concentration of the active ingredient(s) in the selected medium can be determined empirically according to procedures known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0587] During formulation, the compositions of this disclosure will be administered in a manner suitable for the administered formulation and in a therapeutically / prophylactically effective amount. The dosage range for the administration of the polynucleotides, RNA, cRNA, or self-replicating RNA of this disclosure is large enough to produce the desired effect. For example, a composition contains an effective amount of self-replicating RNA. In one example, a composition contains a therapeutically effective amount of polynucleotides, RNA, cRNA, or self-replicating RNA. In another example, a composition contains a prophylactically effective amount of polynucleotides, RNA, cRNA, or self-replicating RNA.

[0588] 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 the severity of the disease, and can be determined by those skilled in the art. If complications occur, the dosage may be adjusted by the individual physician.

[0589] The dosage can vary over a day or several days, administered at least once a day, from approximately 0.1 mg / kg to approximately 300 mg / kg, for example, from approximately 0.2 mg / kg to approximately 200 mg / kg, for example, from approximately 0.5 mg / kg to approximately 20 mg / kg.

[0590] In some cases, polynucleotides, RNA, cRNA, and / or self-replicating RNA are administered at an initial (or loading) dose higher than the subsequent (maintenance) dose. For example, polynucleotides, RNA, cRNA, or self-replicating RNA are administered at an initial dose of approximately 10 mg / kg to approximately 30 mg / kg. Then, polynucleotides, RNA, cRNA, or self-replicating RNA are administered at a maintenance dose of approximately 0.0001 mg / kg to approximately 10 mg / kg. The maintenance dose may be administered every 7 to 35 days, for example, every 7, 14, or 28 days.

[0591] In some cases, dose-escalation regimes are used, where polynucleotides, RNA, cRNA, or self-replicating RNA are initially administered at a lower dose than that used in subsequent doses. This dose regime is useful when the subject is experiencing adverse events early on.

[0592] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, increased doses may be administered.

[0593] The subjects may be re-treated with the polynucleotides, RNA, cRNA, or self-replicating RNA of this disclosure. The subjects may be re-treated with polynucleotides, RNA, cRNA, or self-replicating RNA by being given two or more sets of exposures or doses, such as at least about two exposures of the binding protein, e.g., about 2 to 60 exposures, more specifically about 2 to 40 exposures, most specifically about 2 to 20 exposures.

[0594] For example, optional retreatment may be given when signs or symptoms of the disease return.

[0595] In another example, any retreatment may be given at defined intervals. For instance, subsequent exposures may be administered at various intervals, such as approximately 24–28 weeks, 48–56 weeks, or longer. For example, such exposures may be administered at intervals of approximately 24–26 weeks, 38–42 weeks, or 50–54 weeks, respectively.

[0596] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, increased doses may be administered.

[0597] In another example, for subjects experiencing adverse reactions, the initial (or loading) dose may be divided over several days of the week or over a number of consecutive days.

[0598] The administration of polynucleotides, RNA, cRNA, or self-replicating RNA by the methods of this disclosure may be continuous or intermittent, depending, for example, the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. The administration of polynucleotides, RNA, cRNA, or self-replicating RNA may be essentially continuous over a pre-selected period, or it may be a series of intervald doses, for example, either during or after the onset of a condition.

[0599] Adjuvants and additional ingredients The pharmaceutical compositions according to this disclosure may include additional adjuvants or immunopotentiators. An adjuvant or immunopotentiator is a substance that modifies the action of the main component. Therefore, those skilled in the art will understand that the terms “adjuvant” and “immunopotentiator” may be used interchangeably. In one example, an adjuvant or immunopotentiator is a substance that enhances the immune response of a target to an antigen. Suitable adjuvants or immunopotentiators will be obvious to those skilled in the art, and include, for example, aluminum-containing adjuvants (e.g., amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate), AS04, MF59, AS01 B , or including CpG 1018.

[0600] For example, the immunogenic compositions described herein include an adjuvant for enhancing immunogenicity. For example, the adjuvant is selected from the group consisting of Freund's adjuvants, incomplete Freund's adjuvants, aluminum phosphate, aluminum hydroxide, GMCSP, BCG, MDP compounds, e.g., thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), RIBI, MPL, trehalose dimicholate (TDM), Novasomes®, QS21, Quil A (and their derivatives and components), calcium phosphate, calcium hydroxide, zinc hydroxide, MHC antigens, PolyI:C, MF59, glycolipid analogs, octodecyl esters of amino acids, muramyl dipeptides, polyphosphazenes, lipoproteins, ISCOM matrix, DC-Chol, ODA, cytokines, and other adjuvants and derivatives thereof. For example, the adjuvant is MF59. In one example, MF59 is administered concurrently with the administration of the composition or vaccine of the Disclosure. In another example, MF59 is administered immediately prior to, preceding, or following the administration of the composition or vaccine of the Disclosure.

[0601] For example, an adjuvant has one or more of the following effects: - To increase the magnitude or function of the antibody response, - To increase cell-mediated immunity, - Inducing mucosal immunity, and / or - Reduce the antigen dose.

[0602] For example, a composition according to the present disclosure comprises an additional RNA encoding (i) one or more antigens, (ii) one or more immunoenhancing agents, (iii) one or more chemoattractants, and / or (iv) one or more targeting molecules.

[0603] As used herein, “immunostimulant” means any molecule capable of enhancing the immune response in a subject. Examples of immunostimulants include cytokines, chemokines, and immunostimulants. In the example, one or more immunostimulants are selected from the group consisting of interleukin-12, interleukin-7, interleukin-15, and interleukin-21.

[0604] As used herein, “targeting molecule” means any molecule capable of targeting immune cell surface markers, such as dendritic cells or antigen-presenting cells. In one example, the targeting molecule targets a dendritic cell surface marker. In one example, the targeting molecule is selected from the group consisting of DEC-205, Clec-9A, DC-SIGN, CD11c, DCIR2, Dectin-1 / 2, CD80 / 86, F4 / 80, CIRE, mannose, and CD36. In one example, the targeting molecule is DEC-205.

[0605] In another example, the target molecule is an antibody. In one example, the target molecule is a monoclonal antibody.

[0606] Screening assay Preferred methods for selecting polynucleotides, RNA, cRNA, or self-replicating RNAs of this disclosure are available to those skilled in the art. Assays may be performed, for example, to evaluate the efficiency and effectiveness of polynucleotides, RNA, cRNA, or self-replicating RNAs, including serological tests and immune responses.

[0607] antigen expression In one example, self-replicating RNA is evaluated for the expression of the target polynucleotide(s). In another example, cRNA is evaluated for the expression of the target polynucleotide(s). In yet another example, RNA is evaluated for the expression of the target polynucleotide(s).

[0608] For example, antigen expression is detected using antibodies against the target polynucleotide(s). In one example, the number of cells positive for antigen expression is measured, for example, by fluorescence-activated cell sorting (FACS). In another example, mean fluorescence intensity (MFI) is determined, for example, using FACS. In yet another example, a specific potency value or transfection success rate per unit mass of RNA is calculated.

[0609] Microneutralization assay In one example, self-replicating RNA (naked and / or formulated) is evaluated for antibody response. In another example, cRNA (naked and / or formulated) is evaluated for antibody response. For example, cRNA and / or self-replicating RNA are evaluated using a microneutralization assay. The method for performing a microneutralization assay will be obvious to those skilled in the art. In one example, the microneutralization assay is a short form of assay. In one example, a viral fluorescence focus-based microneutralization assay is performed. In another example, the microneutralization assay is a long form of assay.

[0610] Hemagglutination inhibition (HAI) assay In one example, self-replicating RNA (naked and / or formulated) is evaluated for antibody response. In another example, cRNA (naked and / or formulated) is evaluated for antibody response. For example, cRNA and / or self-replicating RNA are evaluated using a hemagglutination inhibition (HAI) assay. The method for performing a HAI assay is obvious to those skilled in the art and / or is described, for example, in the WHO (2011) Manual for the laboratory diagnosis and virological surveillance of influenza: WHO Press, World Health Organization.

[0611] Antigen-specific T cell response In one example, self-replicating RNA is evaluated for its ability to induce an antigen-specific T cell response. In another example, cRNA is evaluated for its ability to induce an antigen-specific T cell response. Methods for evaluating the induction of an antigen-specific T cell response are apparent to those skilled in the art and / or are described herein.

[0612] For example, antigen-specific T cell detection is performed in spleen cultures. Briefly, the spleen cell culture is established in T cell medium, and the cell culture is either stimulated with the antigen peptide or not. In one example, the antigen-specific T cell response is determined using flow cytometry.

[0613] Neutralization assay The self-replicating RNAs of this disclosure can be screened in vitro for their ability to bind to the SARS-CoV-2 S protein and neutralize the binding of the S protein to ACE2. Suitable assays will be apparent to those skilled in the art and include, for example, the Vero microneutralization assay, the sVNT assay, or the pseudoviral neutralization assay (e.g., using HEK-293T cells or HeLa-ACE2 cells).

[0614] In one example, the neutralization assay is the Vero microneutralization assay. Briefly, SARS-CoV-2 wild-type virus is passaged in Vero cells (i.e., a Vero strain isolated from kidney epithelial cells extracted from African green monkeys). Two-fold serial dilutions of the test protein are passed over 1 hour at 100 TCID. 50 SARS-CoV-2 (i.e., the median infectious dose for tissue culture) is incubated with Vero cells, and residual viral infectivity is evaluated in Vero cells, for example, on day 5, to determine the viral cytotoxic effect. Neutralizing antibody titers are calculated using the Reed / Muench method as described above (Houser et al., 2016, Subbarao et al. 2004).

[0615] In one example, the neutralization assay is a surrogate neutralization assay (sVNT). Briefly, plate wells are coated with hACE2 protein in a carbonate-bicarbonate coated buffer (e.g., pH 9.6). Test proteins and pre-incubated HRP-conjugated SARS-CoV-2 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 antigens are removed by washing. The colorimetric signal is developed by an enzymatic reaction of HRP with a chromogenic substrate, e.g., 3,3',5,5'-tetramethylbenzidine (TMB). In one example, absorbance readings at 450 nm and 570 nm are obtained.

[0616] In one example, neutralization is a pseudoviral neutralization assay. Briefly, an HIV reporter virus pseudotyped with the SARS-CoV-2 S protein is produced by co-transfection of the SARS-2-CoV-2 spike plasmid, together with a viral backbone plasmid (e.g., pDR-NL Δenv FLUC), into, for example, HEK-293T cells. The pseudovirus is collected after transfection and clarified by filtration. The viral stock titer, reported as the relative luciferase unit infectious dose (RLU), is calculated by limiting dilution infection in Hela-hACE2 cells, measuring luciferase activity as a readout of viral infection.

[0617] Methods of treatment or prevention This disclosure provides a method for using the immunogenic composition or pharmaceutical composition of this disclosure as a vaccine.

[0618] This disclosure also provides methods for treating or preventing a disease or condition in a subject, comprising administering an immunogenic composition or pharmaceutical composition of this disclosure. For example, the disease or condition is a respiratory viral infection such as influenza, SARS-CoV-2 infection, COVID-19, or respiratory syncytial virus (RSV). In another example, the disease or condition is acute respiratory distress syndrome (ARDS).

[0619] influenza Influenza, also known as "the flu," is an infectious disease caused by the influenza virus. Symptoms can range from mild to severe, with the most common being high fever, runny nose, sore throat, muscle and joint pain, headache, cough, and fatigue. Symptoms typically begin two days after exposure to the virus, and most symptoms last for less than a week. Complications of influenza can include viral pneumonia, secondary bacterial pneumonia, sinus infections, and exacerbation of pre-existing health problems such as asthma or heart failure. Viral pneumonia can also lead to acute respiratory distress syndrome (ARDS).

[0620] It will be apparent to those skilled in the art that there are currently four influenza viruses: A, B, C, and D. Influenza A virus is the most common influenza virus, infecting humans, animals, and birds, while influenza B virus infection occurs mainly in humans. Influenza C virus infection does not cause any severe symptoms in humans or mammals, and influenza D has so far infected only pigs and cattle.

[0621] Therefore, in some examples of this disclosure, the subject has an influenza virus infection. In one example, the subject has influenza. In some examples, influenza is associated with ARDS. In one example, the method of this disclosure can be used to treat or prevent ARDS in a subject suffering from an influenza virus infection. In one example, the method of this disclosure can be used to treat or prevent ARDS in a subject suffering from influenza.

[0622] For example, the methods described herein include identifying a subject who has or is suspected of having influenza. In this example, the subject may have one or more of the above symptoms and may be classified as having mild or severe influenza.

[0623] Coronavirus disease 2019 (COVID-19) This disclosure provides, for example, methods for treating or preventing COVID-19. This disclosure also provides, for example, methods for treating or preventing SARS-CoV-2 infection. In some examples of this disclosure, subjects have SARS-CoV-2 infection but do not have clinically diagnosed COVID-19.

[0624] COVID-19 is an infectious disease caused by SARS-CoV-2. First identified in December 2019 in Wuhan, Hubei Province, China, it has become an ongoing pandemic. Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. While the majority of cases present with mild symptoms, some progress to ARDS (Acute Respiratory Disorders of Severity). The time from exposure to symptom onset is typically about 5 days, but can range from 2 to 14 days.

[0625] Therefore, in some cases, the subjects have SARS-CoV-2 infection. In one case, the subjects have COVID-19. In particular, severe COVID-19 often causes ARDS. The methods of this disclosure can be used to treat or prevent ARDS in subjects suffering from COVID-19.

[0626] Complications associated with COVID-19 also include sepsis or pneumonia. Therefore, complications including sepsis or pneumonia are also targeted for treatment with RNA (e.g., the self-replicating RNA, compositions, or vaccines of this disclosure).

[0627] Complications associated with COVID-19 also include sepsis or pneumonia. Therefore, complications including sepsis or pneumonia are also targeted for treatment with RNA (e.g., the self-replicating RNA, compositions, or vaccines of this disclosure).

[0628] In one example, the methods described herein include identifying a subject who has or is suspected of having SARS-CoV-2. In this example, a subject may have one or more of the above-described symptoms and may be classified as having SARS-CoV-2.

[0629] Acute respiratory distress syndrome (ARDS) This disclosure provides, for example, a method for treating or preventing ARDS in a subject.

[0630] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltration, severe hypoxemia, and disruption of the alveolar-capillary barrier (i.e., pulmonary vascular leakage), leading to non-cardiogenic pulmonary edema. Currently, there is no effective pharmacological treatment.

[0631] Infectious etiologies, including influenza and coronavirus infections, are the primary causes of ARDS. Therefore, in one example of this disclosure, ARDS is associated with influenza or coronavirus infection. For example, ARDS is associated with influenza. In another example, ARDS is associated with coronavirus infection, such as SARS-CoV-2 infection.

[0632] ARDS are classified according to the Berlin definition, including the following: (1) Presentation of clinically invasive respiratory symptoms or within one week of onset, (2) Acute hypoxic respiratory failure, as determined by a PaO2 / FiO2 ratio of 300 mmHg or less at at least 5 cm of continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP), where PaO2 is the partial pressure of oxygen in arterial blood and FiO2 is the fraction of inspired oxygen, (3) Bilateral shadows on chest radiographs that are not fully explained by exudate, coagulation, or atelectasis, and (4) Edema / respiratory failure not fully explained by heart failure or fluid overload.

[0633] For example, the subject has or is suffering from ARDS (i.e., the subject meets the Berlin definition of ARDS). For instance, the subject requires treatment (i.e., needs it).

[0634] For example, a subject may have or be suffering from symptoms associated with ARDS. The symptoms associated with ARDS, and methods for identifying subjects at risk of developing ARDS, are evident to those skilled in the art and / or are described herein. For example, a subject may have one or more or all of the following symptoms: a) A respiratory rate exceeding 30 breaths per minute, b) Oxygen saturation (SpO2) of 93% or less in indoor air, c) Ratio of arterial oxygen partial pressure to the fraction of inhaled oxygen below 300 mmHg (PaO2 / FiO2), d) SpO2 / FiO2 ratio less than 218, and e) More than 50% of the lungs covered by radiographs.

[0635] Currently, ARDS is classified as mild, moderate, or severe based on the associated increased mortality rate. The severity of ARDS can be classified according to the Berlin definition as follows: (i) Mild ARDS: PaO2 / FiO2 of 200-300 mmHg on CPAP or PEEP at least 5 cm, (ii) Moderate ARDS: PaO2 / FiO2 of 100-200 mmHg on at least 5 cm PEEP, (iii) Severe ARDS: PaO2 / FiO2 less than 100 mmHg on at least 5 cm PEEP.

[0636] In one example, ARDS is mild ARDS. In another example, ARDS is moderate ARDS. In yet another example, ARDS is severe ARDS.

[0637] For example, the methods described herein include identifying a subject who has or is suspected of having ARDS. In this example, the subject may have one or more of the above symptoms and may be classified as having mild or severe ARDS.

[0638] The method disclosed herein can be used to prevent the onset of ARDS in addition to existing treatments for ARDS. Therefore, in one example, the subject does not have ARDS.

[0639] Respiratory syncytial virus (RSV) This disclosure provides, for example, a method for treating or preventing RSV, or for delaying its progression. RSV is an enclosed, unsegmented, negative-strand RNA virus belonging to the Paramyxoviridae family of the genus Pneumovirus. In order to infect host cells, paramyxoviruses such as RSV require the fusion of the viral membrane with the host cell membrane, as do other enveloped viruses such as influenza viruses.

[0640] In one example, a subject has or is suffering from symptoms associated with RSV. Symptoms associated with RSV, and methods for identifying subjects at risk of developing RSV, are obvious to those skilled in the art and / or are described herein. For example, a subject has one or more or all of the following symptoms indicating mild RSV: a) Nasal congestion or runny nose, b) dry cough, c) slight fever, d) Sore throat, e) sneezing, f) Headache, Or in severe cases: a) Short, shallow, and rapid breathing, b) Difficulty breathing, that is, the chest muscles and skin are pulled inward with each breath. c) Cough; d) anorexia; e) Abnormal fatigue (lethargy), f) Irritability.

[0641] Therefore, in one example, RSV is mild RSV. In another example, RSV is severe RSV.

[0642] For example, the methods described herein include identifying a subject who has or is suspected of having RSV. In this example, the subject may have one or more of the symptoms described above and may be classified as having mild or severe RSV.

[0643] The method disclosed herein can be used to prevent the onset of RSV in addition to existing treatments for RSV. Therefore, in one example, the subject does not have RSV.

[0644] Another example provided is a method for treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in an infant, comprising administering an immunogenic composition, pharmaceutical composition, or vaccine disclosed herein to the infant.

[0645] Infants who have or are suspected of having RSV may exhibit one or more or all of the following symptoms that indicate RSV: a) irritability; b) Decreased activity, c) loss of appetite; d) apnea; e) Fever, f) Runny nose, g) Cough that has progressed to wheezing or difficulty breathing.

[0646] In one example, the methods described herein include identifying an infant who has or is suspected of having RSV. In this example, the infant may have one or more of the symptoms described above.

[0647] kit One example of this disclosure provides a kit containing the self-replicating RNA of this disclosure useful for the treatment or prevention of the diseases or disorders described above. Another example of this disclosure provides a kit containing the cRNA of this disclosure useful for the treatment or prevention of the diseases or disorders described above. Another example of this disclosure provides a kit containing the RNA of this disclosure useful for the treatment or prevention of the diseases or disorders described above. Another example of this disclosure provides a kit containing the polynucleotide of this disclosure useful for the treatment or prevention of the diseases or disorders described above.

[0648] For example, the kit includes (a) optionally a container containing self-replicating RNA in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and (b) a package insert with instructions for treating or preventing a disease or disorder in the subject (e.g., SARS-CoV-2 infection, COVID-19, RSV, influenza, ARDS).

[0649] For example, the kit includes (a) optionally a container containing RNA in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and (b) a package insert with instructions for treating or preventing a disease or disorder in the subject (e.g., SARS-CoV-2 infection, COVID-19, RSV, influenza, ARDS).

[0650] In this example of the Disclosure, the accompanying information is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold or contain the composition effective for the disease or disorder of the Disclosure and may have a sterile access port (for example, the container may be a vial with a stopper that can be pierced by an intravenous solution bag or a subcutaneous injection needle). At least one activator in the composition is a polynucleotide, RNA, self-replicating RNA, and / or cRNA. The label or accompanying information indicates that the composition is used to treat subjects suitable for treatment, e.g., subjects having or susceptible to SARS-CoV-2 infection, COVID-19, RSV, influenza, ARDS, or COVID-19-related sepsis or pneumonia, and provides specific guidance on dosage and treatment intervals, as well as any other medicinal products. The kit may further include additional containers containing pharmaceutically acceptable diluent buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0651] This disclosure includes the following non-limiting embodiments. [Examples]

[0652] Example 1: Generation of self-replicating RNA A DNA template encoding self-replicating RNA can be produced in competent Escherichia coli cells transformed with a DNA plasmid. Individual bacterial colonies can be isolated, and the resulting plasmid DNA can be amplified in E. coli cultures. After fermentation, plasmid DNA can be isolated using the Maxiprep DNA kit and linearized by restriction digestion. Restriction enzymes can then be removed using phenol / chloroform extraction and ethanol precipitation.

[0653] mRNA can be prepared by in vitro transcription from a linearized DNA template using T7 RNA polymerase. The DNA template can then be removed by DNase digestion. Enzymatic capping can be performed with cap 0 to provide functional mRNA. The resulting mRNA can then be purified and resuspended in nuclease-free water.

[0654] Example 2: In vitro characterization of self-replicating RNA Next, the self-replicating RNA produced in Example 1 can be used to evaluate the expression of the target gene, which is expressed in the form of an antigen.

[0655] Two-fold serial dilutions of unformulated (naked) or LNP-formulated auto-amplified mRNA constructs can be electroporated or transfected into baby hamster kidney (BHK) cell lines. After approximately 17–19 hours, cells can be harvested and stained for antigen expression using anti-antigen antibodies. The number of cells positive for antigen expression and the mean fluorescence intensity (MFI) can be measured by FACS. The data can be analyzed to calculate a specific potency value (probability of transfection success per unit mass of RNA) and the generated MFI.

[0656] The in vitro activity and potency of unformulated RNA and LNPs can be determined by FACS based on antigen co-expression, and can be expressed in terms of readouts such as FACS potency, encapsulation efficiency, SAM recovery, size, PDI, zeta potential, conductivity, concentration, and endotoxin levels.

[0657] Antibody response To evaluate the antibody response, serum can be collected from immunized mice and tested by microneutralization assays and, for example, hemagglutination inhibition assays.

[0658] For all serological assays, serum can be similarly treated with Vibrio cholerae neuraminidase (Denka Seiken Co., Ltd., Tokyo, Japan), which is also known as a receptor-destroying enzyme (RDE), and diluted to a 1:10 starting dilution with PBS. Sheep serum for H5N1 virus (FDA / CBER Kensington lot nu.H5-Ag-1115) can be used, for example, as a positive control serum for influenza antigen.

[0659] Microneutralization assay Short and long forms of microneutralization assays are performed in a qualified mammalian cell line (proprietary 33016-PF Madin-Darby Canine Kidney (MDCK)).

[0660] Short form of microneutralization assay (MN assay SF) Viral fluorescence focus-based microneutralization (FFA MN) assays can be performed using an in-house developed protocol. RDE-treated test mouse samples and positive control serum can be thermally inactivated, diluted to a 1:40 initial dilution in PBS, and then serially diluted fourfold using a U-bottom 96-well plate (BD Falcon) in neutralization medium (consisting of minimal essential medium D-MEM (GIBCO) supplemented with 1% BSA (Rockland, BSA-30), 100 U / mL penicillin, and 100 ug / mL streptomycin (GIBCO)). The virus can be diluted in neutralization medium to approximately 1,000–1,500 fluorescence focus units (FFU) / well (20,000–30,000 FFU / mL) and added to the diluted serum in a 1:1 ratio.

[0661] After incubation at 37°C in 5% CO2 for 2 hours, plates containing MDCK 33016-PF cells (half-area 96-well plates, Corning) can be inoculated with this mixture and incubated overnight at 37°C in 5% CO2 for 16-18 hours. MDCK 33016-PF cells are seeded 6-8 hours early in cell growth medium (consisting of D-MEM supplemented with 10% HyClone fetal bovine serum-FBS (Gibco), 100 U / mL penicillin, and 100 U / mL streptomycin) at a rate of 3.0E4 / well (3.0E6 / plate). After overnight incubation and before immunostaining, cells can be fixed with a cold mixture of acetone and methanol.

[0662] Next, the virus can be visualized using a separate 1-hour incubation at room temperature of Alexa Fluor 488 goat anti-mouse IgG(H+L)Ab (Invitrogen catalog number A11001) diluted in PBS buffer containing a monoclonal antibody specific to the target viral protein and 0.05% tween-20 (Sigma) and 2% BSA (fraction V, Calbiome, 2960, 1194C175). The viral protein can be quantified by a CTL Immunospot analyzer (Cellular Technology Limited, Shaker Heights, Cleveland, OH) using a fluorescein isothiocyanate (FITC) fluorescence filter set at excitation and emission wavelengths of 482 and 536 nm. Fluorescence foci can be enumerated using the Immunospot 7.0.12.1 Professional Analyzer DC software with a custom analysis module.

[0663] Long form of microneutralization assay (MN assay LF) The MN assay LF will be performed using a protocol developed in-house. RDE-treated test mouse samples and positive control serum are heat-inactivated, diluted to a 1:40 initial dilution in PBS, and serially diluted twofold using a U-bottom 96-well plate (BD Falcon) in neutralization medium (30% used growth medium (Irvine Scientific) and 70% infection medium (protein-free medium - 33016 MDCK PFM; GIBCO)) supplemented with 100 U / mL penicillin, 100 ug / mL streptomycin (GIBCO), and 0.33 ug / mL TPCK-trypsin (TPCK-treated, tosylphenylalanyl chloromethyl ketone, Sigma). The target virus is diluted to 100 TCID (tissue culture infectious dose) per well in neutralization medium and added to the diluted serum in a 1:1 ratio. Pre-diluted serum samples are incubated sequentially with the virus at 37°C in 5% CO2 for 1 hour. In the inoculation step, MDCK Plates containing 33016-PF cells (96-well cell culture plates, Costar) were sterilized 1 day prior in antibiotic-free cell growth medium (Irvine Scientific) with a 3.0 E4 / Well(3.0 E6 Seeds are seeded on plates, washed with sterile PBS, then infected with this mixture and incubated at 37°C in 5% CO2 for 1 hour. Infection is stopped by aspirating the antibody / virus mixture, cells are washed with sterile PBS, inoculated with neutralizing medium (100 ul / well) containing 2x serial dilutions of antibody, and incubated at 37°C in 5% CO2 for 5 days. In the final "readout" step, virus detection is performed by quantification of the virus using 0.5% turkey erythrocytes (Lampire Biological Laboratories). Absence of infectivity constitutes a positive neutralization reaction, indicating the presence of virus-specific antibodies in the serum sample.

[0664] Numbered descriptions in this disclosure This disclosure provides at least the following numbered statements:

[0665] 1. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) RNA comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), RNA in which each nucleotide sequence is operablely linked to a regulatory element.

[0666] 2. The RNA is arranged in the order from 5' to 3', (a) The nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) The nucleotide sequence encoding an antigen from influenza, (c) RNA as described in Description 1, comprising the nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV).

[0667] 3. The RNA is arranged in the order from 5' to 3', (a) The nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) The nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) RNA as described in Description 1, comprising the nucleotide sequence encoding an antigen from influenza.

[0668] 4. The RNA is arranged in the order from 5' to 3', (a) The nucleotide sequence encoding an antigen from influenza, (b) The nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (c) RNA as described in Description 1, comprising the nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0669] 5. The RNA described in any one of descriptions 1 to 4, wherein the regulatory element is selected from the group consisting of a promoter, a Kozak consensus sequence, and an IRES.

[0670] 6. The RNA is (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) RNA as described in Description 1, comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably ligated to an IRES or SG promoter.

[0671] 7. The RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, (c) RNA as described in description 6, comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably ligated to an IRES or SG promoter.

[0672] 8. The RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) RNA as described in description 6, comprising a nucleotide sequence encoding a third antigen from influenza, operably ligated to an IRES or SG promoter.

[0673] 9. The RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) RNA as described in Description 6, comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably ligated to an IRES or SG promoter.

[0674] 10. The RNA described above is a monocistronic RNA, as described in any one of descriptions 1 to 9.

[0675] 11. The RNA described above is a polycistronic RNA, as described in any one of descriptions 1 to 9.

[0676] 12. The RNA described in description 5, wherein the promoter is a subgenome (SG) promoter.

[0677] 13. The RNA described in description 12, wherein the SG promoter is a minimal SG promoter or an extended SG promoter.

[0678] 14. The RNA described in description 13, wherein the extended SG promoter is extended at the 5' end by a nucleotide occurring in the sequence encoding a non-structural protein of the RNA virus.

[0679] 15. The RNA described in description 13, wherein the minimum SG promoter is encoded by the sequence shown in Sequence ID No. 1.

[0680] 16. The RNA described in description 13, wherein the extended SG promoter is encoded by the sequence shown in Sequence ID No. 5.

[0681] 17. The IRES is an RNA described in any one of descriptions 5-9, 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), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (elF4G), death-related protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF-B), Antennapedia, X-linked apoptosis inhibitor (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or a combination thereof.

[0682] 18. The RNA described in description 17, wherein the EMCV IRES is a wild-type IRES encoded by the sequence shown in Sequence ID No. 4.

[0683] 19. RNA described in any one of descriptions 1 to 18, wherein the antigen is expressed at substantially the same level.

[0684] 20. RNA described in any one of descriptions 1 to 19, wherein the antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a nucleocapsid (N) or spike (S) protein, which is optionally encoded by the sequence shown in SEQ ID NO: 21 or 22.

[0685] 21. The RNA described in any one of descriptions 1 to 20, wherein the antigen from influenza is from influenza A, influenza B, or influenza C, and is optionally encoded by the sequence shown in Sequence ID No. 19 or 20.

[0686] 22. RNA as described in any one of descriptions 1 to 21, wherein the antigen from RSV is an RSV surface glycoprotein selected from fusion (F), pre-fusion (Pre F), glycoprotein (G), small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P, and L, and non-structural proteins NS1 and NS2, which is optionally encoded by the sequence shown in Sequence ID No. 17 or 18.

[0687] 23. The RNA described in any one of descriptions 1 to 22, wherein the RNA is self-replicating RNA or cRNA.

[0688] 24. The self-replicating RNA described in description 23, which is derived from an alphavirus.

[0689] 25. The RNA described in description 24, wherein the alphavirus is selected from the group consisting of Semliki Forest Virus (SFV), Sindobis Virus (SIN), and Venezuelan Encephalitis Virus (VEE), and combinations thereof.

[0690] 26. An immunogenic composition comprising RNA as described in any one of descriptions 1 to 25.

[0691] 27. The immunogenic composition according to description 26, wherein the RNA is a self-replicating RNA, and the composition comprises a plurality of self-replicating RNAs, each self-replicating RNA encoding a different polypeptide antigen sequence.

[0692] 28. The immunogenic composition according to description 27, wherein the RNA is a self-replicating RNA, and the composition comprises a plurality of self-replicating RNAs, each self-replicating RNA encoding the same polypeptide antigen sequence.

[0693] 29. A pharmaceutical composition comprising an immunogenic composition described in any one of descriptions 26 to 28 and a pharmaceutically acceptable carrier.

[0694] 30. The pharmaceutical composition according to description 29, further comprising lipid nanoparticles (LNPs), polymer microparticles, or an oil-in-water emulsion.

[0695] 31. The pharmaceutical composition according to description 30, wherein the RNA is encapsulated in, bound to, or adsorbed to LNPs, polymer microparticles, or an oil-in-water emulsion.

[0696] 32. A pharmaceutical composition according to any one of descriptions 29 to 31, wherein each RNA is formulated together in the LNP.

[0697] 33. A pharmaceutical composition according to any one of descriptions 29 to 31, wherein each RNA is separately formulated in the LNP.

[0698] 34. An immunogenic composition according to any one of descriptions 26-28 or a pharmaceutical composition according to any one of descriptions 29-33, for use as a vaccine.

[0699] 35. A vaccine comprising an immunogenic composition described in any one of descriptions 26-28 or a pharmaceutical composition described in any one of descriptions 29-33.

[0700] 36. A polynucleotide that encodes RNA as described in any one of descriptions 1-25.

[0701] 37. The polynucleotide according to description 36, wherein the polynucleotide is recombinant DNA.

[0702] 38. The polynucleotide described in description 37, wherein the recombinant DNA is a plasmid.

[0703] 39.a) The nucleotide sequence encoding the first antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, c) A polynucleotide comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

[0704] 40. The polynucleotides are arranged in the order from 5' to 3', a) The nucleotide sequence encoding the first antigen from SARS-CoV-2, b) The nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, c) The polynucleotide according to description 39, comprising the nucleotide sequence encoding a third antigen from RSV, operably linked to an IRES or SG promoter.

[0705] 41. The polynucleotides are arranged in the order from 5' to 3', a) The nucleotide sequence encoding a third antigen from RSV, b) The nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to an IRES or SG promoter, c) The polynucleotide according to description 39, comprising the nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter.

[0706] 42. The polynucleotides are arranged in the order from 5' to 3', a) The nucleotide sequence encoding a second antigen from influenza, b) The nucleotide sequence encoding a third antigen from RSV, operably linked to an IRES or SG promoter, c) The polynucleotide according to description 39, comprising the nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to an IRES or SG promoter.

[0707] 43.a) The nucleotide sequence encoding the first antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), b) A nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, c) Conventional mRNA (cRNA) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably ligated to an IRES or SG promoter.

[0708] 44. The cRNA is arranged in the order from 5' to 3', a) The nucleotide sequence encoding the first antigen from SARS-CoV-2, b) The nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter, c) The cRNA described in description 43, comprising the nucleotide sequence encoding a third antigen from RSV, operably ligated to an IRES or SG promoter.

[0709] 45. The cRNA is arranged in the order from 5' to 3', a) The nucleotide sequence encoding a third antigen from RSV, b) The nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to an IRES or SG promoter, c) The cRNA described in description 43, comprising the nucleotide sequence encoding a second antigen from influenza, operably ligated to an IRES or SG promoter.

[0710] 46. ​​The cRNA is arranged in the order from 5' to 3', a) The nucleotide sequence encoding a second antigen from influenza, b) The nucleotide sequence encoding a third antigen from RSV, operably linked to an IRES or SG promoter, c) The cRNA described in description 43, comprising the nucleotide sequence encoding a first antigen from SARS-CoV-2, operably ligated to an IRES or SG promoter.

[0711] The nucleotide sequence of 47.(a) is operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, the polynucleotide described in any one of descriptions 39 to 42 or the cRNA described in any one of descriptions 43 to 56.

[0712] 48. A method for treating, preventing, or delaying the progression of a disease or condition in a subject requiring treatment or prevention or delay of its progression, the method comprising administering to the subject RNA described in any one of descriptions 1 to 25, an immunogenic composition described in any one of descriptions 26 to 28, a pharmaceutical composition described in any one of descriptions 29 to 33, or a vaccine described in description 35.

[0713] 49. Use of RNA as described in any one of descriptions 1 to 25, an immunogenic composition as described in any one of descriptions 26 to 28, a pharmaceutical composition as described in any one of descriptions 29 to 33, or a vaccine as described in description 35 in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of a disease or condition in a subject who requires treatment or prevention of the disease or condition or delaying its progression.

[0714] 50. RNA as described in any one of descriptions 1 to 25, an immunogenic composition as described in any one of descriptions 26 to 28, a pharmaceutical composition as described in any one of descriptions 29 to 33, or a vaccine as described in description 35, for use in treating, preventing, or delaying the progression of a disease or condition in subjects requiring treatment or prevention of the progression thereof.

[0715] 51. RNA, pharmaceutical composition, immunogenic composition...

Claims

1. (a) Nucleotide sequences encoding antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) A nucleotide sequence encoding a second antigen from influenza, (c) RNA comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), RNA in which each nucleotide sequence is operablely linked to a regulatory element.

2. The RNA is arranged in the order from 5' to 3', (a) The nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) The nucleotide sequence encoding an antigen from influenza, (c) The RNA according to claim 1, comprising the nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV).

3. The RNA is arranged in the order from 5' to 3'. (a) The nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (b) The nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) The RNA according to claim 1, comprising the nucleotide sequence encoding an antigen from influenza.

4. The RNA is arranged in the order from 5' to 3'. (a) The nucleotide sequence encoding an antigen from influenza, (b) The nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), (c) The RNA according to claim 1, comprising the nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

5. The RNA according to claim 1, wherein the regulatory element is selected from the group consisting of a promoter, a Kozak consensus sequence, and IRES.

6. The RNA mentioned above, (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES or SG promoter, (c) The RNA according to claim 1, comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

7. The RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES or SG promoter, (c) The RNA according to claim 6, comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

8. The RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from respiratory syncytial virus (RSV), operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter, (c) The RNA according to claim 6, comprising a nucleotide sequence encoding a third antigen from influenza, operably linked to an IRES or SG promoter.

9. The RNA is arranged in the order from 5' to 3'. (a) A nucleotide sequence encoding an antigen from influenza, operably linked to the SG promoter, (b) A nucleotide sequence encoding a second antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter, (c) The RNA according to claim 6, comprising a nucleotide sequence encoding a third antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), operably linked to an IRES or SG promoter.

10. The RNA according to claim 1, wherein the RNA is a monocistronic RNA.

11. The RNA according to claim 1, wherein the RNA is a polycistronic RNA.

12. The RNA according to claim 5, wherein the promoter is a subgenome (SG) promoter.

13. The RNA according to claim 12, wherein the SG promoter is a minimal SG promoter or an extended SG promoter.

14. The RNA according to claim 13, wherein the extended SG promoter is extended at its 5' end by a nucleotide generated in a sequence encoding a non-structural protein of the RNA virus.

15. The RNA according to claim 13, wherein the minimum SG promoter is encoded by the sequence shown in SEQ ID NO:

1.

16. The RNA according to claim 13, wherein the extended SG promoter is encoded by the sequence shown in Sequence ID No.

5.

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

18. The RNA according to claim 17, wherein the EMCV IRES is a wild-type IRES encoded by the sequence shown in Sequence ID No.

4.

19. The RNA according to claim 1, wherein the antigen is expressed at substantially the same level.

20. The RNA according to claim 1, wherein the antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a nucleocapsid (N) or spike (S) protein, optionally encoded by the sequence shown in SEQ ID NO: 21 or 22.

21. The RNA according to claim 1, wherein the antigen from influenza is from influenza A, influenza B, or influenza C, which is optionally encoded by the sequence shown in SEQ ID NO: 19 or 20.

22. The RNA according to claim 1, wherein the antigen from RSV is an RSV surface glycoprotein selected from fusion (F), pre-fusion (Pre F), glycoprotein (G), small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P, and L, and non-structural proteins NS1 and NS2, which are optionally encoded by the sequence shown in SEQ ID NO: 17 or 18.

23. The RNA according to claim 1, wherein the RNA is self-replicating RNA or cRNA.

24. The RNA according to claim 23, wherein the self-replicating RNA is derived from an alphavirus.

25. The RNA according to claim 24, wherein the alphavirus is selected from the group consisting of Semliki Forest Virus (SFV), Sindobis Virus (SIN), Venezuelan Encephalitis Virus (VEE), and combinations thereof.

26. An immunogenic composition comprising the RNA described in claim 1.

27. The immunogenic composition according to claim 26, wherein the RNA is a self-replicating RNA, and the composition comprises a plurality of self-replicating RNAs, each self-replicating RNA encoding a different polypeptide antigen sequence.

28. The immunogenic composition according to claim 27, wherein the RNA is a self-replicating RNA, and the composition comprises a plurality of self-replicating RNAs, each self-replicating RNA encoding the same polypeptide antigen sequence.

29. A pharmaceutical composition comprising the immunogenic composition according to claim 26 and a pharmaceutically acceptable carrier.

30. The pharmaceutical composition according to claim 29, further comprising lipid nanoparticles (LNPs), polymer microparticles, or an oil-in-water emulsion.

31. The pharmaceutical composition according to claim 30, wherein the RNA is encapsulated in, bound to, or adsorbed to LNPs, polymer microparticles, or an oil-in-water emulsion.

32. The pharmaceutical composition according to claim 29, wherein each RNA is formulated together in the LNP.

33. The pharmaceutical composition according to claim 29, wherein each RNA is formulated separately in the LNP.

34. An immunogenic composition according to claim 26 or a pharmaceutical composition according to claim 29 for use as a vaccine.

35. A vaccine comprising the immunogenic composition described in claim 26 or the pharmaceutical composition described in claim 29.

36. A polynucleotide encoding RNA as described in claim 1.

37. The polynucleotide according to claim 36, wherein the polynucleotide is recombinant DNA.

38. The polynucleotide according to claim 37, wherein the recombinant DNA is a plasmid.

39. a) The nucleotide sequence encoding the first antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES or SG promoter, c) A polynucleotide comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably linked to an IRES or SG promoter.

40. The polynucleotides are arranged in the order from 5' to 3', a) The nucleotide sequence encoding the first antigen from SARS-CoV-2, b) The nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES or SG promoter, c) The polynucleotide according to claim 39, comprising the nucleotide sequence encoding a third antigen from RSV, operably linked to an IRES or SG promoter.

41. The polynucleotides are arranged in the order from 5' to 3', a) The nucleotide sequence encoding a third antigen from RSV, b) The nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to the IRES or SG promoter, c) The polynucleotide according to claim 39, comprising the nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter.

42. The polynucleotides are arranged in the order from 5' to 3', a) The nucleotide sequence encoding a second antigen from influenza, b) The nucleotide sequence encoding a third antigen from RSV, operably linked to the IRES or SG promoter, c) The polynucleotide according to claim 39, comprising the nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to an IRES or SG promoter.

43. a) The nucleotide sequence encoding the first antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), b) A nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES or SG promoter, c) Conventional mRNA (cRNA) comprising a nucleotide sequence encoding a third antigen from respiratory syncytial virus (RSV), operably ligated to an IRES or SG promoter.

44. The cRNA is arranged in the order from 5' to 3', a) The nucleotide sequence encoding the first antigen from SARS-CoV-2, b) The nucleotide sequence encoding a second antigen from influenza, operably linked to the IRES or SG promoter, c) the cRNA according to claim 43, comprising the nucleotide sequence encoding a third antigen from RSV, operably linked to an IRES or SG promoter.

45. The cRNA is arranged in the order from 5' to 3', a) The nucleotide sequence encoding a third antigen from RSV, b) The nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to the IRES or SG promoter, c) The cRNA according to claim 43, comprising the nucleotide sequence encoding a second antigen from influenza, operably linked to an IRES or SG promoter.

46. The cRNA is arranged in the order from 5' to 3', a) The nucleotide sequence encoding a second antigen from influenza, b) The nucleotide sequence encoding a third antigen from RSV, operably linked to the IRES or SG promoter, c) The cRNA according to claim 43, comprising the nucleotide sequence encoding a first antigen from SARS-CoV-2, operably linked to an IRES or SG promoter.

47. The polynucleotide according to claim 39 or the cRNA according to claim 43, wherein the nucleotide sequence in (a) is operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, an SG promoter, and combinations thereof.

48. A method for treating, preventing, or delaying the progression of a disease or condition in a subject requiring treatment or prevention of such disease or condition, wherein the method comprises administering the RNA described in claim 1 to the subject.

49. The use of RNA according to claim 1 in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of a disease or condition in a subject who requires treatment or prevention of such disease or condition or delay of its progression.

50. RNA according to claim 1, for use in treating or preventing a disease or condition or delaying its progression in subjects requiring treatment or prevention of a disease or condition or delaying its progression.

51. RNA, a pharmaceutical composition, an immunogenic composition, or a vaccine for the method according to claim 48, the use according to claim 49, or the use according to claim 50, wherein the disease or condition is selected from the group consisting of influenza, RSV, SARS-CoV-2 infection, and / or COVID-19.

52. A method for inducing an immune response in a subject, the method comprising administering the RNA described in claim 1 to the subject in need thereof.

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

54. The RNA according to claim 1, for use in inducing an immune response in subjects requiring induction of an immune response.

55. A pharmaceutical composition, immunogenic composition, or vaccine for the method according to claim 52, the use according to claim 53, or the use according to claim 54, wherein the immune response is a humoral and / or cell-mediated immune response.

56. A method for reducing the viral load in a subject, comprising administering the RNA described in claim 1 to the subject in need of such reduction.

57. The use of RNA according to claim 1 in the preparation of a pharmaceutical product for reducing viral load in a subject requiring reduction of viral load.

58. The RNA according to claim 1, for use in reducing viral load in subjects requiring reduction of viral load.

59. A pharmaceutical composition, immunogenic composition, or vaccine for the method according to any one of claims 48, 51, 52, 55, or 56, the use according to any one of claims 49, 51, 53, 55, or 57, or the use according to any one of claims 50, 51, 54, 55, or 58, wherein the subject is a human being 18 years of age or older.

60. A method for treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in an infant, comprising administering the RNA described in claim 1 to the infant.

61. Use of the RNA described in 1 in the preparation of a pharmaceutical product for treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in infants.

62. RNA according to claim 1 for use in treating, preventing, or delaying the progression of respiratory syncytial virus (RSV) in infants.

63. An immunogenic composition, pharmaceutical composition, or vaccine for the method according to claim 60, the use according to claim 61, or the use according to claim 62, wherein the infant is approximately 1 to 12 months of age.

64. A pharmaceutically acceptable composition, immunogenic composition, or vaccine for the use described in any one of claims 48, 51, 52, 55, 56, 59, or 60, the use described in any one of claims 49, 51, 53, 55, 57, 59, or 61, or the use described in any one of claims 50, 51, 54, 55, 58, 59, or 62, wherein the vaccine or composition is administered in a single-dose regimen.

65. A pharmaceutically acceptable composition, immunogenic composition, or vaccine for use according to any one of claims 48, 51, 52, 55, 56, 59, or 60, wherein the composition or vaccine is administered in two, three, or four dose regimens, with the doses administered at intervals of about one, two, or three months; a use according to any one of claims 49, 51, 53, 55, 57, 59, or 61; or a use according to any one of claims 50, 51, 54, 55, 58, 59, or 62.

66. It's a kit, (a) RNA according to claim 1, (b) The instruction manual, and optionally, (c) A kit comprising a pharmaceutically acceptable carrier, excipient, or diluent.

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