Vaccine composition

Abstract

This disclosure relates to antigen-encoding RNAs and other polynucleotides that can be used in vaccine compositions. In some examples, RNA also encodes chemoattractants such as chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), or CXC-motif chemokine ligand 11 (CXCL11). This disclosure also relates to the use of RNAs and compositions in methods for inducing an immune response in a subject.

Description

Technical Field

[0001] Related Application Data This application claims priority from U.S. Patent Application No. 63 / 500,378, filed May 5, 2023, entitled "Vaccine Compositions I" and U.S. Application No. 63 / 500,377, filed May 5, 2023, entitled "Vaccine compositions II", the entire contents of which are incorporated herein by reference.

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

[0003] The present disclosure relates to RNA and / or protein-based immune boosters for use in vaccine compositions.

Background Art

[0004] Bacterial, viral, and parasitic infections are widespread in humans and animals. Diseases caused by these infectious agents are often resistant to antimicrobial drug therapies, leaving no effective treatment means. Therefore, vaccinological approaches are increasingly used to control infectious diseases. Whole infectious pathogens can be rendered suitable for use in vaccine formulations after chemical inactivation or appropriate genetic manipulation. Alternatively, protein subunits of the pathogen can be expressed in a recombinant expression system and purified for use in vaccine formulations. Vaccines can be made more effective by enhancing immunogenicity and / or delaying the release of antigen from the injection site.

[0005] Conventional vaccines generally consist of crude preparations of inactivated, killed, or modified live pathogenic microorganisms. Impurities associated with these cultures of pathological microorganisms can act as adjuvants or immunoenhancing agents to enhance the immune response. However, immunity induced by vaccines using pathological microorganisms or homogeneous preparations of purified protein subunits as antigens is often poor. Therefore, the addition of certain exogenous substances acting as adjuvants or immunoenhancing agents is necessary. Furthermore, synthetic and subunit vaccines are expensive to manufacture, and the addition of immunoenhancing agents potentially allows for the use of lower doses of antigen to stimulate a similar immune response, thereby reducing the cost of vaccine production. Thus, the efficacy of some injectable drugs can be significantly increased when combined with immunoenhancing agents.

[0006] Immunostimulants are generally used to increase the magnitude or function of the antibody response, enhance cell-mediated immunity, induce mucosal immunity, and / or reduce antigen dose. For example, the first reported immunostimulant was Freund's complete adjuvant (FCA), which contained a water-in-oil emulsion and an extract of Mycobacterium. However, FCAs are poorly tolerated and can cause uncontrolled inflammation. Since the discovery of FCAs more than 80 years ago, efforts have been made to reduce the undesirable side effects of immunostimulants. Other materials used as immunostimulants include metal oxides (e.g., aluminum hydroxide), alum, inorganic chelates of salts, gelatin, various paraffinic oils, synthetic resins, alginates, mucoids and polysaccharide compounds, caseins, and blood-derived substances such as fibrin coagulation. While these substances are generally effective in stimulating the immune system, they are unsuitable for use in vaccines due to adverse effects in the host, such as abscess production, organ damage, carcinogenicity, and allergic responses. Undesirable pharmaceutical properties such as rapid or inadequate dispersion from the injection site, or material swelling, have also been reported.

[0007] Therefore, those skilled in the art will understand that the development of new immunostimulants for use in vaccine compositions remains necessary. [Overview of the Initiative]

[0008] This disclosure is based on the inventors' discovery that combining an antigen with a chemoattractant in an immunogenic composition such as a vaccine can enhance the target immune response to the immunogenic composition.

[0009] Accordingly, our findings provide a basis for RNA comprising a nucleotide sequence encoding a chemoattractant and a nucleotide sequence encoding a target antigen. Our findings also provide a basis for immunogenic compositions comprising (i) RNA comprising a nucleotide sequence encoding a target antigen and a nucleotide sequence encoding a chemoattractant, and (ii) a chemoattractant polypeptide. Our findings also provide a basis for immunogenic compositions comprising (i) RNA comprising a nucleotide sequence encoding a target antigen and (ii) a chemoattractant polypeptide. Furthermore, our findings provide a basis for methods of using the immunogenic compositions of this disclosure to treat, prevent, or delay the progression of a disease or disorder in a subject (e.g., a disease caused by a respiratory viral infection such as influenza, SARS-CoV-2 infection, COVID-19, or ARDS).

[0010] Accordingly, the present disclosure provides a polynucleotide comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and a second nucleotide sequence encoding a chemoattractant operably linked to the regulatory element.

[0011] The disclosure also provides RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element.

[0012] The disclosure also provides a cRNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and a second nucleotide sequence encoding a chemoattractant operably linked to the regulatory element.

[0013] The disclosure also provides a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and a second nucleotide sequence encoding a chemoattractant operably linked to the regulatory element.

[0014] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) A nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) comprising a nucleotide sequence encoding a chemoattractant operably linked to a regulatory element.

[0015] In another example, self-replicating RNA is in the order from 5' to 3'. a) A nucleotide sequence encoding a chemoattractant operably linked to a regulatory element, and b) comprising a nucleotide sequence encoding an antigen operably linked to a regulatory element.

[0016] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) A nucleotide sequence encoding an antigen operably linked to the SG promoter, and b) comprising a nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0017] In another example, self-replicating RNA is in the order from 5' to 3'. a) A nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), and b) It contains a nucleotide sequence encoding an antigen operably linked to the SG promoter.

[0018] In one example, the self-replicating RNA, in the order from 5' to 3', a) A nucleotide sequence encoding a chemoattractant operably linked to the SG promoter, and b) It contains a nucleotide sequence encoding an antigen operably linked to a regulatory element selected from the group consisting of the SG promoter and an internal ribosome entry site (IRES).

[0019] In one example, the self-replicating RNA, in the order from 5' to 3', a) A nucleotide sequence encoding a chemoattractant operably linked to the SG promoter, and b) It contains a nucleotide sequence encoding an antigen operably linked to the SG promoter.

[0020] In one example, the chemoattractant is selected from the group consisting of chemokine, C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), and C-X-C motif chemokine ligand 11 (CXCL11).

[0021] In some examples, the chemoattractant is chemokine.

[0022] In some examples, the self-replicating RNA, in the order from 5' to 3', a) A nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) It contains a nucleotide sequence encoding chemokine operably linked to a regulatory element.

[0023] In some examples, the self-replicating RNA, in the order from 5' to 3', a) A nucleotide sequence encoding chemokine operably linked to a regulatory element, and b) comprising a nucleotide sequence encoding an antigen operably linked to a regulatory element.

[0024] In some examples, self-replicating RNA is arranged in the order from 5' to 3'. a) A nucleotide sequence encoding an antigen operably linked to the SG promoter, and b) A nucleotide sequence encoding chemerin operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0025] In some examples, self-replicating RNA is arranged in the order from 5' to 3'. a) A nucleotide sequence encoding chemerin operably linked to the SG promoter, and b) A nucleotide sequence encoding an antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

[0026] In some examples, self-replicating RNA is arranged in the order from 5' to 3'. a) A nucleotide sequence encoding chemerin operably linked to the SG promoter, and b) Includes a nucleotide sequence encoding an antigen operably linked to the SG promoter.

[0027] Accordingly, an example of the present disclosure provides a polynucleotide comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and a second nucleotide sequence operably linked to the regulatory element encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11).

[0028] The disclosure also provides RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence operably ligated to the regulatory element encoding a chemoattractant selected from the group consisting of chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), and CXC-motif chemokine ligand 11 (CXCL11).

[0029] The disclosure also provides a cRNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence operably ligated to the regulatory element encoding a chemoattractant selected from the group consisting of chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), and CXC-motif chemokine ligand 11 (CXCL11).

[0030] The disclosure also provides a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence operably ligated to the regulatory element encoding a chemoattractant selected from the group consisting of chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), and CXC-motif chemokine ligand 11 (CXCL11).

[0031] 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.

[0032] In one example, the nucleotide sequence encoding a chemoattractant is operably linked to the same regulatory element as the nucleotide sequence encoding the antigen.

[0033] In one example, a polynucleotide, RNA, cRNA, or self-replicating RNA contains a second antigen. In another example, a polynucleotide, RNA, cRNA, or self-replicating RNA contains a second and a third antigen.

[0034] The disclosure also provides a polynucleotide comprising a) a first nucleotide sequence encoding a first polypeptide of interest, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of a subgenome (SG) promoter and an internal ribosome entry site (IRES).

[0035] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding the first polypeptide of interest, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0036] In one example, the polynucleotide comprises a first nucleotide sequence encoding a chemoattractant operably linked from 5' to 3' to a) a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0037] The disclosure also provides a polynucleotide comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0038] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0039] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0040] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the target antigen, and b) a second nucleotide sequence encoding a chemerin operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0041] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0042] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding the target antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to the SG promoter.

[0043] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to the SG promoter.

[0044] 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.

[0045] Accordingly, the present disclosure provides an RNA comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0046] In one example, the RNA includes a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0047] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0048] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, and b) a second nucleotide sequence encoding a target antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0049] The disclosure also provides a cRNA comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0050] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0051] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0052] The disclosure further provides a self-replicating RNA comprising a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0053] In one example, the self-replicating RNA includes a) a first nucleotide sequence encoding a first antigen of interest, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0054] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0055] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, and b) a second nucleotide sequence encoding a target antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0056] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the target antigen, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0057] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0058] In one example, the self-replicating RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding the target antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to the SG promoter.

[0059] In one example, the self-replicating RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to the SG promoter.

[0060] In one example, the first nucleotide sequence is operably ligated to a regulatory element. In another example, the regulatory element is operably ligated to the 5' end of the first nucleotide sequence. In one example, the regulatory element is selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof. For example, the regulatory element is a Kozak consensus sequence. In another example, the regulatory element is an IRES. In one example, the nucleotide sequence encoding a chemoattractant is operably ligated to an IRES located 3' relative to the nucleotide sequence encoding the chemoattractant. In another example, the regulatory element is an SG promoter.

[0061] For example, the Kozak consensus sequence contains or consists of the sequence shown in sequence number 37 (GCCACC). For example, the Kozak consensus sequence consists of the sequence shown in sequence number 37 (GCCACC). For example, the Kozak consensus sequence contains the sequence shown in sequence number 37 (GCCACC). For example, the Kozak consensus sequence is ACCATGG.

[0062] For example, the Kozak consensus sequence contains or consists of the sequence (ACCATGG) shown in sequence number 38. For example, the Kozak consensus sequence consists of the sequence (ACCATGG) shown in sequence number 38. For example, the Kozak consensus sequence contains the sequence (ACCATGG) shown in sequence number 38. For example, the Kozak consensus sequence is shown in sequence number 38 (ACCATGG).

[0063] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0064] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0065] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0066] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0067] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0068] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to the SG promoter.

[0069] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding an operably ligated chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target antigen operably ligated to the SG promoter.

[0070] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0071] In one example, the RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0072] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0073] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0074] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0075] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0076] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to the SG promoter.

[0077] In one example, the RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter f, and b) a second nucleotide sequence encoding the target antigen operably ligated to the SG promoter.

[0078] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0079] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0080] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0081] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0082] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0083] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen that is optionally operably ligated to the IRES, and b) a second nucleotide sequence encoding chemerin that is operably ligated to the IRES.

[0084] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably and optionally ligated to the IRES, and b) a second nucleotide sequence encoding the target antigen operably ligated to the IRES.

[0085] This disclosure also provides a composition comprising a cRNA encoding a first antigen of interest and a cRNA encoding chemerin.

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

[0087] In one example, the self-replicating RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0088] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0089] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0090] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0091] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0092] In one example, the self-replicating RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to the SG promoter.

[0093] In one example, the self-replicating RNA contains, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to the SG promoter.

[0094] In one example, the first nucleotide sequence is operably ligated to the Kozak consensus sequence.

[0095] In one example, the first nucleotide sequence is operably ligated to a Kozak consensus sequence and an SG promoter. For instance, the Kozak consensus sequence is operably ligated to the 5' end of an SG promoter, which in turn is operably ligated to the 5' end of the first nucleotide sequence.

[0096] In one example, the first nucleotide sequence is operably ligated to the Kozak consensus sequence and the IRES. For instance, the Kozak consensus sequence is operably ligated to the 5' end of the IRES, which in turn is operably ligated to the 5' end of the first nucleotide sequence.

[0097] In one example, the first nucleotide sequence is operably linked to the SG promoter.

[0098] In one example, the first nucleotide sequence is operably ligated to the IRES.

[0099] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0100] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0101] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0102] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0103] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0104] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably linked to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0105] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0106] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract operably linked to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0107] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0108] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0109] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably linked to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0110] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably linked to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0111] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably linked to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0112] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0113] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0114] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably linked to an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0115] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0116] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably linked to an SG promoter, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0117] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0118] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0119] This disclosure provides a polynucleotide comprising: a) a first nucleotide sequence encoding a target first antigen operably linked to an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0120] In one example, the polynucleotide comprises, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably linked to the IRES, and b) a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0121] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably linked to the IRES, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0122] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0123] In one example, the polynucleotide includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably linked to an IRES, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0124] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0125] In one example, the RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0126] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0127] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0128] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0129] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0130] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0131] In one example, the RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0132] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0133] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0134] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0135] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0136] This disclosure provides an RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0137] In one example, the RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0138] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0139] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0140] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0141] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding the target chemerin operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0142] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0143] In one example, the RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0144] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0145] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0146] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0147] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0148] This disclosure provides RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0149] In one example, the RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the IRES, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and the IRES.

[0150] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and the IRES.

[0151] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0152] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0153] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to an IRES, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0154] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0155] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0156] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding an adjuvant operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0157] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0158] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding an adjuvant operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0159] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0160] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0161] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target chemoattractant operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0162] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0163] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target chemoattractant operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0164] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0165] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0166] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract operably linked to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0167] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0168] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0169] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0170] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0171] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0172] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0173] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0174] This disclosure provides a cRNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0175] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0176] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant operably ligated to an IRES, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0177] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0178] In one example, the cRNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to an IRES, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0179] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0180] In one example, the self-replicating RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0181] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0182] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0183] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0184] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0185] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0186] In one example, self-replication includes a) a first nucleotide sequence encoding a target first antigen operably linked to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably linked to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0187] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0188] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0189] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0190] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0191] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0192] In one example, the self-replicating RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0193] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0194] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0195] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0196] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemerin operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0197] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an SG promoter; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0198] In one example, the self-replicating RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0199] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0200] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0201] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding chemerin operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0202] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to the SG promoter, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0203] This disclosure provides a self-replicating RNA comprising: a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES; and b) a second nucleotide sequence encoding a chemoattractant operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0204] In one example, the self-replicating RNA includes a) a first nucleotide sequence encoding a target first antigen operably ligated to the IRES, and b) a second nucleotide sequence encoding a chemoattractant selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11), operably ligated to a regulatory element selected from the group consisting of the SG promoter and the IRES.

[0205] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and the IRES.

[0206] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0207] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a second nucleotide sequence encoding a chemerin operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0208] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding chemerin operably ligated to an IRES, and b) a second nucleotide sequence encoding the target antigen operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0209] In one example, a polynucleotide is a bicistronic RNA. For example, a polynucleotide is a bicistronic cRNA. In another example, a polynucleotide is a bicistronic self-replicating mRNA. For example, self-replicating RNA is a bicistronic self-replicating RNA.

[0210] In one example, the second nucleotide sequence is operably ligated to the IRES.

[0211] In one example, the second nucleotide sequence is operably ligated to the SG promoter.

[0212] For example, a polynucleotide is a multicistronic RNA. For example, a polynucleotide is a multicistronic cRNA. For example, cRNA is a multicistronic cRNA. In another example, a polynucleotide is a multicistronic self-replicating mRNA. For example, self-replicating RNA is a multicistronic self-replicating mRNA.

[0213] 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.

[0214] In one example, the SG promoter is either a minimum SG promoter or an extended SG promoter.

[0215] 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 SEQ ID NO: 32. For example, the minimal SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 32.

[0216] 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.

[0217] In one example, the SG promoter is extended at its 5' end by 51 or fewer nucleotides occurring in 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 in 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: 32, which is extended at its 5' end by 51 or fewer nucleotides occurring in 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: 36.

[0218] 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).

[0219] 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: 32, 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: 36. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 33.

[0220] In one example, the SG promoter is extended at its 5' end by approximately 31 nucleotides arising from 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: 32, which is extended at its 5' end by 31 nucleotides arising from 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: 36. In yet another example, the extended SG promoter is encoded by a sequence containing or consisting of the sequence shown in SEQ ID NO: 34.

[0221] For example, the extended SG promoter contains a repeating sequence corresponding to nucleotides 66-75 of SEQ ID NO: 36. For instance, the extended SG promoter is encoded by a sequence containing nucleotides 50-75 and nucleotides 66-101 of SEQ ID NO: 36. For example, the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 46.

[0222] In some examples, the extended SG promoter is encoded by the sequence shown in one of sequence numbers 33, 34, 36, or 46.

[0223] In some examples, the extended SG promoter is encoded by the sequence shown in one of sequence numbers 33, 34, 36, 46, 51, or 52. In some examples, the extended SG promoter is encoded by the sequence shown in sequence number 51 or 52.

[0224] 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 inhibitor of apoptosis (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or combinations thereof.

[0225] 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: 35.

[0226] In one example, the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, are codon-optimized.

[0227] In one example, the G / C content of the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, is modified.

[0228] For example, the G / C content of the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, increases by at least 5% compared to the G / C content of the unmodified sequence. For instance, the G / C content of the first and / or second nucleotide sequence, and / or one or more additional nucleotide sequences, increases by at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to the G / C content of the unmodified sequence.

[0229] For example, a polynucleotide contains at least one chemically modified nucleotide.

[0230] 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).

[0231] 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.

[0232] 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: 44.

[0233] 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.

[0234] 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 SEQ ID NOs.40-44.

[0235] 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 identical. In another example, two or more 5'-UTRs are different.

[0236] 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.

[0237] 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.

[0238] 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: 45.

[0239] 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.

[0240] 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.

[0241] In one example, a nucleotide sequence containing 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] For example, a polyA sequence contains 36 consecutive adenosine nucleotides. For instance, a polyA sequence includes the sequence shown in Sequence ID No. 47.

[0246] 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.

[0247] For example, a break linker has a nucleotide length of 10-50, 50-100, or 100-150.

[0248] 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.

[0249] 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. 39. For example, the interrupted linker contains or consists of the nucleotide sequence GCAUAUGACU.

[0250] 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.

[0251] 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: 39, and a further polyA sequence containing 70 consecutive adenosine nucleotides.

[0252] For example, polynucleotides are arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) The first nucleotide sequence encoding the target antigen, d) A second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a regulatory element selected from the group consisting of SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0253] For example, polynucleotides are arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) A first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11. d) A second nucleotide sequence encoding the target antigen, operably linked to a regulatory element selected from the group consisting of SG promoters and IRESs, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0254] In one example, RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) A first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11. d) A second nucleotide sequence encoding the target antigen, operably linked to a regulatory element selected from the group consisting of SG promoters and IRESs, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0255] In one example, RNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) The first nucleotide sequence encoding the target antigen, d) A second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a regulatory element selected from the group consisting of SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0256] In one example, the cRNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) The first nucleotide sequence encoding the target antigen, d) A second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a regulatory element selected from the group consisting of SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0257] In one example, the cRNA is arranged in the order from 5' to 3'. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) A first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11. d) A second nucleotide sequence encoding the target antigen, operably linked to a regulatory element selected from the group consisting of SG promoters and IRESs, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0258] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) The first nucleotide sequence encoding the target antigen, d) A second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a regulatory element selected from the group consisting of SG promoter and IRES, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0259] In one example, self-replicating RNA is structured from 5' to 3' in that order. a) 5'-UTR, its fragments and / or variants, b) A regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof. c) A first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11. d) A second nucleotide sequence encoding the target antigen, operably linked to a regulatory element selected from the group consisting of SG promoters and IRESs, e) 3'-UTR, its fragments and / or variants, and f) 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.

[0260] For example, the self-replicating RNA of this disclosure is in the order from 5' to 3', a) A first nucleotide sequence encoding a target antigen operably linked to the minimal SG promoter, and a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to the minimal SG promoter, or b) A first nucleotide sequence encoding a target antigen operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an extended SG promoter, or c) comprising a first nucleotide sequence encoding a target antigen operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a wild-type EMCV IRES.

[0261] For example, the self-replicating RNA of this disclosure is in the order from 5' to 3', a) A first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to the minimal SG promoter, and a second nucleotide sequence encoding a target antigen, operably linked to the minimal SG promoter, or b) A first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a target antigen, operably linked to an extended SG promoter, or c) comprising a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding a target antigen, operably ligated to a wild-type EMCV IRES.

[0262] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a target antigen operably ligated to a minimal SG promoter, and a second nucleotide sequence operably ligated to the minimal SG promoter, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, encoding a chemoattractant.

[0263] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a first nucleotide sequence operably ligated to a minimal SG promoter, encoding a chemoattractant optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, and a second nucleotide sequence operably ligated to a minimal SG promoter, encoding a target antigen.

[0264] For example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a target antigen operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 32, and a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 32.

[0265] For example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 32, and a second nucleotide sequence encoding a target antigen, operably ligated to a minimal SG promoter containing the sequence shown in SEQ ID NO: 32.

[0266] In one example, the self-replicating RNA of this disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a target antigen operably ligated to a minimal SG promoter, and a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an extended SG promoter.

[0267] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a first nucleotide sequence operably ligated to a minimal SG promoter, encoding a chemoattractant optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, and a second nucleotide sequence operably ligated to an extended SG promoter, encoding a target antigen.

[0268] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a target antigen operably ligated to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 32, and a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an extended SG promoter encoded by the sequence shown in SEQ ID NO: 33.

[0269] In one example, the self-replicating RNA of the present disclosure comprises, in the 5' to 3' order, a first nucleotide sequence encoding a chemotactic substance, optionally selected from the group consisting of chemokine, CXCL9, CXCL10, and CXCL11, operably linked to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 32, and a second nucleotide sequence encoding a target antigen operably linked to an extended SG promoter encoded by the sequence shown in SEQ ID NO: 33.

[0270] In one example, the self-replicating RNA of the present disclosure comprises, in the 5' to 3' order, a first nucleotide sequence encoding a target antigen operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a chemotactic substance, optionally selected from the group consisting of chemokine, CXCL9, CXCL10, and CXCL11, operably linked to a wild-type EMCV IRES.

[0271] In one example, the self-replicating RNA of the present disclosure comprises, in the 5' to 3' order, a first nucleotide sequence encoding a chemotactic substance, optionally selected from the group consisting of chemokine, CXCL9, CXCL10, and CXCL11, operably linked to a minimal SG promoter, and a second nucleotide sequence encoding a target antigen operably linked to a wild-type EMCV IRES.

[0272] In one example, the self-replicating RNA of the present disclosure comprises, in the 5' to 3' order, a first nucleotide sequence encoding a target antigen operably linked to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 32, and a second nucleotide sequence encoding a chemotactic substance, optionally selected from the group consisting of chemokine, CXCL9, CXCL10, and CXCL11, operably linked to a wild-type EMCV IRES encoded by the sequence shown in SEQ ID NO: 35.

[0273] In one example, the self-replicating RNA of the present disclosure comprises, in 5' to 3' order, a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a minimal SG promoter encoded by the sequence shown in SEQ ID NO: 32, and a second nucleotide sequence encoding a target antigen, operably ligated to a wild-type EMCV IRES encoded by the sequence shown in SEQ ID NO: 35.

[0274] In one example, RNA further includes a 5' end cap structure.

[0275] For example, the 5' end cap structure is an endogenous cap or an analogue of it.

[0276] For example, the 5'-terminal cap structure contains guanine or a guanine analog.

[0277] 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.

[0278] For example, the 5'-terminal cap structure is ligated to the 5' end of the RNA by a 5'-5'-triphosphate bond or a 5'-5'-phosphorothioate bond.

[0279] 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 (VEEV), and combinations thereof.

[0280] In one example, the self-replicating RNA originates from the Semliki Forest Virus (SFV).

[0281] In one example, the self-replicating RNA originates from the Sindbis virus (SIN).

[0282] In one example, the self-replicating RNA originates from the Venezuelan encephalitis virus (VEEV).

[0283] In one example, the antigen is a viral antigen. For example, the viral antigen is from a respiratory virus. In one example, the respiratory virus is selected from a group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumonia virus, rhinovirus, coronavirus, adenovirus, and bocavirus. In another example, the respiratory virus is selected from a group consisting of bronchiolitis, pneumonia, croup, SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), acute respiratory distress syndrome (ARDS), and combinations thereof.

[0284] In one example, the viral antigen is derived from the influenza virus.

[0285] In one example, the viral antigen is derived from a respiratory syncytial virus.

[0286] In one example, the viral antigen is derived from the parainfluenza virus.

[0287] In one example, the viral antigen is derived from the metapneumonia virus.

[0288] In one example, the viral antigen is from rhinovirus.

[0289] In one example, the viral antigen is from coronavirus.

[0290] In one example, the viral antigen is from adenovirus.

[0291] In one example, the viral antigen is from bocavirus.

[0292] In one example, the viral antigen is from bronchiolitis.

[0293] In one example, the viral antigen is from pneumonia.

[0294] In one example, the viral antigen is from croup.

[0295] In one example, the viral antigen is from SARS-CoV-2 infection.

[0296] In one example, the viral antigen is from coronavirus disease 2019 (COVID-19).

[0297] In one example, the viral antigen is from acute respiratory distress syndrome (ARDS).

[0298] In one example, the antigen is a viral antigen from influenza virus or coronavirus.

[0299] In one example, the self-replicating RNA is from VEEV, and the antigen is the H5 antigen from influenza. For example, a self-replicating RNA suitable for use in combination with a chemoattractant polypeptide is shown in SEQ ID NO: 48 or 49. In this example, the self-replicating RNA shown in SEQ ID NO: 48 or 49 may be used in combination with a chemoattractant polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, or 21. In another example, the Disclosure intends to use self-replicating RNA having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs. 48 or 49, and chemoattractant polypeptides having amino acid sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs. 3, 6, 9, 12, 15, 18, or 21.

[0300] In one example, the nucleotide sequence encoding the chemoattractant is selected from the group consisting of chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), and CXC-motif chemokine ligand 11 (CXCL11).

[0301] In one example, the nucleotide sequence encoding a chemoattractant is chemerin.

[0302] In one example, the nucleotide sequence encoding a chemoattractant is CXCL9.

[0303] In one example, the nucleotide sequence encoding a chemoattractant is CXCL10.

[0304] In one example, the nucleotide sequence encoding a chemoattractant is CXCL11.

[0305] For example, the mRNA sequence encoding a chemoattractant contains a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any one of any one of sequence numbers 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28.

[0306] For example, the DNA sequence encoding a chemoattractant contains a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any one of any one of sequence numbers 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.

[0307] This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure. This disclosure further provides immunogenic compositions comprising the RNA of this disclosure. For example, this disclosure provides immunogenic compositions comprising the cRNA of this disclosure. This disclosure also provides immunogenic compositions comprising the self-replicating RNA of this disclosure. For example, compositions of this disclosure can induce an immune response in a subject when administered. For example, administration of a composition induces a humoral and / or cell-mediated immune response. In one example, a composition induces a humoral immune response in a subject. For example, a humoral immune response is an antibody-mediated immune response. In another example, a composition induces a cell-mediated immune response. For example, a cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells.

[0308] In one example, the immunogenic composition of this disclosure comprises a plurality of polynucleotides, each polynucleotide encoding a different polypeptide sequence. In another example, the immunogenic composition of this disclosure comprises a plurality of RNAs, each RNA encoding a different polypeptide sequence. In yet another example, the immunogenic composition of this disclosure comprises a plurality of cRNAs, each cRNA encoding a different polypeptide sequence. In one example, the immunogenic composition comprises a plurality of multi-cistronic self-replicating RNAs, each multi-cistronic self-replicating RNA encoding a polypeptide of the target antigen and a chemoattractant as described herein. In yet another example, the immunogenic composition of this disclosure comprises a plurality of self-replicating mono-cistronic RNAs, each self-replicating RNA encoding a different polypeptide sequence. For example, one self-replicating RNA encodes a polypeptide of the target antigen, and different self-replicating RNAs encode a chemoattractant as described herein.

[0309] The Disclosure also provides immunogenic compositions comprising the polynucleotides of the Disclosure and chemoattractant polypeptides. For example, the chemoattractant polypeptide is selected from the group consisting of chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), and CXC-motif chemokine ligand 11 (CXCL11).

[0310] For example, immunogenic compositions are (i) a polynucleotide comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and (ii) Contains a chemoattractant polypeptide.

[0311] In another example, immunogenic compositions, (i) RNA containing a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and (ii) Contains a chemoattractant polypeptide.

[0312] In another example, immunogenic compositions, (i) RNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and (ii) comprising a chemoattractant polypeptide selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11).

[0313] In another example, immunogenic compositions, (i) a cRNA containing a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and (ii) Contains a chemoattractant polypeptide.

[0314] In another example, immunogenic compositions, (i) a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and (ii) comprising a chemoattractant polypeptide selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11).

[0315] In one example, the regulatory element is selected from a group consisting of a promoter, optionally 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 an SG promoter.

[0316] For example, the polynucleotide of the immunogenic composition includes an SG promoter and a first nucleotide sequence encoding an antigen operably linked to a regulatory element selected from IRES and / or Kozak consensus sequences.

[0317] In another example, the RNA of the immunogenic composition includes an SG promoter, as well as a first nucleotide sequence encoding an antigen operably linked to a regulatory element selected from IRES and / or Kozak consensus sequences.

[0318] In another example, the cRNA of the immunogenic composition includes an SG promoter, as well as a first nucleotide sequence encoding an antigen operably linked to a regulatory element selected from IRES and / or Kozak consensus sequences.

[0319] In another example, the self-replicating RNA of the immunogenic composition includes an SG promoter, as well as a first nucleotide sequence encoding an antigen operably linked to a regulatory element selected from IRES and / or Kozak consensus sequences.

[0320] For example, immunogenic compositions, (i) a polynucleotide comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and a second nucleotide sequence operably linked to the regulatory element, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, encoding a chemoattractant, and (ii) A chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0321] In another example, immunogenic compositions, (i) RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence operably ligated to the regulatory element, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, encoding a chemoattractant, and (ii) A chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0322] In another example, immunogenic compositions, (i) a cRNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence operably ligated to the regulatory element, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, encoding a chemoattractant, and (ii) A chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0323] In another example, immunogenic compositions, (i) a self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably ligated to a regulatory element, and a second nucleotide sequence operably ligated to the regulatory element, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, encoding a chemoattractant, and (ii) A chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0324] Therefore, this disclosure is, (i) RNA comprising (a) a first nucleotide sequence encoding a target first antigen, and (b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a regulatory element selected from the group consisting of an SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0325] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0326] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, and b) a second nucleotide sequence encoding a target antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0327] This disclosure further states that (i) a) a first nucleotide sequence encoding a target first antigen, and b) a self-replicating RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably linked to a regulatory element selected from the group consisting of an SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0328] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, which is operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0329] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, and b) a second nucleotide sequence encoding a target antigen, operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0330] This disclosure further states that (i) a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0331] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0332] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0333] This disclosure is, (i) a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of the Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a self-replicating RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0334] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0335] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, SG promoter, and combinations thereof; and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and IRES.

[0336] This disclosure is, (i) RNA comprising (a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and (b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0337] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0338] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0339] This disclosure is, (i) RNA comprising (a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and (b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0340] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0341] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding the target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0342] This disclosure is, (i) RNA comprising (a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and IRES, and (b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0343] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0344] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0345] This disclosure is, (i) a) RNA comprising a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0346] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0347] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0348] This disclosure is, (i) RNA comprising (a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and (b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0349] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and the IRES.

[0350] In one example, the RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0351] This disclosure is, (i) a) self-replicating RNA comprising a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0352] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0353] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a Kozak consensus sequence, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0354] This disclosure is, (i) a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and b) a self-replicating RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0355] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0356] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and the SG promoter, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0357] This disclosure is, (i) a) self-replicating RNA comprising a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0358] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to a Kozak consensus sequence and an IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0359] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the Kozak consensus sequence and IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0360] This disclosure is, (i) a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a self-replicating RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES, and (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0361] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0362] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to the SG promoter, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of the SG promoter and IRES.

[0363] This disclosure is, (i) a) a first nucleotide sequence encoding a target first antigen operably ligated to an IRES, and b) a self-replicating RNA comprising a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES, (ii) To provide an immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

[0364] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a target first antigen operably ligated to the IRES, and b) a second nucleotide sequence encoding a chemoattractant, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to a regulatory element selected from the group consisting of the SG promoter and the IRES.

[0365] In one example, the self-replicating RNA includes, in 5' to 3' order, a) a first nucleotide sequence encoding a chemoattract, optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11, operably ligated to an IRES, and b) a second nucleotide sequence encoding a target antigen, operably ligated to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0366] In one example, the chemoattractant polypeptide is selected from the group consisting of chemerin, CXC motif chemokine ligand 9 (CXCL9), CXC motif chemokine ligand 10 (CXCL10), and CXC motif chemokine ligand 11 (CXCL11).

[0367] One example of a chemoattractant polypeptide is chemerin.

[0368] In one example, the chemoattractant polypeptide is CXCL9.

[0369] One example is the chemoattractant polypeptide CXCL10.

[0370] One example is the chemoattractant polypeptide CXCL11.

[0371] For example, a chemoattractant polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, or 21.

[0372] 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.

[0373] 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.

[0374] 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.

[0375] In one example, RNA encoding a chemoattractant and RNA encoding an antigen are contained within the same LNP.

[0376] For example, this composition, (i) One or more antigens, (ii) One or more immunostimulants, (iii) One or more chemiactive substances, and / or (iv) Further comprising additional RNA encoding one or more target molecules.

[0377] For example, the additional RNA may be contained within the same LNP as the RNA encoding the chemoattractant and / or the RNA encoding the antigen, or within a separate LNP from the RNA encoding the chemoattractant and the RNA encoding the antigen.

[0378] 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.

[0379] In one example, the LNP further contains cationic lipids. In another example, the LNP does not contain cationic lipids.

[0380] 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.

[0381] In one example, the pharmaceutical composition further comprises an oil-in-water emulsion. For example, polynucleotides are encapsulated in the oil-in-water emulsion. In another example, RNA is encapsulated in the oil-in-water emulsion. For example, cRNA is encapsulated in the oil-in-water emulsion. For example, self-replicating RNA is encapsulated in the oil-in-water emulsion. For example, polynucleotides are bound to the oil-in-water emulsion. In another example, RNA is bound to the oil-in-water emulsion. For example, cRNA is bound to the oil-in-water emulsion. In yet another example, self-replicating RNA is bound to the oil-in-water emulsion. In a further example, self-replicating RNA is adsorbed onto the oil-in-water emulsion. In a further example, self-replicating RNA is resuspended in the oil-in-water emulsion.

[0382] This disclosure also provides immunogenic compositions or pharmaceutical compositions of this disclosure for use as vaccines.

[0383] 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.

[0384] For example, DNA is a plasmid.

[0385] This disclosure provides a method for treating, preventing, or slowing the progression of a disease or condition in a subject, comprising administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need. In one example, this disclosure provides a method for treating a disease or condition in a subject, comprising administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need. In another example, this disclosure provides a method for preventing a disease or condition in a subject, comprising administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need. In yet another example, this disclosure provides a method for slowing the progression of a disease or condition in a subject, comprising administering the immunogenic composition or pharmaceutical composition of this disclosure to a subject in need.

[0386] In one example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for the treatment, prevention, or delay of the progression of a disease or condition in a subject that requires such treatment or prevention. For example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for the treatment of a disease or condition in a subject that requires such treatment or prevention. In another example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for the prevention of a disease or condition in a subject that requires such prevention. In yet another example, the Disclosure provides the use of the polynucleotides of the Disclosure in the manufacture of a pharmaceutical product for the delay of the progression of a disease or condition in a subject that requires such prevention.

[0387] In one example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for the treatment, prevention, or delay of the progression of a disease or condition in a subject that requires such treatment or prevention. For example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for the treatment of a disease or condition in a subject that requires such treatment or prevention. In another example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for the prevention of a disease or condition in a subject that requires such prevention. In yet another example, the Disclosure provides the use of RNA in the manufacture of a pharmaceutical product for the delay of the progression of a disease or condition in a subject that requires such prevention.

[0388] In one example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for the treatment, prevention, or delay of the progression of a disease or condition in a subject that requires such treatment or prevention. For example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for the treatment of a disease or condition in a subject that requires such treatment or prevention. In another example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for the prevention of a disease or condition in a subject that requires such prevention. In yet another example, the Disclosure provides the use of the cRNA in the manufacture of a pharmaceutical product for the delay of the progression of a disease or condition in a subject that requires such prevention.

[0389] In one example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for use in subjects requiring treatment, prevention, or delay of the progression of a disease or condition. For example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for use in subjects requiring treatment of a disease or condition. In another example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for use in subjects requiring prevention of a disease or condition. In yet another example, the Disclosure provides the use of the self-replicating RNA in the manufacture of a pharmaceutical product for use in subjects requiring delay of the progression of a disease or condition.

[0390] In one example, the Disclosure provides a self-replicating RNA for use in implementing the treatment, prevention, or delay of the progression of a disease or condition in a subject that requires such treatment or prevention. For example, the Disclosure provides a self-replicating RNA for use in implementing the treatment of a disease or condition in a subject that requires such treatment. In another example, the Disclosure provides a self-replicating RNA for use in implementing the prevention of a disease or condition in a subject that requires such prevention. In yet another example, the Disclosure provides a self-replicating RNA for use in implementing the delay of the progression of a disease or condition in a subject that requires such prevention.

[0391] 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.

[0392] In one example, the disease or condition is selected from a group consisting of influenza, COVID-19, respiratory syncytial virus (RSV), and acute respiratory distress syndrome (ARDS).

[0393] For example, the present disclosure provides a method for inducing an immune response in a subject, comprising administering to a subject in need of such response RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0394] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a pharmaceutical product for inducing an immune response in subjects requiring such induction.

[0395] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in inducing an immune response in subjects requiring the induction of an immune response.

[0396] 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.

[0397] For example, administration of RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD4 T cell-mediated immune response.

[0398] For example, administration of RNA (e.g., self-replicating RNA), pharmaceutical compositions, immunogenic compositions, or vaccines disclosed herein induces a CD8 T cell-mediated immune response.

[0399] For example, administration of RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein induces a CD4 and CD8 T cell-mediated immune response.

[0400] 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.

[0401] 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-.

[0402] 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-.

[0403] 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-.

[0404] For example, the present disclosure provides a method for reducing the viral load in a subject having a viral infection, comprising administering to the subject having a viral infection RNA (e.g., self-replicating RNA), a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0405] For example, this disclosure provides the use of RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical product for reducing the viral load in a subject having a viral infection.

[0406] For example, this disclosure provides RNA (e.g., self-replicating RNA), a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in reducing the viral load in a subject having a viral infection.

[0407] 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, 30 years old, 40 years old, 50 years old, 60 years old, 70 years old, 80 years old, or about 90 years old. In yet another example, the subjects are under 2 years old, under 18 months old, under 12 months old, under 6 months old, or under 3 months old.

[0408] In one example, the composition or vaccine described herein is administered in a single-dose regimen. In another example, the composition is administered in two, three, or four-dose regimens, with doses administered at intervals of approximately one, two, or three months.

[0409] This disclosure also provides polynucleotides encoding the self-replicating RNA of this disclosure. For example, the polynucleotide is recombinant DNA. In one example, the recombinant DNA is a plasmid. In one example, the plasmid contains the sequence shown in SEQ ID NO: 50.

[0410] The Disclosure also provides a kit comprising, optionally, at least one self-replicating RNA of the Disclosure in a delivery system and / or a pharmaceutically acceptable carrier or diluent, packaged with instructions for use in treating, preventing or delaying the progression of a disease or disorder in a subject.

[0411] The Disclosure also provides, optionally, a kit containing at least one self-replicating RNA of the Disclosure in a delivery system and / or a pharmaceutically acceptable carrier or diluent, packaged with instructions for administering the RNA to subjects suffering from or at risk of suffering from a disease or disorder.

[0412] In one example, the self-replicating RNA, RNA, immunogenic composition, or pharmaceutical composition of this disclosure is supplied in a vial. In another example, the RNA, self-replicating RNA, immunogenic composition, or pharmaceutical composition of this disclosure is supplied in a syringe.

[0413] Any discussion of documents, acts, materials, devices, articles, etc., contained herein shall not be deemed to acknowledge that any or all of these matters constitute common knowledge in the art relating to this disclosure, such as forming part of the foundation of the prior art or existing prior to the respective priority dates of the attached claims. [Table 1-1] [Table 1-2] [Modes for carrying out the invention]

[0414] 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.

[0415] 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.

[0416] This disclosure is not limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0417] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specified. 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).

[0418] 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.

[0419] 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).

[0420] 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 literature from sources such as al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0421] The term "and / or," for example, "X and / or Y," is understood to mean "X and Y" or "X or Y," and is considered to provide explicit support for both meanings or either of them.

[0422] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to mean that they include the elements, elements, or steps, or groups of elements, elements, or steps described herein, but not that they exclude any other elements, elements, or steps, or groups of elements, elements, or steps.

[0423] Where used herein, the term “derived from” shall be interpreted as indicating that a particular integer 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 integer may be developed or used from a particular source, but not necessarily directly from that source.

[0424] Selected definition 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 RNA 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.

[0425] As used herein, the terms “conventional mRNA,” “cRNA,” or “non-amplified RNA” refer to RNA that is a construct enabling the expression of heterologous RNA and proteins, but which cannot be amplified within a host cell.

[0426] As used herein, the term “monocistronic” with respect to self-replicating RNA refers to RNA that encodes a single polypeptide.

[0427] As used herein, the term “multicistronic” (also known as “multicistronic”) refers to RNA that codes for two or more polypeptides, with respect to polynucleotides, RNA, cRNA, and / or self-replicating RNA. The term encompasses “bisistronic” (or “disistronic,” i.e., coding for two polypeptides), “tricistronic” (i.e., coding for three polypeptides) molecules, and RNA that can code for more than three polypeptides. “Bicistronic” means a single nucleic acid that can code for two different polypeptides from different regions of the nucleic acid.

[0428] 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 LNPs, liposomes, macromolecular microparticles, or oil-in-water emulsions.

[0429] 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.

[0430] As used herein, the term “chemoattractant” refers to a molecule or structure that attracts immune cells by binding to receptors on leukocytes, which cause their stimulation, polarization, and movement.

[0431] 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 from pathogens such as viruses, bacteria, fungi, protozoa, plants, or tumors.

[0432] 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.

[0433] 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).

[0434] 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.

[0435] 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).

[0436] The term "recombinant" shall be understood to mean a product of artificial genetic modification.

[0437] 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.

[0438] As used herein, a subject “at risk” of developing a disease or condition may or may not have a detectable disease or symptom of the disease, and may or may not exhibit a detectable disease or symptom of the disease prior to treatment as disclosed herein. “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 disease or condition.

[0439] As used herein, the terms “to treat,” “to cure,” or “to treat” include administering the RNA or composition described herein to reduce or eliminate at least one symptom of a particular disease or condition.

[0440] As used herein, the terms “prevent,” “prevent,” or “prevention” include providing prevention of the onset or recurrence of a particular disease or condition in an individual. An individual may have a predisposition or risk of developing a disease but may not yet have been diagnosed with the disease.

[0441] As used herein, the phrase "delay the progression of ~" includes reducing or delaying the progression of a disease or condition and / or at least one symptom of a disease or condition in an individual.

[0442] "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., by 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.

[0443] 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 the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the RNA.

[0444] 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.

[0445] As used herein, the term “subject” shall be interpreted as meaning any animal, including humans, for example, mammals. Illustrative subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0446] Chemoattractants Chemoattractants are molecules that attract immune cells by binding to receptors on leukocytes, which trigger their stimulation, polarization, and movement. The movement of leukocytes toward high concentrations of chemotactic substances is called chemotaxis. Thus, chemotactic substances favorably attract immune cells to sites where antigens are expressed from polynucleotides or RNAs of this disclosure, thereby enhancing the immunogenicity of the polynucleotides or RNAs.

[0447] The RNA-encoded chemokines described herein will be apparent to those skilled in the art and include, for example, chemerins, CXCL9 (also known as MIG), CXCL10 (also known as IP-10), CXCL11 (also known as I-TAC or IP-9), and other chemokines known to have chemokine properties and to act as immunostimulants or adjuvants.

[0448] In some cases, the chemoattractant is chemerin. Chemerin (also known as retinoic acid receptor-responsive protein 2 (RARRES2), tazarotene-inducible gene 2 protein (TIG2), or RAR-responsive protein TIG2) is a protein encoded by the RARRES2 gene in humans. Chemerin is a 14kDa protein that acts as a ligand for the G protein-coupled receptor CMKLR1 (also known as ChemR23). Chemerin is secreted in an inactive form as prochemerin and is activated by inflammatory and coagulative serine proteases by cleavage of its C-terminus.

[0449] The term chemerin includes active or inactive versions of chemerin. The inactive version, or substitute, of chemerin may also be called “prochemerin.” Prochemerin is activated by cleavage of its C-terminus by inflammatory and coagulative serine proteases. The term chemerin also includes variants of chemerin, e.g., cleavage or variant forms of chemerin, provided that the cleavage or variant form of chemerin functions as the active version (i.e., chemoattractant) of chemerin. Sequence information for the full-length human chemerin amino acid sequence can be found, for example, using GenBank accession numbers CAG46789.1 or NP 002880.1. Sequence information for the full-length human chemerin gene sequence can be found, for example, using GenBank gene ID number 5919, or the full-length human chemerin mRNA sequence can be found, for example, using GenBank accession numbers CR542026.1 or CR541992.1 or NM_002889.4.

[0450] Those skilled in the art will understand that chemerins can be found in a variety of species. Non-limiting examples include mice (NP 018128.1), cattle (NP 001039485.1), wild boars (NP 001116658.1), goats (NP 001299622.1), rats (NP 001013445.1), chickens (NP 001264405.1), hamsters (NP 001231216.1), orangutans (NP 001127631.1), and frogs (NP 001135487.1), as well as the corresponding DNA and mRNA encoding their amino acid sequences.

[0451] This disclosure also covers the use of CXCL9 (also known as MIG), CXCL10 (also known as IP-10), and CXCL11 (also known as I-TAC or IP-9). These chemokines belong to the CXC chemokine family, which play a role in inducing chemotaxis, promoting leukocyte differentiation and proliferation, and causing extratissue leakage. Each of CXCL9, CXCL10, and CXCL11 acts to modulate the migration, differentiation, and activation of immune cells via the chemokine receptor CXCR3 receptor. Immunoreactivity results from the recruitment of immune cells such as cytotoxic lymphocytes (CTLs), natural killer (NK) cells, NKT cells, and macrophages. Th1 polarization also activates immune cells in response to IFN-γ. In the context of this disclosure, the use of any of chemerin, CXCL9, CXCL10, or CXCL11 is assumed to result in an adjuvant effect that may include one or more of the following: - To increase the magnitude or function of the antibody response, - To increase cell-mediated immunity, - Inducing mucosal immunity, and / or - Reduce the antigen dose.

[0452] This disclosure provides nucleic acids encoding chemerin, CXCL9, CXCL10, and CXCL11 for use in vaccine compositions.

[0453] For example, the mRNA sequence encoding a chemoattractant contains a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the polynucleotide sequence shown in any one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28.

[0454] For example, the DNA sequence encoding a chemoattractant includes a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of the polynucleotide sequence shown in any one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.

[0455] For example, the mRNA sequence encoding CXCL9 is a polynucleotide sequence: AUGAAGAAGAGCGGCGUGCUGUUCCUGCUGGGCAUCAUCCUGCUGGUGCUGAUCGGCGUGCAGGGCACCCCCGUGGUGAGGAAGGGCAGGUGCAGCUGCAUCAGCACCAACCAGGGCACCAUCCACCUGCAGAGCCUGAAGGACCUGAAGCAGUUCGCCCCCAGCCCCAGCUGCGAGAAGAUCGAGAUCAUCG CCACCCUGAAGAACGGCGUGCAGACCUGCCUGAACCCCGACAGCGCCGACGUGAAGGAGCUGAUCAAGAAGUGGGAGAAGCAGGUGAGCCAGAAGAAGAAGCAGAAGAACGGCAAGAAGCACCAGAAGAAGAAGGUGCUGAAGGUGAGGAAGAGCCAGAGGAGCAGGCAGAAGAAGACCACC (SEQ ID NO: 1).

[0456] For example, the DNA sequence encoding CXCL9 is a polynucleotide sequence: ATGAAGAAGAGCGGCGTGCTGTTCCTGCTGGGCATCATCCTGCTGGTGCTGATCGGCGTGCAGGGCACCCCCGTGGTGAGGAAGGGCAGGTGCAGCTGCATCAGCACCAACCAGGGCACCATCCACCTGCAGAGCCTGAAGGACCTGAAGCAGTTCGCCCCCAGCCCCAGCTGCGAGAAGATCGAGATCATCG CCACCCTGAAGAACGGCGTGCAGACCTGCTGAACCCCGACAGCGCCGACGTGAAGGAGCTGATCAAGAAGTGGGAGAAGCAGGTGAGCCAGAAGAAGAAGCAGAAGAACGGCAAGAAGCACCAGAAGAAGAAGGTGCTGAAGGTGAGGAAGAGCCAGAGGAGCAGGCAGAAGAAGACCACC (SEQ ID NO: 2).

[0457] For example, the mRNA sequence encoding CXCL10 is a polynucleotide sequence: AUGAACCAGACCGCCAUCCUGAUCUGCUGCUGAUCUUCUGACCCUGAGCGGCAUCCAGGGCGUGCCCCUGAGCAGGACCGUGAGGUGCACCUGCAUCAGCAUCAGCAACCAGCCCGUGAACCCCAGGAGCCUGGAGAAGCUGGAGAUCAU CCCCGCCAGCCAGUUCUGCCCCAGGGUGGAGAUCAUCGCCACCAUGAAGAAGAAGGGCGAGAAGAGGUGCCUGAACCCCGAGAGCAAGGCCAUCAAGAACCUGCUGAAGGCCGUGAGCAAGGAGAGGAGCAAGAGGAGCCCC (SEQ ID NO: 4).

[0458] For example, the DNA sequence encoding CXCL10 is a polynucleotide sequence: ATGAACCAGACCGCCATCCTGATTCTGCTGCCTGATCTTCCTGACCCTGAGCGGCATCCAGGGCGTGCCCCTGAGCAGGACCGTGAGGTGCACCTGCATCAGCATCAGCAACCAGCCCGTGAACCCCAGGAGCCTGGAGAAGCTGGAGATCAT CCCCGCCAGCCAGTTCTGCCCCAGGGTGGAGATCATCGCCACCATGAAGAAGAAGGGCGAGAAGAGGTGCCTGAACCCCGAGAGCAAGGCCATCAAGAACCTGCTGAAGGCCGTGAGCAAGGAGAGGAGCAAGAGGAGCCCC (SEQ ID NO: 5).

[0459] For example, the mRNA sequence encoding CXCL11 is a polynucleotide sequence: AUGAGCGUGAAGGGCAUGGCCAUCGCCCUGGCCGUGAUCCUGUGCGCCACCGUGGUGCAGGGCUUCCCCAUGUUCAAGAGGGGCAGGUGCCUGUGCAUCGGCCCCGGCGUGAAGGCCGUGAAGGUGGCCGACAUCGAGAAGGCCAG CAUCAUGUACCCCAGCAACAACUGCGACAAGAUCGAGGUGAUCAUCACCCUGAAGGAGAACAAGGGCCAGAGGUGCCUGAACCCCAAGAGCAAGCAGGCCAGGCUGAUCAUCAAGAAGGUGGAGAGGAAGAACUUC (SEQ ID NO: 7).

[0460] For example, the DNA sequence encoding CXCL11 is a polynucleotide sequence: ATGAGCGTGAAGGGCATGGCCATCGCCCTGGCCGTGATCCTGTGCGCCACCGTGGTGCAGGGCTTCCCCATGTTCAAGAGGGGCAGGTGCCTGTGCATCGGCCCCGGCGTGAAGGCCGTGAAGGTGGCCGACATCGAGAAGGCCAG CATCATGTACCCCAGCAACAACTGCGACAAGATCGAGGTGATCATCACCCTGAAGGAGAACAAGGGCCAGAGGTGCCTGAACCCCAAGAGCAAGCAGGCCAGGCTGATCATCAAGAAGGTGGAGAGGAAGAACTTC (SEQ ID NO: 8).

[0461] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: AUGAGGAGGCUGCUGAUCCCCCUGGCCCUGUGGCUGGGCGCCGUGGGCGUGGGCGUGGCCGAGCUGACCGAGGCCCAGAGGAGGGGCCUGCAGGUGGCCCUGGAGGAGUUCCACAAGCAC CCCCCCGUGCAGUGGGCCUUCCAGGAGACCAGCGUGGAGAGCGCCGUGGACACCCCCUUCCCCGCCGGCAUCUUCGUGAGGCUGGAGUUCAAGCUGCAGCAGACCAGCUGCAGGAAGAGG GACUGGAAGAAGCCCGAGUGCAAGGUGAGGCCCAACGGCAGGAAGAGGAAGUGCCUGGCCUGCAUCAAGCUGGGCAGCGAGGACAAGGUGCUGGGCAGGCUGGUGCACUGCCCCAUCGAG ACCCAGGUGCUGAGGGAGGCCGAGGAGCACCAGGAGACCCAGUGCCUGAGGUGCAGAGGGCCGGCGAGGACCCCCACAGCUUCUACUUCCCCGGCCAGUUCGCCUUCAGCAAGGCCCUG Includes CCCAGGAGC (SEQ ID NO: 10).

[0462] For example, the DNA sequence encoding chemerin is a polynucleotide sequence: ATGAGGAGGCTGCTGATCCCCCTGGCCCTGTGGCTGGGCGCCGTGGGCGTGGGCGTGGCCGAGCTGACCGAGGCCCAGAGGAGGGGCCTGCAGGTGGCCCTGGAGGAGTTCCACAAGCACCCCCC CGTGCAGTGGGCCTTCCAGGAGACCAGCGTGGAGAGCGCCGTGGACACCCCCTTCCCCGCCGGCATCTTCGTGAGGCTGGAGTTCAAGCTGCAGCAGACCAGCTGCAGGAAGAGGGACTGGAAGA AGCCCGAGTGCAAGGTGAGGCCCAACGGCAGGAAGAGGAAGTGCCTGGCCTGCATCAAGCTGGGCAGCGAGGACAAGGTGCTGGGCAGGCTGGTGCACTGCCCCATCGAGACCCAGGTGCTGAGG GAGGCCGAGGAGCACCAGGAGACCCAGTGCCTGAGGGTGCAGAGGGCCGGCGAGGACCCCCACAGCTTCTACTTCCCCGGCCAGTTCGCCTTCAGCAAGGCCCTGCCCAGGAGC (SEQ ID NO: 11).

[0463] Sequence IDs 10 and 11 encode human prochemerin containing a 20-amino acid hydrophobic signal peptide.

[0464] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: GAGCUGACCGAGGCCCAGAGGAGGGGCCUGCAGGUGGCCCUGGAGGAGUUCCACAAGCACCCCCCCGUGCAGUGGGCCUUCCAGGAGACCAGCGUGGAGAGCGCCGUGGACACCCCCUUCCCCGCCGGCAUCUUCGUGAGGCUGGAGUUCAAGCUGCAGCAGACCAGCUGCAGGAAGAGGGACUGGAAGAAGCCCGAGUGCAAGGUGAGGCCCAACGGCAGGAAGAGGAAGUGCCUGGCCUGCAUCAAGCUGGGCAGCGAGGACAAGGUGCUGGGCAGGCUGGUGCACUGCCCCAUCGAGACCCAGGUGCUGAGGGAGGCCGAGGAGCACCAGGAGACCCAGUGCCUGAGGGUGCAGAGGGCCGGCGAGGACCCCCACAGCUUCUACUUCCCCGGCCAGUUCGCCUUCAGCAAGGCCCUGCCCAGGAGC It contains (Sequence No. 13).

[0465] In one example, the DNA sequence encoding kemerine is the polynucleotide sequence: GAGCTGACCGAGGCCCAGAGGAGGGGCCTGCAGGTGGCCCTGGAGGAGTTCCACAAGCACCCCCCCGTGCAGTGGGCCTTCCAGGAGACCAGCGTGGAGAGCGCCGTGGACACCCCCTTCCCCGCCGGCATCTTCGTGAGGCTGGAGTTCAAGCTGCAGCAGACCAGCTGCAGGAAGAGGGACTGGAAGAAGCCCGAGTGCAAGGTGAGGCCCAACGGCAGGAAGAGGAAGTGCCTGGCCTGCATCAAGCTGGGCAGCGAGGACAAGGTGCTGGGCAGGCTGGTGCACTGCCCCATCGAGACCCAGGTGCTGAGGGAGGCCGAGGAGCACCAGGAGACCCAGTGCCTGAGGGTGCAGAGGGCCGGCGAGGACCCCCACAGCTTCTACTTCCCCGGCCAGTTCGCCTTCAGCAAGGCCCTGCCCAGGAGC (Sequence No. 14).

[0466] Sequence IDs 13 and 14 encode mature human prochemerin with the signal peptide removed.

[0467] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: CUGACCGAGGCCCAGAGGAGGGGCCUGCAGGUGGCCCUGGAGGAGUUCCACAAGCACCCCCCCGUGCAGUGGGCCUUCCAGGAGACCAGCGUGGAGAGCGCCGUGGACACCCCCUUCCCCGCCGGCAUCUUCGUGAGGCUGGAGUUCAAGCUGCAGCAGACCAGCUGCAGGAAGAGGGACUGGAAGAAGCCCGAGUGCAAGGUGAGGCCC AACGGCAGGAAGAGGAAGUGCCUGGCCUGCAUCAAGCUGGGCAGCGAGGACAAGGUGCUGGGCAGGCUGGUGCACUGCCCCAUCGAGACCCAGGUGCUGAGGGAGGCCGAGGAGCACCAGGAGACCCAGUGCCUGAGGUGCAGAGGGCCGGCGAGGACCCCCACAGCUUCUACUUCCCCGGCCAGUUCGCCUUCAGC (SEQ ID NO: 16).

[0468] For example, the DNA sequence encoding chemerin is a polynucleotide sequence: GAGCTGACCGAGGCCCAGAGGAGGGGCCTGCAGGTGGCCCTGGAGGAGTTCCACAAGCACCCCCCCGTGCAGTGGGCCTTCCAGGAGACCAGCGTGGAGAGCGCGTGGACACCCCCTTCCCCGCCGGCATCTTCGTGAGGCTGGAGTTCAAGCTGCAGCAGACCAGCTGCAGGAAGAGGGACTGGAAGAAGCCCGAGTGCAAGGTGAGGCCCAACGGCA GGAAGAGGAAGTGCCTGGCCTGCATCAAGCTGGGCAGCGAGGACAAGGTGCTGGGCAGGCTGGTGCACTGCCCCATCGAGACCCAGGTGCTGAGGGAGGCCGAGGAGCACCAGGAGACCCAGTGCCTGAGGGTGCAGAGGGGCCGGCGAGGACCCCCACAGCTTCTACTTCCCCGGCCAGTTCGCCTTCAGCAAGGCCCTGCCCAGGAGC (SEQ ID NO: 17).

[0469] Sequence IDs 16 and 17 encode mature human chemerin with the signal peptide removed.

[0470] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: AUGCGCCGCCUGCUGAUUCCGCUGGCGCUGUGGCUGGGCGCGGUGGGGCGUGGGCGUGGCGGAACUGACCGAAGCGCAGCGCCGCGGCCUGCAGGUGGCGCUGGAAGAAUUUCAUAAACAU CCGCCGGUGCAGUGGGCGUUUCAGGAAACCAGCGUGGAAAGCGCGGUGGAUACCCCGUUUCCGGCGGGCAUUUUUGUGCGCCUGGAAUUUAAACUGCAGCAGACCAGCUGCCGCAAACGCG AUUGGAAAAAACCGGAAUGCAAAGUGCGCCCGAACGGCCGCAAACGCAAAUGCCUGGCGUGCAUUAAACUGGGCAGGAAGAUAAAGUGCUGGGCCGCCUGGUGCAUUGCCCGAUUGAAAC CCAGGUGCUGCGCGAAGCGGAAGAACAUCAGGAAACCCAGUGCCUGCGCUGCAGCGCGCGGGCGAAGAUCCGCAUAGCUUUUAUUUUCCGGGCCAGUUUGCGUUUAGC (SEQ ID NO: 19).

[0471] For example, the DNA sequence encoding chemerin is a polynucleotide sequence: ATGCGCCGCCTGCTGATTCCGCTGGCGCTGTGGCTGGGCGCGGTGGGCGTGGGCGTGGCGGAACTGACCGAAGCGCAGCGCCGCGGCCTGCAGGTGGCGCTGGAAGAATTTCATAAACAT CCGCCGGTGCAGTGGGCGTTTCAGGAAACCAGCGTGGAAAGCGCGGTGGATACCCCGTTTCCGGCGGGCATTTTTGTGCGCTGGAATTTAAACTGCAGCAGACCAGCTGCCGCAAACGCG ATTGGAAAAAACCGGAATGCAAAGTGCGCCCGAACGGCCGCAAACGCAAATGCCTGGCGTGCATTAAACTGGGCAGCGAAGATAAAGTGCTGGGCCGCCTGGTGCATTGCCCGATTGAAAC CCAGGTGCTGCGCGAAGCGGAAGAACATCAGGAAACCCAGTGCCTGCGCGTGCAGGCGCGGGCGAAGATCCGCATAGCTTTTATTTTCCGGGCCAGTTTGCGTTTAGC (SEQ ID NO: 20).

[0472] Sequence IDs 19 and 20 encode, in one embodiment, a human chemerin sequence that exhibits the highest activity, comprising the removal of six amino acids from the chemerin polypeptide ("chemerin-157").

[0473] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: It contains GAGCUGAGCGAGACCCAGAGGAGGAGCCUGCAGGUGGCCCUGGAGGAGUUCCACAAGCACCCCCCCGUGCAGCUGGCCUUCCAGGAGAUCGGCGUGGACAGGGCCGAGGAGGUGCUGUUCAGCGCCGGCACCUUCGUGAGGCUGGAGUUCAAGCUGCAGCAGACCAACUGCCCCAAGAAGGACUGGAAGAAGCCCGAGUGCACCAUCAAGCCCAACGGCAGGAGGAGGAAGUGCCUGGCCUGCCUGAAGAUGGACCCCAAGGGCAAGAUCCUGGGCAGGAUCGUGCACUGCCCCAUCCUGAAGCAGGGCCCCCAGGACCCCCAGGAGCUGCAGUGCAUCAAGAUCGCCCAGGCCGGCGAGGACCCCCACGGCUACUUCCUGCCCGGCCAGUUCGCCUUCAGCAGGGCCCUGAGGACCAAG (SEQ ID NO: 22).

[0474] In one example, the DNA sequence encoding kemerine is the polynucleotide sequence: It contains GAGCTGAGCGAGACCCAGAGGAGGAGCCTGCAGGTGGCCCTGGAGGAGTTCCACAAGCACCCCCCCGTGCAGCTGGCCTTCCAGGAGATCGGCGTGGACAGGGCCGAGGAGGTGCTGTTCAGCGCCGGCACCTTCGTGAGGCTGGAGTTCAAGCTGCAGCAGACCAACTGCCCCAAGAAGGACTGGAAGAAGCCCGAGTGCACCATCAAGCCCAACGGCAGGAGGAGGAAGTGCCTGGCCTGCCTGAAGATGGACCCCAAGGGCAAGATCCTGGGCAGGATCGTGCACTGCCCCATCCTGAAGCAGGGCCCCCAGGACCCCCAGGAGCTGCAGTGCATCAAGATCGCCCAGGCCGGCGAGGACCCCCACGGCTACTTCCTGCCCGGCCAGTTCGCCTTCAGCAGGGCCCTGAGGACCAAG (SEQ ID NO: 23).

[0475] Sequence IDs 22 and 23 encode mature mouse prochemelin.

[0476] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: GAGCUGAGCGAGACCCAGAGGAGGAGCCUGCAGGUGGCCCUGGAGGAGUUCCACAAGCACCCCCCCGUGCAGCUGGCCUUCCAGGAGAUCGGCGUGGACAGGGCCGAGGAGGUGCUGUUCAGGCCGGCACCUUCGUGAGGCUGGAGUUCAAGCUGCAGCAGACCAACUGCCCCAAGAAGGACUGGAAGAAGCCCGAGUGCACCAUC AAGCCCAACGGCAGGAGGAGGAAGUGCCUGGCCUGCAUCAAGAUGGACCCCAAGGGCAAGAUCCUGGGCAGGAUCGUGCACUGCCCCAUCCUGAAGCAGGGCCCCCAGGACCCCCAGGAGCUGCAGUGCAUCAAGAUCGCCCAGGCCGGCGAGGACCCCCACGGCUACUUCCUGCCCGGCCAGUUCGCCUUCAGC (SEQ ID NO: 25).

[0477] For example, the DNA sequence encoding chemerin is a polynucleotide sequence: GAGCTGAGCGAGACCCAGAGGAGGAGCCTGCAGGTGGCCCTGGAGGAGTTCCACAAGCACCCCCCCGTGCAGCTGGCCTTCCAGGAGATCGGCGTGGACAGGGCCGAGGAGGTGCTGTTCAGCGCCGGCACCTTCGTGAGGCTGGAGTTCAAGCTGCAGCAGACCAACTGCCCCAAGAAGGACTGGAAGAAGCCCGAGTGCACCATC AAGCCCAACGGCAGGAGGAGGAAGTGCCTGGCCTGCATCAAGATGGACCCCAAGGCAAGATCCTGGGCAGGATCGTGCACTGCCCCATCCTGAAGCAGGGCCCCCAGGACCCCCAGGAGCTGCAGTGCATCAAGATCGCCCAGGCCGGCGAGGACCCCACGGCTACTTCCTGCCCGGCCAGTTCGCCTTCAGC (SEQ ID NO: 26).

[0478] Sequence IDs 25 and 26 encode mature mouse chemerin.

[0479] For example, the mRNA sequence encoding chemerin is a polynucleotide sequence: AUGAAGUGCCUGCUGAUCAGCCUGGCCCUGUGGCUGGGCACCGUGGGCACCAGGGGCACCGAGCCCGAGCUGAGCGAGACCCAGAGGAGGAGCCUGCAGGUGGCCCUGGAGGAGUUCCAC AAGCACCCCCCCGUGCAGCUGGCCUUCCAGGAGAUCGGCGUGGACAGGGCCGAGGAGGUGCUGUUCAGCGCCGGCACCUUCGUGAGGCUGGAGUUCAAGCUGCAGCAGACCAACUGCCCC AAGAAGGACUGGAAGAAGCCCGAGUGCACCAUCAAGCCCAACGGCAGGAGGAGGAAGUGCCUGGCCUGCAUCAAGAUGGACCCCAAGGGCAAGAUCCUGGGCAGGAUCGUGCACUGCCCC AUCCUGAAGCAGGGCCCCAGGACCCCAGGAGCUGCAGUGCAUCAAGAUCGCCCAGGCCGGCGAGGACCCCCACGGCUACUUCCUGCCCGGCCAGUUCGCCUUCAGC (SEQ ID NO: 28).

[0480] For example, the DNA sequence encoding chemerin is a polynucleotide sequence: ATGAAGTGCCTGCTGATCAGCCTGGCCCTGTGGCTGGGCACCGTGGGCACCAGGGGCACCGAGCCCGAGCTGAGCGAGACCCAGAGGAGGAGCCTGCAGGTGGCCCTGGAGGAGTTCCAC AAGCACCCCCCCGTGCAGCTGGCCTTCCAGGAGATCGGCGTGGACAGGGCCGAGGAGGTGCTGTTCAGCGCCGGCACCTTCGTGAGGCTGGAGTTCAAGCTGCAGCAGACCAACTGCCCC AAGAAGGACTGGAAGAAGCCCGAGTGCACCATCAAGCCCAACGGCAGGAGGAGGAAGTGCCTGGCCTGCATCAAGATGGACCCCAAGGGCAAGATCCTGGGCAGGATCGTGCACTGCCCC ATCCTGAAGCAGGGCCCCCAGGACCCCCAGGAGCTGCAGTGCATCAAGATCGCCCAGGCCGGCGAGGACCCCCACGCTACTTCCTGCCCGGCCAGTTCGCCTTCAGC (SEQ ID NO: 29).

[0481] Sequence IDs 28 and 29 encode mouse prochemerin sequences containing a 20-amino acid hydrophobic signal peptide.

[0482] For example, Kemerin has a sequence that has at least 70% identity with SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, or SEQ ID NO: 28. For example, Kemerin may have approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 10, 13, 16, 19, 22, 25, or 28.

[0483] The Disclosure also provides immunogenic and pharmaceutical compositions comprising the polynucleotides, RNA, cRNA, or self-replicating RNA of the Disclosure, and chemoattractant polypeptides optionally selected from the group consisting of chemerin, CXC-motif chemokine ligand 9 (CXCL9), CXC-motif chemokine ligand 10 (CXCL10), and CXC-motif chemokine ligand 11 (CXCL11).

[0484] For example, a chemoattractant polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity with any one of SEQ ID NOs: 3, SEQ ID NOs: 6, SEQ ID NOs: 9, SEQ ID NOs: 12, SEQ ID NOs: 15, SEQ ID NOs: 18, SEQ ID NOs: 21, SEQ ID NOs: 24, SEQ ID NOs: 27, or SEQ ID NOs: 30.

[0485] For example, the chemerin, CXCL9, CXCL10, or CXCL11 polypeptide contains an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30. For example, chemerin, CXCL9, CXCL10, or CXCL11 polypeptides may have approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, or SEQ ID NO: 30.

[0486] For example, the CXCL9 polypeptide has the following amino acid sequence: Includes MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT (Sequence ID 3).

[0487] For example, the CXCL10 polypeptide has the following amino acid sequence: Includes MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP (Sequence ID 6).

[0488] For example, the CXCL11 polypeptide has the following amino acid sequence: Includes MSVKGMAIALAVILCATVVQGFPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLIIKKVERKNF (Sequence ID 9)

[0489] An exemplary chemoattractant is chemerin. In some examples, chemerin contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any one of SEQ ID NOs: 12, SEQ ID NOs: 15, SEQ ID NOs: 18, SEQ ID NOs: 21, SEQ ID NOs: 24, SEQ ID NOs: 27, SEQ ID NOs: 30, or SEQ ID NOs: 31.

[0490] For example, chemerin polypeptides have the following amino acid sequence: Includes MRRLLIPLALWLGAVGVGVAELTEAQRRGLQVALEEFHKHPPVQWAFQETSVESAVDTPFPAGIFVRLEFKLQQTSCRKRDWKKPECKVRPNGRKRKCLACIKLGSEDKVLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFSKALPRS (Sequence ID 12).

[0491] This sequence is a human prochemerin containing a 20-amino acid hydrophobic signal peptide.

[0492] In another example, the chemerin polypeptide has the following amino acid sequence: Includes ELTEAQRRGLQVALEEFHKHPPVQWAFQETSVESAVDTPFPAGIFVRLEFKLQQTSCRKRDWKKPECKVRPNGRKRKCLACIKLGSEDKVLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFSKALPRS (Sequence ID 15).

[0493] This sequence is mature human prochemerin with the signal peptide removed.

[0494] In another example, the chemerin polypeptide has the following amino acid sequence: Includes LTEAQRRGLQVALEEFHKHPPVQWAFQETSVESAVDTPFPAGIFVRLEFKLQQTSCRKRDWKKPECKVRPNGRKRKCLACIKLGSEDKVLGRLVHCPIETQVLREAEEHQETQ CLRVQRAGEDPHSFYFPGQFAFS (Sequence ID 18).

[0495] This sequence is mature human chemerin with the signal peptide removed.

[0496] In another example, the chemerin polypeptide has the following amino acid sequence: Includes MRRLLIPLALWLGAVGVGVAELTEAQRRGLQVALEEFHKHPPVQWAFQETSVESAVDTPFPAGIFVRLEFKLQQTSCRKRDWKKPECKVRPNGRKRKCLACIKLGSEDKVLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFS (Sequence ID 21).

[0497] In one embodiment, this human sequence includes the removal of six amino acids from the chemerin polypeptide ("chemerin-157"), which exhibits the highest activity.

[0498] In another example, the amino acid sequence of Kemelin is: Includes ELSETQRRSLQVALEEFHKHPPVQLAFQEIGVDRAEEVLFSAGTFVRLEFKLQQTNCPKKDWKKPECTIKPNGRRRKCLAClKMDPKGKILGRIVHCPILKQGPQDPQELQCIKIAQ AGEDPHGYFLPGQFAFSRALRTK (Sequence ID 24).

[0499] This sequence is mature mouse prochemerin.

[0500] In another example, the chemerin polypeptide has the following amino acid sequence: Includes ELSETQRRSLQVALEEFHKHPPVQLAFQEIGVDRAEEVLFSAGTFVRLEFKLQQTNCPKKDWKKPECTIKPNGRRRKCLACIKMDPKGKILGRIVHCPILKQGPQDPQELQCIKIAQ AGEDPHGYFLPGQFAFS (Sequence ID 27).

[0501] This sequence is mature mouse chemerin.

[0502] In another example, the chemerin polypeptide has the following amino acid sequence: Includes MKCLLISLALWLGTVGTRGTEPELSETQRRSLQVALEEFHKHPPVQLAFQEIGVDRAEEVLFSAGTFVRLEFKLQQTNCPKKDWKKPECTIKPNGRRRKCLACIKMDPKGKILGRIVHCPILKQGPQDPQELQCIKIAQAGEDPHGYFLPGQFAFS (Sequence ID 30).

[0503] This sequence is a mouse prochemerin sequence containing a 20-amino acid hydrophobic signal peptide.

[0504] In another example, a preferred control chemerin polypeptide has the following amino acid sequence: Includes MRRLLIPLALWLGAVGVGVAELTEAQRRGLQVALEEFHKHPPVQWAFQETSVESAVDTPFPAGIFVRLEFKLQQTSCRKRDWKKPECKVRPNGRKRKCLACIKLGSEDKVLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFSFAFQGPFY* (Sequence ID 31).

[0505] This sequence is the reverse of human chemerin ("chemerin-157").

[0506] In certain embodiments, the chemerin, CXCL9, CXCL10, or CXCL11 polypeptide may be pegylated for improved systemic half-life and reduced administration frequency. In one embodiment, PEG may be added to the chemerin, CXCL9, CXCL10, or CXCL11 polypeptide. Thus, the compositions of the present disclosure may comprise the chemerin, CXCL9, CXCL10, or CXCL11 polypeptide, comprising PEG. In one embodiment, PEG may be selected from the group consisting of PEG-10K, PEG-20K, and PEG-40K. Methods for conjugating PEG to proteins are standard in the art. See, for example, Kolate et al, Journal of Controlled Release 2014;192(28):67-81, which is incorporated herein by reference in its entirety.

[0507] This disclosure covers analogues of chemerin, CXCL9, CXCL10, and CXCL11 in other organisms, and is not limited to human analogues. Homologes can be found in other species by methods known in the art. For example, sequence similarity may be determined by conventional algorithms, which typically allow for the introduction of a small number of gaps to achieve the best fit. In particular, the "identity percentage" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). Such algorithms are incorporated into the BLASTN and BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches may be performed using the BLASTN program to obtain nucleotide sequences homologous to the nucleic acid molecules of this disclosure.

[0508] Similarly, BLAST protein search may be performed using the BLASTX program to obtain amino acid sequences homologous to the polypeptides of this disclosure. To obtain gapped alignment for comparison purposes, gapped BLAST is used as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., BLASTX and BLASTN) are used.

[0509] antigen Suitable antigens for use as described herein will be obvious to those skilled in the art and include, for example, proteins and peptides derived from any pathogen. For example, the antigen may be a virus, bacterium, fungus, or protist.

[0510] Viral antigens In one example, the antigen is a viral antigen.

[0511] The 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 (pneumoviruses (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 (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, coxsate) Key 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), Flavius 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.

[0512] In one example, the antigen is a viral antigen from a respiratory virus. Respiratory viral antigens that can be encoded by self-replicating RNA are obvious to those skilled in the art and include, for example, proteins and peptides from orthomyxoviruses (e.g., influenza A, B, and C), paramyxoviridae viruses (pneumoviruses (e.g., respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, and turkey rhinotracheitis virus), paramyxoviruses (PIV), and metapneumonia viruses, e.g., human metapneumonia virus (hMPV) and trimeta pneumonia virus (aMPV)), picornaviruses (e.g., rhinovirus), and 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), and mouse hepatitis virus (MHV)).

[0513] In one example, the viral antigen is derived from the influenza virus.

[0514] In one example, the viral antigen is derived from respiratory syncytial virus. RSV is an enclosed, non-segmented, negative-strand RNA virus belonging to the Paramyxoviridae family of the genus Pneumovirus. To infect host cells, paramyxoviruses such as RSV, like other enveloped viruses such as influenza virus and HIV, require fusion of the viral membrane with the host cell membrane. In the case of RSV, a conserved fusion protein (RSV-F glycoprotein) fuses the viral membrane and the cell membrane by coupling irreversible protein refolding with membrane juxtaposition. In current models 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 into its stable post-fusion conformation.

[0515] In one example, the antigen may be derived from the RSV surface glycoprotein 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.

[0516] Suitable RSV-F antigens for inclusion in the immunogenic compositions described herein, either in RNA-encoded form or as polypeptides, include RSV-F glycoproteins and RSV-F glycoprotein variants. Suitable RSV-F glycoprotein variants include, for example, full-length F proteins and cleavage variants such as soluble ectodomains, 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. Full-length and cleavage RSV-F glycoproteins (glycoprotiens), 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, the disclosure of which is incorporated herein by reference in its entirety.

[0517] In one example, the viral antigen is derived from the parainfluenza virus.

[0518] In one example, the viral antigen is derived from the metapneumonia virus.

[0519] In one example, the viral antigen is derived from rhinovirus.

[0520] In one example, the viral antigen is derived from a coronavirus. In this example, the coronavirus may be SARS-CoV-2, and the antigen may be 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.

[0521] In one example, the viral antigen is derived from an adenovirus.

[0522] In one example, the viral antigen is derived from the bocavirus.

[0523] In one example, the antigen may be derived from a single strain (i.e., monovalent) of the influenza virus, or from multiple strains (i.e., polyvalent).

[0524] In one example, the antigen is derived from influenza A, B, and / or C virus strains.

[0525] 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.

[0526] 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.

[0527] 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.

[0528] In one example, the antigen is from an influenza A virus strain having the same hemagglutinin subtype. In another example, the antigen is from an influenza A virus strain having a different hemagglutinin subtype. In one example, the antigen is an influenza A virus strain with H1, H2, H3, H5, H6, H7, or H9 subtypes. For example, the antigen is H1 hemagglutinin, or H2 hemagglutinin, or H3 hemagglutinin, or H5 hemagglutinin, or H6 hemagglutinin, or H7 hemagglutinin, or H9 hemagglutinin. For example, the antigen is an influenza A virus strain with H5 subtype (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.

[0529] In one example, the antigen is from an influenza A virus strain having the same neuraminidase subtype. In another example, the antigen is from an influenza A virus strain having a different neuraminidase subtype. In one example, the antigen is an influenza A virus strain with N1, N2, N3, N7, or N9 subtypes. For example, the antigen is N1 neuraminidase, or N2 neuraminidase, or N3 neuraminidase, or N7 neuraminidase, or N9 neuraminidase. For example, the antigen is an influenza A virus strain with N1 neuraminidase subtype. In one example, the N1 neuraminidase is from the A / turkey / Turkey / 1 / 2005 strain. In another example, the N2 neuraminidase is from the A / Delaware / 39 / 2019 virus strain.

[0530] In one example, the composition of the present disclosure comprises one or more antigens. In one example, one or more antigens are H5 hemagglutinin proteins and / or N1 neuraminidase proteins. For example, the first antigen is an H5 hemagglutinin subtype influenza A virus strain, and the second antigen is an N1 neuraminidase subtype influenza A virus strain.

[0531] Infections such as influenza and coronavirus infections are major causes of ARDS. Therefore, in one example of this disclosure, ARDS is associated with influenza or coronavirus infection, such as SARS-CoV infection. In another 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.

[0532] bacterial antigen In one example, the antigen is a bacterial antigen.

[0533] The various types of antibiotics include Neisseria meningitides, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Burkholderia sp., Burkholderia sp influenzae, Clostridium tetani, Clostridium perfringens, Clostridium botulinums, Cornynebacterium diphtheriae, Pseudomonas aeruginosa, Legionella pneumophila, Coxiella burnetii, Brucella sp. (B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, and 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.

[0534] fungal antigen In one example, the antigen is a fungal antigen.

[0535] Suitable fungal antigens are apparent to those skilled in the art and include, 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*.

[0536] Protazoan Antigen In one example, the antigen is a protazoan antigen.

[0537] Suitable protazoane antigens are obvious to those skilled in the art and include, for example, proteins and peptides from Entamoeba histolytica, Giardia lambli, Cryptosporidium parvum, Cyclospora cayatanensis, and Toxoplasma.

[0538] Other molecules For example, a composition according to this disclosure comprises (i) one or more antigens, (ii) one or more immunoenhancing agents, (iii) one or more chemoattractants, and / or (iv) one or more RNAs encoding targeting molecules.

[0539] As used herein, “immunostimulant” means any molecule capable of enhancing the immune response in a subject. Examples of immunostimulants include cytokines, chemokines, and other immunostimulants. In the example, one or more immunostimulants are selected from the group consisting of interleukin-12, interleukin-7, interleukin-15, and interleukin-21.

[0540] 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.

[0541] In another example, the target molecule is an antibody. In one example, the target molecule is a monoclonal antibody.

[0542] Polynucleotides As used herein, the term “polynucleotide” refers to a molecular chain of nucleotides chemically linked by a series of ester bonds (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.

[0543] 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.

[0544] 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.

[0545] The polynucleotides described herein may include modifications. For example, a modification is a chemical modification of one or more nucleotides in a nucleotide sequence. For instance, at least one naturally occurring nucleotide in the polynucleotide may be replaced by a chemically modified nucleotide (e.g., pseudouridine (ψ) and 1-methylpseudridine (m1ψ)).

[0546] In one example, the modification involves increasing the G / C content of the nucleotide sequence.

[0547] In one example, the modification involves codon optimization of the nucleotide sequence.

[0548] 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.

[0549] The polynucleotides of this disclosure include DNA and RNA (e.g., mRNA).

[0550] Deoxyribonucleic acid (DNA) One example of a polynucleotide is DNA (for example, a DNA vector).

[0551] It will be apparent to those skilled in the art that the DNA of this disclosure may further include 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.

[0552] 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.

[0553] 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.

[0554] In one example, the disclosure provides a transcribed polynucleotide comprising a first nucleotide sequence encoding a first antigen of interest, and a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of, for example, an SG promoter and an IRES. For example, the polynucleotide is a DNA plasmid comprising the first and second nucleotide sequences, and optionally, one or more nucleotide sequences encoding one or more antigens of interest.

[0555] 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.

[0556] RNA This disclosure relates to RNA, for example mRNA, and other polynucleotides comprising nucleotide sequences encoding at least one chemoattractant and at least one antigen disclosed herein.

[0557] In some examples, polynucleotides are RNA. As used herein, the term “RNA (ribonucleic acid)” refers to a single-stranded molecular chain of nucleotides chemically linked by a series of ester bonds (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 obvious 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.

[0558] Conventional (non-replicating) RNA The RNAs of this disclosure encompass non-replicating mRNA (also referred to as conventional mRNA (cRNA) or non-amplified mRNA). Those skilled in the art will understand that the cRNAs of this disclosure include, in 5' to 3' order, a 5' cap structure, a 5'-UTR, fragments and / or variants thereof, a first nucleotide sequence encoding a first antigen of interest, a second nucleotide sequence encoding a second antigen of interest, a 3'-UTR, and a 3' tailing sequence (e.g., a polyadenylation signal or one or more poly-A tails). The cRNAs of this disclosure may further include an intratranslational ribosome entry site (e.g., a Kozak consensus sequence or IRES) operably linked to a chemoattractant or antigen of interest. In one example, this disclosure provides monocistronic cRNA. In one example, this disclosure provides multicistronic cRNA.

[0559] self-replicating RNA The RNAs in this disclosure encompass self-replicating RNA (also known as self-amplifying RNA or sa-mRNA). In one example, RNA is self-replicating RNA (also known as a replicon). In one example, this disclosure provides monocistronic self-replicating RNA. In one example, this disclosure provides multicistronic self-replicating RNA.

[0560] 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.

[0561] 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.

[0562] 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).

[0563] It will be apparent to those skilled in the art that RNA suitable for use in this disclosure may also include a 5′ untranslated region (5′-UTR), a 3′ untranslated region (3′UTR), and / or coding or translation sequences. In addition, RNA may include a 5′ cap structure, a terminal nucleotide, a stem-loop (e.g., a histone stem-loop), and a 3′ tailing sequence (e.g., a polyadenylation signal or one or more poly-A tails). In another example, self-replicating RNA includes the 5′ and 3′ UTRs of an RNA virus. It will be apparent to those skilled in the art 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.

[0564] 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.

[0565] 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.

[0566] 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.

[0567] Alphavirus For example, the self-replicating RNA of this disclosure is derived from (or based on) an alphavirus.

[0568] 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 ORF 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.

[0569] 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.

[0570] 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).

[0571] 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 SEQ ID NO: 32.

[0572] 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 genomic sequences from two or more alphaviruses (as described, for example, by Perri et al, (2003) J. Virol. 77(19):10394-403).

[0573] Adjustment element This disclosure provides monocistronic mRNA (e.g., cRNA or self-replicating RNA) comprising a nucleotide sequence encoding a chemoattractant or antigen. For example, this disclosure provides monocistronic self-replicating RNA comprising a nucleotide sequence encoding a chemoattractant, or an antigen operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an internal ribosome entry site (IRES), or a subgenome (SG) promoter.

[0574] In one example, the present disclosure provides a monocistronic self-replicating RNA comprising a nucleotide sequence encoding a chemoattractant or an antigen, operably ligated to an SG promoter.

[0575] The disclosure also provides a multicistronic mRNA (e.g., cRNA or self-replicating RNA) comprising a first nucleotide sequence encoding a chemoattractant and a second nucleotide sequence encoding a target antigen. In one example, the nucleotide sequence encoding the target chemoattractant or antigen is operably ligated to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, an SG promoter, and combinations thereof.

[0576] In one example, a first nucleotide sequence encoding a chemoattractant and a second nucleotide sequence encoding a target antigen are operably linked to a regulatory element selected from the group consisting of an SG promoter and an IRES.

[0577] In one example, the present disclosure provides a multi-cistronic self-replicating RNA comprising a first nucleotide sequence encoding a chemoattractant operably ligated to an SG promoter, and a second nucleotide sequence encoding an antigen operably ligated to a regulatory element to a promoter selected from the group consisting of SG promoters and IRESs.

[0578] 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.

[0579] 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.

[0580] 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.

[0581] In one example, the self-replicating RNA contains an SG promoter from any alphavirus.

[0582] In one example, the polynucleotide of the Disclosure is a multicistronic mRNA containing two or more nucleotide sequences encoding a chemoattractant and a target antigen. In one example, each of the two or more nucleotide sequences is operaby-linked to an SG promoter. If two or more SG promoters are present in the RNA of the Disclosure, the promoters may be the same or different. For example, the two or more SG promoters may originate from the same alphavirus. In another example, the two or more SG promoters may originate from different alphaviruses.

[0583] If two or more SG promoters are present in the self-replicating RNA of this disclosure, the promoters may be the same or different. For example, two or more SG promoters may originate from the same alphavirus. In another example, two or more SG promoters may originate from different alphaviruses.

[0584] Exemplary SG promoter sequences are encoded by sequences SEQ ID NOs. 32, 33, 34, 36, 46, 51, and 52.

[0585] Internal ribosome entry site (IRES) IRES sequences suitable for use as disclosed herein will be obvious to those skilled in the art and / or are described herein.

[0586] In one example, IRES originates from encephalomyocarditis virus (EMCV). For instance, IRES is a wild-type IRES derived from EMCV.

[0587] In one example, IRES is derived from fibroblast growth factor 1A (FGF1A)IRES.

[0588] 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).

[0589] 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.

[0590] 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: 37 (GCCACC). In another example, the Kozak consensus sequence is shown in SEQ ID NO: 38 (ACCATGG). In one example, the Kozak consensus sequence is ACCATGG. In another example, the Kozak consensus sequence is ACCATG.

[0591] 5'Untranslated region (5'UTR) For example, the RNA of this disclosure includes a 5'-UTR, such as the 5'UTR of an RNA virus. For example, the self-replicating RNA includes a 5'-UTR of an RNA virus.

[0592] 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).

[0593] 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.

[0594] 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: 44.

[0595] 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.

[0596] microRNA binding site As used herein, the term “microRNA binding site” refers to a sequence within a polynucleotide (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.

[0597] 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.

[0598] Suitable miRNA binding sites for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0599] 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.

[0600] AU Rich Element (ARE) As used herein, the terms “AU-rich element (ARE)” or “AU-rich element(s) (AREs)” 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.

[0601] 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.

[0602] 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.

[0603] 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 in length.

[0604] 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.

[0605] 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.

[0606] For example, a GC-rich element is at least 50% cytosine.

[0607] For example, a GC-rich element is at least 60% cytosine.

[0608] For example, a GC-rich element is at least 70% cytosine.

[0609] In one example, the GC-rich element contains the nucleotide sequence CCCCGGCGCC. In another example, the GC-rich element contains the nucleotide sequence CCCCGGC. In yet another example, the GC-rich element contains the nucleotide sequence GGCCCCGCGGCGCCCCGCG.

[0610] In one example, the GC-rich element contains the nucleotide sequence shown in SEQ ID NOs. 40. In another example, the GC-rich element contains the nucleotide sequence shown in SEQ ID NO. 41. In yet another example, the GC-rich element contains the nucleotide sequence shown in SEQ ID NO. 42 (CCCCGGC).

[0611] stem loop As used herein, the term “stem-loop” refers to a nucleotide sequence containing an intramolecular base pairing of two adjacent complete or partially 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 partially reverse-complementary sequences separated by a shorter sequence that constructs the loop into a stem-loop structure.

[0612] 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.

[0613] 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.

[0614] In one example, the loop in a stem-loop has a length of 4 nucleotides.

[0615] 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. 43.

[0616] 3' Untranslated region (3'-UTR) For example, the RNA of this disclosure includes a 3' untranslated region (3'-UTR).

[0617] 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).

[0618] 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.

[0619] 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: 45.

[0620] 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.

[0621] 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.

[0622] Stop codon As used herein, the term “stop codon” refers to a trinucleotide sequence within mRNA that signals the cessation of ribosome-mediated protein synthesis.

[0623] 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.

[0624] For example, a polynucleotide contains two consecutive stop codons with the sequence UGAUGA.

[0625] For example, a polynucleotide contains two consecutive stop codons with the sequence UAAUAG.

[0626] 3' Tailing arrangement The RNAs disclosed herein may contain one or more 3' tailing sequences located at the 3' end of the 3' UTR.

[0627] As used herein, the terms “3' tailing sequence” or “3' tailing sequence(s)” refer to a nucleotide sequence (e.g., a polyadenylation signal) that induces the addition of a non-coding nucleotide to the 3' end of mRNA or to 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.

[0628] 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 binds 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.

[0629] 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.

[0630] 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).

[0631] Suitable polyA sequences for use in this disclosure will be obvious to those skilled in the art and / or are described herein. For example, a polyA sequence comprises a sequence of adenosine nucleotides of any length (e.g., 10 to 300). For example, a polyA sequence comprises 36 consecutive adenosine nucleotides. For example, a polyA sequence comprises the sequence shown in Sequence ID No. 47.

[0632] In one example, the polyA sequence contains consecutive adenosine nucleotides separated by one or more interrupting linkers. In another example, the polyA sequence contains consecutive adenosine nucleotides without interrupting linkers.

[0633] 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).

[0634] Suitable polyadenylation signals for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0635] 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.

[0636] In one example, the 3' tailing sequence contains a polyadenylation signal but does not contain a poly(A) sequence.

[0637] G-quadruplex As used herein, the terms “G quadruple” or “G4” refer to nucleotide sequences rich in guanine residues that form a quadruple-stranded secondary structure. For example, a G quadruple is a cyclic hydrogen-bonded array of four guanine nucleotides formed by a G-rich sequence in both DNA and RNA.

[0638] 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 bound to a G quadruplex to produce a poly(AG) quartet.

[0639] 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).

[0640] Suitable polyC sequences for use in this disclosure will be obvious to those skilled in the art and / or are described herein.

[0641] 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.

[0642] 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.

[0643] 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.

[0644] 5' Cap In one example, the mRNA according to this disclosure includes a 5' terminal cap structure.

[0645] 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.

[0646] 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.

[0647] Typically, endogenous mRNA is 5'-capped with guanosine via a (5)'-ppp-(5)'-triphosphate bond 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).

[0648] In one example, the polynucleotides of this disclosure include an endogenous cap.

[0649] 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.

[0650] For example, the polynucleotides of this disclosure include analogues of endogenous caps (also referred to as cap analogues).

[0651] As used herein, the term “its analog” in the context of endogenous caps or “cap analogs” refers to synthetic 5' caps. Cap analogs can be used to produce 5' cap mRNA in in vitro transcription reactions. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or conjugated to nucleotides (e.g., the 5' terminal nucleotide of mRNA). Exemplary cap analogs 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 analog is N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (i.e., anti-reverse cap analog (ARCA)).

[0652] 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.

[0653] 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 bound to the 5' end of mRNA via an α-phosphorothieate bond. The method of binding the modified nucleotide to the 5' end of mRNA will be obvious to those skilled in the art. For example, a Vaccina Capping Enzyme (New England Biolabs) is used.

[0654] 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.

[0655] In one example, a first nucleotide sequence containing the 5'-UTR and / or a fragment thereof is modified. Modification of the first nucleotide sequence containing the 5'-UTR and / or a fragment thereof results in a variant of the 5'-UTR and / or a fragment thereof.

[0656] 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®).

[0657] 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.

[0658] In one example, the G / C content increases in the first and / or second nucleotide sequences encoding the first and / or second antigen of interest. In another example, the G / C content increases in the first and / or second nucleotide sequences encoding the first and / or second antigen of interest, and / or in one or more additional nucleotide sequences encoding one or more antigens of interest. The modification(s) in the first and / or second nucleotide sequences, and / or one or more nucleotide sequences, utilizes 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 encoding the same amino acid.

[0659] In one example, the G / C content increases in one or more nucleotide sequences of polynucleotides that do not encode the target antigen. For example, the G / C content increases in the 5'-UTR, its fragments, and / or variants. For example, the G / C content increases in the 3'-UTR, its fragments, and / or variants.

[0660] For example, a polynucleotide contains at least one chemically modified nucleotide.

[0661] 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).

[0662] Pharmaceutical composition This disclosure provides immunogenic compositions comprising the polynucleotides of this disclosure.

[0663] This disclosure provides immunogenic compositions comprising the RNA of this disclosure.

[0664] This disclosure also provides immunogenic compositions comprising the cRNA of this disclosure.

[0665] This disclosure further provides immunogenic compositions comprising the self-replicating RNA of this disclosure.

[0666] This disclosure also provides a pharmaceutical composition comprising the immunogenic composition of this disclosure and a pharmaceutically acceptable carrier.

[0667] 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.

[0668] 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.

[0669] Lipid nanoparticles In one example, the pharmaceutical composition of this disclosure further comprises LNP.

[0670] The terms “lipid nanoparticles” or “LNPs” refer to any lipid composition, including but not limited to liposomes or vesicles, in which aqueous volumes are encapsulated by amphiphilic lipid bilayers (e.g., single; monolayer, or multiple; multilayer) micelle-like lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles, while solid lipid nanoparticles may lack a lipid bilayer.

[0671] 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.

[0672] 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.

[0673] For example, LNPs include PEG-lipids. For instance, the 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.

[0674] 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.

[0675] 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.

[0676] 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)butonoate (LKY750). One example of a phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,diene-1-yloxyl)benzyl-4-(dimethylamino)butonoate (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.

[0677] Polymer fine particles In one example, the pharmaceutical composition of this disclosure further comprises polymer microparticles.

[0678] Those skilled in the art will recognize that various polymers can form microparticles for encapsulating or adsorbing the polynucleotides, cRNAs, and / or self-replicating RNAs of this disclosure. The use of substantially non-toxic polymers will obviously mean that the particles are safe, and the use of biodegradable polymers will obviously mean that the particles can be metabolized after delivery to avoid long-term persistence. Useful polymers are also sterilizable to aid in the preparation of pharmaceutical-grade formulations.

[0679] 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.

[0680] Oil-in-water cationic emulsion For example, the pharmaceutical composition of this disclosure further comprises an oil-in-water cationic emulsion.

[0681] 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.

[0682] 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).

[0683] 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).

[0684] Pharmacologically acceptable carriers Preferably, in a composition or method for administering the polynucleotides, RNA, cRNA, and / or self-replicating RNA of the present disclosure, the polynucleotides, RNA, cRNA, and / or self-replicating RNA are combined with a pharmaceutically acceptable carrier as understood in the art. Thus, an example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the polynucleotides, RNA, cRNA, and / or self-replicating RNA (and an optional delivery system) of the present disclosure combined with a pharmaceutically acceptable carrier.

[0685] Generally, "carrier" means any solid or liquid filler, binder, diluent, encapsulating agent, 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).

[0686] The polynucleotides, RNA, cRNA, 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 self-replicating RNA is administered parenterally, such as intramuscular, subcutaneous, or intravenous. For example, the polynucleotides, RNA, cRNA, and / or self-replicating RNA may be administered intramuscularly. In another example, the polynucleotides, RNA, cRNA, and / or self-replicating RNA are administered parenterally, such as intramuscular, subcutaneous, or intravenous. For example, the polynucleotides, RNA, cRNA, and / or self-replicating RNA are administered intramuscularly.

[0687] The formulation of the administered polynucleotides, RNA, cRNA, and / or self-replicating RNA will vary depending on the chosen route of administration and formulation (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition containing the administered polynucleotides, RNA, cRNA, and / or self-replicating RNA can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, for example, those containing saline and a buffer medium. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate solution, or fixative oil. Those skilled in the art will know of a variety of suitable aqueous carriers, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles may contain a variety of additives, preservatives, or fluid, nutrient, 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 modifiers, 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.

[0688] The optimal concentration of the active ingredient(s) in the selected culture medium can be determined empirically according to procedures known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0689] When formulated, the compositions of this disclosure will be administered in a manner compatible with the administered formulation and in a therapeutically / prophylactically effective amount. The dosage range for the administration of the polynucleotides, RNA, cRNA, and / or self-replicating RNA of this disclosure is large enough to produce the desired effect. For example, a composition contains an effective amount of polynucleotides, RNA, cRNA, and / or self-replicating RNA. In one example, the composition contains a therapeutically effective amount of self-replicating RNA. In another example, the composition contains a prophylactically effective amount of self-replicating RNA. In one example, the composition contains an effective amount of RNA. In one example, the composition contains a therapeutically effective amount of RNA. In another example, the composition contains a prophylactically effective amount of RNA.

[0690] The dosage should not be so high as to cause adverse side effects. Generally, the dosage varies depending on the patient's age, condition, sex, and the severity of the disease, and can be determined by those skilled in the art. The dosage may be adjusted by the individual physician in the event of any complications.

[0691] 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.

[0692] In some cases, polynucleotides, RNA, cRNA, and self-replicating RNA are administered at a higher initial (or loading) dose than the subsequent (maintenance) dose. For example, polynucleotides, RNA, cRNA, and / 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.

[0693] 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.

[0694] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, increased doses may be administered.

[0695] 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.

[0696] For example, optional retreatment may be given when signs or symptoms of the disease return.

[0697] 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.

[0698] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, increased doses may be administered.

[0699] 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.

[0700] 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.

[0701] Adjuvant The pharmaceutical compositions according to this disclosure may contain additional adjuvants or immunopotentiating agents. An adjuvant or immunopotentiating agent is a substance that modifies the action of the main component. Therefore, those skilled in the art will understand that the terms “adjuvant” and “immunopotentiating agent” may be used interchangeably. For example, an adjuvant or immunopotentiating agent is a substance that enhances the immune response of a target to an antigen. Suitable adjuvants or immunopotentiating agents will be apparent 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.

[0702] Method of production Preferred methods for the production of polynucleotides, RNA, cRNA, or self-replicating RNA according to this disclosure will be obvious to those skilled in the art and / or are described herein.

[0703] 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.

[0704] For example, plasmid DNA suitable for use according to this disclosure has the sequence described in SEQ ID NO: 50, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 50.

[0705] Regarding sequence number 50, base pairs 1-44 contain the 5'UTR, base pairs 45-1649 contain nsP1, base pairs 1650-4031 contain nsP2, base pairs 4032-5681 contain nsp3, base pairs 5682-7526 contain nsp4, base pairs 7513-7536 contain the subgenome promoter, base pairs 7562-9253 contain the turkey HA antigen, base pairs 9276-9594 contain the 3'UTR, base pairs 9823-10490 contain ColE1, base pairs 10634-11449 contain the kanamycin-resistant transposon, and base pairs 11867-11883 contain the T7 promoter.

[0706] 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.

[0707] In one example, mRNA is prepared by in vitro transcription from a linearized DNA template using RNA polymerase (e.g., T7 RNA polymerase). After in vitro transcription, the DNA template is removed by DNase digestion. Those skilled in the art will understand that synthetic mRNA capping is performed to correct mRNA processing and contribute to mRNA stabilization. In one example, mRNA is enzymatically 5'-capped. For example, the 5' cap is either a cap 0 structure or a cap 1 structure. In one example, the 5' cap is a cap 0 structure, for example, the 5'-cap (i.e., cap 0) consists of inverted 7-methylguanosine attached to the remainder of the mRNA via a 5'-5' triphosphate crosslink. In another example, the 5' cap is a cap 1 structure, for example, the 5'-cap (i.e., cap 1) consists of cap 0 having an additional methylation at the 2'O position of the start nucleotide.

[0708] 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.

[0709] Screening assay Preferred methods for selecting polynucleotides, RNAs, cRNAs, or self-replicating RNAs of this disclosure are available to those skilled in the art. Assays may be performed to evaluate the efficiency and effectiveness of RNAs, for example, including serological and immune responses.

[0710] Polypeptide expression In one example, self-replicating RNA is evaluated for the expression of target atigen and chemoattractants.

[0711] For example, antigen expression is detected using antibodies against the target antigen. 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.

[0712] Microneutralization assay In one example, self-replicating RNA (naked and / or formulated) is evaluated for antibody response. For example, self-replicating RNA is 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.

[0713] Hemagglutination inhibition (HAI) assay In one example, self-replicating RNA (naked and / or formulated) is evaluated for antibody response. For example, self-replicating RNA is 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.

[0714] Antigen-specific T cell response In one example, self-replicating RNA 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.

[0715] 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.

[0716] Neutralization assay The self-replicating RNAs of this disclosure may be screened in vitro for their ability to bind to antigens, such as the SARS-CoV-2 S protein RBD, and to neutralize the binding of the S protein RBD 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).

[0717] 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).

[0718] 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-RBD 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.

[0719] In one example, neutralization is a pseudoviral neutralization assay. For example, an HIV reporter virus pseudotyped with the SARS-2 spike 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 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.

[0720] Methods of treatment or prevention This disclosure provides a method for using the immunogenic composition or pharmaceutical composition of this disclosure as a vaccine.

[0721] 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, COVID-19, or respiratory syncytial virus (RSV). In one example, the disease or condition is acute respiratory distress syndrome (ARDS).

[0722] 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).

[0723] 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.

[0724] Therefore, in some examples of this disclosure, the subjects have an influenza virus infection. In one example, the subjects have influenza. In particular, 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 another example, the method of this disclosure can be used to treat or prevent ARDS in a subject suffering from influenza.

[0725] 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.

[0726] Coronavirus disease 2019 (COVID-19) This disclosure provides, for example, a method for treating or preventing COVID-19.

[0727] 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.

[0728] COVID-19 is an infectious disease caused by SARS-CoV-2. First identified in December 2019 in Wuhan, Hubei Province, China, it has resulted in 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. Currently, there is no vaccine or specific antiviral treatment for COVID-19, and management includes symptomatic treatment, supportive care, isolation, and experimental measures.

[0729] Therefore, in some cases, the subjects have SARS-CoV-2 infection. In one case, the subjects have COVID-19, for example, severe COVID-19. Severe COVID-19, in particular, often causes ARDS. The methods of this disclosure can be used to treat or prevent ARDS in subjects suffering from severe COVID-19.

[0730] For example, the methods described herein include identifying a subject who has or is suspected of having SARS-CoV-2. In this example, the subject may have one or more of the above symptoms and may be classified as having mild or severe SARS-CoV-2.

[0731] Acute respiratory distress syndrome (ARDS) This disclosure provides, for example, a method for treating or preventing ARDS in a subject.

[0732] 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.

[0733] 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 infection. In one example, ARDS is associated with SARS-CoV-2 infection.

[0734] ARDS are classified according to the Berlin definition, including the following: (1) Presentation of a clinical attack of respiratory symptoms or within one week of onset, (2) Acute 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.

[0735] For example, the subject has or is suffering from ARDS (i.e., the subject meets the Berlin definition of ARDS). For example, the subject needs treatment (i.e., needs it).

[0736] In one example, a subject has or is suffering from symptoms associated with ARDS. The symptoms associated with ARDS, and methods for identifying subjects at risk of developing ARDS, are apparent 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: 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.

[0737] 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.

[0738] In one example, ARDS is mild ARDS. In another example, ARDS is moderate ARDS. In yet another example, ARDS is severe ARDS.

[0739] 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.

[0740] 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.

[0741] Respiratory syncytial virus (RSV) This disclosure provides, for example, a method for treating or preventing RSV.

[0742] In one example, a subject has or is suffering from symptoms associated with RSV. The 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 - the chest muscles and skin are pulled inward with each breath. c) Cough; d) anorexia; e) Abnormal fatigue (lethargy), f) Irritability.

[0743] Therefore, in one example, RSV is mild RSV. In another example, RSV is severe RSV.

[0744] 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.

[0745] The method described 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.

[0746] kit Another example of the present disclosure provides a kit containing the self-replicating RNA of the present disclosure that is useful for treating or preventing the diseases or disorders described above.

[0747] Another example of the present disclosure provides a kit containing the self-replicating RNA of the present disclosure that is useful for treating or preventing the diseases or disorders described above.

[0748] 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 use to treat or prevent a disease or disorder in a subject (e.g., influenza, COVID-19, or ARDS).

[0749] For example, the kit includes (a) optionally a container containing cRNA in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and (b) a package insert with instructions for use to treat or prevent a disease or disorder in a subject (e.g., influenza, COVID-19, or ARDS).

[0750] 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 self-replicating RNA, RNA, cRNA, or polynucleotide. The label or accompanying information indicates that the composition is used to treat subjects who are therapeutically suitable, e.g., subjects who have or are susceptible to developing influenza, influenza virus infection, SARS-CoV-2 infection, COVID-19, and / or ARDS, 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. [Examples]

[0751] Example 1: Generation of self-replicating RNA DNA templates 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.

[0752] 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.

[0753] 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.

[0754] 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. Approximately 17–19 hours later, cells can be harvested and stained for HA, NA, NS1, NP, or M1 antigen expression using anti-HA, anti-NA, anti-NS1, anti-NP, or anti-M1 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 (the probability of successful transfection per unit mass of RNA) and the generated MFI.

[0755] The in vitro activity and potency of unformulated RNA and LNPs can be determined by FACS based on antigen co-expression and expressed in readout values ​​such as FACS potency, encapsulation efficiency, SAM recovery, size, PDI, Zeta potential, conductivity, concentration, and endotoxin level.

[0756] Antibody response To evaluate the antibody response, serum can be collected at the end of the study and tested by microneutralization assays and hemagglutination inhibition assays.

[0757] For all serological assays, serum can be treated in the same manner 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.H...

Claims

1. A self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and a second nucleotide sequence encoding a chemoattractant operably linked to the regulatory element.

2. The self-replicating RNA according to claim 1, wherein the chemical attractant is selected from the group consisting of chemerin, C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), and C-X-C motif chemokine ligand 11 (CXCL11).

3. The self-replicating RNA is in the order from 5' to 3', a) A nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) The self-replicating RNA according to claim 1 or 2, comprising a nucleotide sequence encoding a chemoattractant operably linked to a regulatory element.

4. The self-replicating RNA is in the order from 5' to 3', a) A nucleotide sequence encoding a chemoattractant operably linked to a regulatory element, and b) The self-replicating RNA according to claim 1 or 2, comprising a nucleotide sequence encoding an antigen operably linked to a regulatory element.

5. The self-replicating RNA according to any one of claims 1 to 4, wherein the regulatory element is selected from the group consisting of a promoter, IRES, and Kozak consensus sequence.

6. The self-replicating RNA is in the order from 5' to 3', a) A nucleotide sequence encoding an antigen operably linked to the SG promoter, and b) The self-replicating RNA according to claim 5, comprising a nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

7. The self-replicating RNA is in the order from 5' to 3', a) A nucleotide sequence encoding a chemoattractant operably linked to the SG promoter, and b) The self-replicating RNA according to claim 5, comprising a nucleotide sequence encoding an antigen operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES).

8. The self-replicating RNA according to claim 5, wherein the promoter is a subgenome (SG) promoter.

9. The self-replicating RNA according to claim 8, wherein the SG promoter is a minimal SG promoter or an extended SG promoter.

10. The self-replicating RNA according to claim 9, 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.

11. The self-replicating RNA according to claim 9, wherein the minimum SG promoter is encoded by the sequence shown in SEQ ID NO:

32.

12. The self-replicating RNA according to claim 9, wherein the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 33, 34, 36, 46, 51, or 52.

13. The self-replicating RNA according to claim 5, wherein the nucleotide sequence encoding the chemoattractant is operably linked to an IRES located 5' with respect to the nucleotide sequence encoding the chemoattractant.

14. The self-replicating RNA according to claim 5 or 13, wherein the IRES is an IRES 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 inhibitor of apoptosis (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or a combination thereof.

15. The self-replicating RNA according to claim 14, wherein the EMCV IRES is a wild-type IRES encoded by the sequence shown in Sequence ID No.

35.

16. The self-replicating RNA according to any one of claims 1 to 15, wherein the self-replicating RNA is a monocistronic self-replicating RNA.

17. The self-replicating RNA according to any one of claims 1 to 15, wherein the self-replicating RNA is a polycistronic self-replicating RNA.

18. The self-replicating RNA according to any one of claims 1 to 17, wherein the antigen is selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumonia virus, rhinovirus, coronavirus, adenovirus, and bocavirus.

19. The self-replicating RNA according to claim 2, wherein the chemical attractant is a chemerin having a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or about 100% sequence identity with respect to any one of sequence numbers 10, 13, 16, or 19.

20. The self-replicating RNA according to claim 19, wherein the chemerin nucleotide has the sequence described in SEQ ID NO:

19.

21. The self-replicating RNA according to claim 2, wherein the chemical attractant is selected from the group consisting of CXCL9, CXCL10, and CXCL11, each having a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity with SEQ ID NOs: 1, 4, or 7.

22. The self-replicating RNA according to any one of claims 1 to 21, wherein the self-replicating RNA is derived from an alphavirus.

23. The self-replicating RNA according to claim 22, wherein the alphavirus is selected from the group consisting of Semliki Forest Virus (SFV), Sindobis Virus (SIN), Venezuelan Encephalitis Virus (VEEV), and combinations thereof.

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

25. An immunogenic composition comprising the self-replicating RNA described in any one of claims 1 to 24.

26. The immunogenic composition according to claim 25, comprising a plurality of self-replicating RNAs as described in any one of claims 1 to 24, wherein each self-replicating RNA encodes a different polypeptide sequence.

27. The immunogenic composition according to claim 25, comprising a plurality of self-replicating RNAs as described in any one of claims 1 to 24, wherein each self-replicating RNA encodes the same polypeptide sequence.

28. The immunogenic composition according to any one of claims 25 to 27, wherein the immunogenic composition further comprises a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

29. a) Self-replicating RNA comprising a first nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) An immunogenic composition comprising a chemoattractant polypeptide optionally selected from the group consisting of chemerin, CXCL9, CXCL10, and CXCL11.

30. (i) One or more antigens, (ii) One or more immunostimulants, (iii) One or more chemoattractants, and / or (iv) The immunogenic composition according to any one of claims 25 to 29, further comprising additional RNA encoding one or more targeting molecules.

31. The immunogenic composition according to claim 29 or 30, wherein the regulatory element is a promoter, an internal ribosome entry site (IRES), a Kozak consensus sequence, or a combination thereof.

32. The immunogenic composition according to claim 31, wherein the promoter is a subgenome (SG) promoter.

33. The immunogenic composition according to claim 32, wherein the SG promoter is a minimum SG promoter or an extended SG promoter.

34. The immunogenic composition according to claim 32, wherein the minimum SG promoter is encoded by the sequence shown in SEQ ID NO:

32.

35. The immunogenic composition according to claim 32, wherein the extended SG promoter is encoded by the sequence shown in SEQ ID NO: 33, 34, 36, 46, 51, or 52.

36. The immunogenic composition according to claim 31, wherein the nucleotide sequence encoding a chemoattractant is operably linked to an IRES located at 5' with respect to the nucleotide sequence encoding the chemoattractant.

37. The immunogenic composition according to claim 36, 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 inhibitor of apoptosis (XIAP or Apaf-1), immunoglobulin heavy chain binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or a combination thereof.

38. The immunogenic composition according to claim 37, wherein the EMCV IRES is a wild-type IRES encoded by the sequence shown in Sequence ID No.

35.

39. The immunogenic composition according to any one of claims 29 to 38, wherein the antigen is selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumonia virus, rhinovirus, coronavirus, adenovirus, and bocavirus.

40. The immunogenic composition according to any one of claims 29 to 39, wherein the chemical attractant polypeptide is selected from the group consisting of chemerin, C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), and C-X-C motif chemokine ligand 11 (CXCL11).

41. The immunogenic composition according to claim 40, wherein the chemical attractant polypeptide is a chemerin comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any one of SEQ ID NOs: 12, 15, 18, or 21.

42. The immunogenic composition according to claim 41, wherein the chemerin polypeptide has the amino acid sequence shown in SEQ ID NO:

21.

43. The immunogenic composition according to claim 40, wherein the chemical attractant polypeptide is CXCL9, CXCL10, or CXCL11, each containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity with any one of SEQ ID NOs: 3, 6, or 9.

44. The immunogenic composition according to any one of claims 29 to 43, wherein the self-replicating RNA is derived from an alphavirus.

45. The immunogenic composition according to claim 44, wherein the alphavirus is selected from the group consisting of Semliki Forest Virus (SFV), Sindbisvirus (SIN), Venezuelan Encephalitis Virus (VEE), and combinations thereof.

46. The immunogenic composition according to claim 45, wherein the alphavirus is VEEV, and the antigen is an H5 antigen from influenza, preferably having a sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO:

49.

47. A pharmaceutical composition comprising an immunogenic composition according to any one of claims 25 to 46 and a pharmaceutically acceptable carrier.

48. The pharmaceutical composition according to claim 47, further comprising lipid nanoparticles (LNPs), polymer microparticles, or an oil-in-water emulsion.

49. The pharmaceutical composition according to claim 48, wherein the self-replicating RNA is encapsulated in, bound to, or adsorbed to LNPs, polymer microparticles, or an oil-in-water emulsion.

50. The pharmaceutical composition according to any one of claims 47 to 49, wherein each RNA is formulated together in the LNP.

51. The pharmaceutical composition according to any one of claims 47 to 50, wherein each RNA is separately formulated in the LNP.

52. An immunogenic composition according to any one of claims 25 to 46, or a pharmaceutical composition according to any one of claims 48 to 51, for use as a vaccine.

53. A vaccine comprising a pharmaceutical composition according to any one of claims 48 to 51, or an immunogenic composition according to any one of claims 25 to 46.

54. A polynucleotide encoding the self-replicating RNA described in any one of claims 1 to 24.

55. The polynucleotide according to claim 54, wherein the polynucleotide is recombinant DNA.

56. The polynucleotide according to claim 55, wherein the recombinant DNA is a plasmid.

57. The polynucleotide according to claim 56, wherein the plasmid comprises the sequence shown in Sequence ID No.

50.

58. It is a polynucleotide, a) A first nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) comprising a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), A polynucleotide in which the chemical attractant is optionally selected from the group consisting of chemerin, C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), and C-X-C motif chemokine ligand 11 (CXCL11).

59. The polynucleotides are arranged in the order from 5' to 3', a) A first nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) The polynucleotide according to claim 58, comprising a second nucleotide sequence encoding a chemoattractant operably linked to an IRES or SG promoter.

60. Conventional mRNA (cRNA), a) A first nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) comprising a second nucleotide sequence encoding a chemoattractant operably linked to a regulatory element selected from the group consisting of an SG promoter and an internal ribosome entry site (IRES), A conventional mRNA (cRNA) in which, at the discretion of selection, the chemical attractant is selected from the group consisting of chemerin, C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), and C-X-C motif chemokine ligand 11 (CXCL11).

61. The cRNA is arranged in the order from 5' to 3', a) A first nucleotide sequence encoding an antigen operably linked to a regulatory element, and b) The cRNA according to claim 60, comprising a second nucleotide sequence encoding a chemoattractant operably linked to an IRES or SG promoter.

62. The polynucleotide according to any one of claims 54 to 59, or the cRNA according to claim 60 or 61, wherein the first nucleotide sequence is operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, IRES, an SG promoter, and combinations thereof.

63. A method for carrying out treatment or prevention or delay of the progression of a disease or condition in a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53.

64. Use of a pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53 in the manufacture of a pharmaceutical product for carrying out treatment or prevention or delay of progression of a disease or condition in a subject that requires such treatment or prevention or delay of progression.

65. A pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53, used for use in subjects requiring treatment or prevention or delay of the progression of a disease or condition.

66. A method for inducing an immune response in a subject, comprising administering to the subject in need of such response the pharmaceutical composition according to any one of claims 47 to 51, the immunogenic composition according to any one of claims 25 to 46, or the vaccine according to claim 53.

67. Use of a pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53 in the manufacture of a pharmaceutical product for inducing an immune response in a subject requiring such induction.

68. A pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53, for use in inducing an immune response in subjects requiring induction of an immune response.

69. A self-replicating RNA, pharmaceutical composition, immunogenic composition, or vaccine for the method according to claim 66, the use according to claim 67, or the use according to claim 68, wherein the immune response is a humoral and / or cell-mediated immune response.

70. A method for reducing the viral load in a subject, comprising administering to the subject in need of such reduction the pharmaceutical composition described in any one of claims 47 to 51, the immunogenic composition described in any one of claims 25 to 46, or the vaccine described in claim 53.

71. The use of a pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53 in the preparation of a pharmaceutical for carrying out a reduction in viral load in a subject that requires such reduction.

72. A pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53, for use in subjects requiring a reduction in viral load.

73. A pharmaceutical composition, immunogenic composition, or vaccine for the method according to any one of claims 63, 66, 69, or 70, the use according to any one of claims 64, 67, 69, or 71, or the use according to any one of claims 65, 68, 69, or 72, wherein the subject is a human being 18 years of age or older.

74. A pharmaceutically acceptable composition, immunogenic composition, or vaccine for the use described in any one of claims 63, 66, 69, or 70, the use described in any one of claims 64, 67, 69, or 71, or the use described in any one of claims 65, 68, 69, or 72, wherein the vaccine or composition is administered in a single-dose regimen.

75. A pharmaceutically acceptable composition, immunogenic composition, or vaccine for the use described in any one of claims 63, 66, 69, or 70, wherein the vaccine or composition is administered in two, three, or four dose regimens, with the doses administered at intervals of about one, two, or three months; the use described in any one of claims 64, 67, 69, or 71; or the use described in any one of claims 65, 68, 69, or 72.

76. It's a kit, (a) Self-replicating RNA according to any one of claims 1 to 24, a pharmaceutical composition according to any one of claims 47 to 51, an immunogenic composition according to any one of claims 25 to 46, or a vaccine according to claim 53 (b) the user manual, and optionally, (c) A kit comprising a pharmaceutically acceptable carrier, excipient, or diluent.

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