RNA-encoding virus-like particles and uses thereof

Abstract

The present disclosure relates to compositions comprising one or more ribonucleic acids (RNAs) encoding influenza virus-like particle (VLP)-forming elements. The present disclosure further provides uses of the compositions.

Description

[Technical Field]

[0001] Related application data This application claims priority from U.S. Patent Application No. 63 / 370,725 (filed August 8, 2022, entitled "RNA-Encoded Virus-Like Particles and Uses Thereof"), the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to compositions comprising one or more ribonucleic acids (RNAs) encoding influenza virus-like particle (VLP)-forming elements. The present disclosure further provides uses of the compositions. [Background technology]

[0004] Influenza virus infection is a serious threat to human health and life. The World Health Organization (WHO) estimates that approximately 1 billion cases of influenza occur each year, of which 3 to 5 million are severe cases and 290,000 to 650,000 result in influenza-related respiratory deaths. Currently, influenza infections are treated with antiviral drugs or other medications.

[0005] Influenza virus vaccines rely on the induction of antibodies, which protect against infection by neutralizing virions or blocking the virus from entering cells. The humoral immune response targets viral surface proteins. Various adjuvants and immunopotentiators have been included in vaccine formulations to enhance immune responses (e.g., antibody responses) against influenza virus surface proteins. However, safety and efficacy issues remain.

[0006] Currently, the most common method for producing influenza vaccines is an egg-based manufacturing process. This process requires significant time to optimize virus growth in eggs and also requires a significant number of eggs to produce sufficient vaccine quantities (especially during a pandemic). Furthermore, given the long development period, vaccine strain selection occurs before the vaccine is available, making it difficult to adapt to changes in the virus. Influenza vaccines have also been produced using cell-based manufacturing processes (involving the cultivation of mammalian cells (Madin-Darby canine kidney cells) instead of eggs) and virus-based manufacturing processes (involving recombinant viruses (e.g., baculoviruses encoding influenza antigens)).

[0007] There remains a need for the development of specific and efficient influenza vaccines for the treatment or prevention of influenza that can be produced more quickly than current egg-based techniques. Although nucleic acid-based vaccines offer distinct advantages over current egg-based, cell-based, and virus-based manufacturing platforms, several challenges remain. For example, nucleic acid-based vaccines that produce antigenic portions of pathogens are often poorly recognized by the immune system, which can limit the ability of nucleic acid-based vaccines to induce strong and sustained immune responses. Therefore, it will be apparent to those skilled in the art that there is a need for influenza virus vaccines that improve the induction of immune responses in subjects. Summary of the Invention

[0008] The present disclosure is based on the inventors' search for compositions with improved ability to elicit an immune response against influenza virus in a subject. Specifically, the inventors have determined that including a sequence encoding the influenza virus matrix-1 (M1) structural protein, along with the influenza virus antigens hemagglutinin (HA) and neuraminidase (NA), in the ribonucleic acid(s) (RNA(s)) of the composition enables the expression of influenza virus virus-like particles (VLPs). This is expected to elicit improved recognition of influenza virus antigens by the immune system compared to airborne influenza virus antigens (e.g., HA alone). Furthermore, the inventors have determined that the stability of VLPs produced from the compositions described herein can be increased by increasing the incorporation of M1 protein into VLPs. The inventors have also determined that the efficient release of VLPs, for example, from cells, can be increased by increasing the incorporation of NA protein into VLPs.

[0009] The inventors' findings provide the basis for compositions comprising one or more ribonucleic acids (RNAs) encoding the VLP-forming elements of influenza virus: hemagglutinin (HA), neuraminidase (NA), and matrix-1 (M1) proteins, and for methods of using the compositions to treat, prevent, or delay the progression of influenza in a subject.

[0010] Accordingly, the present disclosure provides compositions comprising one or more ribonucleic acids (RNAs), each RNA comprising, in 5' to 3' order: a) a first nucleotide sequence comprising a 5'-untranslated region (5'-UTR), a fragment and / or a variant thereof; b) one or more nucleotide sequence(s) encoding virus-like particle (VLP) forming elements; c) a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR), a fragment and / or a variant thereof; the VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein; The composition includes a nucleotide sequence encoding each of the HA protein, the NA protein, and the M1 protein.

[0011] In one example, when introduced into a cell, the expression level of the M1 protein is higher than the expression level of the HA protein and / or the NA protein. As exemplified herein, the inventors have shown that increasing the expression level of the M1 protein increases the production of intact VLPs and / or the stability of the resulting VLPs.

[0012] The present disclosure provides a method for increasing the production of intact VLPs and / or the stability of VLPs, the method comprising introducing a nucleotide sequence encoding an influenza virus matrix-1 (M1) protein into a composition, the composition comprising one or more ribonucleic acids (RNAs), each RNA being in 5' to 3' order: a) a first nucleotide sequence comprising a 5'-untranslated region (5'-UTR), a fragment and / or a variant thereof; b) one or more nucleotide sequence(s) encoding virus-like particle (VLP) forming elements; c) a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR), a fragment and / or a variant thereof; the VLP-forming elements are selected from the hemagglutinin (HA) protein, neuraminidase (NA) protein, and M1 protein of influenza viruses; The composition comprises a nucleotide sequence encoding each of an HA protein, an NA protein, and an M1 protein; After introducing the RNA(s) into cells, the M1 protein is expressed at a higher level than the HA and / or NA proteins.

[0013] The present disclosure further provides a method for increasing the efficient release of virus-like particles (VLPs), the method comprising introducing a nucleotide sequence encoding an influenza virus neuraminidase (NA) protein into a composition, the composition comprising one or more ribonucleic acids (RNAs), each RNA being arranged in 5' to 3' order as follows: a) a first nucleotide sequence comprising a 5'-untranslated region (5'-UTR), a fragment and / or a variant thereof; b) one or more nucleotide sequence(s) encoding virus-like particle (VLP) forming elements; c) a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR), a fragment and / or a variant thereof; the VLP-forming elements are selected from the hemagglutinin (HA) protein, NA protein, and M1 protein of influenza viruses; The composition comprises a nucleotide sequence encoding each of an HA protein, an NA protein, and an M1 protein; After introducing the RNA(s) into cells, the NA protein is expressed at a higher level than the M1 or HA protein.

[0014] The present disclosure also provides a method for increasing the stability and efficient release of virus-like particles (VLPs), the method comprising introducing a nucleotide sequence encoding an influenza virus matrix-1 (M1) protein and a nucleotide sequence encoding a neuraminidase (NA) protein into a composition, the composition comprising one or more ribonucleic acids (RNAs), each RNA being arranged in 5' to 3' order: a) a first nucleotide sequence comprising a 5'-untranslated region (5'-UTR), a fragment and / or a variant thereof; b) one or more nucleotide sequence(s) encoding virus-like particle (VLP) forming elements; c) a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR), a fragment and / or a variant thereof; the VLP-forming elements are selected from the hemagglutinin (HA) protein, NA protein, and M1 protein of influenza viruses; The composition comprises a nucleotide sequence encoding each of an HA protein, an NA protein, and an M1 protein; Introducing nucleotide sequences encoding the M1 and NA proteins into the composition increases the stability and efficient release of VLPs produced from the composition.

[0015] In one example, the composition comprises RNA comprising a nucleotide sequence encoding an HA protein, a nucleotide sequence encoding an NA protein, and a nucleotide sequence encoding an M1 protein.

[0016] In one example, the composition comprises RNA comprising, in 5' to 3' order, a nucleotide sequence encoding an HA protein, a nucleotide sequence encoding an NA protein, and a nucleotide sequence encoding an M1 protein. In one example, the sequence encoding the NA protein is linked to an extended subgenomic promoter, and the sequence encoding the M1 protein is linked to an extended subgenomic promoter. In one example, the sequence encoding the NA protein is linked to extended subgenomic promoter v2, and the sequence encoding the M1 protein is linked to subgenomic promoter v1.

[0017] In one example, the composition comprises a first and a second RNA, wherein the first RNA comprises a nucleotide sequence encoding an HA protein, an NA protein, or an M1 protein, and the second RNA comprises a) HA protein and M1 protein, b) the NA protein and the M1 protein, or c) It comprises a nucleotide sequence encoding a combination of an HA protein and an NA protein, and the first RNA and the second RNA encode different VLP-forming elements.

[0018] For example, the first RNA comprises a nucleotide sequence encoding an HA protein, and the second RNA comprises a nucleotide sequence encoding an M1 protein and a nucleotide sequence encoding an NA protein. For example, the first RNA comprises a nucleotide sequence encoding an NA protein, and the second RNA comprises a nucleotide sequence encoding an M1 protein and a nucleotide sequence encoding an HA protein. For example, the first RNA comprises a nucleotide sequence encoding an M1 protein, and the second RNA comprises a nucleotide sequence encoding an HA protein and a nucleotide sequence encoding an NA protein.

[0019] In one example, the composition comprises a first RNA comprising a nucleotide sequence encoding an HA protein, a second RNA comprising a nucleotide sequence encoding an NA protein, and a third RNA comprising a nucleotide sequence encoding an M1 protein.

[0020] In one example, the disclosure provides a composition comprising RNA comprising sequences encoding an HA protein, an NA protein, and an M1 protein.

[0021] In one example, the order of the coding sequences (5'-3') is a sequence encoding the HA protein, a sequence encoding the NA protein, and a sequence encoding the M1 protein. In one example, the sequence encoding the NA protein and the sequence encoding the M1 protein are each linked to a subgenomic promoter. For example, the sequence encoding the NA protein is linked to subgenomic promoter v2, and the sequence encoding the M1 protein is linked to subgenomic promoter v1. For example, the sequence encoding the NA protein is linked to subgenomic promoter v2, and the sequence encoding the M1 protein is linked to subgenomic promoter v2.

[0022] In one example, the disclosure provides a composition comprising RNA comprising sequences encoding an HA protein, an NA protein, an M1 protein, and an M2 protein.

[0023] In one example, the order of the coding sequences (5'-3') is a sequence encoding the M1 protein, a sequence encoding the M2 protein, a sequence encoding the NA protein, and a sequence encoding the HA protein. In one example, the sequence encoding the M1 protein, the sequence encoding the NA protein, and the sequence encoding the M1 protein are each linked to a subgenomic promoter. In one example, the sequence encoding the M2 protein, the sequence encoding the NA protein, and the sequence encoding the HA protein are each linked to a subgenomic promoter.

[0024] In the above example, an exemplary HA protein is the H5 protein. An exemplary NA protein is the N1 protein.

[0025] In one example, a composition of the disclosure comprises a nucleotide sequence encoding an HA protein, an NA protein, and an M1 protein, a) the HA protein is an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18 protein; b) The NA protein is an N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, or N11 protein.

[0026] For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N1 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N2 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N3 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1, H1, and N6 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N7 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N8 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N9 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N10 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H1 protein, and the N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N8 protein.For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H2 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H3 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N5 protein.For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H4 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H5 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N2 protein.For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H6 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N10 protein.For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H7 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H8 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N7 protein.For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H9 protein, and an N10 protein. The composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N11 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N2 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N3 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N4 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N5 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N6 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N7 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N8 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N9 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H10 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H10 protein, and the N11 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N1 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N2 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N3 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N6 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N7 protein.For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N8 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N9 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N10 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H11 protein, and the N11 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N1 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N2 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N3 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N6 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H12 protein, and the N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H12 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H12 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H12 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H12 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H13 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H13 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H13 protein, and an N3 protein.For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N6 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N7 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N8 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N9 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N10 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H13 protein, and the N11 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N1 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N2 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N3 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N6 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N7 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N8 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N9 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N10 protein.For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H14 protein, and the N11 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N1 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N2 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N3 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N6 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N7 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N8 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N9 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H15 protein, and the N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H15 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H16 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H16 protein, and an N2 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H16 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H16 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H16 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H16 protein, and an N6 protein.For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H16 protein, and the N7 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H16 protein, and the N8 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H16 protein, and the N9 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H16 protein, and the N10 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H16 protein, and the N11 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H17 protein, and the N1 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H17 protein, and the N2 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H17 protein, and the N3 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H17 protein, and the N4 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H17 protein, and the N5 protein. For example, the composition comprises a nucleotide sequence encoding the M1 protein, the H17 protein, and the N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H17 protein, and an N7 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H17 protein, and an N8 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H17 protein, and an N9 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H17 protein, and an N10 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H17 protein, and an N11 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N1 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N2 protein.For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N3 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N4 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N5 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N6 protein. For example, the composition comprises a nucleotide sequence encoding an M1 protein, an H18 protein, and an N7 protein. For example, the composition encodes an M1 protein, an H18 protein, and an N8 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H18 protein, and an N9 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H18 protein, and an N10 protein. For example, the composition includes a nucleotide sequence encoding an M1 protein, an H18 protein, and an N11 protein.

[0027] In one example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0028] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0029] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0030] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0031] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0032] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0033] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0034] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0035] for example, a) the nucleotide sequence encoding the HA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0036] In one example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) the nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0037] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0038] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0039] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0040] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0041] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0042] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0043] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0044] for example, a) the nucleotide sequence encoding the HA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0045] In one example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0046] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0047] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0048] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0049] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0050] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0051] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0052] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0053] for example, a) the nucleotide sequence encoding the HA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0054] In one example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) the nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0055] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0056] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) the nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0057] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0058] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) the nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0059] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0060] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) the nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0061] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0062] for example, a) the nucleotide sequence encoding the HA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) the nucleotide sequence encoding the M1 protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0063] In one example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0064] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0065] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0066] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0067] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0068] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0069] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0070] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0071] for example, a) the nucleotide sequence encoding the HA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0072] In one example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0073] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0074] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0075] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0076] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0077] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0078] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0079] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0080] for example, a) the nucleotide sequence encoding the HA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0081] In one example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) the nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0082] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0083] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0084] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0085] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0086] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0087] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0088] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0089] for example, a) the nucleotide sequence encoding the HA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0090] In one example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0091] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0092] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0093] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0094] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0095] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0096] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0097] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0098] for example, a) the nucleotide sequence encoding the HA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0099] In one example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0100] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0101] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0102] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0103] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0104] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0105] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0106] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0107] for example, a) the nucleotide sequence encoding the HA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0108] In one example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 66 or 70.

[0109] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0110] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0111] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0112] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0113] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0114] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0115] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:66.

[0116] for example, a) the nucleotide sequence encoding the HA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:70.

[0117] In one example, a) the nucleotide sequence encoding the HA protein is selected from the nucleotide sequences set forth in SEQ ID NO: 64 or 71; b) the nucleotide sequence encoding the NA protein is selected from the nucleotide sequences set forth in SEQ ID NO: 65 or 72; c) The nucleotide sequence encoding the M1 protein is selected from the nucleotide sequences set forth in SEQ ID NO: 66 or 70.

[0118] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:66.

[0119] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:70.

[0120] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:66.

[0121] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 64; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:70.

[0122] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:66.

[0123] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 65; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:70.

[0124] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:66.

[0125] for example, a) the nucleotide sequence encoding the HA protein comprises the nucleotide sequence set forth in SEQ ID NO: 71; b) the nucleotide sequence encoding the NA protein comprises the nucleotide sequence set forth in SEQ ID NO: 72; c) The nucleotide sequence encoding the M1 protein comprises the nucleotide sequence set forth in SEQ ID NO:70.

[0126] In one example, the VLP-forming elements are derived from the same influenza virus.

[0127] In one example, two or more of the VLP-forming elements are derived from different influenza viruses, for example, two of the VLP-forming elements are derived from different influenza viruses, or for example, three of the VLP-forming elements are derived from different influenza viruses.

[0128] In one example, one or more RNA(s) comprise one or more additional nucleotide sequences encoding influenza virus matrix-2 (M2), nucleoprotein (NP), and / or nonstructural (NS) proteins, wherein the one or more additional nucleotide sequences are located 3' or 5' to the one or more nucleotide sequence(s) encoding VLP-forming elements. For example, one or more RNA(s) comprise an additional nucleotide sequence encoding matrix-2 (M2). For example, one or more RNA(s) comprise an additional nucleotide sequence encoding nucleoprotein (NP) protein. For example, one or more RNA(s) comprise an additional nucleotide sequence encoding a nonstructural (NS) protein. For example, the NS protein is nonstructural 1 (NS1) protein. For example, the NS protein is nonstructural 2 (NS2) protein. For example, one or more RNA(s) comprise an additional nucleotide sequence encoding an M2 protein and an additional nucleotide sequence encoding an NP protein. For example, one or more RNA(s) comprise an additional nucleotide sequence encoding an M2 protein and an additional sequence encoding an NS protein. For example, one or more RNA(s) include an additional nucleotide sequence encoding the NP protein and an additional sequence encoding the NS protein. For example, one or more RNA(s) include an additional nucleotide sequence encoding the M2 protein, an additional sequence encoding the NP protein, and an additional sequence encoding the NS protein.

[0129] In one example, a) the nucleotide sequence encoding the NP protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0130] In one example, a) the nucleotide sequence encoding the NP protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 91% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0131] In one example, a) the nucleotide sequence encoding the NP protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 92% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0132] In one example, a) the nucleotide sequence encoding the NP protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 93% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0133] In one example, a) the nucleotide sequence encoding the NP protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0134] In one example, a) the nucleotide sequence encoding the NP protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0135] In one example, a) the nucleotide sequence encoding the NP protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 96% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0136] In one example, a) the nucleotide sequence encoding the NP protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0137] In one example, a) the nucleotide sequence encoding the NP protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0138] In one example, a) the nucleotide sequence encoding the NP protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 69; b) the nucleotide sequence encoding the NS protein is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:68.

[0139] In one example, a) the nucleotide sequence encoding the NP protein comprises the nucleotide sequence set forth in SEQ ID NO: 69; b) The nucleotide sequence encoding the NS protein comprises the nucleotide sequence set forth in SEQ ID NO:68.

[0140] In one example, the nucleoprotein (NP) and / or nonstructural (NS) proteins are from the same influenza virus.

[0141] In one example, the nucleoprotein (NP) and / or nonstructural (NS) proteins are from different influenza viruses.

[0142] In one example, the first nucleotide sequence comprises the 5'-UTR of haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha 1 collagen (Col1A), alpha 6 collagen (Col6A), alpha 1-antitrypsin (SERPINA1), alpha 1-antichymotrypsin (SERPINA3), arachidonate 5-lipoxygenase (ALOX5), tyrosine hydroxylase (TH gene), tumor protein P53-induced protein 3 (TP5313), fragments thereof, and / or variants thereof. In one example, the first nucleotide sequence includes the 5'-UTR of haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), alpha 1 collagen (Col1A), alpha 6 collagen (Col6A), alpha 1-antitrypsin (SERPINA1), alpha 1-antichymotrypsin (SERPINA3), arachidonate 5-lipoxygenase (ALOX5), tyrosine hydroxylase (TH gene), tumor protein P53-inducible protein 3 (TP5313), alphavirus, or a fragment and / or variant thereof. For example, the first nucleotide sequence includes the 5'-UTR of haptoglobin (HP). For example, the first nucleotide sequence includes the 5'-UTR of fibrinogen beta chain (FGB). For example, the first nucleotide sequence includes the 5'-UTR of haptoglobin-related protein (HPR). For example, the first nucleotide sequence includes the 5'-UTR of albumin (ALB). For example, the first nucleotide sequence includes the 5'-UTR of complement component 3 (C3). For example, the first nucleotide sequence includes the 5'-UTR of fibrinogen alpha chain (FGA). For example, the first nucleotide sequence includes the 5'-UTR of alpha 1 collagen (Col1A). For example, the first nucleotide sequence includes the 5'-UTR of alpha 6 collagen (Col6A). For example, the first nucleotide sequence includes the 5'-UTR of alpha-1 antitrypsin (SERPINA1).For example, the first nucleotide sequence includes the 5'-UTR of alpha-1-antichymotrypsin (SERPINA3). For example, the first nucleotide sequence includes the 5'-UTR of arachidonate 5-lipoxygenase (ALOX5). For example, the first nucleotide sequence includes the 5'-UTR of tyrosine hydroxylase (TH gene). For example, the first nucleotide sequence includes the 5'-UTR of tumor protein P53-inducible protein 3 (TP5313). For example, the first nucleotide sequence includes the 5'-UTR of an alphavirus. For example, the first nucleotide sequence includes the 5'-UTR of Venezuelan equine encephalitis virus.

[0143] In one example, the 5'-UTR, its fragments, and / or variants are 40 to 2000 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 40 to 100 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 100 to 250 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 250 to 500 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 500 to 750 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 750 to 1000 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 1000 to 1250 nucleotides in length. For example, the 5'-UTR, its fragments, and / or variants are 1250 to 1500 nucleotides in length. For example, the 5'-UTR, fragments and / or variants thereof are 1500 to 1750 nucleotides in length. For example, the 5'-UTR, fragments and / or variants thereof are 1750 to 2000 nucleotides in length.

[0144] In one example, the 5'-UTR, fragment, and / or variant thereof comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 6-19, 60, and 76. For example, the 5'-UTR, fragment, and / or variant thereof comprises a nucleotide sequence that is 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 6-19, 60, and 76. For example, the 5'-UTR, fragment, and / or variant thereof comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 6. For example, the 5'-UTR, fragment, and / or variant thereof comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 7. For example, the 5'-UTR, fragment, and / or variant thereof comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 8. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 9. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 10. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 11. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 12. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 13. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 14.For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 15. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 16. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 17. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 18. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 19. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 60. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 76.

[0145] In one example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 6-19, 60, and 76. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 6. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 7. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 8. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 9. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 10. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 11. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 12. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 13. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 14. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 15. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 16.For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 17. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 18. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 19. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 60. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 76.

[0146] In one example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 6-19, 60, and 76. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 6. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 7. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 8. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 9. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 10. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 11. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 12. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 13. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 14. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 15. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 16.For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 17. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 18. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 19. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 60. For example, the 5'-UTR, fragments and / or variants thereof comprise a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 76.

[0147] In one example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 6-19 or 60. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 6. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 7. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 8. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 9. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 10. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 11. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 12. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 13. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 14. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 15. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 16. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 17. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 18. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 19. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 60. For example, the 5'-UTR, fragments and / or variants thereof comprise the nucleotide sequence set forth in SEQ ID NO: 76.

[0148] In one example, the 5'-UTR is a synthetic 5'-UTR.

[0149] In one example, the 5'-UTR is a synthetic 5'-UTR comprising a nucleotide sequence at least 90% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 20-39 or 62. For example, the synthetic 5'-UTR comprises a nucleotide sequence that is 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 20-39 or 62. For example, the synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 20. For example, the synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 21. For example, the synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 22. For example, the synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 23. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 24. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 25. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 26. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 27. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 28. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 29. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 30. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 31. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 32.For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 33. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 34. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 35. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 36. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 37. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 38. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 39. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 62.

[0150] In one example, the 5'-UTR is a synthetic 5'-UTR comprising a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 20-39 or 62. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 20. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 21. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 22. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 23. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 24. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 25. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 26. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 27. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 28. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 29. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 30. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 31. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 32. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 33. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 34.For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 35. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 36. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 37. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 38. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 39. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 62.

[0151] In one example, the 5'-UTR is a synthetic 5'-UTR comprising a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 20-39 or 62. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 20. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 21. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 22. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 23. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 24. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 25. For example, the synthetic 5'-UTR comprises a nucleotide sequence at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 26. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 27. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 28. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 29. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 30. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 31. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 32. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 33. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 34.For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 35. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 36. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 37. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 38. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 39. For example, a synthetic 5'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 62.

[0152] In one example, the 5'-UTR is a synthetic 5'-UTR comprising or consisting of the nucleotide sequence set forth in any one of SEQ ID NOs: 20 to 39 or 62. For example, the synthetic 5'-UTR comprises ... SEQ ID NO: 20. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 21. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 22. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 23. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 24. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 25. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 26. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 27. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 28. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 29. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 30. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 31. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 32. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 33. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 34. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 35. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 36. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 37. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 38. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 39. For example, the synthetic 5'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 62.

[0153] For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 20 to 39 or 62. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 20. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 21. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 22. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 23. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 24. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 25. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 26. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 27. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 28. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 29. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 30. For example, a synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 31. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 32. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 33. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 34. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 35. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 36. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 37. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 38. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 39. For example, the synthetic 5'-UTR consists of the nucleotide sequence set forth in SEQ ID NO: 62.

[0154] In one example, the first nucleotide sequence comprises a combination of two or more 5'-UTRs, fragments and / or variants thereof. In one example, two or more 5'-UTRs are the same. In one example, two or more 5'-UTRs are different.

[0155] In one example, the first nucleotide sequence comprises at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, or a combination thereof. For example, the first nucleotide sequence comprises a microRNA binding site. For example, the first nucleotide sequence comprises an AU-rich element (ARE). For example, the first nucleotide sequence comprises a GC-rich element. For example, the first nucleotide sequence comprises a stem-loop.

[0156] In one example, a translation initiation sequence selected from the group consisting of a Kozak consensus sequence, an internal ribosome entry site (IRES), a subgenomic (SG) promoter, and combinations thereof, is operably linked to the 5' end of one or more nucleotide sequence(s) encoding VLP-forming elements and / or one or more additional nucleotide sequences encoding nucleoprotein (NP) and / or nonstructural (NS) proteins. For example, a Kozak consensus sequence is operably linked to the 5' end of one or more nucleotide sequence(s) encoding VLP-forming elements and / or one or more additional nucleotide sequences encoding nucleoprotein (NP) and / or nonstructural (NS) proteins. For example, an IRES is operably linked to the 5' end of one or more nucleotide sequence(s) encoding VLP-forming elements and / or one or more additional nucleotide sequences encoding nucleoprotein (NP) and / or nonstructural (NS) proteins. For example, the SG promoter is operably linked to the 5' end of one or more nucleotide sequence(s) encoding VLP-forming elements and / or one or more additional nucleotide sequences encoding nucleoprotein (NP) and / or nonstructural (NS) proteins.

[0157] In one example, the Kozak consensus sequence comprises or consists of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In one example, the Kozak consensus sequence comprises the sequence set forth in SEQ ID NO: 1. In one example, the Kozak consensus sequence comprises the sequence set forth in SEQ ID NO: 1. In one example, the Kozak consensus sequence comprises the sequence set forth in SEQ ID NO: 2. In one example, the Kozak consensus sequence comprises the sequence set forth in SEQ ID NO: 2.

[0158] In one example, the IRES is derived from poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (eIF4G), death-associated protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF B), antennapedia, X-linked inhibitor of apoptosis (XIAP or Apaf-1), immunoglobulin heavy chain-binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or a combination thereof. For example, the IRES is derived from poliovirus (PV). For example, the IRES is derived from a human enterovirus. For example, the IRES is an IRES from foot-and-mouth disease virus (FMDV). For example, the IRES is an IRES from hepatitis C virus (HCV). For example, the IRES is an IRES from classical swine fever virus (CSFV). For example, the IRES is an IRES from murine leukemia virus (MLV). For example, the IRES is an IRES from simian immunodeficiency virus (SIV). For example, the IRES is an IRES from eukaryotic translation initiation factor 4G (eIF4G). For example, the IRES is an IRES from death-associated protein 5 (DAP5). For example, the IRES is an IRES from cellular Myc (c-Myc). For example, the IRES is an IRES from NF-κB inhibitor (NRF). For example, the IRES is an IRES from vascular endothelial growth factor (VEGF). For example, the IRES is an IRES from fibroblast growth factor (FGF-2). For example, the IRES is an IRES from platelet-derived growth factor B (PDGF B). For example, the IRES is an IRES from antennapedia. For example, the IRES is an IRES from X-linked inhibitor of apoptosis (XIAP or Apaf-1). For example, the IRES is an IRES from immunoglobulin heavy chain-binding protein BiP. For example, the IRES is an IRES from fibroblast growth factor 1a (FGF1A).For example, the IRES is an IRES from GTX.

[0159] In one example, the IRES comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 57 or 58. For example, the IRES comprises a nucleotide sequence that is 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 57 or 58. For example, the IRES comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 57. For example, the IRES comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 58.

[0160] In one example, the IRES comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 57 or 58. For example, the IRES comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 57. For example, the IRES comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 58.

[0161] In one example, the IRES comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 57 or 58. For example, the IRES comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 57. For example, the IRES comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 58.

[0162] In one example, the IRES comprises the nucleotide sequence set forth in SEQ ID NO: 57 or 58. For example, the IRES comprises the nucleotide sequence set forth in SEQ ID NO: 57. For example, the IRES comprises the nucleotide sequence set forth in SEQ ID NO: 58.

[0163] In one example, the SG promoter is a minimal SG promoter or an extended SG promoter.

[0164] In one example, the SG promoter is a minimal SG promoter. For example, the minimal SG promoter is a native SG promoter. For example, the minimal SG promoter is the minimum sequence required for initiation of transcription. In one example, the minimal native SG promoter is 49 nucleotides in length. In another example, the extended SG promoter comprises or consists of SG promoter v1. In one example, the minimal native SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 3. In one example, the SG promoter consists of the sequence set forth in SEQ ID NO: 3.

[0165] In one example, the SG promoter is an extended SG promoter. For example, the extended SG promoter is extended at the 5' end by nucleotides present in the sequence encoding the nonstructural protein of an RNA virus. In one example, the extended SG promoter is extended at the 5' end by nucleotides present in the sequence encoding the alphavirus NSP4. In one example, the extended SG promoter comprises a minimal SG promoter, which is extended at the 5' end by nucleotides present in the sequence encoding the nonstructural protein of an RNA virus. In one example, the extended SG promoter comprises the sequence set forth in SEQ ID NO: 3, which is extended at the 5' end by nucleotides present in the sequence encoding the nonstructural protein of an RNA virus.

[0166] In one example, the SG promoter is extended at the 5' end by 51 or fewer nucleotides present in the sequence encoding the nonstructural proteins. In one example, the extended SG promoter is a minimal SG promoter extended at the 5' end by 51 or fewer nucleotides present in the sequence encoding the nonstructural proteins.

[0167] In one example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 3, extended at the 5' end by 51 or fewer nucleotides present in the sequence encoding the nonstructural protein. For example, the extended SG promoter is 100 nucleotides or less in length. In one example, the extended SG promoter comprises or consists of nucleotides 2-101 of SEQ ID NO: 4.

[0168] In one example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 4. For example, the extended SG promoter comprises the sequence set forth in SEQ ID NO: 4. For example, the extended SG promoter consists of the sequence set forth in SEQ ID NO: 4.

[0169] In another example, the extended SG promoter comprises or consists of SG promoter v2. In another example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 75. For example, the extended SG promoter comprises the sequence set forth in SEQ ID NO: 75. For example, the extended SG promoter consists of the sequence set forth in SEQ ID NO: 75.

[0170] In another example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 86. For example, the extended SG promoter comprises the sequence set forth in SEQ ID NO: 86. For example, the extended SG promoter consists of the sequence set forth in SEQ ID NO: 86.

[0171] In another example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 87. For example, the extended SG promoter comprises the sequence set forth in SEQ ID NO: 87. For example, the extended SG promoter consists of the sequence set forth in SEQ ID NO: 87.

[0172] In one example, the extended SG promoter comprises the sequence set forth in SEQ ID NO:75 or SEQ ID NO:86 or SEQ ID NO:87.

[0173] The present disclosure further provides a method for the preparation of a 5' to 3' a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding an influenza virus neuraminidase (NA) protein operably linked to a second subgenomic (SG) promoter; and c) providing a composition comprising an RNA comprising a nucleotide sequence encoding the matrix-1 (M1) protein of influenza virus operably linked to a third subgenomic (SG) promoter;

[0174] In one example, a) a nucleotide sequence encoding an influenza virus hemagglutinin (HA) protein operably linked to a first subgenomic (SG) promoter; b) a nucleotide sequence encoding an influenza virus neuraminidase (NA) protein operably linked to a second subgenomic (SG) promoter; and c) A composition is provided, comprising an RNA comprising a nucleotide sequence encoding the matrix-1 (M1) protein of influenza virus operably linked to a third subgenomic (SG) promoter.

[0175] In one example, the RNA is self-replicating RNA or conventional mRNA.

[0176] In one example, the first subgenomic (SG) promoter comprises a native subgenomic (SG) promoter.

[0177] In one example, the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 75. In one example, the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 3. In one example, the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 75.

[0178] In one example, the third subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 75. In one example, the third subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 3. In one example, the third subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 75.

[0179] In one example, the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 75, and the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 3. In one example, the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 3, and the second subgenomic (SG) promoter comprises the sequence set forth in SEQ ID NO: 75.

[0180] In one example, the RNA comprises a 5' UTR (e.g., as defined herein). In one example, the RNA comprises a 3' UTR (e.g., as defined herein). In one example, the RNA comprises a tailing sequence (e.g., as defined herein). In one example, the RNA comprises a 5' cap (e.g., as defined herein).

[0181] The present disclosure also provides a method for increasing the stability of a virus-like particle (VLP), the method comprising introducing into a composition a nucleotide sequence encoding the matrix-1 (M1) protein of influenza virus, the composition comprising the following nucleotide sequence in 5' to 3' order: a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding the neuraminidase (NA) protein of an influenza virus operably linked to a second subgenomic (SG) promoter; c) RNA comprising a nucleotide sequence encoding an M1 protein of an influenza virus operably linked to a third subgenomic (SG) promoter; Introducing a nucleotide sequence encoding the M1 protein into the composition increases the stability of VLPs produced from the composition.

[0182] The present disclosure further provides a method for increasing the efficient release of virus-like particles (VLPs), the method comprising introducing into a composition a nucleotide sequence encoding the neuraminidase (NA) protein of an influenza virus, the composition comprising: a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding the NA protein of an influenza virus operably linked to a second subgenomic (SG) promoter; c) RNA comprising a nucleotide sequence encoding the matrix-1 (M1) protein of influenza virus operably linked to a third subgenomic (SG) promoter; Introducing a nucleotide sequence encoding an NA protein into the composition increases the efficient release of VLPs produced from the composition.

[0183] The present disclosure also provides a method for increasing the stability and efficient release of virus-like particles (VLPs), the method comprising introducing into a composition a nucleotide sequence encoding the matrix-1 (M1) protein and a nucleotide sequence encoding the neuraminidase (NA) protein of influenza virus, the composition comprising: a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding the NA protein of an influenza virus operably linked to a second subgenomic (SG) promoter; c) RNA comprising a nucleotide sequence encoding an M1 protein of an influenza virus operably linked to a third subgenomic (SG) promoter; Introducing nucleotide sequences encoding the M1 and NA proteins into the composition increases the stability and efficient release of VLPs produced from the composition.

[0184] In one example, a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus is operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof.

[0185] In one example, a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus is operably linked to a first SG promoter.

[0186] In one example, the first SG promoter and the second SG promoter are the same.

[0187] In one example, the first SG promoter and the second SG promoter are different.

[0188] In one example, the second SG promoter and the third SG promoter are the same.

[0189] In one example, the second SG promoter and the third SG promoter are different.

[0190] In one example, the first SG promoter and the third SG promoter are the same.

[0191] In one example, the first SG promoter and the third SG promoter are different.

[0192] In one example, the first SG promoter, the second SG promoter, and the third SG promoter are the same.

[0193] In one example, the first SG promoter, the second SG promoter, and the third SG promoter are different.

[0194] In one example, the first subgenomic promoter comprises a minimal native SG promoter. In one example, the first subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO: 3. In one example, the first subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3.

[0195] In one example, the second subgenomic promoter comprises a native SG promoter. In one example, the second subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO:3. In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:3. In one example, the second subgenomic promoter comprises an extended SG promoter. In one example, the second subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO:75. In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:75.

[0196] In one example, the third subgenomic promoter comprises a native SG promoter. In one example, the third subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO: 3. In one example, the third subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3. In one example, the third subgenomic promoter comprises an extended SG promoter. In one example, the third subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO: 75. In one example, the third subgenomic promoter consists of the sequence set forth in SEQ ID NO: 75.

[0197] In one example, the first subgenomic promoter consists of the sequence set forth in SEQ ID NO:3, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:3, and the third subgenomic promoter consists of the sequence set forth in SEQ ID NO:3.

[0198] In one example, the first subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO: 75, and the third subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3. In one example, the first subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO: 75, and the third subgenomic promoter consists of the sequence set forth in SEQ ID NO: 75. In one example, the first subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3, and the third subgenomic promoter consists of the sequence set forth in SEQ ID NO: 75.

[0199] In one example, the second nucleotide sequence comprises creatine kinase, globin, α-actin, albumin, granulocyte colony-stimulating factor (G-CSF), collagen, ribophorin I (RPNI), low-density lipoprotein receptor-related protein 1 (LRP1), cardiotrophin-like cytokine factor 1 (CLCF1), calreticulin (Calr), procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 (Plodl), nucleobindin 1 (Nucbl), amino-terminal split enhancer (AES), the 3'-UTR of human mitochondrial 12S rRNA (mtRNR1), fragments and / or variants thereof. For example, the second nucleotide sequence comprises the 3'-UTR of creatine kinase, fragments and / or variants thereof. In one example, the second nucleotide sequence comprises the 3'-UTR, fragments and / or variants thereof, of creatine kinase, globin, α-actin, albumin, granulocyte colony-stimulating factor (G-CSF), collagen, ribophorin I (RPNI), low-density lipoprotein receptor-related protein 1 (LRP1), cardiotrophin-like cytokine factor 1 (CLCF1), calreticulin (Calr), procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 (Plodl), nucleobindin 1 (Nucbl), amino-terminal split enhancer (AES), human mitochondrial 12S rRNA (mtRNR1), or alphavirus. For example, the second nucleotide sequence comprises the 3'-UTR, fragments and / or variants thereof, of globin. For example, the second nucleotide sequence comprises the 3'-UTR, fragments and / or variants thereof, of α-actin. For example, the second nucleotide sequence includes the 3'-UTR of albumin, a fragment and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of granulocyte colony-stimulating factor (G-CSF), a fragment and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of collagen, a fragment and / or a variant thereof. For example, the second nucleotide sequence includes ribophorin I (RPNI), a fragment and / or a variant thereof.For example, the second nucleotide sequence includes the 3'-UTR of low-density lipoprotein receptor-related protein 1 (LRP1), a fragment thereof, and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of cardiotrophin-like cytokine factor 1 (CLCF1), a fragment thereof, and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of calreticulin (Calr), a fragment thereof, and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 (Plodl), a fragment thereof, and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of nucleobindin 1 (Nucbl), a fragment thereof, and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of amino-terminal split enhancer (AES), a fragment thereof, and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of human mitochondrial 12S rRNA (mtRNR1), a fragment and / or a variant thereof. For example, the second nucleotide sequence includes the 3'-UTR of an alphavirus. For example, the second nucleotide sequence includes the 3'-UTR of Venezuelan equine encephalitis virus. For example, the second nucleotide sequence includes the 3'-UTR of Sindbis virus.

[0200] In one example, the 3'-UTR is 40 to 400 nucleotides in length. For example, the 3'-UTR is 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, 225 to 250, 250 to 275, 275 to 300, 300 to 325, 325 to 350, 350 to 375, or 375 to 400 nucleotides in length. For example, the 3'-UTR is 40 to 50 nucleotides in length. For example, the 3'-UTR is 50 to 60 nucleotides in length. For example, the 3'-UTR is 60 to 70 nucleotides in length. For example, the 3'-UTR is 70 to 80 nucleotides long. For example, the 3'-UTR is 80 to 90 nucleotides long. For example, the 3'-UTR is 90 to 100 nucleotides long. For example, the 3'-UTR is 100 to 125 nucleotides long. For example, the 3'-UTR is 125 to 150 nucleotides long. For example, the 3'-UTR is 150 to 175 nucleotides long. For example, the 3'-UTR is 175 to 200 nucleotides long. For example, the 3'-UTR is 200 to 225 nucleotides long. For example, the 3'-UTR is 225 to 250 nucleotides long. For example, the 3'-UTR is 250 to 275 nucleotides long. For example, the 3'-UTR is 275 to 300 nucleotides long. For example, the 3'-UTR is 300 to 325 nucleotides long. For example, the 3'-UTR is 325 to 350 nucleotides long. For example, the 3'-UTR is 350 to 375 nucleotides in length. For example, the 3'-UTR is 375 to 400 nucleotides in length.

[0201] In one example, 3'-UTR is at least 40 nucleotides in length.For example, 3'-UTR is at least 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95, or 100, or 110, or 120, or 130, or 140, or 150, or 160, or 170, or 180, or 190, or 200, or 210, or 220, or 230, or 240, or 250, or 260, or 270, or 280, or 290, or 300, or 310, or 320, or 330, or 340, or 350, or 360, or 370, or 380, or 390 nucleotides in length.For example, 3'-UTR is at least 45 nucleotides in length. For example, the 3'-UTR is at least 50 nucleotides in length. For example, the 3'-UTR is at least 55 nucleotides in length. For example, the 3'-UTR is at least 60 nucleotides in length. For example, the 3'-UTR is at least 65 nucleotides in length. For example, the 3'-UTR is at least 70 nucleotides in length. For example, the 3'-UTR is at least 75 nucleotides in length. For example, the 3'-UTR is at least 80 nucleotides in length. For example, the 3'-UTR is at least 85 nucleotides in length. For example, the 3'-UTR is at least 90 nucleotides in length. For example, the 3'-UTR is at least 95 nucleotides in length. For example, the 3'-UTR is at least 100 nucleotides in length. For example, the 3'-UTR is at least 110 nucleotides in length. For example, the 3'-UTR is at least 120 nucleotides in length. For example, the 3'-UTR is at least 130 nucleotides in length. For example, the 3'-UTR is at least 140 nucleotides in length. For example, the 3'-UTR is at least 150 nucleotides in length. For example, the 3'-UTR is at least 160 nucleotides in length. For example, the 3'-UTR is at least 170 nucleotides in length. For example, the 3'-UTR is at least 180 nucleotides in length. For example, the 3'-UTR is at least 190 nucleotides in length. For example, the 3'-UTR is at least 200 nucleotides in length.For example, the 3'-UTR is at least 210 nucleotides in length. For example, the 3'-UTR is at least 220 nucleotides in length. For example, the 3'-UTR is at least 230 nucleotides in length. For example, the 3'-UTR is at least 240 nucleotides in length. For example, the 3'-UTR is at least 250 nucleotides in length. For example, the 3'-UTR is at least 260 nucleotides in length. For example, the 3'-UTR is at least 270 nucleotides in length. For example, the 3'-UTR is at least 280 nucleotides in length. For example, the 3'-UTR is at least 290 nucleotides in length. For example, the 3'-UTR is at least 300 nucleotides in length. For example, the 3'-UTR is at least 310 nucleotides in length. For example, the 3'-UTR is at least 320 nucleotides in length. For example, the 3'-UTR is at least 330 nucleotides in length. For example, the 3'-UTR is at least 340 nucleotides in length. For example, the 3'-UTR is at least 350 nucleotides in length. For example, the 3'-UTR is at least 360 nucleotides in length. For example, the 3'-UTR is at least 370 nucleotides in length. For example, the 3'-UTR is at least 380 nucleotides in length. For example, the 3'-UTR is at least 390 nucleotides in length.

[0202] In one example, the 3'-UTR is 40, or 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95, or 100, or 110, or 120, or 130, or 140, or 150, or 160, or 170, or 180, or 190, or 200, or 210, or 220, or 230, or 240, or 250, or 260, or 270, or 280, or 290, or 300, or 310, or 320, or 330, or 340, or 350, or 360, or 370, or 380, or 390, or 400 nucleotides in length. For example, the 3'-UTR is 40 nucleotides in length. For example, the 3'-UTR is 45 nucleotides in length. For example, the 3'-UTR is 50 nucleotides long. For example, the 3'-UTR is 55 nucleotides long. For example, the 3'-UTR is 60 nucleotides long. For example, the 3'-UTR is 65 nucleotides long. For example, the 3'-UTR is 70 nucleotides long. For example, the 3'-UTR is 75 nucleotides long. For example, the 3'-UTR is 80 nucleotides long. For example, the 3'-UTR is 85 nucleotides long. For example, the 3'-UTR is 90 nucleotides long. For example, the 3'-UTR is 95 nucleotides long. For example, the 3'-UTR is 100 nucleotides long. For example, the 3'-UTR is 110 nucleotides long. For example, the 3'-UTR is 120 nucleotides long. For example, the 3'-UTR is 130 nucleotides long. For example, the 3'-UTR is 140 nucleotides long. For example, the 3'-UTR is 150 nucleotides long. For example, the 3'-UTR is 160 nucleotides long. For example, the 3'-UTR is 170 nucleotides long. For example, the 3'-UTR is 180 nucleotides long. For example, the 3'-UTR is 190 nucleotides long. For example, the 3'-UTR is 200 nucleotides long. For example, the 3'-UTR is 210 nucleotides long. For example, the 3'-UTR is 220 nucleotides long. For example, the 3'-UTR is 230 nucleotides long. For example, the 3'-UTR is 240 nucleotides long. For example, the 3'-UTR is 250 nucleotides long.For example, the 3'-UTR is 260 nucleotides long. For example, the 3'-UTR is 270 nucleotides long. For example, the 3'-UTR is 280 nucleotides long. For example, the 3'-UTR is 290 nucleotides long. For example, the 3'-UTR is 300 nucleotides long. For example, the 3'-UTR is 310 nucleotides long. For example, the 3'-UTR is 320 nucleotides long. For example, the 3'-UTR is 330 nucleotides long. For example, the 3'-UTR is 340 nucleotides long. For example, the 3'-UTR is 350 nucleotides long. For example, the 3'-UTR is 360 nucleotides long. For example, the 3'-UTR is 370 nucleotides long. For example, the 3'-UTR is 380 nucleotides long. For example, the 3'-UTR is 390 nucleotides long. For example, the 3'-UTR is 400 nucleotides long.

[0203] In one example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 40-56, 63, 77, and 79. For example, the 3'-UTR, fragment, and / or variant thereof comprises a nucleotide sequence that is 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 40-56, 63, 77, and 79. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 40. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 41. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 42. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 43. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 44. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 45. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 46. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 47. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 48. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 49. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 50. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 51. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 52.For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 53. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 54. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 55. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 56. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 63. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 77. For example, the 3'-UTR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 79.

[0204] In one example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 40-56, 63, 77, and 79. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 40. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 41. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 42. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 43. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 44. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 45. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 46. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 47. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 48. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 49. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 50. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 51. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 52. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 53. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 54.For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 55. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 56. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 63. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 77. For example, the 3'-UTR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 79.

[0205] In one example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 40-56, 63, 77, and 79. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 40. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 41. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 42. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 43. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 44. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 45. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 46. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 47. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 48. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 49. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 50. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 51. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 52. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 53. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 54.For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 55. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 56. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 63. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 77. For example, the 3'-UTR comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 79.

[0206] In one example, the 3'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 40 to 56, 63, 77, and 79. For example, the 3'-UTR comprises the nucleotide sequence set forth in SEQ ID NO: 40. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 41. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 42. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 43. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 44. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 45. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 46. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 47. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 48. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 49. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 50. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 51. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 52. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 53. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 54. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 55. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 56. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 63. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 77. For example, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 79.

[0207] In one example, the second nucleotide sequence comprises a 3'-CSE of Venezuelan equine encephalitis virus (VEEV) or Sindbis virus (SIN).

[0208] In one example, the second nucleotide sequence comprises a combination of two or more 3'-UTRs. In one example, two or more 3'-UTRs are the same. In one example, two or more 3'-UTRs are different.

[0209] In one example, the second nucleotide sequence 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, a 3'CSE from an alphavirus, or a combination thereof. In one example, the second nucleotide sequence 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. For example, the second nucleotide sequence comprises a microRNA binding site. For example, the second nucleotide sequence comprises an AU-rich element (ARE). For example, the second nucleotide sequence comprises a GC-rich element. For example, the second nucleotide sequence comprises a triple helix. For example, the second nucleotide sequence comprises a stem-loop. For example, the second nucleotide sequence comprises one or more stop codons. For example, the second nucleotide sequence comprises one or more stop codons located at the 5' end of the 3'-UTR. For example, the second nucleotide sequence comprises a 3'CSE from an alphavirus. For example, the second nucleotide sequence comprises the 3'-CSE of Venezuelan equine encephalitis virus (VEEV) or Sindbis virus (SIN). For example, the second nucleotide sequence comprises the 3'-CSE of Venezuelan equine encephalitis virus (VEEV). For example, the second nucleotide sequence comprises the 3'-CSE of Sindbis virus (SIN).

[0210] In one example, the one or more RNAs comprise a third nucleotide sequence that includes one or more 3' tailing sequences located at the 3' end of the second nucleotide sequence.

[0211] In one example, the one or more 3' tailing sequences are selected from the group consisting of a polyA sequence, a polyadenylation signal, a G-quadruplex, a polyC sequence, a stem-loop, and combinations thereof. For example, the third nucleotide sequence comprises a polyA sequence. For example, the third nucleotide sequence comprises a polyadenylation signal. For example, the third nucleotide sequence comprises a G-quadruplex. For example, the third nucleotide sequence comprises a polyC sequence. For example, the third nucleotide sequence comprises a stem-loop. For example, the third nucleotide sequence comprises a polyA sequence and a G-quadruplex.

[0212] In one example, the one or more 3' tailing sequences include one or more polyA sequences, each containing 10 to 300 consecutive adenosine nucleotides. For example, the one or more polyA sequences each contain 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, 225 to 250, 250 to 275, or 275 to 300 consecutive adenosine nucleotides. For example, the one or more polyA sequences each contain 10 to 20 consecutive adenosine nucleotides. For example, the one or more polyA sequences each contain 20 to 30 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 30 to 40 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 40 to 50 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 50 to 60 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 60 to 70 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 70 to 80 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 80 to 90 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 90 to 100 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 100 to 125 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 125 to 150 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 150 to 175 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 175 to 200 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 200 to 225 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 225 to 250 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 250 to 275 consecutive adenosine nucleotides.For example, the one or more polyA sequences each contain 275 to 300 consecutive adenosine nucleotides.

[0213] In one example, each of the one or more polyA sequences contains 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 10 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 20 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 30 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 40 consecutive adenosine nucleotides. For example, each of the one or more polyA sequences contains 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, the one or more polyA sequences each contain 250 consecutive adenosine nucleotides. For example, the one or more polyA sequences each contain 275 consecutive adenosine nucleotides. For example, the one or more polyA sequences each contain 300 consecutive adenosine nucleotides.

[0214] In one example, the one or more polyA sequences are separated by an interrupted linker, e.g., the third nucleotide sequence including the one or more 3' tailing sequences includes, in 5' to 3' order, consecutive adenosine nucleotides, an interrupted linker, and further consecutive adenosine nucleotides.

[0215] In one example, the interruption linker is 10 to 50, or 50 to 100, or 100 to 150 nucleotides in length. For example, the interruption linker is 10 to 50 nucleotides in length. For example, the interruption linker is 50 to 100 nucleotides in length. For example, the interruption linker is 100 to 150 nucleotides in length.

[0216] In one example, the interruption linker is at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95, or 100, or 110, or 120, or 130, or 140, or 150 nucleotides in length. For example, the interruption linker is 1 nucleotide in length. For example, the interruption linker is 2 nucleotides in length. For example, the interruption linker is 3 nucleotides in length. For example, the interruption linker is 4 nucleotides in length. For example, the interruption linker is 5 nucleotides in length. For example, the interruption linker is 6 nucleotides in length. For example, the interruption linker is 7 nucleotides in length. For example, the interruption linker is 8 nucleotides in length. For example, the interruption linker is 9 nucleotides in length. For example, the interruption linker is 10 nucleotides in length. For example, the interruption linker is 11 nucleotides in length. For example, the interruption linker is 12 nucleotides in length. For example, the interruption linker is 13 nucleotides in length. For example, the interruption linker is 14 nucleotides in length. For example, the interruption linker is 15 nucleotides in length. For example, the interruption linker is 16 nucleotides in length. For example, the interruption linker is 17 nucleotides in length. For example, the interruption linker is 18 nucleotides in length. For example, the interruption linker is 19 nucleotides in length. For example, the interruption linker is 20 nucleotides in length. For example, the interruption linker is 25 nucleotides in length. For example, the interruption linker is 30 nucleotides in length. For example, the interruption linker is 35 nucleotides in length. For example, the interruption linker is 40 nucleotides in length. For example, the interruption linker is 45 nucleotides in length. For example, the interruption linker is 50 nucleotides in length. For example, the interruption linker is 55 nucleotides in length. For example, the interruption linker is 60 nucleotides in length. For example, the interruption linker is 65 nucleotides in length. For example, the interruption linker is 70 nucleotides in length.For example, the interruption linker is 75 nucleotides in length. For example, the interruption linker is 80 nucleotides in length. For example, the interruption linker is 85 nucleotides in length. For example, the interruption linker is 90 nucleotides in length. For example, the interruption linker is 95 nucleotides in length. For example, the interruption linker is 100 nucleotides in length. For example, the interruption linker is 110 nucleotides in length. For example, the interruption linker is 120 nucleotides in length. For example, the interruption linker is 130 nucleotides in length. For example, the interruption linker is 140 nucleotides in length. For example, the interruption linker is 150 nucleotides in length.

[0217] In one example, the interrupted linker comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 59. For example, the interrupted linker comprises the nucleotide sequence set forth in SEQ ID NO: 59. For example, the interrupted linker consists of the nucleotide sequence set forth in SEQ ID NO: 59.

[0218] In one example, the one or more RNAs comprising the third nucleotide sequence comprise, in 5'→3' order, consecutive adenosine nucleotides, an interrupted linker, and more consecutive adenosine nucleotides.

[0219] In one example, the one or more RNAs comprising the third nucleotide sequence comprise, in 5'→3' order, consecutive adenosine nucleotides, an interrupted linker comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 59, and further consecutive adenosine nucleotides.

[0220] In one example, the one or more RNAs comprising the third nucleotide sequence comprise, in 5' to 3' order, 30 consecutive adenosine nucleotides, an interrupted linker comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 59, and 70 consecutive adenosine nucleotides.

[0221] In one example, the one or more RNAs comprise at least one chemically modified nucleotide.

[0222] In one example, the chemically modified nucleotide is selected from the group consisting of N6,2'-O-dimethyl-adenosine (m6Am), 5-methyluridine (m5U), N4-acetylcytidine (ac4C), 2-thiocytidine (s2C), 2-thiouridine (s2U), 5-methylcytidine (m5C), N6-methyladenosine (m6a), pseudouridine (ψ), 1-methylpseudouridine (m1ψ), and combinations thereof. For example, the chemically modified nucleotide is N6,2'-O-dimethyl-adenosine (m6Am). For example, the chemically modified nucleotide is 5-methyluridine (m5U). For example, the chemically modified nucleotide is N4-acetylcytidine (ac4C). For example, the chemically modified nucleotide is 2-thiocytidine (s2C). For example, the chemically modified nucleotide is 2-thiouridine (s2U). For example, a chemically modified nucleotide is 5-methylcytidine (mC). For example, a chemically modified nucleotide is N-methyladenosine (ma). For example, a chemically modified nucleotide is pseudouridine (ψ). For example, a chemically modified nucleotide is 1-methylpseudouridine (mψ).

[0223] In one example, one or more RNAs are conventional mRNA (cRNA) or self-amplifying mRNA (sa-mRNA). For example, the mRNA is cRNA. For example, the mRNA is sa-mRNA.

[0224] In one example, the sa-mRNA comprises one or more nucleotide sequences from an alphavirus selected from the group consisting of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEEV), and combinations thereof. For example, the sa-mRNA comprises one or more nucleotide sequences from an alphavirus of Semliki Forest virus (SFV). For example, the sa-mRNA comprises one or more nucleotide sequences from an alphavirus of Sindbis virus (SIN). For example, the sa-mRNA comprises one or more nucleotide sequences from an alphavirus of Venezuelan equine encephalitis virus (VEEV).

[0225] In one example, the sa-mRNA includes one or more nucleotide sequences encoding a nonstructural protein (NSP) from an alphavirus.

[0226] In one example, the sa-mRNA comprises one or more nucleotide sequences comprising or consisting of the SG promoter of an alphavirus.

[0227] In one example, the sa-mRNA comprises one or more nucleotide sequences derived from an alphavirus sequence encoding a nonstructural protein (NSP) and one or more nucleotide sequences comprising or consisting of the SG promoter of the alphavirus.

[0228] In one example, the RNA further comprises a 5'-end cap structure.

[0229] In one example, the 5'-terminal cap structure is an endogenous cap or an analog thereof. For example, the 5'-terminal cap structure is an endogenous cap. For example, the 5'-terminal cap structure is an analog of an endogenous cap.

[0230] In one example, the 5'-terminal cap structure comprises guanine or a guanine analog thereof. For example, the 5'-terminal cap structure comprises guanine. For example, the 5'-terminal cap structure comprises a guanine analog of guanine.

[0231] In one 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), Cap1, and Cap2. For example, the 5'-terminal cap structure is an anti-reverse cap analog (ARCA). For example, the 5'-terminal cap structure is N7,2'-O-dimethylguanosine (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 Cap1. For example, the 5'-end cap structure is Cap2.

[0232] In one example, the 5'-terminal cap structure is linked to the 5'-end of the RNA by a 5'-5'-triphosphate bond or a 5'-5' phosphorothioate bond. For example, the 5'-terminal cap structure is linked to the 5'-end of the RNA by a 5'-5'-triphosphate bond. For example, the 5'-terminal cap structure is linked to the 5'-end of the RNA by a 5'-5' phosphorothioate bond.

[0233] In one example, one or more RNAs are formulated in lipid nanoparticles (LNPs). For example, the RNA is encapsulated in the LNPs. In another example, the RNA is bound to the LNPs. For example, the RNA is absorbed into the LNPs.

[0234] In one example, the LNP further comprises a PEG-lipid, a structured lipid, and / or a neutral lipid. For example, the LNP further comprises a PEG-lipid. For example, the LNP further comprises a structured lipid. For example, the LNP further comprises a neutral lipid.

[0235] In one example, the LNP comprises an ionizable lipid. For example, the ionizable lipid is a cationic lipid. For example, the ionizable lipid is a zwitterionic lipid.

[0236] In one example, the LNP does not include an ionizable lipid.

[0237] In one example, each RNA is formulated together in LNP. For example, a composition containing a first, second, and / or third RNA is formulated together in LNP. For example, in a composition containing a first RNA and a second RNA, the first RNA and the second RNA are formulated together in LNP. For example, in a composition containing a first, second, and third RNA, the first, second, and third RNA are formulated together in LNP.

[0238] In one example, each RNA is formulated separately into an LNP. For example, a composition containing a first, second, and / or third RNA is formulated separately into an LNP. For example, in a composition containing a first and second RNA, the first and second RNAs are formulated into separate LNPs. For example, in a composition containing a first, second, and third RNA, the first, second, and third RNAs are each formulated into separate LNPs. For example, in a composition containing a first, second, and third RNA, the first and second RNAs are formulated together in one LNP, and the third RNA is formulated in another LNP. For example, in a composition containing a first, second, and third RNA, the first and third RNAs are formulated together in an LNP, and the second RNA is formulated in another LNP. For example, in a composition containing a first, second, and third RNA, the third and second RNAs are formulated together in an LNP, and the first RNA is formulated in another LNP.

[0239] In one example, the composition is an immunogenic composition.

[0240] In one example, the composition is a pharmaceutical composition.

[0241] The present disclosure further provides a pharmaceutical composition comprising an immunogenic composition of the present disclosure and a pharmaceutically acceptable carrier.

[0242] In one example, the present disclosure provides an immunogenic or pharmaceutical composition of the present disclosure for use as a vaccine.

[0243] In one example, the immunogenic or pharmaceutical composition of the disclosure is for use in treating or preventing or delaying the progression of influenza and / or influenza virus infection.

[0244] In one example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating, preventing, or delaying the progression of influenza and / or influenza virus infection. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating influenza and / or influenza virus infection. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in preventing influenza and / or influenza virus infection. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in delaying the progression of influenza and / or influenza virus infection.

[0245] In one example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating, preventing, or delaying the progression of influenza and / or influenza virus infection. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating influenza and / or influenza virus infection. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in preventing influenza and / or influenza virus infection. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in delaying the progression of influenza and / or influenza virus infection.

[0246] The present disclosure also provides for the use of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating or preventing or delaying the progression of influenza and / or an influenza virus infection in a subject in need thereof. In one example, the present disclosure provides for the use of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating influenza and / or an influenza virus infection in a subject in need thereof. In one example, the present disclosure provides for the use of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for preventing influenza and / or an influenza virus infection in a subject in need thereof. In one example, the present disclosure provides for the use of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating influenza and / or an influenza virus infection and / or delaying the progression of influenza and / or an influenza virus infection in a subject in need thereof.

[0247] The present disclosure also provides a method for treating, preventing, or delaying the progression of influenza and / or influenza virus infection in a subject, the method comprising administering an immunogenic or pharmaceutical composition of the present disclosure to a subject in need thereof. For example, the present disclosure provides a method for treating influenza and / or influenza virus infection in a subject, the method comprising administering an immunogenic or pharmaceutical composition of the present disclosure to a subject in need thereof. For example, the present disclosure provides a method for preventing influenza and / or influenza virus infection in a subject, the method comprising administering an immunogenic or pharmaceutical composition of the present disclosure to a subject in need thereof. The present disclosure also provides a method for delaying the progression of influenza and / or influenza virus infection in a subject, the method comprising administering an immunogenic or pharmaceutical composition of the present disclosure to a subject in need thereof.

[0248] In one example of any of the methods described herein, the composition, immunogenic composition, or pharmaceutical composition of the present disclosure is administered before or after the onset of influenza and / or influenza virus infection in a subject. In one example of any of the methods described herein, the composition, immunogenic composition, or pharmaceutical composition of the present disclosure is administered before the onset of influenza and / or influenza virus infection in a subject. In one example of any of the methods described herein, the composition, immunogenic composition, or pharmaceutical composition of the present disclosure is administered after the onset of influenza and / or influenza virus infection in a subject.

[0249] The present disclosure provides a method of inducing an immune response to influenza and / or influenza virus infection in a subject, the method comprising administering to a subject in need thereof a composition, immunogenic composition, or pharmaceutical composition of the present disclosure.

[0250] The present disclosure also provides the use of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for inducing an immune response in a subject in need thereof.

[0251] In one example, the composition, immunogenic composition, or pharmaceutical composition of the present disclosure induces a humoral and / or cell-mediated immune response. In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response, such as 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 a further example, the T cells are CD4 and CD8 T cells.

[0252] In one example, administration of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure induces a CD4 T cell-mediated immune response.

[0253] In one example, administration of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure induces a CD8 T cell-mediated immune response.

[0254] In one example, administration of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure induces a CD4 and CD8 T cell-mediated immune response.

[0255] The present disclosure also provides a method of expressing a virus-like particle (VLP) in a subject, the method comprising administering to the subject a composition, immunogenic composition, or pharmaceutical composition of the present disclosure.

[0256] The present disclosure further provides the use of a composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for expressing a VLP in a subject in need thereof.

[0257] The present disclosure also provides an immunogenic or pharmaceutical composition of the present disclosure for use in a method for expressing a VLP in a subject in need thereof.

[0258] The present disclosure also provides kits comprising at least one composition of the present disclosure.

[0259] In one example, the kit includes a composition of the present disclosure, optionally in a delivery system and / or a pharmaceutically acceptable carrier or diluent, packaged with instructions for use in treating, preventing, or delaying influenza and / or influenza virus infection in a subject in need thereof. For example, the RNA is mRNA. For example, the mRNA is cRNA or sa-mRNA. In one example, the kit includes at least one RNA of the present disclosure, optionally in a delivery system and / or a pharmaceutically acceptable carrier or diluent, packaged with instructions for administering the mRNA to a subject suffering from or at risk of suffering from influenza and / or influenza virus infection.

[0260] In one example, the composition, immunogenic composition, or pharmaceutical composition of the disclosure is supplied in a vial, hi another example, the immunogenic or pharmaceutical composition of the disclosure is supplied in a syringe.

[0261] Any discussion herein of a translation initiation sequence (e.g., a Kozak consensus sequence, an IRES, or an SG promoter), a 5'-UTR, a VLP, an M1, an HA, an NA, and / or a 3'-UTR should be understood to include fragments and / or variants of the translation initiation sequence (e.g., a Kozak consensus sequence, an IRES, or an SG promoter), a 5'-UTR, a VLP, an M1, an HA, an NA, and / or a 3'-UTR. [Brief explanation of the drawings]

[0262] [Figure 1] Western blot images of (A) BHK cell culture lysates and (B) BHK cell culture supernatants used to generate VLPs from four different VLP mRNA constructs probed for HA, NA, M1, and GAPDH proteins. [Figure 2]1 is a graphical representation showing the expression of HA, NA, and M1 proteins in (A) BHK cell culture lysates and (B) BHK cell culture supernatants used to generate VLPs from four different VLP mRNA constructs, as quantified from the Western blot images in FIG. 1 , and the expression of M1 protein in (C) BHK cell culture lysates and (D) BHK cell culture supernatants.

[0263] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0264] Although the sequence listing refers to DNA sequences, the disclosure of this application is to be understood to include their RNA equivalents and their complementary sequences, unless the context clearly dictates otherwise. DETAILED DESCRIPTION OF THE INVENTION

[0265] General Throughout this specification, unless expressly stated otherwise or the context requires otherwise, references to a single step, a single composition, a group of steps, or a group of compositions shall be interpreted as encompassing one and more (i.e., one or more) of those steps, compositions, steps, or compositions.

[0266] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described, and it is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any combination or any two or more of such steps or features.

[0267] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only, and functionally equivalent objects, compositions, and methods are expressly intended to be within the scope of the present disclosure.

[0268] Any example of the present disclosure shall be construed as applying mutatis mutandis to any other example of the present disclosure, unless expressly stated otherwise. In other words, any embodiment of the present disclosure may be combined with any other embodiment of the present disclosure (except where mutually exclusive).

[0269] Any example of the present disclosure disclosing a specific feature or group of features, or a method or method step, is deemed to provide explicit support for disclaiming the specific feature or group of features, or method or method step.

[0270] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0271] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained in the literature, for example, in the following sources: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M.G. Lover and B.D.H.Means (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F.M.A. Subetal et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all previous updates); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988); and JE Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all previous updates).

[0272] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as expressly endorsing both meanings or either meaning.

[0273] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" should be understood to include a stated element, integer, or step, or group of elements, integers, or steps, but not to exclude any other element, integer, or step, or group of elements, integers, or steps.

[0274] As used herein, the term "derived from" shall be construed to indicate that the specified integral may be obtained from (but not necessarily obtained directly from) a particular source. Similarly, the term "based on" shall be construed to indicate that the specified integral may be developed or used from (but not necessarily obtained directly from) a particular source.

[0275] Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification shall not be construed as an admission that any or all of such matters formed part of the prior art or were common general knowledge in the fields relevant to this disclosure by virtue of their existence before the priority date of each of the appended claims.

[0276] Selected Definitions As used herein, the term "ribonucleic acid" or "RNA" refers to a molecular chain of nucleotides chemically linked by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar in the adjacent nucleotide. In one example, the RNA is mRNA. For example, the mRNA is conventional mRNA (cRNA) or self-amplifying RNA (sa-mRNA).

[0277] As used herein, the terms "conventional mRNA" or "cRNA" or "unamplified RNA" refer to constructs that allow for the expression of heterologous RNA and proteins, but RNA that cannot be amplified in a host cell.

[0278] As used herein, the term "self-replicating RNA" refers to an RNA virus-based construct that has been engineered to allow for the expression of heterologous mRNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified within a host cell and express a desired gene product within the host cell.

[0279] As used herein, the term "multicistronic" (also known as "polycistronic") with respect to polynucleotides, RNA, cRNA, and / or self-replicating RNA refers to RNA that encodes two or more polypeptides. This term encompasses "bicistronic" (or "dicistronic," i.e., encoding two polypeptides) and "tricistronic" (i.e., encoding three polypeptides) molecules. "Bicistronic" refers to a single nucleic acid that can encode two different polypeptides from different regions of the nucleic acid.

[0280] As used herein, the term "naked" refers to a nucleic acid that is substantially free of other macromolecules, such as lipids, polymers, or proteins. Naked nucleic acids (e.g., self-amplifying RNAs) are not formulated with other macromolecules to improve cellular uptake. Thus, naked nucleic acids are not encapsulated, absorbed, or bound to lipid nanoparticles (LNPs), liposomes, polymeric microparticles, or oil-in-water emulsions.

[0281] 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 a defined activity of the full-length nucleotide sequence or polypeptide. In one example, the defined activity is inducing an immune response in a subject administered with a composition of the present disclosure.

[0282] As used herein, the term "variant" refers to a nucleotide sequence (e.g., a VLP-forming element) or polypeptide (e.g., an antigenic polypeptide) with one or more nucleotide sequence(s) or amino acid sequence(s) difference(s) relative to a reference nucleotide sequence of a polypeptide disclosed herein, which maintains a defined activity of the nucleotide sequence or polypeptide. The one or more nucleotide sequence(s) or amino acid sequence(s) difference(s) result from one or more modifications made to the nucleotide sequence or polypeptide of the present disclosure. In one example, the modification is a chemical modification of one or more nucleotide(s) of the nucleotide sequence. For example, at least one naturally occurring RNA nucleotide is replaced with a chemically modified nucleotide (e.g., pseudouridine (ψ) and 1-methylpseudouridine (m1ψ)). In one example, the modification includes increasing the G / C content of the nucleotide sequence. In one example, the modification includes codon optimization of the nucleotide sequence. In one example, the defined activity is inducing an immune response in a subject administered with a composition of the present disclosure. For example, the variant is a variant of a VLP-forming element, for example, a variant of the H5 protein and / or the N1 protein of an influenza virus.

[0283] In one example, a variant has at least 70%, or 75%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to a VLP-forming element and / or fragment thereof. In one example, a variant has at least 70%, or 75%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to a VLP-forming element and / or fragment thereof. The nucleotide sequence or polypeptide variants disclosed herein can have one or more nucleotide(s) or amino acid(s) deleted or replaced with a different nucleotide(s) or amino acid(s). In one example, the substitutions are conservative substitutions. Those skilled in the art will understand that a conservative substitution with respect to a polypeptide involves replacing an amino acid in a polypeptide with a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, and size). In one example, the substitution is a non-conservative substitution.

[0284] As used herein, the terms "encode," "encodes," or "encoding" refer to a region of RNA that can undergo translation into a polypeptide.

[0285] As used herein, the term "antigen" refers to a molecule or structure containing one or more epitopes that induce, elicit, enhance, or boost a cellular and / or humoral immune response. Antigens can include, for example, proteins and peptides derived from pathogens such as viruses, bacteria, fungi, protozoa, plants, or tumors. For example, antigens are derived from a gene of interest.

[0286] As used herein, the term "nucleotide sequence" or "nucleic acid sequence" is understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. By convention, sequences are presented from the 5' to the 3' end unless otherwise specified.

[0287] As used herein, the term "operably linked" means positioning a translation initiation sequence (e.g., a Kozak consensus sequence, an internal ribosome entry site (IRES), a subgenomic (SG) promoter) or a stability element (e.g., a 5'-UTR) relative to a nucleic acid such that expression of the nucleic acid is controlled or regulated by the sequence or element. For example, a translation initiation sequence can be operably linked to the 5' end of one or more nucleotide sequence(s) encoding VLP-forming elements.

[0288] The term "polypeptide" or "polypeptide chain" is understood to mean a series of consecutive amino acids linked by peptide bonds. For example, a protein shall be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). A series of polypeptide chains may be covalently linked by suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0289] The term "recombinant" shall be understood to mean the product of artificial genetic recombination.

[0290] As used herein, the term "lipid nanoparticle" or "LNP" refers to a lipid-based particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising a compound of any formula described herein. In embodiments, LNPs are formulated into compositions for delivering polynucleotides to desired targets, such as cells, tissues, organs, and tumors. For example, lipid nanoparticles or LNPs, any lipid composition, can be selected from, but not limited to, liposomes or vesicles in which the aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; unilamellar or multilamellar; multilamellar), micelle-like lipid nanoparticles with a non-aqueous core, and solid lipid nanoparticles (solid lipid nanoparticles do not have a lipid bilayer). As one skilled in the art will appreciate, LNPs can be formed by mixing nucleic acid and lipids in an appropriate solvent system. For example, LNPs can be formed using techniques known to those skilled in the art, such as microfluidic mixing (e.g., using a herringbone mixer), rapid mixing, T-shaped mixing, etc. As used herein, the term "stability" in the context of VLPs shall be understood to refer to the ability of the VLP to maintain its structure and / or function. The inventors have determined that using the compositions of the present disclosure to produce and incorporate higher levels of M1 protein into VLPs (compared to VLPs with lower levels of M1 protein incorporation) also increased the ability of the VLPs to remain intact, making these VLPs more stable.

[0291] As used herein, the term "release" in the context of a VLP shall be understood to refer to the release of the VLP from the sialic acid receptor to which it is bound. HA proteins are known to bind to one or more sialic acid receptors (e.g., by expression from a cell). The inventors have determined that producing and incorporating a high level of NA protein into VLPs using the compositions of the present disclosure (compared to VLPs with low levels of NA protein incorporation) also increases the ability of the VLPs to be released from the cell. As used herein, the terms "disease," "disorder," or "condition" refer to a disruption or interference with normal function.

[0292] As used herein, a subject "at risk" of having or developing an influenza virus infection and / or influenza may or may not have detectable disease or symptoms of influenza virus infection and / or influenza, and may or may not have exhibited detectable disease or symptoms of influenza virus infection and / or influenza prior to treatment according to the present disclosure. "At risk" indicates that the subject has one or more risk factors, which are measurable parameters that correlate with developing influenza virus infection and / or influenza, as known in the art and / or described herein.

[0293] As used herein, the terms "treating," "treat," or "treatment" include administering an RNA or composition described herein to thereby reduce or eliminate at least one symptom of the specified disease or condition.

[0294] As used herein, the terms "preventing," "prevent," or "prevention" include providing protection against the occurrence or recurrence of a particular disease or condition in an individual who may be susceptible to or at risk of developing the disease, but who has not yet been diagnosed with the disease.

[0295] As used herein, the phrase "slowing the progression of" includes reducing or slowing the progression of a disease or condition, and / or at least one symptom of a disease or condition, in an individual.

[0296] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired result. For example, the desired result can be a therapeutic or prophylactic result. An effective amount can be provided by one or more administrations. In some examples of the present disclosure, the term "effective amount" refers to the amount necessary to effect treatment of a disease or condition as described herein. In some examples of the present disclosure, the term "effective amount" refers to the amount necessary to effect a change associated with a disease or condition as described herein. An effective amount can vary depending on the disease or condition being treated or the factor to be modified, and can also vary depending on the weight, age, ethnic background, sex, health, and / or physical condition of the mammal being treated, as well as other factors. Typically, an effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined by routine trial and experimentation by a medical professional. Thus, the term should not be construed to limit the present disclosure to specific amounts (e.g., weight and RNA count). An effective amount can be administered in a single dose or in doses repeated once or several times during the treatment period.

[0297] A "therapeutically effective amount" is at least the minimum concentration required to produce a measurable improvement in a specific disease or condition. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, weight, and the ability of the RNA of the present disclosure to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the RNA.

[0298] As used herein, the term "prophylactically effective amount" shall be taken to mean an amount of RNA of the present disclosure sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a disease or disorder.

[0299] As used herein, the term "subject" shall be taken to mean any animal (e.g., mammal), including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0300] composition As used herein, the terms "virus-like particle," "VLP," "virus-like particle," or "VLP" shall be taken to mean a protein- and lipid-based multi-subunit structure composed of virus-like particle (VLP)-forming elements and resembling the shape and / or size of a virus particle, but containing no viral genetic material. A VLP or VLPs displays antigens presenting conformational epitopes that elicit a T-cell and / or B-cell immune response, but is unable to replicate and / or infect host cells.

[0301] As used herein, the term "virus-like particle-forming element" or "VLP-forming element" refers to viral structural and antigenic proteins that have the ability to self-assemble to form a VLP or VLPs. For example, the VLP-forming element is selected from the hemagglutinin (HA), neuraminidase (NA), and matrix-1 (M1) proteins of influenza viruses.

[0302] The composition of the present application comprises one or more ribonucleic acids (RNAs) encoding influenza virus-like particle (VLP)-forming elements. The VLP-forming elements are selected from the structural polypeptide M1 protein and the antigenic polypeptides NA protein and HA protein. The influenza virus structural protein (i.e., M1 protein) generated from one or more RNAs self-assembles with the influenza virus antigenic proteins (i.e., NA protein and HA protein) to generate virus-like particles (VLPs).

[0303] influenza virus The VLP-forming elements, fragments and / or variants thereof of the present disclosure are those of influenza viruses.

[0304] Influenza viruses are members of the Orthomyxoviriadae family and represent enveloped viruses containing segmented, negative-sense single-stranded RNA. Those skilled in the art will recognize that there are currently four types of influenza viruses (A, B, C, and D). Influenza A viruses are the most common strains of influenza viruses that infect humans, animals, and birds, while influenza B virus infections occur most often in humans. Influenza C virus infections do not cause any serious symptoms in humans or mammals, and influenza D viruses have so far only infected pigs and cattle.

[0305] Exemplary influenza A virus subtypes would be apparent to those skilled in the art. In one example, the influenza A virus is an influenza A (H1N1) or influenza A (H3N2) subtype. For example, the influenza A virus is an influenza A (H1N1) subtype. For example, the influenza A virus is an influenza A (H3N2) subtype. For example, the influenza A virus is an A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) pdm09 virus. For example, the influenza A virus is an A / Hawaii / 70 / 2019 (H1N1) pdm09 virus. For example, the influenza A virus is an A / Brisbane / 02 / 2018 (H1N1) pdm09 virus. For example, the influenza A virus is an A / Hong Kong / 2671 / 2019 (H3N2)-like virus. For example, an influenza A virus is the A / Kansas / 14 / 2017 (H3N2) virus. For example, an influenza A virus is the A / Victoria / 2570 / 2019 (H1N1) virus. For example, an influenza A virus is the A / Wisconsin / 588 / 2019 (H1N1) virus. For example, an influenza A virus is the A / Cambodia / e0826360 / 2020 (H3N2) virus. For example, an influenza A virus is the A / South Australia / 34 / 2019 (H3N2) virus. For example, an influenza A virus is the A / Brisbane / 02 / 2018 (H1N1) virus.

[0306] Exemplary strains of influenza B viruses will be apparent to those skilled in the art. In one example, the influenza B virus is a virus of the B / Yamagata or B / Victoria lineage. For example, the influenza B virus is a virus of the B / Yamagata lineage. For example, the influenza B virus is a virus of the B / Victoria lineage. For example, the influenza B virus is a virus of the B / Washington / 02 / 2019 (B / Victoria lineage) virus. For example, the influenza B virus is a virus of the B / Colorado / 06 / 2017 (B / Victoria lineage) virus. For example, the influenza B virus is a virus of the B / Phuket / 3073 / 2013 (Yamagata lineage) virus.

[0307] Influenza A and B viruses contain a viral envelope composed of glycoproteins hemagglutinin (HA) and neuraminidase (NA) proteins within a lipid bilayer membrane. The HA and NA proteins form spike-like structures on the surface of the viral envelope. The HA protein functions to initiate infection of host cells by binding to sialic acid-containing receptors on the host cell surface. The NA protein functions to enable influenza viruses to be released from infected host cells. Underneath the lipid bilayer membrane of influenza A and B viruses is a scaffold formed by matrix-1 (M1) protein, which provides strength and rigidity to the viral envelope. Other components of influenza A and B viruses include nucleoprotein (NP) and nonstructural proteins (NS).

[0308] The viral envelope of influenza A virus further contains the ion channel matrix-2 (M2) protein.

[0309] The viral envelope of influenza B virus further contains the ion channel influenza B matrix protein 2 (BM2) and the NB protein.

[0310] Ribonucleic acid (RNA) The RNA of the present disclosure encompasses non-self-amplifying mRNA (also referred to as conventional mRNA (cRNA)) and self-amplifying RNA (sa-mRNA). Typically, cRNA comprises, in 5' to 3' order, a 5' cap structure, a 5'-UTR, a nucleotide sequence encoding a polypeptide of interest, a 3'-UTR, a fragment and / or variant thereof, and a tailing sequence (e.g., a polyadenylation signal or polyA tail). In the present disclosure, the polypeptide of interest is a VLP-forming element selected from the hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein of influenza virus. The cRNA of the present disclosure may further comprise a translational internal ribosome entry site (e.g., a Kozak consensus sequence or IRES).

[0311] The sa-mRNA of the present disclosure comprises one or more features of a cRNA, but further comprises a nucleotide sequence encoding a nonstructural protein (NSP), which enables the sa-mRNA to direct its own amplification. For example, the sa-mRNA comprises an NSP derived from (or based on) an RNA virus (e.g., an alphavirus).

[0312] The sa-mRNA typically also contains a subgenomic (SG) promoter, which, when bound to a nucleotide sequence encoding the NSP and / or polypeptide of interest, drives expression of the NSP and / or polypeptide of interest. Because the sa-mRNA is a plus-strand, it can be directly translated after delivery to a cell without the need for an intervening replication step (e.g., reverse transcription). Once introduced into a cell, the NSP of the sa-mRNA is expressed and binds to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The replicase complex is a component of the sa-mRNA and amplifies the original RNA to produce both antisense and sense transcripts, resulting in the generation of multiple daughter RNAs and subsequent production of the encoded polypeptide of interest.

[0313] Those skilled in the art will understand that sa-mRNA is mRNA based on the genomic RNA of an RNA virus (e.g., an alphavirus). Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middleburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus also includes chimeric alphaviruses, which contain genomic sequences from more than one alphavirus (described in Perri et al. (2003) J. Virol. 77(19):10394-403).

[0314] In one example, a self-replicating RNA of the present disclosure comprises nonstructural proteins of an RNA virus, 5' and 3' untranslated regions (UTRs), and a native subgenomic promoter.

[0315] In one example, the self-replicating RNA comprises one or more nonstructural proteins of an RNA virus. For example, the RNA comprises at least one or more genes selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase, and other nonstructural viral proteins. For example, the self-replicating RNA comprises a viral replicase (or viral polymerase).

[0316] In another example, the self-replicating RNA comprises the 5'- and 3'-terminal UTRs of an RNA virus. In one example, the self-replicating RNA comprises a 5'-CSE. In one example, the 3'-CSE forms part of the 3'-terminal UTR. In one example, the self-replicating RNA comprises a 5'-CSE. In one example, the 5'-CSE is a 51-nucleotide conserved sequence element (CSE). In one example, the 5'-CSE is located within the nsp1 coding sequence.

[0317] The self-replicating RNA of the present disclosure is incapable of inducing the production of infectious viral particles, for example, the self-replicating RNA of the present disclosure does not contain viral genes encoding structural proteins necessary for the production of viral particles.

[0318] In one example, the self-replicating RNA is derived from or based on an alphavirus. Suitable alphaviruses will be apparent to those skilled in the art and / or are described herein.

[0319] In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus (e.g., a positive-strand RNA virus). Suitable positive-strand RNA viruses suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.

[0320] Alphaviruses In one example, the self-replicating RNA of the present disclosure is derived from (or based on) an alphavirus.

[0321] Alphaviruses are the only genus in the family Togaviridae and are enveloped viruses with a positive-sense, single-stranded RNA genome. Those skilled in the art will understand that an alphavirus genome contains two open reading frames (ORFs): nonstructural and structural. The first ORF encodes four nonstructural proteins (NSP1, NSP2, NSP3, and NSP4) required for viral RNA transcription and replication. The second encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and E1, which assemble as a heterodimer. Viral membrane-anchored surface glycoproteins are involved in receptor recognition and entry into target cells via membrane fusion.

[0322] In one example, a self-replicating RNA of the present disclosure includes a viral replicase (or viral polymerase). For example, the viral replicase is an alphavirus replicase (e.g., the alphavirus protein NSP4). In one example, a self-replicating RNA of the present disclosure includes NSP1, NSP2, NSP3, and NSP4.

[0323] In one example, a self-replicating RNA of the present disclosure does not encode one or more alphavirus structural proteins (e.g., capsid and / or envelope glycoproteins), e.g., the self-replicating RNA is incapable of producing RNA-containing alphavirus virions (i.e., infectious virus particles).

[0324] In one example, the self-replicating RNA comprises a native alphavirus SG promoter (also referred to as "SGP"). For example, the native alphavirus SG promoter is a minimal SG promoter (i.e., the minimal sequence required for transcription initiation), and comprises the sequence set forth in SEQ ID NO:3.

[0325] Those of skill in the art will be aware of alphaviruses suitable for use in the present disclosure. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEE; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus also includes chimeric alphaviruses (described in Perri et al., (2003) J. Virol. 77(19):10394-403) that contain genomic sequences from two or more alphaviruses. In one example, the alphavirus includes VEE (e.g., Trinidad donkey, TC83CR). In one example, the alphavirus includes TC-83 (e.g., represented by Genbank accession number L01443.1). In one example, the alphavirus includes Sindbis virus.

[0326] 5' untranslated region (5'-UTR) The present disclosure provides an RNA comprising a first nucleotide sequence comprising a 5'-untranslated region (5'-UTR).

[0327] As used herein, the term "5'-untranslated region" or "5'-UTR" refers to the non-coding region of an mRNA located 5' to the translation initiation codon (eg, AUG).

[0328] Exemplary 5'-UTRs include, for example, 5'-UTRs comprising or consisting of nucleotides derived from the 5'-UTR of a gene selected from the group consisting of haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha 1 collagen (Col1A), alpha 6 collagen (Col6A), alpha 1-antitrypsin (SERPINA1), alpha 1-antichymotrypsin (SERPINA3), arachidonate 5-lipoxygenase (ALOX5), tyrosine hydroxylase (TH gene), tumor protein P53-induced protein 3 (TP5313), fragments thereof, and / or variants thereof.

[0329] In one example, the 5'UTR comprises or consists of a nucleotide sequence derived from the 5'-UTR of an alphavirus, such as an alphavirus as defined herein. In one example, the 5'UTR comprises or consists of a nucleotide sequence derived from the 5'-UTR of Venezuelan equine encephalitis virus (VEEV) or a modified form thereof (e.g., Trinidad donkey, TC-83CR, or TC-83). For example, the 5'UTR comprises or consists of the sequence set forth in SEQ ID NO: 76.

[0330] In one example, the first nucleotide sequence comprises at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, and combinations thereof.

[0331] microRNA binding site As used herein, the term "microRNA binding site" refers to a sequence within a microRNA (miRNA) that has sufficient complementarity with all or a region of the miRNA to interact with, associate with, or bind to the miRNA.

[0332] As used herein, the term "microRNA" or "miRNA" refers to a 19-25 nucleotide long non-coding RNA that binds to the 5'-UTR of an RNA and down-regulates gene expression (e.g., by inhibiting translation). The presence of a microRNA binding site(s) in the first nucleotide of an RNA of the present disclosure may function to inhibit translation of the 5'-UTR.

[0333] Suitable miRNA binding sites for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0334] In one example, the miRNA binding site includes binding sites in tissue-specific microRNAs or those that regulate biological processes. For example, miRNAs in liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). For example, a microRNA (miR-132) that regulates biological processes such as angiogenesis. Further exemplary miRNAs and miRNA binding sites are disclosed in US Patent Application No. US 14 / 043,927.

[0335] AU Rich Element (ARE) As used herein, the term "AU-rich element (ARE)" or "AU-rich element (ARE)" refers to a region of a nucleotide sequence that contains a stretch of adenosine (A) and uridine (U). Exemplary AREs include, for example, AREs derived from cytoplasmic myc (c-myc), myoblast determination protein 1 (myoD), c-Jun, myogenin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α), or a combination thereof.

[0336] In one example, the ARE contains a specific binding site for HuR (also known as Elavl1), which is known to bind to the ARE and increase mRNA stability.

[0337] GC-rich elements As used herein, the term "GC-rich element" refers to a nucleotide sequence that has a large amount of guanine (G) and / or cytosine (C) relative to adenine (A) and thymine (T) / uracil (U). The presence of GC-rich elements in RNA (e.g., mRNA) can stabilize the mRNA.

[0338] In one example, the GC-rich element comprises a sequence that is 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 nucleotides in length.

[0339] In one example, a GC-rich element contains 30% to 40%, or 40% to 50%, or 50% to 60%, or 60% to 70% cytosines. For example, a GC-rich element contains 30% to 40% cytosines. For example, a GC-rich element contains 40% to 50% cytosines. For example, a GC-rich element contains 50% to 60% cytosines. For example, a GC-rich element contains 60% to 70% cytosines.

[0340] In one example, a GC-rich element contains 30%, or 40%, or 50%, or 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.

[0341] In one example, the GC-rich element is at least 50% cytosine.

[0342] In one example, the GC-rich element is at least 60% cytosine.

[0343] In one example, the GC-rich element is at least 70% cytosine.

[0344] In one example, the GC-rich element comprises the nucleotide sequence CCCCGGCGCC. In another example, the GC-rich element comprises the nucleotide sequence CCCCGGC. In a further example, the GC-rich element comprises the nucleotide sequence GCGCCCCGCGGCGCCCCGCG.

[0345] In one example, the GC-rich element comprises the nucleotide sequence set forth in SEQ ID NOs: 89-91. In one example, the GC-rich element comprises the nucleotide sequence (CCCCGGCGCC) set forth in SEQ ID NO: 89. In another example, the GC-rich element comprises the nucleotide sequence (CCCCGGC) set forth in SEQ ID NO: 90. In a further example, the GC-rich element comprises the nucleotide sequence set forth in SEQ ID NO: 91 (GCGCCCCGCGGCGCCCCGCG).

[0346] stem loop As used herein, the term "stem-loop" refers to a nucleotide sequence that contains two adjacent fully or partially reverse complementary sequences that intramolecularly base pair to form a stem-loop. Stem-loops occur in single-stranded DNA or, more commonly, in RNA. Stem-loops, which may also be referred to as hairpins or hairpin loops, usually consist of a stem and a terminal loop within a continuous sequence, with the stem formed by two adjacent fully or partially reverse complementary sequences separated by a short sequence that organizes the loop into a stem-loop structure.

[0347] The stability of the paired stem-loop is determined by its length, the number of mismatches or bulges it contains, and the nucleotide composition of the paired region.

[0348] In one example, the loop of the stem-loop is 3 to 10 nucleotides in length, for example, 3 to 8, or 3 to 7, or 3 to 6, or 4 to 5 nucleotides in length.

[0349] In one example, the loop of the stem-loop is 4 nucleotides in length.

[0350] In one example, the stem loop is a histone stem loop, for example, the histone stem loop comprises or consists of the nucleotide sequence set forth in SEQ ID NO:5.

[0351] Translation initiation sequence(s) In one example, the RNA further comprises a translation initiation sequence. In one example, the translation initiation sequence is linked to the 3' end of the 5'-UTR. In one example, the translation initiation sequence is operably linked to one or more coding sequences. For example, the translation initiation sequence is operably linked to one or more nucleotide sequence(s) encoding virus-like particle (VLP)-forming elements. As used herein, the term "translation initiation sequence" refers to a nucleic acid sequence that initiates translation of an encoded polypeptide (e.g., a VLP-forming element) within a polynucleotide (e.g., an RNA).

[0352] Suitable translation initiation sequence(s) will be apparent to those skilled in the art and / or are described herein, for example, the translation initiation sequence is selected from the group consisting of a Kozak consensus sequence, an internal ribosome entry site (IRES), a subgenomic (SG) promoter, and combinations thereof.

[0353] When the RNA(s) of the present disclosure comprise a translation initiation sequence operably linked to the 5' end of one or more nucleotide sequence(s) encoding VLP-forming elements and / or one or more additional nucleotide sequences encoding nucleoprotein (NP) and / or nonstructural (NS) proteins, each translation initiation sequence can be the same or different. In one example, the translation initiation sequences are two or more SG promoters. In one example, the two or more SG promoters are derived from the same alphavirus. In another example, the two or more SG promoters are derived from different alphaviruses. In one example, the translation initiation sequence is one SG promoter and two or more IRES. In one example, the two or more IRES are the same. In one example, the two or more IRES are different. In one example, the translation initiation sequence is one IRES and two or more SG promoters.

[0354] In one example, the sa-mRNA comprises, in 5' to 3' order: (i) a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4; (ii) a first translation initiation sequence operably linked to the 5' end of a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a second translation initiation sequence operably linked to the 5' end of a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; and (iv) a third translation initiation sequence operably linked to the 5' end of a nucleotide sequence encoding a third virus-like particle (VLP)-forming element. The first, second, and third VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein, and the sa-mRNA contains nucleotide sequences encoding each of the HA protein, NA protein, and M1 protein.

[0355] In one example, the sa-mRNA comprises, in 5' to 3' order: (i) a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4; (ii) a first subgenomic (SG) promoter or a first IRES operably linked to the 5' end of a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a second SG promoter or a second IRES operably linked to the 5' end of a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; and (iv) a third SG promoter or a third IRES operably linked to the 5' end of a nucleotide sequence encoding a third virus-like particle (VLP)-forming element. The first, second, and third VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein, and the sa-mRNA contains nucleotide sequences encoding each of the HA protein, NA protein, and M1 protein.

[0356] For example, the sa-mRNA comprises, in 5' to 3' order: (i) a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4; (ii) a first subgenomic (SG) promoter operably linked to the 5' end of a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a second IRES operably linked to the 5' end of a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; and (iv) a third IRES operably linked to the 5' end of a nucleotide sequence encoding a third virus-like particle (VLP)-forming element; The first, second, and third VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein, and the sa-mRNA contains nucleotide sequences encoding each of the HA protein, NA protein, and M1 protein.

[0357] For example, the sa-mRNA comprises, in 5' to 3' order: (i) a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4; (ii) a first SG promoter operably linked to the 5' end of a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a second SG promoter operably linked to the 5' end of a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; and (iv) a third SG promoter operably linked to the 5' end of a nucleotide sequence encoding a third virus-like particle (VLP)-forming element; The first, second, and third VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein, and the sa-mRNA contains nucleotide sequences encoding each of the HA protein, NA protein, and M1 protein.

[0358] In one example, the first SGP comprises or consists of a native SGP. In one example, the second SGP comprises or consists of the sequence set forth in SEQ ID NO:3 and the third SGP comprises or consists of the sequence set forth in SEQ ID NO:3. In one example, the second SGP comprises or consists of the sequence set forth in SEQ ID NO:3 and the third SGP comprises or consists of the sequence set forth in SEQ ID NO:75. In one example, the second SGP comprises or consists of the sequence set forth in SEQ ID NO:75 and the third SGP comprises or consists of the sequence set forth in SEQ ID NO:4.

[0359] For example, the sa-mRNA comprises, in 5' to 3' order: (i) a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4; (ii) a first SG promoter operably linked to the 5' end of a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a second IRES operably linked to the 5' end of a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; and (iv) a third SG promoter operably linked to the 5' end of a nucleotide sequence encoding a third virus-like particle (VLP)-forming element; The first, second, and third VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein, and the sa-mRNA contains nucleotide sequences encoding each of the HA protein, NA protein, and M1 protein.

[0360] For example, the sa-mRNA comprises, in 5' to 3' order: (i) a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4; (ii) a first SG promoter operably linked to the 5' end of a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a second SG promoter operably linked to the 5' end of a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; and (iv) a third IRES operably linked to the 5' end of a nucleotide sequence encoding a third virus-like particle (VLP)-forming element; The first, second, and third VLP-forming elements are selected from the influenza virus hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein, and the sa-mRNA contains nucleotide sequences encoding each of the HA protein, NA protein, and M1 protein.

[0361] In one example, the first VLP-forming element is the hemagglutinin (HA) protein of influenza virus, the second VLP-forming element is the neuraminidase (NA) protein of influenza virus, and the third VLP-forming element is the matrix-1 (M1) protein of influenza virus.

[0362] In one example, the first SGP is a native SGP, the first VLP-forming element is an influenza virus hemagglutinin (HA) protein, the second SGP comprises or consists of the sequence set forth in SEQ ID NO: 3, the second VLP-forming element is an influenza virus neuraminidase (NA) protein, the third SGP comprises or consists of the sequence set forth in SEQ ID NO: 3, and the third VLP-forming element is an influenza virus matrix-1 (M1) protein. In one example, the first SGP is a native SGP, the first VLP-forming element is an influenza virus hemagglutinin (HA) protein, the second SGP comprises or consists of the sequence set forth in SEQ ID NO: 3, the second VLP-forming element is an influenza virus neuraminidase (NA) protein, the third SGP comprises or consists of the sequence set forth in SEQ ID NO: 75, and the third VLP-forming element is an influenza virus matrix-1 (M1) protein. In one example, the first SGP is a native SGP, the first VLP-forming element is the hemagglutinin (HA) protein of influenza virus, the second SGP comprises or consists of the sequence set forth in SEQ ID NO: 75, the second VLP-forming element is the neuraminidase (NA) protein of influenza virus, the third SGP comprises or consists of the sequence set forth in SEQ ID NO: 3, and the third VLP-forming element is the matrix-1 (M1) protein of influenza virus.

[0363] In one example, the sa-mRNA comprises a 5' UTR followed by a nucleotide sequence(s) encoding an alphavirus nonstructural protein selected from NSP1, NSP2, NSP3, and NSP4. In one example, the sa-mRNA comprises a 3' UTR. In one example, the sa-mRNA comprises a polyA tail.

[0364] Kozak consensus sequence As used herein, the term "Kozak consensus sequence" refers to a nucleotide sequence identified within eukaryotic genes that contains an initiation codon (also called a translation initiation codon) recognized by the ribosome, thereby facilitating translation of the gene.

[0365] Exemplary Kozak consensus sequences are known in the art and / or described herein. In one example, the Kozak consensus sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1. In one example, the Kozak consensus sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 2. In one example, the Kozak consensus sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 85. For example, the Kozak sequence is ACCATG. For example, the Kozak sequence is ACCAUGG. For example, the Kozak sequence is ACCATGG.

[0366] Internal ribosome entry site (IRES) As used herein, the term "internal ribosome entry site" or "IRES" refers to a sequence of nucleotides within an RNA to which a ribosome or a component thereof (e.g., the 40S subunit of the ribosome) can bind. An IRES does not necessarily contain a nucleic acid (e.g., an initiation codon; AUG) that directs translation of the RNA.

[0367] Exemplary IRES include, for example, IRESs derived from poliovirus (PV), human enterovirus, foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), eukaryotic translation initiation factor 4G (eIF4G), death-associated protein 5 (DAP5), cellular Myc (c-Myc), NF-κB inhibitor (NRF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factor B (PDGF B), antennapedia, X-linked inhibitor of apoptosis (XIAP or Apaf-1), immunoglobulin heavy chain-binding protein BiP, or fibroblast growth factor 1a (FGF1A), GTX, or a combination thereof.

[0368] In one example, the IRES is derived from encephalomyocarditis virus (EMCV). For example, the IRES is a wild-type IRES from EMCV. For example, the wild-type EMCV IRES comprises the sequence set forth in SEQ ID NO: 88.

[0369] In one example, the IRES is derived from the fibroblast growth factor 1A (FGF1A) IRES.

[0370] In addition, synthetic IRES elements have been described that can be designed according to methods known in the art to mimic the function of naturally occurring IRES elements (see Chappell, SA et al. Proc. Natl Acad. Sci. USA (2000) 97(4):1536-41).

[0371] In one example, the mRNA comprises, in 5' to 3' order: (i) a first nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof; (ii) a nucleotide sequence encoding a first virus-like particle (VLP)-forming element; (iii) a nucleotide sequence encoding a second virus-like particle (VLP)-forming element; (iv) a nucleotide sequence encoding a third virus-like particle (VLP)-forming element; and (v) a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR), fragments and / or variants thereof.

[0372] the first, second, and third VLP-forming elements are selected from the hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein of influenza virus; the RNA comprises a nucleotide sequence encoding each of an HA protein, an NA protein, and an M1 protein; Internal ribosome entry sites (IRES) (a) between a first nucleotide sequence and a nucleotide sequence encoding a first VLP-forming element, and / or (b) between the nucleotide sequences encoding the first and second VLP-forming elements, and / or (c) located between the nucleotide sequence encoding the second VLP-forming element and the nucleotide sequence encoding the third VLP-forming element;

[0373] For example, an IRES is located between a first nucleotide sequence and a nucleotide sequence encoding a first VLP-forming element. For example, an IRES is located between a nucleotide sequence encoding a first VLP-forming element and a nucleotide sequence encoding a second VLP-forming element. For example, an IRES is located between a nucleotide sequence encoding a second VLP-forming element and a nucleotide sequence encoding a third VLP-forming element.

[0374] For example, an IRES may be (a) between a first nucleotide sequence and a nucleotide sequence encoding a first VLP-forming element; (b) between the nucleotide sequences encoding the first VLP-forming element and the second VLP-forming element; and (c) located between the nucleotide sequence encoding the second VLP-forming element and the nucleotide sequence encoding the third VLP-forming element;

[0375] For example, an IRES may be (a) between a first nucleotide sequence and a nucleotide sequence encoding a first VLP-forming element; and (b) located between the nucleotide sequences encoding the first and second VLP-forming elements;

[0376] For example, an IRES may be (a) between a nucleotide sequence encoding a first VLP-forming element and a second VLP-forming element; and (b) located between a nucleotide sequence encoding a second VLP-forming element and a nucleotide sequence encoding a third VLP-forming element;

[0377] For example, an IRES may be (a) between a first nucleotide sequence and a nucleotide sequence encoding a first VLP-forming element; and (b) located between a nucleotide sequence encoding a second VLP-forming element and a nucleotide sequence encoding a third VLP-forming element;

[0378] Subgenomic (SG) promoter As used herein, the term "subgenomic promoter" (also known as a "junction region" promoter) refers to a promoter that directs the expression of a heterologous nucleotide sequence and regulates the expression of a protein.

[0379] SG promoters suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0380] In one example, the polynucleotide of the present disclosure comprises an SG promoter derived from any alphavirus. For example, the RNA (e.g., cRNA or self-replicating RNA) of the present disclosure comprises an SG promoter derived from any alphavirus. In one example, the self-replicating RNA comprises an SG promoter derived from any alphavirus.

[0381] In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. For example, a native SG promoter is a promoter native to the RNA virus (e.g., alphavirus) from which the promoter is derived and / or based.

[0382] In one example, the native SG promoter is a minimal SG promoter. For example, the minimal SG promoter is the minimum sequence required for initiation of transcription. In one example, the minimal SG promoter comprises the sequence set forth in SEQ ID NO: 3.

[0383] In one example, the native SG promoter is an extended SG promoter. For example, the extended SG promoter is a minimal SG promoter extended at the 5' end by nucleotides present in the sequence encoding a nonstructural protein (e.g., NSP4) of an RNA virus (e.g., an alphavirus). In one example, the extended SG promoter is a minimal SG promoter extended at the 5' end by nucleotides present in the sequence encoding alphavirus NSP4. In one example, the extended SG promoter is a minimal SG promoter extended at the 5' end by 51 or fewer nucleotides present in the sequence encoding alphavirus NSP4. In one example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 3, extended at the 5' end by 51 or fewer nucleotides present in the sequence encoding a nonstructural protein (e.g., an alphavirus NSP4). For example, the extended SG promoter is 100 nucleotides long or less. In one example, the extended SG promoter comprises or consists of nucleotides 2 to 101 of SEQ ID NO: 4.

[0384] In one example, the SG promoter is extended at the 5' end by about 5 to about 20 nucleotides, e.g., about 5, or about 10, or about 12, or about 15, or about 20 nucleotides, present within the sequence encoding the nonstructural protein (e.g., alphavirus NSP4). In another example, the SG promoter is extended at the 5' end by about 20 to about 35 nucleotides, e.g., about 25, or about 27, or about 30, or about 35 nucleotides, present within the sequence encoding the nonstructural protein (e.g., alphavirus NSP4).

[0385] In one example, the SG promoter is extended at the 5' end by approximately 12 nucleotides present in the sequence encoding a nonstructural protein (e.g., alphavirus NSP4). In one example, the extended SG promoter comprises the sequence set forth in SEQ ID NO:3, extended at the 5' end by 12 nucleotides present in the sequence encoding a nonstructural protein (e.g., alphavirus NSP4). For example, the extended SG promoter is 61 nucleotides or less in length. In one example, the extended SG promoter comprises or consists of nucleotides 41-101 of SEQ ID NO:4. In another example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO:75.

[0386] In one example, the SG promoter is extended at the 5' end by approximately 31 nucleotides present within the sequence encoding a nonstructural protein (e.g., alphavirus NSP4). In one example, the extended SG promoter comprises the sequence set forth in SEQ ID NO:3, extended at the 5' end by 31 nucleotides present within the sequence encoding a nonstructural protein (e.g., alphavirus NSP4). For example, the extended SG promoter is 80 nucleotides or less in length. In one example, the extended SG promoter comprises or consists of nucleotides 22-101 of SEQ ID NO:4. In another example, the extended SG promoter comprises or consists of the sequence set forth in SEQ ID NO:86.

[0387] In one example, the SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 3. In one example, the SG promoter comprises or consists of the sequence set forth in SEQ ID NO: 75.

[0388] 3' untranslated region (3'-UTR) The present disclosure provides RNAs comprising a second nucleotide sequence comprising a 3'-untranslated region (3'-UTR), fragments and / or variants thereof.

[0389] As used herein, the term "3'-UTR" refers to the region of an mRNA located 3' to the translation termination codon (i.e., stop codon). In one example, the 3'-UTR is located 3' to the translation termination codon of the nucleotide sequence encoding the last VLP-forming element in the mRNA (e.g., if the mRNA encodes three VLP-forming elements, the nucleotide sequence encoding the third VLP-forming element).

[0390] Exemplary 3'-UTRs include, for example, the 3'-UTRs of arachidonate 5-lipoxygenase (ALOX5), alpha I collagen (COL1A1), tyrosine hydroxylase (TH) genes, amino-terminal split enhancer (AES), and human mitochondrial 12S rRNA (mtRNR1), as well as fragments and / or variants thereof.

[0391] In one example, the 3'UTR is derived from or based on an alphavirus 3'UTR. In one example, the 3'UTR is the 3'UTR of an alphavirus (e.g., an alphavirus as defined herein). In one example, the 3'UTR is a native alphavirus 3'UTR. For example, a native 3'UTR is a 3'UTR native to the RNA virus (e.g., alphavirus) from which the 3'UTR is derived and / or based. For example, a native 3'UTR is a 3'UTR native to the RNA virus from which the NSP is derived. In one example, the 3'UTR is a heterologous alphavirus 3'UTR.

[0392] In one example, the 3'UTR is the 3'UTR of Venezuelan equine encephalitis virus (VEEV) or a modified form thereof. For example, the 3'UTR comprises the sequence set forth in SEQ ID NO: 77. In one example, the 3'UTR is the 3'UTR of Sindbis virus (SINV) or a modified form thereof. For example, the 3'UTR comprises the sequence set forth in SEQ ID NO: 79.

[0393] In one example, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of an albumin gene. In one example, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of a vertebrate α-globin gene. For example, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of a mammalian α-globin gene. For example, the 3'-UTR comprises or consists of a nucleotide sequence derived from the 3'-UTR of a human α-globin gene.

[0394] In one example, the 3'-UTR of the present 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.

[0395] In one example, the 3'UTR comprises or consists of a conserved sequence element (CSE). In one example, the 3'UTR comprises or consists of a 3'CSE. In one example, the 3'CSE is derived from or based on an alphavirus 3'CSE. In one example, the 3'CSE is a 3'CSE of an alphavirus (e.g., an alphavirus as defined herein). In one example, the 3'CSE is a 3'CSE of Venezuelan equine encephalitis virus (VEEV) or a modified form thereof. For example, the 3'CSE comprises the sequence set forth in SEQ ID NO: 78. In one example, the 3'CSE is a 3'CSE of Sindbis virus (SINV) or a modified form thereof. For example, the 3'CSE comprises the sequence set forth in SEQ ID NO: 80.

[0396] stop codon As used herein, the term "stop codon" refers to a trinucleotide sequence within an mRNA that signals the ribosome to stop protein synthesis.

[0397] In one example, the second nucleotide sequence comprises one or more stop codons attached to the 5' end of the 3'-UTR, fragment and / or variant thereof, e.g., the stop codons are selected from UAG, UAA, and UGA.

[0398] In one example, the RNA contains two consecutive stop codons containing the sequence UGAUGA.

[0399] In one example, the RNA contains two consecutive stop codons containing the sequence UAAUAG.

[0400] 3' tailing sequence The RNA of the present disclosure comprises a third nucleotide sequence comprising one or more 3' tailing sequences located at the 3' end of the third nucleotide sequence.

[0401] As used herein, the term "3' tailing sequence" or "3' tailing sequences" refers to a nucleotide sequence that directs the addition of non-coding nucleotides to the 3' end of an mRNA (e.g., a polyadenylation signal) or a nucleotide sequence located at the 3' end of an mRNA (e.g., a polyA sequence). Those skilled in the art will understand that a 3' tailing sequence and / or the product of a 3' tailing sequence within an mRNA functions to stabilize the mRNA and / or protect the mRNA from degradation.

[0402] As used herein, the term "interruption linker" with respect to a polyA or polyC sequence of the present disclosure refers to a single nucleotide or nucleotide sequence that binds to and interrupts a stretch of consecutive adenosine or cytosine nucleotides within the polyA or polyC sequence. For example, an interruption linker for a polyA sequence is a single nucleotide or nucleotide sequence that consists of or includes nucleotides other than adenosine nucleotides. For example, an interruption linker for a polyC sequence is a single nucleotide or nucleotide sequence that consists of or includes nucleotides other than cytosine nucleotides.

[0403] In one example, the 3' tailing sequence is selected from the group consisting of a polyA sequence, a polyadenylation signal, a G-quadruplex, a polyC sequence, a stem loop, and combinations thereof.

[0404] Poly(A) sequence As used herein, the term "poly A sequence" refers to a nucleotide sequence of adenine (A) located at the 3' end of an mRNA. In the context of the present disclosure, the poly A sequence can be located within an mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating mRNA by transcription of a vector).

[0405] Suitable polyA sequences for use in the present disclosure will be apparent to those of skill in the art and / or are described herein. In one example, the polyA sequence comprises consecutive (i.e., consecutive) adenosine nucleotides of any length (e.g., 10-300). In one example, the polyA sequence comprises consecutive adenosine nucleotides separated by one or more interrupted linkers.

[0406] In one example, the polyA sequence comprises consecutive adenosine nucleotides without interrupting linkers.

[0407] Polyadenylation signal As used herein, the term "polyadenylation signal" refers to a nucleotide sequence that induces polyadenylation. Polyadenylation is typically understood to be the addition of a polyA sequence to RNA (e.g., to a premature mRNA to produce a mature mRNA). The polyadenylation signal can be located within the nucleotide sequence at the 3' end of the RNA (e.g., mRNA) to be polyadenylated.

[0408] Suitable polyadenylation signals for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0409] In one example, the polyadenylation signal comprises a hexamer of adenine and uracil / thymidine nucleotides. In one example, the hexamer sequence comprises or consists of AAUAAA.

[0410] In one example, the third nucleotide sequence comprising the 3' tailing sequence comprises a polyadenylation signal but does not comprise a polyA sequence.

[0411] G-quadruplex As used herein, the term "G-quadruplex" or "G4" refers to a guanine residue-rich nucleotide sequence that forms a four-stranded secondary structure. For example, a G-quadruplex is a cyclic, hydrogen-bonded sequence of four guanine nucleotides formed by G-rich sequences in both DNA and RNA.

[0412] In one example, the third nucleotide sequence comprises a polyA sequence and a G-quadruplex, e.g., the third nucleotide sequence comprises a polyA sequence linked to a G-quadruplex to produce a polyAG quadruplex.

[0413] Poly C sequence As used herein, the term "polyC sequence" refers to a nucleotide sequence of cytosine (C) located at the 3' end of an mRNA. In the context of the present disclosure, the polyC sequence may be located within an mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating mRNA by transcription of a vector).

[0414] Suitable polyC sequences for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0415] In one example, the one or more 3' tailing sequences comprise one or more poly-C sequences each containing 10 to 300 consecutive cytosine nucleotides, for example, the poly-C sequences each containing 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. In one example, the 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.

[0416] In one example, the one or more poly-C sequences are separated by an interrupted linker, e.g., the third nucleotide sequence including the one or more 3' tailing sequences includes, in 5'→3' order, consecutive cytosine nucleotides, an interrupted linker, and further consecutive cytosine nucleotides.

[0417] In one example, the interruption linker is 10 to 50, or 50 to 100, or 100 to 150 nucleotides in length. For example, the interruption linker is at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95, or 100, or 110, or 120, or 130, or 140, or 150 nucleotides in length.

[0418] 5' cap structure In one example, the present disclosure provides an mRNA comprising a 5'-end cap structure.

[0419] As used herein, the term "5' cap structure" refers to a structure at the 5' end of an mRNA that is involved in nuclear export and binds to mRNA cap-binding protein (CBP). The 5' cap structure is known to stabilize mRNA by allowing CBP to bind to poly(A)-binding protein to form mature mRNA. Thus, the presence of a 5' cap structure in the mRNA of the present disclosure may further increase the stability of the mRNA compared to mRNA without a 5' cap.

[0420] Exemplary 5' cap structures include, for example, 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), Cap1, and Cap2.

[0421] Typically, endogenous mRNAs are 5'-capped with guanosine via a (5)'-ppp-(5)'-triphosphate linkage attached to the 5'-terminal nucleotide of the mRNA. The guanosine cap can then be methylated to 7-methylguanosine (m7G) to generate 7mG(5')ppp(5')N,pN2p (Cap0 structure), where N represents the first and second 5'-terminal nucleotides of the mRNA. The cap0 structure can be further 2'-O-methylated to generate 7mG(5')ppp(5')NlmpNp (Cap1) and / or 7mG(5')-ppp(5')NlmpN2mp (Cap2).

[0422] In one example, the RNA of the present disclosure includes an endogenous cap.

[0423] As used herein, the term "endogenous cap" refers to a 5' cap synthesized within a cell. For example, an endogenous cap is a natural or wild-type 5' cap. For example, an endogenous cap is a Cap0, Cap1, or Cap2 structure.

[0424] In one example, an RNA of the present disclosure includes an analog of the endogenous cap (also referred to as a cap analog).

[0425] As used herein, the term "analog thereof" or "cap analog" in the context of an endogenous cap refers to a synthetic 5' cap. Cap analogs can be used to generate 5'-capped mRNA in in vitro transcription reactions. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or attached to a nucleotide (e.g., the 5'-terminal nucleotide of an mRNA). Exemplary cap analogs are commercially available, including, for example, 3'-O-Me-m7G(5')ppp(5')G, G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs). In one example, the cap analog is N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (i.e., anti-reverse cap analog (ARCA)).

[0426] In one example, the 5' cap structure is a non-hydrolyzable cap structure, which can prevent decapping of the mRNA and increase the half-life of the mRNA.

[0427] In one example, the non-hydrolyzable cap structure comprises 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 attached to the 5' end of the mRNA via an α-phosphorothioate bond. Methods for attaching the modified nucleotide to the 5' end of the mRNA would be apparent to those skilled in the art. For example, Vaccinia Capping Enzyme (New England Biolabs) can be used.

[0428] qualification In one example, the RNA of the present disclosure comprises one or more modification(s). Typically, modifications are introduced into RNA to increase the translation efficiency and / or stability of the RNA (e.g., mRNA). Suitable modifications to RNA will be apparent to those skilled in the art and / or are described herein.

[0429] In one example, a first nucleotide sequence comprising a 5'-UTR and / or a fragment thereof is modified. By modifying the first nucleotide sequence comprising a 5'-UTR and / or a fragment thereof, a variant of the 5'-UTR and / or a fragment thereof is obtained.

[0430] In one example, the second nucleotide sequence comprising the 3'-UTR and / or a fragment thereof is modified. By modifying the second nucleotide sequence comprising the 3'-UTR and / or a fragment thereof, a variant of the 3'-UTR and / or a fragment thereof is obtained.

[0431] In one example, the nucleotide sequence encoding the VLP-forming elements and / or fragments thereof is modified. By modifying the nucleotide sequence encoding the VLP-forming elements and / or fragments thereof, variants of the VLP-forming elements and / or fragments thereof are obtained.

[0432] In one example, one or more nucleotide sequence(s) of the RNA are codon-optimized. Methods of codon optimization would be apparent to one of skill in the art and / or are described herein. For example, tools for codon optimization of RNA include, for example, GeneArt GeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).

[0433] In one example, RNA is modified to increase the amount of guanine (G) and / or cytosine (C) in the RNA. The amount of G / C in the RNA (i.e., G / C content) can affect the stability of the RNA. Thus, RNA containing an increased amount of G / C nucleotides is functionally more stable than RNA containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. G / C content is increased by substituting G or C nucleotides for A or T nucleotides.

[0434] In one example, the G / C content is increased in the nucleotide sequence encoding the VLP-forming elements. The modification(s) of the nucleotide sequence encoding the VLP-forming elements utilizes the ability to replace codons containing unfavorable nucleotide combinations (in terms of mRNA stability) with alternative codons encoding the same amino acid or amino acid(s) with similar chemical properties (e.g., conservative amino acid substitutions). For example, the G / C content is increased by replacing codons containing A or T nucleotides with codons containing G or C nucleotides that encode the same amino acid. For example, the G / C content is increased by replacing codons containing A or T nucleotides with codons containing G or C nucleotides that encode similar chemical amino acids.

[0435] In one example, the G / C content is increased in one or more nucleotide sequences of the RNA that do not encode VLP-forming elements, e.g., the G / C content is increased in a second nucleotide sequence that includes a 3'-UTR and / or a fragment thereof, e.g., the G / C content is increased in a first nucleotide sequence that includes a 5'-UTR.

[0436] In one example, the RNA comprises at least one chemically modified nucleotide.

[0437] As used herein, the term "chemically modified" or "chemically modified" in the context of nucleotides refers to a naturally occurring nucleotide (i.e., A, T, C, G, U) that has been modified by the substitution, insertion, or removal of an individual atom or a group of atoms compared to a naturally occurring nucleotide. In one example, at least one naturally occurring RNA nucleotide is replaced with a chemically modified nucleotide. In one example, at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100% of the naturally occurring RNA nucleotides are replaced with chemically modified nucleotides. Chemically modified nucleotides suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein. Exemplary chemically modified nucleotides include, for example, N6,2'-O-dimethyl-adenosine (m6Am), 5-methyluridine (m5U), N4-acetylcytidine (ac4C), 2-thiocytidine (s2C), 2-thiouridine (s2U), 5-methylcytidine (m5C), N6-methyladenosine (m6a), pseudouridine (ψ), and 1-methylpseudouridine (m1ψ).

[0438] Generation method Suitable methods for producing the RNA of the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0439] Plasmid DNA is generated by inserting a nucleotide sequence comprising a 5'-UTR, optionally encoding an NSP (e.g., NSP1, NSP2, NSP3, and NSP4), a nucleotide sequence(s) encoding a VLP-forming element(s), and a nucleotide sequence comprising a 3'UTR into a DNA vector. Suitable DNA vectors for use will be apparent to those skilled in the art, and the nucleotide sequences of the present disclosure can be purchased from any commercial supplier. Insertion of a nucleotide sequence(s) into a DNA vector can be performed using standard methods in the art.

[0440] In one example, mRNA is produced using plasmid DNA. Those skilled in the art will understand that plasmid DNA is relatively stable. Briefly, a DNA plasmid encoding the mRNA of the present disclosure is used to transform competent bacterial cells (e.g., Escherichia coli cells). Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in E. coli cultures.

[0441] In one example, plasmid DNA is isolated after fermentation. For example, the plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other conventional methods known to those skilled in the art. After isolation, the plasmid DNA is linearized by restriction digestion (i.e., using a restriction enzyme). The restriction enzyme is removed using methods known in the art (e.g., including phenol / chloroform extraction and ethanol precipitation).

[0442] In one example, mRNA is produced by in vitro transcription from a linearized DNA template using an RNA polymerase (e.g., T7 RNA polymerase). In some embodiments, the RNA described herein further comprises one or more additional 5' nucleotides (e.g., additional 5' Gs) as an artifact of promoter usage (e.g., the T7 promoter (TAATACGACTCACTATAG, SEQ ID NO: 92)). After in vitro transcription, the DNA template is removed by DNase digestion. Those skilled in the art will appreciate that synthetic RNA capping can be performed to correct mRNA processing and contribute to mRNA stabilization. In one example, the RNA is enzymatically 5'-capped. For example, the 5' cap is a cap0 structure or a cap1 structure. In one example, the 5' cap is a cap0 structure, for example, a 5'-cap (i.e., cap0) consisting of an inverted 7-methylguanosine connected to the remainder of the RNA via a 5'-5' triphosphate bridge. In one example, the 5' cap is a cap1 structure, e.g., a 5'-cap (i.e., cap1) consists of a cap0 with an additional methylation at the 2'0 position of the initiating nucleotide. One skilled in the art will also understand that polyadenylation of mRNA can be performed on mRNAs that contain a polyadenylation sequence.

[0443] 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). In one example, mRNA is purified using anion exchange chromatography. For example, anion exchange chromatography is performed using an anion exchange resin (e.g., MustangQ® membrane (Pall®)). After purification, the mRNA is suspended in, for example, nuclease-free water.

[0444] composition The present disclosure provides an immunogenic composition comprising the mRNA of the present disclosure. For example, the immunogenic composition is a vaccine.

[0445] The present disclosure also provides a pharmaceutical composition comprising an immunogenic composition of the present disclosure and a pharmaceutically acceptable carrier.

[0446] It will be apparent to one of skill in the art and / or as described herein that the mRNA of the present disclosure can be present as naked mRNA or in combination with lipids, polymers, or other delivery systems that facilitate entry into cells.

[0447] delivery system In one example, the pharmaceutical composition of the present disclosure further comprises a lipid nanoparticle (LNP), a polymeric microparticle, and / or an oil-in-water emulsion, e.g., the mRNA is encapsulated, bound to, or adsorbed to the LNP, polymeric microparticle, and / or oil-in-water emulsion.

[0448] lipid nanoparticles In one example, the pharmaceutical composition of the present disclosure further comprises an LNP.

[0449] It will be apparent that the term "lipid nanoparticle" or "LNP" should be understood to refer to any lipid composition, including, but not limited to, liposomes or vesicles in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; unilamellar or multilamellar; multilamellar), micelle-like lipid nanoparticles with a non-aqueous core, and solid lipid nanoparticles (solid lipid nanoparticles do not have a lipid bilayer). Methods for preparing LNPs are known to those skilled in the art and / or described herein. In one example, LNPs are prepared using a staggered herringbone mixer, e.g., as described in U.S. Patent Application Publication No. 2012 / 0276209. In another example, liposomes are prepared using a microfluidic device, e.g., as described in WO2018220553. In one example, LNPs are prepared using a T-shaped mixer.

[0450] Lipid nanoparticles suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein, for example, LNPs comprising ionizable lipids.

[0451] As used herein, the term "ionizable lipid" or "ionizable lipids" refers to a lipid having at least one protonatable or deprotonatable group. For example, the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutrally charged at a second pH (e.g., above physiological pH). For example, the lipid is a cationic lipid.

[0452] Suitable ionizable lipids may have anionic, cationic, or zwitterionic hydrophilic head groups. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. In one example, the lipid is a cationic lipid. Exemplary 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), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxy)benzyl-4-(dimethylamino)butanoate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxy)benzyl-4-(dimethylamino)butanoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids (e.g., dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphocholine). The lipids can be saturated or unsaturated.

[0453] Lipid moieties suitable for use in LNPs will be apparent to those skilled in the art and include, for example, fatty acids, isoprenoids, and combinations thereof. In one example, the lipid moiety is selected from the group consisting of an isoprenoid, a triglyceride, a phospholipid, a cholesteryl ester, and combinations thereof.

[0454] In one example, the lipid nanoparticles further comprise a PEG-lipid, a sterol-structured lipid, and / or a neutral lipid. In one example, the lipid nanoparticles do not comprise a cationic lipid.

[0455] PEG-lipid In one example, the present disclosure provides an LNP comprising a PEGylated lipid.

[0456] It will be clear to those skilled in the art that PEGylated lipid is the lipid modified with polyethylene glycol.Exemplary PEGylated lipid includes but is not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.For example, PEG lipid includes PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipid, and their combinations.

[0457] neutral lipid In one example, the present disclosure provides an LNP comprising a neutral lipid.

[0458] Neutral or zwitterionic lipids suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, 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), 1,2-dipa Hemisuccinoyl-sn-glycero-3-phosphocholine (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-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero Lipids include 1,2-dioleoyl-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. The lipids may be saturated or unsaturated.

[0459] structured lipids In one example, the present disclosure provides an LNP comprising structured lipids.

[0460] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.

[0461] In one example, the structured lipid is a sterol, for example, cholesterol, or in another example, campesterol.

[0462] polymer particles In one example, the pharmaceutical composition of the present disclosure further comprises polymeric microparticles.

[0463] Those skilled in the art will recognize that various polymers can form the microparticles for encapsulating or adsorbing the RNA of the present disclosure.It will be clear that the use of substantially non-toxic polymer means that the particles are safe, and the use of biodegradable polymer means that the particles can be metabolized after delivery to avoid long-term persistence.In addition, useful polymers can be sterilized to support the preparation of pharmaceutical-grade formulations.

[0464] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acid), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinylpyrrolidinone, or polyesteramides, and combinations thereof.

[0465] Oil-in-water emulsion In one example, the pharmaceutical composition of the present disclosure further comprises an oil-in-water emulsion, for example, a cationic oil-in-water emulsion.

[0466] Oils suitable for use in oil-in-water emulsions will be apparent to those skilled in the art and / or are described herein. For example, the emulsion may contain one or more oils derived from, for example, animal (e.g., fish) or vegetable (e.g., nuts, seeds, grains) sources. 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 oil, and corn oil.

[0467] In addition to oil, the oil-in-water emulsion also contains a cationic lipid to promote emulsion formation and stabilization. Suitable cationic lipids will be apparent 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), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl[C16:0]trimethylammoniumpropane (DPTAP), and distearoyltrimethylammoniumpropane (DSTAP).

[0468] In some examples, the oil-in-water emulsion also includes a nonionic surfactant and / or a zwitterionic surfactant. Those skilled in the art would be aware of surfactants suitable for use in the present disclosure. Exemplary surfactants include, but are not limited to, polyoxyethylene sorbitan ester surfactants (e.g., polysorbate 20 and polysorbate 80) and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0469] Pharmaceutically Acceptable Carriers Preferably, in the compositions or methods for administering the mRNA of the present disclosure to a subject, the mRNA is combined with a pharmaceutically acceptable carrier, as understood in the art. Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the mRNA of the present disclosure (and any delivery system, e.g., LNP) combined with a pharmaceutically acceptable carrier.

[0470] Generally, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating material, emulsion, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject (e.g., a human). Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0471] The mRNA of the present disclosure is useful for parenteral, topical, oral or local administration, intramuscular administration, aerosol administration or transdermal administration for preventive or therapeutic treatment.In one example, mRNA is administered parenterally (for example, intramuscularly, subcutaneously or intravenously).For example, RNA is administered intramuscularly.

[0472] Formulations of the mRNA of the present disclosure to be administered will vary depending on the route of administration and formulation (e.g., solution, emulsion, capsule) selected. Suitable pharmaceutical compositions containing the mRNA to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Various suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can include various additives, preservatives, fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition can optionally contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting agents, buffering agents, and toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The mRNA can be stored in a liquid phase or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

[0473] The optimal concentration of the active ingredient(s) in the selected vehicle can be determined empirically according to procedures known to those skilled in the art and depends on the final pharmaceutical formulation desired.

[0474] Once formulated, the compounds of the present disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically / prophylactically effective. The dosage ranges for administration of the molecules of the present disclosure are those sufficient to produce the desired effect. For example, the composition comprises an effective amount of mRNA. In one example, the composition comprises a therapeutically effective amount of mRNA. In another example, the composition comprises a prophylactically effective amount of mRNA.

[0475] The dosage should not be so high as to cause adverse side effects. Generally, the dosage will vary depending on the age, condition, sex, and degree of disease in the patient, but can be determined by those skilled in the art. The dosage can be adjusted by an individual physician if any complications arise.

[0476] Dosages can vary from about 0.1 mg / kg to about 300 mg / kg, for example, about 0.2 mg / kg to about 200 mg / kg, for example, about 0.5 mg / kg to about 20 mg / kg, administered once or more times a day for one to several days.

[0477] In some instances, the mRNA is administered at an initial (or loading) dose, which is higher than subsequent (maintenance) doses. For example, the mRNA is administered at an initial dose of about 10 mg / kg to about 30 mg / kg. The mRNA is then administered at maintenance doses of about 0.0001 mg / kg to about 10 mg / kg. The maintenance doses can be administered every 7 to 35 days (e.g., every 7, 14, or 28 days).

[0478] In some instances, a dose escalation regimen is used, in which mRNA is initially administered at a lower dose than that used in subsequent doses. This administration regimen is useful when the subject first experiences an adverse event.

[0479] For subjects who do not respond adequately to treatment, multiple doses can be administered weekly. Alternatively, or additionally, increasing doses may be administered.

[0480] The subject can be re-treated with mRNA by administering multiple exposures or dose sets (e.g., at least about 2 exposures of mRNA, e.g., about 2-60 exposures, more particularly about 2-40 exposures, most particularly about 2-20 exposures).

[0481] In one example, optional re-treatment can occur when signs or symptoms of the disease recur.

[0482] In one example, optional re-treatment may occur when there are no signs or symptoms of disease recurrence.

[0483] In another example, optional retreatments can be administered at defined intervals. For example, subsequent exposures can be administered at various intervals (e.g., about 3-4 weeks, or 4-12 weeks, or 24-28 weeks, or 48-56 weeks, or longer). For example, such exposures are administered at intervals of about 3-4 weeks, or 4-8 weeks, or 4-12 weeks, or 24-26 weeks, or about 38-42 weeks, or about 50-54 weeks.

[0484] In another example, for subjects experiencing side effects, the initial (or loading) dose can be administered on multiple days, either divided into separate doses or on multiple consecutive days, within a week.

[0485] Administration of mRNA according to the disclosed methods can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of mRNA can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, e.g., during or after the onset of the condition.

[0486] Screening assays Expression of virus-like particle (VLP)-forming elements In one example, a composition containing mRNA(s) is evaluated for expression of VLP-forming elements. For example, antigen expression is detected using an antibody against the VLP-forming elements. 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 quantified, for example, using FACS. In a further example, a specific efficacy value or probability of successful transfection per unit mass of mRNA is calculated.

[0487] Quantification of virus-like particle (VLP) release In one example, a composition containing mRNA(s) is assessed for VLP formation and release from cells expressing the VLP-forming elements. For example, VLP release from the cells is analyzed using antibodies against the VLP-forming elements. In a further example, association between VLP-forming elements is quantified using antibody-mediated co-immunoprecipitation and / or detection of VLP-forming elements (e.g., HA, NA, and M1) in co-immunoprecipitated samples (e.g., by Western blot analysis).

[0488] Microneutralization assay In one example, a composition (naked and / or formulated) comprising mRNA(s) is evaluated for antibody responses. For example, the composition comprising mRNA(s) is evaluated using a microneutralization assay. Methods for performing a microneutralization assay would be apparent to one skilled in the art. In one example, the microneutralization assay is a short-form assay. In one example, a viral fluorescent focus-based microneutralization assay is performed. In another example, the microneutralization assay is a long-form assay.

[0489] Hemagglutination Inhibition (HAI) Assay In one example, compositions (naked and / or formulated) comprising mRNA(s) are assessed for antibody responses. For example, compositions comprising mRNA(s) are assessed using a hemagglutination inhibition (HAI) assay. Methods for performing an HAI assay will be apparent to those skilled in the art and / or are described, for example, in WHO (2011) Manual for the laboratory diagnosis and virological surveillance of influenza: WHO Press, World Health Organization.

[0490] Neuraminidase Inhibition Assay (NAI) Assay In one example, compositions (naked and / or formulated) comprising mRNA(s) are assessed for antibody responses. For example, compositions comprising mRNA(s) are assessed using a neuraminidase inhibition (NAI) assay. Methods for performing an NAI assay would be apparent to one of skill in the art and / or are described, for example, in WHO (2011) Manual for the laboratory diagnosis and virological surveillance of influenza: WHO Press, World Health Organization.

[0491] Antigen-specific T cell response In one example, compositions (naked and / or formulated) comprising mRNA(s) are assessed for their ability to induce an antigen-specific T cell response. Methods for assessing the induction of an antigen-specific T cell response will be apparent to those skilled in the art and / or are described herein.

[0492] For example, the detection of antigen-specific T cell is carried out in spleen culture.Briefly, spleen cell culture is established in T cell medium, and cell culture is stimulated or not stimulated with antigen peptide.In one example, the antigen-specific T cell response is quantified by flow cytometry.

[0493] Methods for generating virus-like particles The compositions and RNAs described herein can be used to produce virus-like particles. The inventors have found that the stability of VLPs produced from the compositions described herein can be increased by increasing the incorporation of M1 protein into the VLPs. The inventors have also found that the efficient release of VLPs, for example from cells, can be increased by increasing the incorporation of NA protein into the VLPs. Thus, the present disclosure provides a method for expressing virus-like particles (VLPs) in a subject, the method comprising administering to the subject an RNA, composition, immunogenic composition, or pharmaceutical composition of the present disclosure.

[0494] The present disclosure further provides the use of an RNA, composition, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for expressing a VLP in a subject in need thereof.

[0495] The present disclosure also provides an RNA, immunogenic composition, or pharmaceutical composition of the present disclosure for use in a method for expressing a VLP in a subject in need thereof.

[0496] The present disclosure also provides a method for increasing the stability of a virus-like particle (VLP), the method comprising introducing into a composition a nucleotide sequence encoding the matrix-1 (M1) protein of influenza virus, the composition comprising the following nucleotide sequence in 5' to 3' order: a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding the neuraminidase (NA) protein of an influenza virus operably linked to a second subgenomic (SG) promoter; c) RNA comprising a nucleotide sequence encoding an M1 protein of an influenza virus operably linked to a third subgenomic (SG) promoter; Introducing a nucleotide sequence encoding the M1 protein into the composition increases the stability of VLPs produced from the composition.

[0497] The present disclosure further provides a method for increasing the efficient release of virus-like particles (VLPs), the method comprising introducing into a composition a nucleotide sequence encoding the neuraminidase (NA) protein of an influenza virus, the composition comprising: a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding the NA protein of an influenza virus operably linked to a second subgenomic (SG) promoter; c) RNA comprising a nucleotide sequence encoding the matrix-1 (M1) protein of influenza virus operably linked to a third subgenomic (SG) promoter; Introducing a nucleotide sequence encoding an NA protein into the composition increases the efficient release of VLPs produced from the composition.

[0498] The present disclosure also provides a method for increasing the stability and efficient release of virus-like particles (VLPs), the method comprising introducing into a composition a nucleotide sequence encoding the matrix-1 (M1) protein and a nucleotide sequence encoding the neuraminidase (NA) protein of influenza virus, the composition comprising: a) a nucleotide sequence encoding the hemagglutinin (HA) protein of an influenza virus operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof, or a first subgenomic (SG) promoter; b) a nucleotide sequence encoding the NA protein of an influenza virus operably linked to a second subgenomic (SG) promoter; c) RNA comprising a nucleotide sequence encoding an M1 protein of an influenza virus operably linked to a third subgenomic (SG) promoter; Introducing nucleotide sequences encoding the M1 and NA proteins into the composition increases the stability and efficient release of VLPs produced from the composition.

[0499] In one example, a nucleotide sequence encoding an influenza virus hemagglutinin (HA) protein is operably linked to a nucleotide sequence comprising a 5'-untranslated region (5'-UTR), fragments and / or variants thereof. In one example, the 5'UTR comprises or consists of the sequence set forth in SEQ ID NO:76.

[0500] In one example, the nucleotide sequence encoding the influenza virus hemagglutinin (HA) protein is operably linked to a first SG promoter. In one example, the first SG promoter is a native subgenomic promoter. In one example, the first SG promoter is derived from an alphavirus. In one example, the first SG promoter is derived from VEEV.

[0501] In one example, the second subgenomic promoter is derived from an alphavirus. In one example, the second subgenomic promoter is derived from VEEV. In one example, the second subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:75. In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:3. In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:75.

[0502] In one example, the third subgenomic promoter is derived from an alphavirus. In one example, the third subgenomic promoter is derived from VEEV. In one example, the third subgenomic promoter comprises or consists of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:75. In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:3. In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO:75.

[0503] In one example, the second subgenomic promoter consists of the sequence set forth in SEQ ID NO: 75, and the third subgenomic promoter consists of the sequence set forth in SEQ ID NO: 3. Without wishing to be bound by theory, the inventors have found that a combination of a second subgenomic promoter consisting of the sequence set forth in SEQ ID NO: 75 and a third subgenomic promoter consisting of the sequence set forth in SEQ ID NO: 3 increases the amount of NA and M1 proteins incorporated into VLPs.

[0504] Treatment or prevention methods The present disclosure provides methods for treating or preventing or delaying the progression of influenza and / or influenza virus infection.

[0505] influenza The present disclosure provides methods for treating, preventing, or delaying the progression of influenza in a subject.

[0506] Influenza (also known as "flu") is an infectious disease caused by the influenza virus. Symptoms can range from mild to severe, with the most common symptoms including 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 often last less than a week. Complications of influenza include viral pneumonia, secondary bacterial pneumonia, sinus infections, and worsening of pre-existing health problems (e.g., asthma or heart failure). Viral pneumonia can also lead to acute respiratory distress syndrome (ARDS).

[0507] Thus, in some examples of the present disclosure, the subject has influenza virus infection. In one example, the subject has influenza. In particular, influenza is associated with ARDS. In one example, the method of the present disclosure can be used to treat or prevent ARDS in a subject suffering from influenza virus infection. In one example, the method of the present disclosure can be used to treat or prevent ARDS in a subject suffering from influenza.

[0508] In one example, the subject is at risk of having an influenza virus infection. In one example, the subject is at risk of having influenza. In particular, influenza is associated with ARDS. In one example, the method of the present disclosure can be used to prevent ARDS in a subject suffering from an influenza virus infection. In one example, the method of the present disclosure can be used to prevent ARDS in a subject suffering from an influenza virus infection. In one example, the method of the present disclosure can be used to delay the progression of ARDS in a subject suffering from an influenza virus infection.

[0509] Acute respiratory distress syndrome (ARDS) The present disclosure provides methods for treating, preventing, or slowing the progression of ARDS in a subject.

[0510] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltrates, severe hypoxemia, and disruption of the alveolar-capillary membrane barrier (i.e., pulmonary vascular leakage), which ultimately leads to noncardiogenic pulmonary edema. Currently, there is no effective pharmacological therapy.

[0511] Infectious causes, including influenza, are the main causes of ARDS.Therefore, in one example of the present disclosure, ARDS is associated with influenza infection.For example, ARDS is associated with influenza.

[0512] ARDS is classified according to the Berlin definition, which includes the following: (1) symptoms within 1 week of the onset of clinical attacks or respiratory symptoms; (2) acute hypoxemic respiratory failure (determined by a PaO2 / FiO2 ratio of 300 mmHg or less with 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 the arteries and FiO2 is the fraction of inspired oxygen); (3) bilateral radiographic opacities of the lungs not adequately explained by exudation, consolidation, or atelectasis; and (4) edema / respiratory failure not adequately explained by cardiac failure or fluid overload;

[0513] In one example, the subject has or is suffering from ARDS (i.e., the subject meets the Berlin definition of ARDS). For example, the subject is in need of (i.e., in need of) treatment.

[0514] In one example, the subject has symptoms associated with ARDS or suffers from symptoms associated with ARDS. Methods for identifying symptoms associated with ARDS and subjects at risk of developing ARDS will be apparent to those skilled in the art and / or are described herein. For example, the subject has one or more or all of the following symptoms: a) respiratory frequency greater than 30 breaths per minute; b) Oxygen saturation (SpO2) on room air is 93% or less; c) The ratio of arterial oxygen partial pressure to inspired oxygen partial pressure (PaO2 / FiO2) is less than 300 mmHg. d) SpO2 / FiO2 ratio less than 218, and e) Radiographic pulmonary infiltrates greater than 50%.

[0515] Currently, ARDS is classified as mild, moderate, or severe, with increasing mortality in that order. The severity of ARDS can be classified according to the Berlin definition as follows:

[0516] (i) Mild ARDS: PaO2 / FiO2 of 200–300 mmHg with at least 5 cm of CPAP or PEEP; (ii) Moderate ARDS: PaO2 / FiO2 of 100–200 mmHg with a PEEP of at least 5 cm, and (iii) Severe ARDS: PaO2 / FiO2 less than or equal to 100 mmHg with at least 5 cm PEEP.

[0517] In one example, the ARDS is mild ARDS. In another example, the ARDS is moderate ARDS. In a further example, the ARDS is severe ARDS.

[0518] The methods of the present disclosure can be used to prevent or delay the onset of ARDS, in addition to treating existing ARDS. Thus, in one example, the subject does not have ARDS.

[0519] In one example, the subject is at risk of developing one or more symptom(s) associated with ARDS.

[0520] kit In one example, the present disclosure provides a kit containing DNA useful for producing the mRNA of the present disclosure. For example, the kit includes a DNA plasmid and components for producing the mRNA. For example, the components for producing the mRNA include sequencing and / or tail PCR primers, transcription reagents (e.g., T7 RNA polymerase), and are packaged with instructions for producing the mRNA of the present disclosure from the DNA.

[0521] In one example, the present disclosure provides a kit comprising an mRNA of the present disclosure useful for treating or preventing influenza virus infection and / or influenza.

[0522] In one example, the kit includes (a) a container containing RNA (optionally in a delivery system and / or a pharmaceutically acceptable carrier or diluent) and (b) a package insert with instructions for treating or preventing influenza and / or influenza virus infection in a subject.

[0523] According to this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition effective for a disease or disorder of the present disclosure and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is RNA. The label or package insert indicates that the composition is used for treating an eligible subject (e.g., a subject suffering from or susceptible to influenza, influenza virus infection, and / or ARDS), along with specific guidance regarding dosage and treatment intervals, as well as any other medications provided. The kit may further include an additional container containing a pharmaceutically acceptable diluent buffer (e.g., 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 standpoint, including other buffers, diluents, filters, needles, and syringes.

[0524] The present disclosure includes the following non-limiting examples. [Example]

[0525] Example 1: Production of RNA Using the H5 and N1 subtypes from A / turkey / Turkey / 1 / 2005 and the M1 matrix protein from PR8X as VLP-forming elements, DNA templates for sa-mRNA were prepared comprising the nucleotide sequence of the 5'-UTR set forth in SEQ ID NO: 76 and the nucleotide sequence of the 3'-UTR set forth in SEQ ID NO: 80. The nucleotide sequences encoding the N1 subtype from A / turkey / Turkey / 1 / 2005 and the M1 matrix protein from PR8X were operably linked to the nucleotide sequence of the extended subgenomic promoter (v2) set forth in SEQ ID NO: 75 or the nucleotide sequence of the alphavirus native subgenomic promoter (v1) set forth in SEQ ID NO: 3. The nucleotide sequences encoding the H5 subtype from A / turkey / Turkey / 1 / 2005 and the N1 subtype from A / turkey / Turkey / 1 / 2005 were operably linked to the nucleotide sequence of the extended subgenomic promoter (v2) set forth in SEQ ID NO: 75 or the nucleotide sequence of the alphavirus native subgenomic promoter (v1) set forth in SEQ ID NO: 3. The following constructs were prepared: NSP1-4.nativeSGP.H5.SGPv1.N1.SGPv1.M1 (F554, SEQ ID NO: 81), NSP1-4.nativeSGP.H5.SGPv1.N1.SGPv2.M1 (F624, SEQ ID NO: 82), NSP1-4.nativeSGP.H5.SGPv2.N1.SGPv1.M1 (F625, SEQ ID NO: 83), and -NSP1-4.nativeSGP.H5.SGPv2.N1.SGPv2.M1 (F626, SEQ ID NO: 84).

[0526] Standard laboratory methods and materials were used to generate the sa-mRNA used herein. A DNA template encoding the sa-mRNA was generated in competent Escherichia coli cells transformed with a DNA plasmid. Individual bacterial colonies were isolated, and the resulting plasmid DNA was amplified in E. coli cultures. After fermentation, the plasmid DNA was isolated using a Maxiprep DNA kit and linearized by restriction digestion. The restriction enzyme was then removed using phenol / chloroform extraction and ethanol precipitation.

[0527] sa-mRNA was generated by in vitro transcription from a linearized DNA template using T7 RNA polymerase. The DNA template was subsequently removed by DNase digestion. Functional mRNA was obtained by enzymatic capping using Cap0. The resulting sa-mRNA was purified and resuspended in nuclease-free water.

[0528] The resulting mRNA was formulated into LNPs to yield the LNP-formulated sa-mRNA construct.

[0529] Example 2: In vitro characterization of self-replicating RNA and virus-like particles (VLPs) Expression levels were evaluated for compositions comprising one or more RNAs containing nucleotide sequences encoding VLP-forming elements produced in Example 1. LNP-formulated mRNA constructs (50 μg) were incubated with a BHK cell line for 48 hours.

[0530] To quantify and characterize VLPs in vitro, cell culture supernatants and cells from BHK cells incubated with the LNP-formulated sa-mRNA construct were harvested. The cell culture supernatants were separated from the BHK cells by centrifugation at 1000 g for 5 minutes. VLPs present in the cell culture supernatants were immunoprecipitated using an anti-HA antibody (monoclonal antibody 15A6) conjugated to protein A beads. Western blot analysis was performed on BHK cell lysates and immunoprecipitated cell culture supernatants using anti-HA antibody (avian influenza A virus H5N3 HA antibody clone AT2B7), anti-NA antibody (influenza A H1N1 NA polyclonal), anti-M1 antibody (influenza A M1 monoclonal clone GA2B), and anti-GAPDH (control) (Figure 1). Figure 1 shows that intact VLPs were produced for all sa-mRNA constructs produced in Example 1 and that the VLPs were released into the supernatant from the BHK cells used to produce the VLPs.

[0531] The levels of three VLP-forming elements (HA, NA, and M1) in cell culture supernatants were quantified and compared among the different sa-mRNA constructs using Western blot densitometry (Figure 2). To further confirm VLP production, electron microscopy was used to visualize VLPs released from cells expressing the VLP-forming elements. The results of the densitometry analysis of the Western blots in Figure 1 are summarized in Tables 1 and 2. [Table 2] [Table 3]

[0532] The sa-mRNA construct F626 produced the highest amount of M1 protein in BHK cells, whereas construct F625 had the highest level of M1 protein incorporated into VLPs (Fig. 2C and 2D). sa-mRNA construct F625 also had the highest levels of HA, NA, and M1 protein incorporation into VLPs (Fig. 2B and Table 2).

[0533] Example 3: Further characterization of self-replicating RNA and virus-like particles (VLPs) antibody response To assess antibody immune responses in preclinical animal models, mice can be immunized with LNP-formulated sa-mRNA constructs at a suitable dose (e.g., 0.1 pg or 0.001 pg) on ​​day 0 and a second dose on day 21. To assess antibody responses, serum is collected at the end of the study (i.e., 42 days after the first vaccination or 21 days after the second vaccination) and tested by microneutralization assay and hemagglutination inhibition assay.

[0534] For all serologi...

Claims

1. A composition comprising one or more ribonucleic acids (RNAs), wherein each RNA is arranged in the order of 5'→3'. a) A first nucleotide sequence comprising the 5'-untranslated region (5'-UTR), its fragment and / or variant, b) One or more nucleotide sequences that encode a virus-like particle (VLP) forming element, c) comprising a second nucleotide sequence including a 3'-untranslated region (3'-UTR), its fragment and / or variant, The VLP-forming element is selected from the hemagglutinin (HA) protein, neuraminidase (NA) protein, and matrix-1 (M1) protein of the influenza virus. The composition comprises a nucleotide sequence encoding the HA protein, the NA protein, and the M1 protein, respectively.

2. The composition according to claim 1, wherein the composition comprises RNA including a nucleotide sequence encoding the HA protein, a nucleotide sequence encoding the NA protein, and a nucleotide sequence encoding the M1 protein.

3. The composition according to claim 1, wherein the composition comprises a first RNA containing a nucleotide sequence encoding the HA protein, a second RNA containing a nucleotide sequence encoding the NA protein, and a third RNA containing a nucleotide sequence encoding the M1 protein.

4. The composition according to claim 1, wherein the one or more RNAs comprise one or more additional nucleotide sequences encoding matrix-2 (M2), nucleoproteins (NPs), and / or non-structural (NS) proteins of the influenza virus, and the one or more additional nucleotide sequences are located on the 3' or 5' side of the one or more nucleotide sequences encoding the VLP-forming elements.

5. In the order of 5' → 3' a) A nucleotide sequence encoding the hemagglutinin (HA) protein of the influenza virus, which is responsively bound to a nucleotide sequence containing the 5'-untranslated region (5'-UTR), its fragments and / or variants, or a first subgenome (SG) promoter. b) A nucleotide sequence encoding the influenza virus neuraminidase (NA) protein, which is admissibly bound to the second subgenome (SG) promoter, and c) A nucleotide sequence encoding the influenza virus matrix-1 (M1) protein, which is admissibly bound to the third subgenome (SG) promoter. The composition according to claim 1, comprising RNA containing the following:

6. The composition according to claim 5, wherein the RNA is sa-mRNA or cRNA.

7. The composition according to claim 5, wherein the nucleotide sequence encoding the hemagglutinin (HA) protein of the influenza virus is admissibly bound to a first subgenome (SG) promoter.

8. The composition according to claim 5, wherein the second subgenome (SG) promoter comprises the sequence described in SEQ ID NO: 3 or SEQ ID NO:

75.

9. The composition according to claim 5, wherein the third subgenome (SG) promoter comprises the sequence described in SEQ ID NO: 3 or SEQ ID NO:

75.

10. The composition according to claim 5, wherein the second subgenome (SG) promoter comprises the sequence described in SEQ ID NO: 75, and the third subgenome (SG) promoter comprises the sequence described in SEQ ID NO:

3.

11. The composition according to any one of claims 1 to 10, wherein the RNA is formulated into lipid nanoparticles (LNPs).

12. The composition according to any one of claims 1 to 10, wherein the composition comprises an immunogenic composition.

13. A pharmaceutical composition comprising the immunogenic composition described in claim 12 and a pharmaceutically acceptable carrier.

14. An immunogenic composition according to claim 12 or a pharmaceutical composition according to claim 13 for use as a vaccine.

15. An immunogenic composition according to claim 12 or a pharmaceutical composition according to claim 13 for use in the treatment, prevention, or delay of the progression of influenza or influenza virus infection.

16. An immunogenic composition according to claim 12 or a pharmaceutical composition according to claim 13 for use in inducing an immune response in a subject requiring such induction.

17. The composition according to claim 16, wherein the immune response is a humoral and / or cell-mediated immune response.

18. An immunogenic composition according to claim 12 or a pharmaceutical composition according to claim 13 for use in a method for inducing VLP expression in a subject that requires it.

19. A composition according to claim 1, an immunogenic composition according to claim 12, or a pharmaceutical composition according to claim 13, for use in inducing an immune response to influenza virus in a subject that requires such response.

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