Influenza virus vaccine

Abstract

The present invention relates, in particular, to an immunogenic composition for use in the treatment or prevention of infection by influenza virus. The immunogenic composition comprises (a) a first nucleic acid encoding the hemagglutinin (HA) antigen of a strain of a first subtype of influenza A virus, (b) a second nucleic acid encoding the HA antigen of a strain of a second subtype of influenza A virus, (c) a third nucleic acid encoding the HA antigen of a first strain of influenza B virus, and (d) a fourth nucleic acid, optionally included, encoding the HA antigen of a second strain of influenza B virus, wherein the immune response is induced against the aforementioned strains of the first and second subtypes of influenza A virus, the aforementioned first strain and optionally included second strain of influenza B virus, and the HA antigen of at least one further HA antigen subtype of influenza A virus (different from any of the HA antigen subtypes of influenza A virus encoded by the nucleic acids present in the composition). [No selection diagram available]

Description

[Technical Field]

[0001] The present invention relates, in particular, to immunogenic compositions for use in the treatment or prevention of influenza virus infection, comprising nucleic acids, preferably mRNA, encoding hemagglutinin (HA) antigens derived from strains of influenza A and B viruses, as well as vaccines and kits or kits of parts comprising the same. The present invention also relates to methods for inducing an immune response to influenza viruses, and methods for treating or preventing disorders caused by influenza viruses. [Background technology]

[0002] background Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family (NCBI classification ID: 11308), and are subdivided into, for example, alpha-influenza viruses (the genus including influenza A virus) and beta-influenza viruses (the genus including influenza B virus), which are prevalent in all regions of the world. Influenza viruses often cause acute respiratory illness during localized outbreaks or seasonal epidemics, and sometimes during pandemics. Typical influenza outbreaks lead to an increased incidence of pneumonia and lower respiratory tract disease, thereby increasing hospitalization and mortality rates. The elderly or those with underlying chronic diseases are most likely to develop such complications, but infants and young children can also contract serious illnesses. Influenza viruses (mainly influenza A and influenza B viruses) have a significant impact on public health worldwide, causing millions of serious illnesses, thousands of deaths, and considerable economic losses each year.

[0003] Influenza viruses, such as influenza A and influenza B viruses, are enveloped viruses containing eight segmented negative-strand RNA molecules that encode 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). Among these viral proteins, the most distinctive are hemagglutinin (HA) and neuraminidase (NA), two large glycoproteins located on the outside of the viral particle. NA is an enzyme involved in the release of progeny viruses from infected cells. HA is a lectin that mediates the binding of the virus to target cells and the entry of the viral genome into those cells.

[0004] Currently, there are 18 described HA (H1-H18) subtypes and 11 described NA (N1-N11) subtypes of influenza A virus, which potentially form 144 combinations of HA and NA. Unlike influenza A viruses, which have a broad range of hosts, influenza B viruses infect humans almost without exception. Influenza B viruses are classified into two distinct lineages: B / Victoria / 2 / 1987-like (B / Victoria lineage) and B / Yamagata / 16 / 1988-like (B / Yamagata lineage) (which has been circulating worldwide since 1983). Influenza B viruses mutate 2-3 times slower than influenza A viruses, yet this still has a significant impact on children and young adults every year.

[0005] The constant emergence of new influenza virus strains due to antigenic drift is the virological cause of seasonal epidemics. Due to its constantly evolving nature, regular updates of the viruses contained in influenza vaccines are necessary to maintain vaccine effectiveness. Public health authorities monitor influenza viruses circulating in humans and update recommended formulations of influenza vaccines twice a year. The published recommendations (usually three or four different influenza virus strains) are used by national vaccine regulatory bodies and pharmaceutical companies for the development, manufacture, and approval of influenza vaccines for the next influenza season.

[0006] Vaccination is currently the most widely used method to prevent influenza outbreaks, particularly in high-risk populations. Multivalent live attenuated influenza vaccines (FLUMIST, AstraZeneca), inactivated influenza vaccines (AFLURIA, FLUAD and FLUCELVAX, Seqirus; FLUARIX and FLULAVAL, GlaxoSmithKline; FLUZONE, Sanofi), or recombinant influenza vaccines (FLUBLOK, Sanofi) are already commercially available for active immunization against diseases caused by influenza A subtype and influenza B viruses contained in the vaccines.

[0007] Because HA is the major influenza virus antigen recognized by neutralizing antibodies, this glycoprotein is currently the focus of approved inactivated and recombinant influenza vaccines. Most of these influenza vaccines are quadrivalent vaccines based on four HAs derived from each of the four strains of influenza virus designated by health authorities to be included in the seasonal vaccine for the year (typically two influenza A subtypes and two influenza B strains), meaning the vaccine is designed to provide protection against those four different strains of influenza virus.

[0008] Current quadrivalent influenza vaccines only elicit antibody responses against the vaccine strain (e.g., homologous immune response) or closely related isolates, and rarely extend to more branched strains within a subtype / lineage (e.g., heterologous immune response) or strains belonging to different subtypes / lineages (e.g., heterosubtype immune response).

[0009] Therefore, there is still a need to obtain immunogenic compositions that can induce a broad, rapid, and robust immune response against influenza viruses. In particular, there is still a need for influenza vaccines that protect individuals from heterologous and heterosubtype strains of influenza virus (i.e., strains not present in the vaccine). [Overview of the Initiative]

[0010] Summary of the Invention In a first aspect, the present invention provides an immunogenic composition for use in the treatment or prevention of infection by influenza virus, wherein the immunogenic composition is (a) The first nucleic acid encoding the hemagglutinin (HA) antigen of the first subtype strain of influenza A virus, (b) The second nucleic acid encoding the HA antigen of the second subtype strain of influenza A virus, (c) The third nucleic acid encoding the HA antigen of the first strain of influenza B virus, and (d) A fourth nucleic acid encoding the HA antigen of a second strain of influenza B virus, which may be included as desired. The composition comprises, wherein the immune response is induced against the aforementioned strains of the first and second subtypes of influenza A virus, the aforementioned first strain and optionally a second strain of influenza B virus, and the HA antigen of at least one further HA antigen subtype of influenza A virus (different from any of the HA antigen subtypes of influenza A virus encoded by nucleic acids present in the composition).

[0011] In a second embodiment, the present invention provides a vaccine for use in the treatment or prevention of infection by influenza virus, comprising an immunogenic composition as defined herein, wherein the immune response is induced as defined herein.

[0012] In a third aspect, the present invention provides a kit or kit of parts for use in the treatment or prevention of infection by influenza virus, wherein the kit or kit of parts comprises nucleic acids, preferably mRNA, and preferably (a), (b), (c), (d), (e) as defined herein. 1 ), (e 2 ), (e 3 ) and / or (e 4 The preparation contains mRNA of ), optionally a liquid vehicle for solubilization, and optionally a technical manual providing information on the administration and dosage of the components, and the immune response is induced as defined herein.

[0013] In a fourth embodiment, the present invention provides a method for inducing an immune response to an influenza virus, the method comprising applying or administering an immunogenic composition as defined herein to a subject requiring such treatment, wherein the immune response is induced as defined herein.

[0014] In a fifth aspect, the present invention provides a method for treating or preventing a disorder caused by an influenza virus, the method comprising applying or administering an immunogenic composition as defined herein to a subject requiring such treatment, the immune response being induced as defined herein.

[0015] A brief explanation of arrays SEQ ID NO: 1: Amino acid sequence of HA derived from A / Michigan / 45 / 2015(H1N1). SEQ ID NO: 2: Amino acid sequence of NA derived from A / Michigan / 45 / 2015(H1N1). SEQ ID NO: 3: Amino acid sequence of HA derived from A / Switzerland / 8060 / 2017(H3N2). SEQ ID NO: 4: Amino acid sequence of NA derived from A / Switzerland / 8060 / 2017(H3N2). The amino acid sequence of HA derived from Sequence ID No. 5:B / Colorado / 06 / 2017. The amino acid sequence of NA derived from Sequence ID No. 6:B / Colorado / 06 / 2017. The amino acid sequence of HA derived from Sequence ID No. 7:B / Phuket / 3073 / 2013. The amino acid sequence of NA derived from Sequence ID No. 8:B / Phuket / 3073 / 2013. SEQ ID NO: 9: Amino acid sequence of HA derived from A / Singapore / INFIMH-16-0019 / 2016(H3N2). SEQ ID NO: 10: Amino acid sequence of NA derived from A / Singapore / INFIMH-16-0019 / 2016(H3N2). SEQ ID NO: 11: Amino acid sequence of HA derived from A / Brisbane / 02 / 2018(H1N1). SEQ ID NO: 12: Amino acid sequence of NA derived from A / Brisbane / 02 / 2018(H1N1). SEQ ID NO: 13: Amino acid sequence of HA derived from A / Kansas / 14 / 2017(H3N2). The amino acid sequence of NA derived from sequence number 14:A / Kansas / 14 / 2017(H3N2). SEQ ID NO: 15: Amino acid sequence of HA derived from A / South Australia / 34 / 2019 (H3N2). SEQ ID NO: 16: Amino acid sequence of NA derived from A / South Australia / 34 / 2019 (H3N2). The amino acid sequence of HA derived from Sequence ID No. 17:B / Washington / 02 / 2019. The amino acid sequence of NA derived from Sequence ID No. 18:B / Washington / 02 / 2019. The amino acid sequence of HA derived from SEQ ID NO: 19:A / Guangdong-Maonan / SWL1536 / 2019(H1N1). The amino acid sequence of NA derived from SEQ ID NO: 20:A / Guangdong-Maonan / SWL1536 / 2019(H1N1). SEQ ID NO: 21: Amino acid sequence of HA derived from A / Hong Kong / 2671 / 2019 (H3N2). The amino acid sequence of NA derived from SEQ ID NO: 22:A / Hong Kong / 2671 / 2019(H3N2). SEQ ID NO: 23: Amino acid sequence of HA derived from A / Hawaii / 70 / 2019(H1N1). The amino acid sequence of NA derived from SEQ ID NO: 24:A / Hawaii / 70 / 2019(H1N1). The amino acid sequence of HA derived from SEQ ID NO: 25:A / Hong Kong / 45 / 2019(H3N2). The amino acid sequence of NA derived from SEQ ID NO: 26:A / Hong Kong / 45 / 2019(H3N2). SEQ ID NO: 27: Amino acid sequence of HA derived from A / Victoria / 2570 / 2019(H1N1). SEQ ID NO: 28: Amino acid sequence of NA derived from A / Victoria / 2570 / 2019(H1N1). SEQ ID NO: 29: Amino acid sequence of HA derived from A / Wisconsin / 588 / 2019(H1N1). SEQ ID NO: 30: Amino acid sequence of NA derived from A / Wisconsin / 588 / 2019(H1N1). The amino acid sequence of HA derived from Sequence ID No. 31:A / Cambodia / e0826360 / 2020(H3N2). The amino acid sequence of NA derived from sequence number 32:A / Cambodia / e0826360 / 2020(H3N2). SEQ ID NO: 33: Amino acid sequence of HA derived from A / Darwin / 9 / 2021(H3N2). SEQ ID NO: 34: Amino acid sequence of NA derived from A / Darwin / 9 / 2021(H3N2). The amino acid sequence of HA derived from Sequence ID No. 35:B / Austria / 1359417 / 2021. The amino acid sequence of NA derived from SEQ ID NO: 36:B / Austria / 1359417 / 2021. SEQ ID NO: 37: Amino acid sequence of HA derived from A / Darwin / 6 / 2021(H3N2). SEQ ID NO: 38: Amino acid sequence of NA derived from A / Darwin / 6 / 2021(H3N2). SEQ ID NO: 39: Amino acid sequence of HA derived from A / Victoria / 4897 / 2022(H1N1). SEQ ID NO: 40: The amino acid sequence of NA derived from A / Victoria / 4897 / 2022(H1N1). SEQ ID NO: 41: Amino acid sequence of HA derived from A / Wisconsin / 67 / 2022(H1N1). SEQ ID NO: 42: Amino acid sequence of NA derived from A / Wisconsin / 67 / 2022(H1N1). SEQ ID NO: 43: Amino acid sequence of HA derived from A / Sydney / 5 / 2021(H1N1). SEQ ID NO: 44: Amino acid sequence of NA derived from A / Sydney / 5 / 2021(H1N1). SEQ ID NO: 45: Amino acid sequence of HA derived from A / Thailand / 8 / 2022(H3N2). The amino acid sequence of NA derived from sequence number 46:A / Thailand / 8 / 2022(H3N2). SEQ ID NO: 47: Amino acid sequence of HA derived from A / Massachusetts / 18 / 2022(H3N2). SEQ ID NO: 48: Amino acid sequence of NA derived from A / Massachusetts / 18 / 2022(H3N2). [Brief explanation of the drawing]

[0016] [Figure 1]Domain structure of the influenza A virus (IAV) HA protein. The HA1 domain includes a fusion (F1), vestigial esterase (VE), and receptor-binding domain (RBD). The HA2 domain includes the HA2 ectodomain, transmembrane region (TM), and cytoplasmic tail (CT). The HA head contains the receptor-binding subdomain and vestigial esterase subdomain. The stalk (also called the "stem") contains the HA1 fusion domain and the HA2 ectodomain. [Figure 2] Figures 2A-B. (A) IFN-α release in human PBMCs stimulated with mRNA-LNP. hPBMCs from four donors were incubated in 96-well flat-bottom plates in triplicates for 24 hours with 10 μg / ml of each mRNA-LNP encoding the HA protein of influenza A / Hawaii / 70 / 2019 (H1N1pdm09). IFNα was measured by ELISA. Values ​​obtained from individual donors (dots) are shown for each group, and the lines represent the geometric mean with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). ns: not significant. (B) HA-encoding mRNA-LNP vaccines containing pseudouridine or N1-methylpseudridine induce substantially lower IFNα levels in mice compared to unmodified mRNA-LNP vaccines. Female Balb / c mice (n = 10 / group) were vaccinated with 5 μg or 0.71 μg of the mRNA-LNP vaccines CVAC21-53-R9973 (unmodified), CVAC21-53-R10736 (pseuduridine), and CVAC21-53-R10737 (N1-methylpseuduridine). Control animals (n = 5 / group) were administered a 0.9% NaCl solution. Serum IFNα levels were measured 18 hours after immunization using ELISA. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). ns: Not significant. [Figure 3] Figures 3A-B. (A) IFN-α release in hPBMCs stimulated with mRNA-LNP vaccine. hPBMCs from four donors were incubated for 24 hours in 3-column 96-well flat-bottom plates with 10 μg / ml of each mRNA-LNP vaccine encoding the NA protein of influenza A / Hong Kong / 45 / 2019 (H3N2). IFNα was measured by ELISA. Values ​​obtained from each donor (dots) are shown for each group, and the lines represent the geometric mean with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown in the graph, and statistical significance is indicated by * (CI does not include 1). ns: not significant. (B) NA-encoding mRNA-LNP vaccines containing pseudouridine or N1-methylpseudridine induce substantially lower IFNα levels in mice compared to unmodified mRNA-LNP vaccines. Female Balb / c mice (n = 10 / group) were vaccinated with either 5 μg or 0.71 μg of mRNA-LNP vaccine. The control group (n = 5 / group) received a 0.9% NaCl solution. Serum IFNα levels were measured using ELISA 18 hours after immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). ns: Not significant. [Figure 4]Figures 4A-B. (A) IFNα release in hPBMCs stimulated with 4-component and 7-component mRNA vaccine formulations of seasonal influenza. hPBMCs from four donors were incubated for 24 hours in 3-column 96-well flat-bottom plates with 10 μg / ml of seasonal influenza 4-component (4HA; unmodified, ψ, and N1-mψ) and 7-component (4HA+3NA; unmodified, ψ, and N1-mψ) mRNA-LNP vaccines. IFNα was measured by ELISA in the cell-free supernatant. Values ​​obtained from individual donors (dots) are shown for each group, and the lines represent the geometric mean (GM) with 95% CI. Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). (B) Seasonal influenza 4-component and 7-component mRNA vaccines containing modified (ψ and N1-mψ) nucleosides induce substantially lower IFNα levels in mice compared to unmodified mRNA vaccines. Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the 4-component (4HA; unmodified, ψ and N1-mψ) mRNA vaccine and 1 μg or 2.84 μg of the 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA vaccine. Control animals (n = 5 / group) were administered a 0.9% NaCl solution. IFNα levels were measured using ELISA in serum samples collected 18 hours after primary immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% CI. The geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by an asterisk (*) (CI does not include 1). [Figure 5A]Figures 5A-D. HI titers induced by 4-component or 7-component seasonal influenza mRNA vaccines containing unmodified or modified nucleosides (ψ and N1-mψ). Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the 4-component (4HA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine and 1 μg or 2.84 μg of the 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received either a 0.9% NaCl solution or one-tenth of the human dose of the approved QIV, FLUARIX or FLUZONE High-Dose. HI titers against influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(H3N2)(B), B / Washington / 02 / 2019(C), and B / Phuket / 3073 / 2013(D) were measured in mouse serum two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean titer (GMT) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows an HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 5B]Figures 5A-D. HI titers induced by 4-component or 7-component seasonal influenza mRNA vaccines containing unmodified or modified nucleosides (ψ and N1-mψ). Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the 4-component (4HA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine and 1 μg or 2.84 μg of the 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received either a 0.9% NaCl solution or one-tenth of the human dose of the approved QIV, FLUARIX or FLUZONE High-Dose. HI titers against influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(H3N2)(B), B / Washington / 02 / 2019(C), and B / Phuket / 3073 / 2013(D) were measured in mouse serum two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean titer (GMT) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows an HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 5C]Figures 5A-D. HI titers induced by 4-component or 7-component seasonal influenza mRNA vaccines containing unmodified or modified nucleosides (ψ and N1-mψ). Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the 4-component (4HA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine and 1 μg or 2.84 μg of the 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received either a 0.9% NaCl solution or one-tenth of the human dose of the approved QIV, FLUARIX or FLUZONE High-Dose. HI titers against influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(H3N2)(B), B / Washington / 02 / 2019(C), and B / Phuket / 3073 / 2013(D) were measured in mouse serum two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean titer (GMT) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows an HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 5D]Figures 5A-D. HI titers induced by 4-component or 7-component seasonal influenza mRNA vaccines containing unmodified or modified nucleosides (ψ and N1-mψ). Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the 4-component (4HA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine and 1 μg or 2.84 μg of the 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received either a 0.9% NaCl solution or one-tenth of the human dose of the approved QIV, FLUARIX or FLUZONE High-Dose. HI titers against influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(H3N2)(B), B / Washington / 02 / 2019(C), and B / Phuket / 3073 / 2013(D) were measured in mouse serum two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean titer (GMT) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows an HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 6]Figures 6A-C. NI titers induced by a 7-component seasonal influenza mRNA vaccine containing unmodified or modified (ψ and N1-mψ) nucleosides. Female Balb / c mice (n = 10 / group) were vaccinated with 1 μg or 2.84 μg of the 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA vaccine. Control animals (n = 5 / group) received either a 0.9% NaCl solution or one-tenth of the human dose of the approved QIV, FLUARIX or FLUZONE High-Dose. NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(H3N2)(B), and B / Washington / 02 / 2019(C) were measured in serum two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean titer (GMT) with a 95% confidence interval (CI). [Figure 7A]Figures 7A-B. Four-component and seven-component seasonal influenza mRNA vaccines induced a T-cell immune response after intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly vaccinated twice on days 0 and 21 with 1.25, 2.5, 5, or 10 μg of the four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine. Control animals received intramuscular administration twice on days 0 and 21 with saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group). T-cell immune responses were analyzed two weeks after a second immunization by intracellular cytokine staining in isolated splenocytes restimulated with a 15-mer duplicate peptide library spanning full-length HA or NA proteins of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09). HA-specific IFNγ+ TNF+ CD4+ T cells (A) and CD8+ T cells (B) were measured. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). [Figure 7B]Figures 7A-B. Four-component and seven-component seasonal influenza mRNA vaccines induced a T-cell immune response after intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly vaccinated twice on days 0 and 21 with 1.25, 2.5, 5, or 10 μg of the four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine. Control animals received intramuscular administration twice on days 0 and 21 with saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group). T-cell immune responses were analyzed two weeks after a second immunization by intracellular cytokine staining in isolated splenocytes restimulated with a 15-mer duplicate peptide library spanning full-length HA or NA proteins of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09). HA-specific IFNγ+ TNF+ CD4+ T cells (A) and CD8+ T cells (B) were measured. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). [Figure 8A]Figures 8A-B. Four-component and seven-component seasonal influenza mRNA vaccines induced a T-cell immune response after intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly immunized twice on days 0 and 21 with 1.25, 2.5, 5, or 10 μg of the four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine. Control animals received intramuscular administration twice on days 0 and 21 with saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group). T-cell immune responses were analyzed two weeks after a second immunization by intracellular cytokine staining in isolated splenocytes restimulated with a 15-mer duplicate peptide library spanning full-length HA or NA proteins of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09). NA-specific IFNγ+ TNF+ CD4+ (A) T cells and CD8+ T cells (B) were measured. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). [Figure 8B]Figures 8A-B. Four-component and seven-component seasonal influenza mRNA vaccines induced a T-cell immune response after intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly immunized twice on days 0 and 21 with 1.25, 2.5, 5, or 10 μg of the four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine. Control animals received intramuscular administration twice on days 0 and 21 with saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group). T-cell immune responses were analyzed two weeks after a second immunization by intracellular cytokine staining in isolated splenocytes restimulated with a 15-mer duplicate peptide library spanning full-length HA or NA proteins of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09). NA-specific IFNγ+ TNF+ CD4+ (A) T cells and CD8+ T cells (B) were measured. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). [Figure 9-1]Figures 9A-D. Unmodified and modified 7-component seasonal influenza mRNA vaccines induced a high-intensity response (HI) in naive (unexposed) ferrets. Female ferrets (n = 6 / group) were immunized twice via the intramuscular (IM) route on days 0 and 28 with 3 μg or 12.5 μg of the 7-component mRNA vaccine containing unmodified nucleosides, or 12.5 μg or 50 μg of the 7-component mRNA vaccine containing modified nucleosides (N1mψ). Two groups were immunized twice via the IM route on days 0 and 28 with human doses of the approved fractional inactivated QIV, FLUARIX or FLUZONE HD. Animals in the negative control group were injected twice with saline via the IM route on days 0 and 28. HI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(B), B / Washington / 02 / 2019(C), and B / Phuket / 3073 / 2013(D) were measured in the serum of vaccinated ferrets 4 weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 9-2]Figures 9A-D. Unmodified and modified 7-component seasonal influenza mRNA vaccines induced a high-intensity response (HI) in naive (unexposed) ferrets. Female ferrets (n = 6 / group) were immunized twice via the intramuscular (IM) route on days 0 and 28 with 3 μg or 12.5 μg of the 7-component mRNA vaccine containing unmodified nucleosides, or 12.5 μg or 50 μg of the 7-component mRNA vaccine containing modified nucleosides (N1mψ). Two groups were immunized twice via the IM route on days 0 and 28 with human doses of the approved fractional inactivated QIV, FLUARIX or FLUZONE HD. Animals in the negative control group were injected twice with saline via the IM route on days 0 and 28. HI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(B), B / Washington / 02 / 2019(C), and B / Phuket / 3073 / 2013(D) were measured in the serum of vaccinated ferrets 4 weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 10-1]Figures 10A-C. Unmodified and modified 7-component seasonal influenza mRNA vaccines induced an NI response in naive ferrets. Female ferrets (n = 6 / group) were immunized twice via the IM route on days 0 and 28 with 3 μg or 12.5 μg of the 7-component mRNA vaccine containing unmodified nucleosides, or 12.5 μg or 50 μg of the 7-component mRNA vaccine containing modified nucleosides (N1mψ). Two groups were immunized twice via the IM route on days 0 and 28 with whole human doses of the approved fractional inactivated QIV, FLUARIX or FLUZONE HD. Animals in the negative control group were injected twice with saline via the intramuscular route on days 0 and 28. NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(B), and B / Washington / 02 / 2019(C) were measured using ELLA in the serum of vaccinated ferrets 4 weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). [Figure 10-2]Figures 10A-C. Unmodified and modified 7-component seasonal influenza mRNA vaccines induced an NI response in naive ferrets. Female ferrets (n = 6 / group) were immunized twice via the IM route on days 0 and 28 with 3 μg or 12.5 μg of the 7-component mRNA vaccine containing unmodified nucleosides, or 12.5 μg or 50 μg of the 7-component mRNA vaccine containing modified nucleosides (N1mψ). Two groups were immunized twice via the IM route on days 0 and 28 with whole human doses of the approved fractional inactivated QIV, FLUARIX or FLUZONE HD. Animals in the negative control group were injected twice with saline via the intramuscular route on days 0 and 28. NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Cambodia / e0826360 / 2020(B), and B / Washington / 02 / 2019(C) were measured using ELLA in the serum of vaccinated ferrets 4 weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). [Figure 11-1]Figures 11A-D. HI responses induced during intramuscular immunization in naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on days 0 and 28 with 12.5 μg and 25 μg of the 4-component seasonal influenza N1mψ mRNA vaccine, and 25 μg and 50 μg of the 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals received either a 0.9% NaCl solution (n = 6 / group) or the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group) intramuscularly on days 0 and 28. HI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not include 1). The dashed line shows the HI titer of 40, which was defined as the surrogate correlate of protection. [Figure 11-2]Figures 11A-D. HI responses induced during intramuscular immunization in naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on days 0 and 28 with 12.5 μg and 25 μg of the 4-component seasonal influenza N1mψ mRNA vaccine, and 25 μg and 50 μg of the 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals received either a 0.9% NaCl solution (n = 6 / group) or the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group) intramuscularly on days 0 and 28. HI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not include 1). The dashed line shows the HI titer of 40, which was defined as the surrogate correlate of protection. [Figure 12-1]Figures 12A-D. MN titers induced during intramuscular immunization of naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on days 0 and 28 with 12.5 μg and 25 μg of the 4-component seasonal influenza N1mψ mRNA vaccine and 25 μg and 50 μg of the 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals were administered intramuscularly on days 0 and 28 either with a 0.9% NaCl solution (n = 6 / group) or with the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group). MN titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not include 1). [Figure 12-2]Figures 12A-D. MN titers induced during intramuscular immunization of naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on days 0 and 28 with 12.5 μg and 25 μg of the 4-component seasonal influenza N1mψ mRNA vaccine and 25 μg and 50 μg of the 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals were administered intramuscularly on days 0 and 28 either with a 0.9% NaCl solution (n = 6 / group) or with the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group). MN titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not include 1). [Figure 13]Figures 13A-D. NI titers induced by intramuscular immunization of naive ferrets with an 8-component seasonal influenza mRNA vaccine. Female ferrets were immunized via the intramuscular route with 25 μg and 50 μg of the 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals received intramuscular administration on day 0 and day 28 of either a 0.9% NaCl solution (n = 6 / group) or the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not include 1). [Figure 14-1]Figures 14A-D. Viral load in respiratory tissue after influenza A / Victoria / 2570 / 2019 (H1N1pdm09) challenge in ferrets. Female ferrets (n = 6 / group) were immunized twice via intramuscular route on day 0 and day 28 with 3 μg or 12.5 μg of a 7-component seasonal influenza mRNA vaccine containing unmodified nucleosides, or 12.5 μg or 50 μg of a 7-component mRNA vaccine containing modified nucleosides (N1mψ). Two groups were immunized twice via intramuscular administration on day 0 and day 28 with human doses of the approved split-inactivated QIV, FLUARIX or FLUZONE HD. Animals in the negative control group were injected twice with saline via intramuscular route on day 0 and day 28. Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (intratracheal and intranasal routes) and euthanized four days after challenge infection. Titer measurements were performed on lung tissue (A) and turbinate (B) samples collected four days after challenge with A / Victoria / 2570 / 2019 (H1N1pdm09). Individual titers are shown by group, with the group mean shown by the solid black line. The limit of detection (LLOD) of the assay depended on the tissue sample weight, and the LLOD range is shown by the dashed line. The severity of bronchiolitis (C) and alveolitis (D) (inflammatory changes in the lung parenchyma of the animals) is averaged by group. Four tissue slides were evaluated for each animal and lung section. Each dot represents the mean of an individual animal, and the lines show the mean of each group with a 95% confidence interval (CI). Differences between groups are shown on the graph, and statistical significance is indicated by an asterisk (*). [Figure 14-2]Figures 14A-D. Viral load in respiratory tissue after influenza A / Victoria / 2570 / 2019 (H1N1pdm09) challenge in ferrets. Female ferrets (n = 6 / group) were immunized twice via intramuscular route on day 0 and day 28 with 3 μg or 12.5 μg of a 7-component seasonal influenza mRNA vaccine containing unmodified nucleosides, or 12.5 μg or 50 μg of a 7-component mRNA vaccine containing modified nucleosides (N1mψ). Two groups were immunized twice via intramuscular administration on day 0 and day 28 with human doses of the approved split-inactivated QIV, FLUARIX or FLUZONE HD. Animals in the negative control group were injected twice with saline via intramuscular route on day 0 and day 28. Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (intratracheal and intranasal routes) and euthanized four days after challenge infection. Titer measurements were performed on lung tissue (A) and turbinate (B) samples collected four days after challenge with A / Victoria / 2570 / 2019 (H1N1pdm09). Individual titers are shown by group, with the group mean shown by the solid black line. The limit of detection (LLOD) of the assay depended on the tissue sample weight, and the LLOD range is shown by the dashed line. The severity of bronchiolitis (C) and alveolitis (D) (inflammatory changes in the lung parenchyma of the animals) is averaged by group. Four tissue slides were evaluated for each animal and lung section. Each dot represents the mean of an individual animal, and the lines show the mean of each group with a 95% confidence interval (CI). Differences between groups are shown on the graph, and statistical significance is indicated by an asterisk (*). [Figure 15]Figures 15A-B. Viral load in respiratory tissue after influenza A / Victoria / 2570 / 2019 (H1N1pdm09) challenge in ferrets. Female ferrets were immunized intramuscularly with 25 μg and 50 μg of 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either 0.9% NaCl solution (n = 6 / group) or the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) at 10∧5 TCID50 (intratracheal and intranasal routes) and euthanized four days after challenge infection. Titer measurements of lung tissue (A) and nasal turbinate (B) samples collected four days after challenge with A / Victoria / 2570 / 2019 (H1N1pdm09). Individual titers are shown by group, with the group mean shown as a solid black line. The limit of detection (LLOD) of the assay depended on the tissue sample weight, and the LLOD range is shown as a dashed line. [Figure 16-1] Figures 16A-C. Body temperature changes in ferrets after influenza A / Victoria / 2570 / 2019 (H1N1pdm09) challenge. Female ferrets were immunized via intramuscular route on days 0 and 28 with 25 μg and 50 μg of 4-component (A) and 8-component (B) seasonal influenza N1 mψ mRNA vaccines (n = 6). The control group received either a 0.9% NaCl solution (n = 6 / group) or a whole-human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group) via intramuscular route on days 0 and 28 (C). Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (via intratracheal and intranasal routes). Mean temperature change (°C) relative to baseline from day 0 to day 4 after challenge is shown for each group. [Figure 16-2]Figures 16A-C. Body temperature changes in ferrets after influenza A / Victoria / 2570 / 2019 (H1N1pdm09) challenge. Female ferrets were immunized via intramuscular route on days 0 and 28 with 25 μg and 50 μg of 4-component (A) and 8-component (B) seasonal influenza N1 mψ mRNA vaccines (n = 6). The control group received either a 0.9% NaCl solution (n = 6 / group) or a whole-human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group) via intramuscular route on days 0 and 28 (C). Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (via intratracheal and intranasal routes). Mean temperature change (°C) relative to baseline from day 0 to day 4 after challenge is shown for each group. [Figure 17-1] Figures 17A-F. Viral titer measurements of pharyngeal swabs (A-C) and nasal swabs (D-F) from day 0 (pre-challenge) to day 4 after challenge with influenza A / Victoria / 2570 / 2019 (H1N1pdm09). Female ferrets were immunized via intramuscular route with 25 μg and 50 μg of 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals received intramuscular administration on day 0 and day 28 of either 0.9% NaCl solution (n = 6 / group) or the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group). Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (intratracheal and intranasal routes). Individual values ​​are shown by group, with the group mean shown as a solid black line. The limit of detection (LLOD) of the assay is shown as a dashed line. [Figure 17-2]Figures 17A-F. Viral titer measurements of pharyngeal swabs (A-C) and nasal swabs (D-F) from day 0 (pre-challenge) to day 4 after challenge with influenza A / Victoria / 2570 / 2019 (H1N1pdm09). Female ferrets were immunized via intramuscular route with 25 μg and 50 μg of 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals received intramuscular administration on day 0 and day 28 of either 0.9% NaCl solution (n = 6 / group) or the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group). Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (intratracheal and intranasal routes). Individual values ​​are shown by group, with the group mean shown as a solid black line. The limit of detection (LLOD) of the assay is shown as a dashed line. [Figure 17-3] Figures 17A-F. Viral titer measurements of pharyngeal swabs (A-C) and nasal swabs (D-F) from day 0 (pre-challenge) to day 4 after challenge with influenza A / Victoria / 2570 / 2019 (H1N1pdm09). Female ferrets were immunized via intramuscular route with 25 μg and 50 μg of 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals received intramuscular administration on day 0 and day 28 of either 0.9% NaCl solution (n = 6 / group) or the approved fractional inactivated QIV FLUARIX (NH22-23) whole human dose (n = 6 / group). Four weeks after final immunization, ferrets were challenged with wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09) 10∧5 TCID50 (intratracheal and intranasal routes). Individual values ​​are shown by group, with the group mean shown as a solid black line. The limit of detection (LLOD) of the assay is shown as a dashed line. [Figure 18-1]Figures 18A-D. Ferrets immunized with 4-component and 8-component seasonal influenza mRNA vaccines showed reduced macroscopic and microscopic pathological changes in the lungs. Female ferrets were immunized via the intramuscular route with 25 μg and 50 μg of 8-component seasonal influenza N1 mψ mRNA vaccine on days 0 and 28 (n = 6). Control animals were administered via the intramuscular route on days 0 and 28 either 0.9% NaCl solution (n = 6 / group) or the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). Four weeks after final immunization, ferrets were challenged (intratracheal and intranasal routes) with 10∧5 TCID50 of wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09). (A) Relative lung weight (RLW) 4 days after challenge with influenza A / Victoria / 2570 / 2019(H1N1pdm09) virus in ferrets. Relative lung weight (RLW) reflects the extent and severity of lung lesions, as lungs in an inflamed and / or infiltrated state are usually heavier than unaffected lungs. Generally, in healthy or mildly pneumoniaized ferrets, RLWs of 1 or less are recorded. Individual percentages are shown by group, with group mean ± SD indicated. The dashed line represents a relative lung weight of 1. (B) Percentage of affected lung tissue 4 days after challenge. Macroscopic lung lesions consisted of pulmonary sclerosis characterized by redness and mild increase in stiffness of the lung parenchyma. The degree of pulmonary sclerosis was assessed based on a visual estimate of the percentage of affected lung tissue. Individual percentages of observed affected lung tissue are shown by group, with group mean ± SD indicated. Severity scores for conduction system inflammation (C) and pneumonia (D) are averaged by group. Individual scores are shown by group, and the group mean is shown by a solid black line. [Figure 18-2]Figures 18A-D. Ferrets immunized with 4-component and 8-component seasonal influenza mRNA vaccines showed reduced macroscopic and microscopic pathological changes in the lungs. Female ferrets were immunized via the intramuscular route with 25 μg and 50 μg of 8-component seasonal influenza N1 mψ mRNA vaccine on days 0 and 28 (n = 6). Control animals were administered via the intramuscular route on days 0 and 28 either 0.9% NaCl solution (n = 6 / group) or the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). Four weeks after final immunization, ferrets were challenged (intratracheal and intranasal routes) with 10∧5 TCID50 of wild-type influenza A / Victoria / 2570 / 2019 (H1N1pdm09). (A) Relative lung weight (RLW) 4 days after challenge with influenza A / Victoria / 2570 / 2019(H1N1pdm09) virus in ferrets. Relative lung weight (RLW) reflects the extent and severity of lung lesions, as lungs in an inflamed and / or infiltrated state are usually heavier than unaffected lungs. Generally, in healthy or mildly pneumoniaized ferrets, RLWs of 1 or less are recorded. Individual percentages are shown by group, with group mean ± SD indicated. The dashed line represents a relative lung weight of 1. (B) Percentage of affected lung tissue 4 days after challenge. Macroscopic lung lesions consisted of pulmonary sclerosis characterized by redness and mild increase in stiffness of the lung parenchyma. The degree of pulmonary sclerosis was assessed based on a visual estimate of the percentage of affected lung tissue. Individual percentages of observed affected lung tissue are shown by group, with group mean ± SD indicated. Severity scores for conduction system inflammation (C) and pneumonia (D) are averaged by group. Individual scores are shown by group, and the group mean is shown by a solid black line. [Figure 19-1]Figures 19A-D. Four-component or seven-component seasonal influenza mRNA vaccines induced HI responses to several heterologous influenza strains during intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were vaccinated twice intramuscularly with 1.25 μg or 10 μg of four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine on days 0 and 21. Control animals received intramuscular administration of either saline (NaCl) (n = 5 / group) or one-tenth of the human dose of approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group) twice on days 0 and 21. HI titers induced by 1.25 μg and 10 μg seasonal influenza mRNA vaccines, as well as FLUARIX and FLUZONE HD [against influenza A / California / 7 / 2009(H1N1pdm09)(A), A / Hong Kong / 2671 / 2019(H3N2)(B), B / Darwin / 7 / 2019(C), and A / Brisbane / 02 / 2018(H1N1pdm09)(D)], were measured in serum collected two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 19-2]Figures 19A-D. Four-component or seven-component seasonal influenza mRNA vaccines induced HI responses to several heterologous influenza strains during intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were vaccinated twice intramuscularly with 1.25 μg or 10 μg of four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine on days 0 and 21. Control animals received intramuscular administration of either saline (NaCl) (n = 5 / group) or one-tenth of the human dose of approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group) twice on days 0 and 21. HI titers induced by 1.25 μg and 10 μg seasonal influenza mRNA vaccines, as well as FLUARIX and FLUZONE HD [against influenza A / California / 7 / 2009(H1N1pdm09)(A), A / Hong Kong / 2671 / 2019(H3N2)(B), B / Darwin / 7 / 2019(C), and A / Brisbane / 02 / 2018(H1N1pdm09)(D)], were measured in serum collected two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). The dashed line shows HI titer = 40, which was defined as the surrogate correlate of protection. [Figure 20]Figures 20A-B. Four-component or seven-component seasonal influenza mRNA vaccines induced a wide range of heterologous and heterosubtype HA-binding antibodies during intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly vaccinated twice with 1.25 μg or 10 μg of four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine on days 0 and 21. Control animals received intramuscular administration of either saline (NaCl) (n = 5 / group) or one-tenth of the human dose of approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group), twice on days 0 and 21. HA-specific antibodies were measured in the serum of vaccinated animals two weeks after the second immunization using a multiplex HA binding assay containing recombinant HA proteins derived from four H1, one H2, one H5, six H3, one H7, one H10(A), two Yamagata strains, and eight Victoria strains(B). In each panel, the heatmap shows a grayscale as a function of the intensity of response between vaccination groups (dark gray = best response, light gray = worst response). [Figure 21-1]Figures 21A-F. Seven-component seasonal influenza mRNA vaccines induced heterogeneous and heterosubtype NI responses during intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly vaccinated twice with 1.25 μg or 10 μg of either a four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine on days 0 and 21. Control animals received intramuscular administration of either saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group), twice on days 0 and 21. The NI titers relative to NA for influenza A / California / 7 / 2009(H1N1pdm09)(A), A / Brevig Mission / 1 / 1918(H1N1)(B), A / Washington / 01 / 2007(H3N2)(C), B / Brisbane / 60 / 2008(D), A / Vietnam / 1194 / 2004(H5N1)(E), and B / Phuket / 3073 / 2013(F) were analyzed using enzyme-linked lectin assay (ELLA) in serum from vaccinated mice collected two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). LLOQ = lower limit of quantification; ULOQ = upper limit of quantitation. [Figure 21-2]Figures 21A-F. Seven-component seasonal influenza mRNA vaccines induced heterogeneous and heterosubtype NI responses during intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly vaccinated twice with 1.25 μg or 10 μg of either a four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine on days 0 and 21. Control animals received intramuscular administration of either saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group), twice on days 0 and 21. The NI titers relative to NA for influenza A / California / 7 / 2009(H1N1pdm09)(A), A / Brevig Mission / 1 / 1918(H1N1)(B), A / Washington / 01 / 2007(H3N2)(C), B / Brisbane / 60 / 2008(D), A / Vietnam / 1194 / 2004(H5N1)(E), and B / Phuket / 3073 / 2013(F) were analyzed using enzyme-linked lectin assay (ELLA) in serum from vaccinated mice collected two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). LLOQ = lower limit of quantification; ULOQ = upper limit of quantitation. [Figure 21-3]Figures 21A-F. Seven-component seasonal influenza mRNA vaccines induced heterogeneous and heterosubtype NI responses during intramuscular immunization in mice. Female Balb / c mice (n = 8 / group) were intramuscularly vaccinated twice with 1.25 μg or 10 μg of either a four-component (4HA) or seven-component (4HA+3NA) seasonal influenza mRNA vaccine on days 0 and 21. Control animals received intramuscular administration of either saline (NaCl) (n = 5 / group) or one-tenth of the human dose of the approved fractional inactivated QIV, FLUARIX (= 6 μg) or FLUZONE HD (= 24 μg) (n = 8 / group), twice on days 0 and 21. The NI titers relative to NA for influenza A / California / 7 / 2009(H1N1pdm09)(A), A / Brevig Mission / 1 / 1918(H1N1)(B), A / Washington / 01 / 2007(H3N2)(C), B / Brisbane / 60 / 2008(D), A / Vietnam / 1194 / 2004(H5N1)(E), and B / Phuket / 3073 / 2013(F) were analyzed using enzyme-linked lectin assay (ELLA) in serum from vaccinated mice collected two weeks after the second immunization. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMRs) between groups are shown on the graph, and statistical significance is indicated by * (CI does not include 1). LLOQ = lower limit of quantification; ULOQ = upper limit of quantitation. [Figure 22-1]Figures 22A-E. Heterogeneous HI responses induced during intramuscular immunization of naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on day 0 and day 28 with 12.5 μg and 25 μg of 4-component seasonal influenza N1mψ mRNA vaccine and 25 μg and 50 μg of 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with a 0.9% NaCl solution (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). HI titers against influenza A / Brisbane / 02 / 2018(A), A / California / 07 / 2009(B), A / South Australia / 34 / 2019(C), B / Darwin / 7 / 2019(D), and A / Hong Kong / 45 / 2019(E) were measured in serum collected from vaccinated animals on day 55. Each dot represents an individual animal, and the line represents the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3; confidence interval (CI) does not include 1). The dashed line shows the HI titers of 40, which were defined as surrogate correlates of protection. [Figure 22-2]Figures 22A-E. Heterogeneous HI responses induced during intramuscular immunization of naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on day 0 and day 28 with 12.5 μg and 25 μg of 4-component seasonal influenza N1mψ mRNA vaccine and 25 μg and 50 μg of 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with a 0.9% NaCl solution (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). HI titers against influenza A / Brisbane / 02 / 2018(A), A / California / 07 / 2009(B), A / South Australia / 34 / 2019(C), B / Darwin / 7 / 2019(D), and A / Hong Kong / 45 / 2019(E) were measured in serum collected from vaccinated animals on day 55. Each dot represents an individual animal, and the line represents the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3; confidence interval (CI) does not include 1). The dashed line shows the HI titers of 40, which were defined as surrogate correlates of protection. [Figure 22-3]Figures 22A-E. Heterogeneous HI responses induced during intramuscular immunization of naive ferrets with 4-component and 8-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly on day 0 and day 28 with 12.5 μg and 25 μg of 4-component seasonal influenza N1mψ mRNA vaccine and 25 μg and 50 μg of 8-component seasonal influenza N1mψ mRNA vaccine (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with a 0.9% NaCl solution (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). HI titers against influenza A / Brisbane / 02 / 2018(A), A / California / 07 / 2009(B), A / South Australia / 34 / 2019(C), B / Darwin / 7 / 2019(D), and A / Hong Kong / 45 / 2019(E) were measured in serum collected from vaccinated animals on day 55. Each dot represents an individual animal, and the line represents the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3; confidence interval (CI) does not include 1). The dashed line shows the HI titers of 40, which were defined as surrogate correlates of protection. [Figure 23-1]Figures 23A-G. Heterogeneous NI responses induced during intramuscular immunization of naive ferrets with an 8-component seasonal influenza mRNA vaccine. Female ferrets were immunized intramuscularly with 25 μg and 50 μg of the 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with 0.9% NaCl buffer (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). NI titers against NA for influenza A / California / 07 / 2009(A), A / Brevig Mission / 1 / 1918(B), A / Vietnam / 1194 / 2004(C), A / Cambodia / e0826360 / 2020(D), A / Washington / 01 / 2007(E), B / Brisbane / 60 / 2008(F), and B / Washington / 02 / 2019(G) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not contain 1). The dashed line shows the NI titers of 40, which were designated as surrogate correlates of protection. [Figure 23-2]Figures 23A-G. Heterogeneous NI responses induced during intramuscular immunization of naive ferrets with an 8-component seasonal influenza mRNA vaccine. Female ferrets were immunized intramuscularly with 25 μg and 50 μg of the 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with 0.9% NaCl buffer (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). NI titers against NA for influenza A / California / 07 / 2009(A), A / Brevig Mission / 1 / 1918(B), A / Vietnam / 1194 / 2004(C), A / Cambodia / e0826360 / 2020(D), A / Washington / 01 / 2007(E), B / Brisbane / 60 / 2008(F), and B / Washington / 02 / 2019(G) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not contain 1). The dashed line shows the NI titers of 40, which were designated as surrogate correlates of protection. [Figure 23-3]Figures 23A-G. Heterogeneous NI responses induced during intramuscular immunization of naive ferrets with an 8-component seasonal influenza mRNA vaccine. Female ferrets were immunized intramuscularly with 25 μg and 50 μg of the 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with 0.9% NaCl buffer (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). NI titers against NA for influenza A / California / 07 / 2009(A), A / Brevig Mission / 1 / 1918(B), A / Vietnam / 1194 / 2004(C), A / Cambodia / e0826360 / 2020(D), A / Washington / 01 / 2007(E), B / Brisbane / 60 / 2008(F), and B / Washington / 02 / 2019(G) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not contain 1). The dashed line shows the NI titers of 40, which were designated as surrogate correlates of protection. [Figure 23-4]Figures 23A-G. Heterogeneous NI responses induced during intramuscular immunization of naive ferrets with an 8-component seasonal influenza mRNA vaccine. Female ferrets were immunized intramuscularly with 25 μg and 50 μg of the 8-component seasonal influenza N1 mψ mRNA vaccine on day 0 and day 28 (n = 6). Control animals were administered intramuscularly on day 0 and day 28 either with 0.9% NaCl buffer (n = 6 / group) or with the whole human dose of approved fractional inactivated QIV FLUARIX (NH22-23) (n = 6 / group). NI titers against NA for influenza A / California / 07 / 2009(A), A / Brevig Mission / 1 / 1918(B), A / Vietnam / 1194 / 2004(C), A / Cambodia / e0826360 / 2020(D), A / Washington / 01 / 2007(E), B / Brisbane / 60 / 2008(F), and B / Washington / 02 / 2019(G) were measured in serum from vaccinated animals collected on day 55. Each dot represents an individual animal, and the line shows the geometric mean (GM) with a 95% confidence interval (CI). Geometric mean ratios (GMR) between groups are shown on the graph, and statistical significance is indicated by * (GMR > 3, CI does not contain 1). The dashed line shows the NI titers of 40, which were designated as surrogate correlates of protection. [Figure 24A] Figures 24A-C: Reactionogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine clinical trial. Figure 24A: Solicited adverse events in subjects at the mRNA dose levels shown at the bottom of the graph. Figure 24B: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly. Figures 24A-B: Percentages of events with grade 0 and vertically increasing grades, above the dose level indicator at the bottom of the graph. Figure 24C: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly, and separated into local and systemic events. Events with grades 0-1 above the dose level indicator at the bottom of the graph. Percentages of grades 0-1 and grade ≥2 are shown. [Figure 24B-1] Figures 24A-C: Reactionogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine clinical trial. Figure 24A: Solicited adverse events in subjects at the mRNA dose levels shown at the bottom of the graph. Figure 24B: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly. Figures 24A-B: Percentages of events with grade 0 and vertically increasing grades, above the dose level indicator at the bottom of the graph. Figure 24C: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly, and separated into local and systemic events. Events with grades 0-1 above the dose level indicator at the bottom of the graph. Percentages of grades 0-1 and grade ≥2 are shown. [Figure 24B-2] Figures 24A-C: Reactionogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine clinical trial. Figure 24A: Solicited adverse events in subjects at the mRNA dose levels shown at the bottom of the graph. Figure 24B: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly. Figures 24A-B: Percentages of events with grade 0 and vertically increasing grades, above the dose level indicator at the bottom of the graph. Figure 24C: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly, and separated into local and systemic events. Events with grades 0-1 above the dose level indicator at the bottom of the graph. Percentages of grades 0-1 and grade ≥2 are shown. [Figure 24C-1]Figures 24A-C: Reactionogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine clinical trial. Figure 24A: Solicited adverse events in subjects at the mRNA dose levels shown at the bottom of the graph. Figure 24B: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly. Figures 24A-B: Percentages of events with grade 0 and vertically increasing grades, above the dose level indicator at the bottom of the graph. Figure 24C: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly, and separated into local and systemic events. Events with grades 0-1 above the dose level indicator at the bottom of the graph. Percentages of grades 0-1 and grade ≥2 are shown. [Figure 24C-2] Figures 24A-C: Reactionogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine clinical trial. Figure 24A: Solicited adverse events in subjects at the mRNA dose levels shown at the bottom of the graph. Figure 24B: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly. Figures 24A-B: Percentages of events with grade 0 and vertically increasing grades, above the dose level indicator at the bottom of the graph. Figure 24C: Solicited adverse events in subjects at the shown mRNA dose levels, separated into young adults and elderly, and separated into local and systemic events. Events with grades 0-1 above the dose level indicator at the bottom of the graph. Percentages of grades 0-1 and grade ≥2 are shown. [Figure 25A]Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25B] Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25C] Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25D]Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25E] Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25F] Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25G]Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 25H] Figures 25A–H: Graphs show the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI). Figures 25A, C, E, and G show the HAI titers for all subjects on days 1, 22, and 183 at the indicated vaccine mRNA dose levels. Data in Figures 25B, D, F, and H are separated into young adults (YA) and older adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figures 25A–B), H3N2 (Figures 25C–D), B / Phuket (Figures 25E–F), and B / Washington (Figures 25G–H). [Figure 26] Serum conversion rate (SCR) from HAI assays. The table in the upper left panel shows the SCR (SCR is defined as follows: if the pre-vaccination titer is <1:10, the post-vaccination titer should be ≥1:40; if the pre-vaccination titer is ≥1:10, the post-vaccination titer should be at least a 4-fold increase from baseline). Data are shown for each coded HA, at each dose level, and for all subjects or separately for young adults and elderly. The graph in the lower left panel shows the overall SCR for each coded HA at each dose level. The graph in the upper right panel shows the SCR for each coded HA at each dose level in young adults. The graph in the lower right panel shows the SCR for each coded HA at each dose level in elderly. [Figure 27] This table shows the percentage of trial subjects who showed a four-fold or greater increase in anti-HA titer by micro-neutralization (MN) assay. The table in the upper left panel shows the percentage of subjects who showed a four-fold or greater increase in anti-HA titer by mN assay. The data is shown for each coded HA, at each dose level, and for all subjects or separately for young adults and elderly. The graph in the lower left panel shows the overall four-fold increase in anti-HA by MN assay for each coded HA at each dose level. The graph in the upper right panel shows the four-fold increase in anti-HA by MN assay for each coded HA at each dose level in young adults. The graph in the lower right panel shows the four-fold increase in anti-HA by MN assay for each coded HA at each dose level in elderly. [Figure 28] This table shows the percentage of trial subjects who showed a four-fold or greater increase in anti-NA titer by enzyme-linked lectin assay (ELLA). The table in the upper left panel shows the percentage of subjects who showed a four-fold or greater increase in anti-NA titer by ELLA assay. The data is shown for each coded HA, at each dose level, and for all subjects or separately for young adults and elderly subjects. The graph in the lower left panel shows the overall four-fold increase in anti-NA by ELLA assay for each coded HA at each dose level. The graph in the upper right panel shows the four-fold increase in anti-NA by ELLA assay for each coded HA at each dose level in young adults. The graph in the lower right panel shows the four-fold increase in anti-NA by ELLA assay for each coded HA at each dose level in elderly subjects. [Figure 29A]Figures 29A-D show the HI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers for influenza A / Victoria / 2570 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Connecticut / 01 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 29B] Figures 29A-D show the HI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers for influenza A / Victoria / 2570 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Connecticut / 01 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 29C] Figures 29A-D show the HI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers for influenza A / Victoria / 2570 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Connecticut / 01 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 29D]Figures 29A-D show the HI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers for influenza A / Victoria / 2570 / 2019(H1N1pdm09)(A), A / Darwin / 6 / 2021(H3N2)(B), B / Connecticut / 01 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 30A] Figures 30A-D show the NI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), influenza A / Cambodia / e0826360 / 2020(H3N2)(B), influenza B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 30B] Figures 30A-D show the NI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), influenza A / Cambodia / e0826360 / 2020(H3N2)(B), influenza B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 30C]Figures 30A-D show the NI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), influenza A / Cambodia / e0826360 / 2020(H3N2)(B), influenza B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 30D] Figures 30A-D show the NI titers induced during immunization of healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers for influenza A / Wisconsin / 588 / 2019(H1N1pdm09)(A), influenza A / Cambodia / e0826360 / 2020(H3N2)(B), influenza B / Austria / 1359417 / 2021(C), and B / Phuket / 3073 / 2013(D) were measured on day 29. [Figure 31A] Figures 31A-D show the percentage of healthy human adults (18-50 years old) who experienced involuntary events (A), local events (B), and systemic events (C) within 7 days of immunization with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control group was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary of each event, including Grade 3 events. [Figure 31B] Figures 31A-D show the percentage of healthy human adults (18-50 years old) who experienced involuntary events (A), local events (B), and systemic events (C) within 7 days of immunization with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control group was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary of each event, including Grade 3 events. [Figure 31C]Figures 31A-D show the percentage of healthy human adults (18-50 years old) who experienced involuntary events (A), local events (B), and systemic events (C) within 7 days of immunization with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control group was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary of each event, including Grade 3 events. [Figure 31D] Figures 31A-D show the percentage of healthy human adults (18-50 years old) who experienced involuntary events (A), local events (B), and systemic events (C) within 7 days of immunization with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control group was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary of each event, including Grade 3 events. [Figure 32] Figure 32 shows the percentage of healthy human adults (18-50 years old) who experienced related spontaneous events within 7 days of immunization with 1-component, 4-component, and 8-component seasonal influenza mRNA vaccine formulations. The control group was influenza D-QIV (FLUARIX, NH2022-23).

[0017] Detailed description of the invention This application is filed together with an electronic sequence listing, which is part of this specification (WIPO standard ST.26). All the information contained in the sequence listing is incorporated herein by reference. Where a “sequence number” is referred to herein, the corresponding nucleic acid (Na) sequence or amino acid (AA) sequence in the sequence listing having the respective identifier is referred to. For many sequences, the sequence listing also provides further details, such as specific structural features, sequence optimizations, and further details regarding GenBank (NCBI) or GISAID (epi) identifiers or their coding capacity. Where a “sequence number” is referred to in another published patent application or patent, the aforementioned sequence, such as an amino acid sequence or nucleic acid sequence, is explicitly incorporated herein by reference. These sequences thus constitute an essential part of the underlying description.

[0018] Immunogenic composition: The protective immune response induced by vaccination against the influenza virus is primarily directed at the viral HA protein, a glycoprotein on the surface of the virus that facilitates the interaction between the virus and host cell receptors.

[0019] The HA protein on the viral surface is a homotrimer of the HA protein monomer, which is cleaved by enzymes to produce an amino-terminal HA1 polypeptide and a carboxy-terminal HA2 polypeptide. Structurally, the hemagglutinin protein consists of several domains: the globular head domain, the stalk domain (also called the stem domain), the transmembrane domain, and the cytoplasmic domain (see Figure 1, Russell et al., 2021).

[0020] It is generally believed that during influenza virus infection of host cells (e.g., eukaryotic cells, e.g., human cells), hemagglutinin proteins recognize and bind to sialic acid receptors on the surface of host cells, thereby promoting viral attachment to the host cells. Following viral endocytosis and endosome acidification, hemagglutinin proteins undergo pH-dependent conformational changes, which allow them to facilitate the fusion of the viral envelope with the endosomal membrane of the host cell and the entry of viral nucleic acids into the host cell.

[0021] The spherical head consists exclusively of the major portion of the HA1 polypeptide, while the stem that anchors the HA protein within the viral lipid envelope is composed of HA2 and a portion of HA1. The spherical head of the HA protein contains two domains: the receptor-binding domain (RBD), which contains the sialic acid binding site, and the trace esterase domain, a smaller region directly beneath the RBD. In general, influenza viruses are classified based on the amino acid sequence of the viral hemagglutinin protein and / or the amino acid sequence of the viral neuraminidase (NA). Differences in amino acid sequences between different subtypes of HA proteins are mainly found in the sequences of the protein's head domain. The amino acid sequence of the stalk domain is considered to be more conserved among HA subtypes compared to the sequence of the head domain. The domains of the HA protein can be predicted using conventional methods known in the art.

[0022] Numerous naturally occurring and experimentally obtained antibodies that bind to and neutralize the HA protein are thought to bind to epitopes within the head domain of HA, inhibiting or reducing the interaction between HA and sialic acid on host cell receptors, thereby preventing or reducing cellular infection. Alternatively, or additionally, neutralizing antibodies may prevent or reduce the fusion of the viral membrane with the endosomal membrane. Such antibodies may bind to epitopes within the stalk domain, thereby suppressing conformational changes of the protein. Antibodies against influenza primarily target variable antigenic sites within the globular head of HA and therefore neutralize only antigenically related viruses. Current trivalent and quadrivalent influenza vaccines induce antibody responses against the vaccine strain (e.g., alloimmune response), or against closely related isolates (but rarely extended to further branched strains within subtypes / lineages) (e.g., hetero- or intrasubtypic immune response), or against strains belonging to different subtypes / lineages (e.g., hetero-subtypic immune response).

[0023] The present inventors have overcome the shortcomings of the prior art by providing an immunogenic composition for use in the treatment or prevention of influenza virus infection. Herein, the immunogenic composition is (a) The first nucleic acid encoding the hemagglutinin (HA) antigen of the first subtype strain of influenza A virus, (b) The second nucleic acid encoding the HA antigen of the second subtype strain of influenza A virus, (c) The third nucleic acid encoding the HA antigen of the first strain of influenza B virus, and (d) A fourth nucleic acid encoding the HA antigen of a second strain of influenza B virus, which may be included as desired. The composition comprises, wherein the immune response is induced against the aforementioned strains of the first and second subtypes of influenza A virus, the aforementioned first strain and optionally a second strain of influenza B virus, and the HA antigen of at least one further HA antigen subtype of influenza A virus (different from any of the HA antigen subtypes of influenza A virus encoded by nucleic acids present in the composition).

[0024] The immunogenic compositions for use according to the present invention have been shown to induce a broad, rapid, and potent cross-reactive immune response to influenza viruses, such as influenza A and / or B.

[0025] In particular, or additionally, immunogenic compositions for use according to the present invention have been found to induce broad, rapid, and potent homogeneous, heterogeneous, and heterosubtype immune responses to influenza viruses, such as influenza A and / or B.

[0026] In particular, or additionally, the immunogenic compositions for use according to the present invention have been found to induce antibody responses not only against vaccine strains and closely related strains, but also against further strains belonging to the same or different subtypes / lineages.

[0027] In particular, or additionally, immunogenic compositions for use according to the present invention have been found to induce an antibody response against influenza virus strains antigenically different from vaccine strains.

[0028] In particular, or additionally, the immunogenic compositions for use according to the present invention have been found to induce an antibody response against influenza virus strains different from any of the strains having HA encoded by mRNA present in the composition.

[0029] In particular, or additionally, the immunogenic compositions for use according to the present invention have been found to induce an antibody response against influenza viruses having a different geographical origin and / or isolation year from any of the strains having HA and / or NA encoded by mRNA present in the composition.

[0030] In particular, or additionally, the immunogenic compositions for use according to the present invention have been found to induce an antibody response to an influenza A virus HA antigen subtype that is different from any of the influenza A virus HA antigen subtypes encoded by the nucleic acids present in the composition.

[0031] In particular, or additionally, the immunogenic compositions for use according to the present invention have been found to induce an antibody response to an influenza A virus HA antigen subtype distinct from any of the influenza B virus strains whose HA antigens are encoded by the nucleic acids present in the composition.

[0032] In particular, or additionally, immunogenic compositions for use according to the present invention have been found to induce cross-reactive binding and functional anti-HA responses.

[0033] In particular, or additionally, immunogenic compositions for use according to the present invention have been found to protect individuals from strains of influenza virus that are not present in the immunogenic composition.

[0034] Particularly, or additionally, immunogenic compositions for use according to the invention have been found to protect an individual from homologous, heterologous and heterosubtypic strains of influenza virus.

[0035] Preferably, immunogenic compositions for use according to the invention have at least some of the following advantageous features. - Translation of the first, second, third and fourth nucleic acids, preferably mRNA, at the site of injection / vaccination (e.g., muscle). - Induction of an immune response at low doses and / or dosing regimens. - Suitability for vaccination of infants and / or neonates or the elderly, particularly the elderly. - Suitability of the composition / vaccine for intramuscular administration. - Rapid onset of immune protection against influenza virus, preferably influenza A virus and / or influenza B virus. - Extension of the duration of the induced immune response against influenza virus, preferably influenza A virus and / or influenza B virus. - Absence of enhancement of viral infection (e.g., influenza virus infection) due to vaccination or immunopathological effects. - Absence of antibody-dependent enhancement (ADE) caused by nucleic acid-based compositions / vaccines. - Absence of overinduction of systemic cytokine or chemokine responses after application of the composition / vaccine, which can result in undesirably high reactogenicity upon injection / vaccination. - Good tolerance of the composition / vaccine, absence of side effects, absence of toxicity. - Advantageous stability properties of nucleic acid-based compositions / vaccines. - Speed, adaptability, simplicity and scalability of nucleic acid-based composition / vaccine production. - Advantageous injection / vaccination regimens that require only low doses of the composition / vaccine for sufficient protection.

[0036] Thus, in a first aspect, the present invention relates to an immunogenic composition for use in the treatment or prevention of influenza virus infection, wherein the immunogenic composition comprises (a) a first nucleic acid encoding a hemagglutinin (HA) antigen of a strain of a first subtype of influenza A virus, (b) a second nucleic acid encoding an HA antigen of a strain of a second subtype of influenza A virus, (c) a third nucleic acid encoding an HA antigen of a first strain of influenza B virus, and (d) optionally, a fourth nucleic acid encoding an HA antigen of a second strain of influenza B virus, and wherein the immune response is induced against the said strains of the first and second subtypes of influenza A virus, the said first strain of influenza B virus and optionally the second strain, and against an HA antigen of at least one further HA antigen subtype of influenza A virus (which is different from any of the HA antigen subtypes of influenza A virus encoded by the nucleic acids present in the composition).

[0037] The terms "hemagglutinin", "hemagglutinin protein" and "HA" are used interchangeably throughout and mean the hemagglutinin protein that may be present on the surface of an influenza virus.

[0038] In some embodiments, the said strain of the first subtype of influenza A virus of (a) is different from the said strain of the second subtype of influenza A virus of (b).

[0039] As is well known in the art, influenza A viruses are classified into subtypes based on the antigenic properties of hemagglutinin (HA) and neuraminidase (NA) surface proteins. Currently, there are 18 described HA subtypes (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18 subtypes) and 11 described NA subtypes (N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11 subtypes), which can potentially form 144 combinations, such as H1N1, H2N2, H3N2, H5N1, H7N9, or H10N8.

[0040] In some embodiments, the first strain of influenza B virus in (c) and the second strain of influenza B virus in (d) are identical.

[0041] In some embodiments, (c) and (d) are identical.

[0042] In some embodiments, the first strain of influenza B virus in (c) and the second strain of influenza B virus in (d) are different.

[0043] In some embodiments, (c) and (d) are different.

[0044] In some embodiments, the aforementioned first and / or second subtypes of influenza A virus are selected from influenza A viruses characterized by HA selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18, preferably from the group consisting of H1, H3, H5, H7, H9, and H10, and more preferably from the group consisting of H1 and H3.

[0045] In some embodiments, the aforementioned first and / or second subtypes of influenza A virus are selected from influenza A viruses characterized by neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11, preferably from the group consisting of N1, N2, and N8, and more preferably from the group consisting of N1 and N2.

[0046] The terms "neuraminidase," "neuraminidase protein," and "NA" are used interchangeably throughout the text and refer to the neuraminidase protein that may be present on the surface of the influenza virus.

[0047] In some embodiments, the first and / or second subtypes of influenza A virus are selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, and H10N8, preferably from H1N1 and H3N2.

[0048] In some embodiments, the first subtype of influenza A virus is a subtype of influenza A group 1, which is appropriately influenza A subtype H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18, and more appropriately H1.

[0049] In some embodiments, the first subtype of the influenza A virus is the influenza A H1N1 subtype.

[0050] In some embodiments, the second subtype of the influenza A virus is a subtype of influenza A group 2, which is preferably influenza A subtypes H3, H4, H7, H10, H14, and H15, and more preferably H3.

[0051] In some embodiments, the aforementioned second subtype of influenza A virus is the influenza A H3N2 subtype.

[0052] In some embodiments, the strain of the first and / or second subtype of influenza A virus is selected from the group consisting of: A / ThailaNd / 8 / 2022(H3N2)-like virus, A / Massachusetts / 18 / 2022(H3N2)-like virus, A / Victoria / 4897 / 2022(H1N1)pdm09-like virus, A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus, A / Sydney / 5 / 2021(H1N1)pd m09 virus, A / Victoria / 2570 / 2019(H1N1)pdm09 virus, A / Darwin / 9 / 2021(H3N2) virus, A / Wisconsin / 588 / 2019(H1N1)pdm09 virus, A / Darwin / 6 / 2021(H3N2) virus, A / Cambodia / e0826360 / 2020(H3N2) virus, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09 virus, A / Hong Viruses such as Kong / 2671 / 2019(H3N2), A / Hawaii / 70 / 2019(H1N1)pdm09, A / Hong Kong / 45 / 2019(H3N2), A / Brisbane / 02 / 2018(H1N1)pdm09, A / Kansas / 14 / 2017(H3N2), A / California / 7 / 2009(H1N1)pdm09, A / Switzerland / 97]5293 / 2013(H3N2), and A / Hong Kong / 4801 / 2014(H3N2) virus, A / Michigan / 45 / 2015(H1N1)pdm09 virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus, A / Switzerland / 8060 / 2017(H3N2) virus, A / Brisbane / 02 / 2018(H1N1)pdm09 virus, A / Kansas / 14 / 2017(H3N2) virus, A / SouthAustralia / 34 / 2019(H3N2) virus, A / Idaho / 07 / 2018(H1N1)pdm09 virus, A / Maine / 38 / 2018(H1N1)pdm09 virus, A / Nebraska / IS / 2018(H1N1)pdm09 virus, A / Nebraska / 14 / 2019(H1N1)pdm09 virus, A / Iowa / 33 / 2019 H1N1)pdm09 virus, A / Arkansas / 28 / 2019 H1N1)pdm09 virus, A / Virginia / 41 / 2019 H1N1)pdm09 virus, A / Minnesota / 60 / 2019 H1N1)pdm09 virus, A / Alabama / 27 / 2019 H1N1)pdm09 virus, A / Iowa / 60 / 2018(H3N2) virus, A / Jamaica / 60361 / 2019(H3N2) virus, A / Florida / 130 / 2019(H3N2) virus, A / Laos / 1789 / 2019(H3N2) virus, A / Vermont / 25 / 2019(H3N2) virus, A / New Jersey / 34 / 2019(H3N2) virus, A / California / 176 / 2019(H3N2) virus, A / Pennsylvania / 1026 / 2019(H3N2) virus, A / Togo / 634 / 2019(H3N2) virus, A / Kenya / 130 / 2019(H3N2) virus, A / Togo / 1307 / 2019(H3N2) virus, A / Ohio / 30 / 2019(H3N2) virus, A / Guatemala / 93 / 2019(H3N2) virus, A / Guatemala / 10 / 2019(H3N2) virus, and A / Hong Kong / 4801 / 2014(H3N2) virus.

[0053] In some embodiments, the first strain of the first subtype of influenza A virus is an H1N1 strain selected from the group consisting of: A / Victoria / 4897 / 2022(H1N1)pdm09-like virus, A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus, A / Sydney / 5 / 2021(H1N1)pdm09-like virus, A / Beijing / 262 / 95(H1N1)-like virus, A / New Caledonia / 20 / 99(H1N1)-like virus, and A / Solomon Islands / 3 / 2006(H1N1) virus, A / Brisbane / 59 / 2007(H1N1) virus, A / California / 7 / 2009(H1N1) virus, A / California / 7 / 2009(H1N1)pdm09 virus, A / Michigan / 45 / 2015(H1N1)pdm09 virus, A / Victoria / 2570 / 2019(H1N1)pdm09 virus Viruses, including A / Wisconsin / 588 / 2019(H1N1)pdm09, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09, A / Hawaii / 70 / 2019(H1N1)pdm09, A / Brisbane / 02 / 2018(H1N1)pdm09, A / Christchurch / 16 / 2010 and A / South Dakota / 6 / 2007, A / Victoria / 4897 / 2022(H1N1)pdm09, A / Wisconsin / 67 / 2022(H1N1)pdm09, and A / Sydney / 5 / 2021(H1N1)pdm09.

[0054] In some embodiments, the second strain of influenza A virus is H3N2 and is selected from the group consisting of: A / Thailand / 8 / 2022(H3N2)-like virus, A / Massachusetts / 18 / 2022(H3N2)-like virus, A / Sydney / 5 / 97(H3N2)-like virus, A / Moscow / 10 / 99(H3N2)-like virus, A / Panama / 2007 / 99, A / Fujian / 411 / 2002(H3N2)-like virus, A / Wyoming / 3 / 2003, A / Kumamoto / 102 / 2002, A / Wellington / 1 / 2004(H3N2)-like virus, A / California / 7 / 2004(H3N2)-like virus, A / New York / 55 / 2004, A / Wisconsin / 67 / 2005 (H3N2) virus, A / Hiroshima / 52 / 2005, A / Brisbane / 10 / 2007 (H3N2) virus, A / Uruguay / 716 / 2007, A / Perth / 16 / 2009 (H3N2) virus, A / Wisconsin / 15 / 2009, A / Victoria / 210 / 2009, A / Victoria / 361 / 2011 (H3N2) virus, A / Ohio / 2 / 2012, A / Maryland / 2 / 2012, A / South Australia / 30 / 2012, A / Brisbane / 1 / 2012, A / Brisbane / 6 / 2012, A(H3N2) virus antigenically similar to the cell-proliferating protozoan virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like virus, A / Darwin / 9 / 2021(H3N2)-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, A / Cambodia / e0826360 / 2020(H3N2)-like virus, A / Hong Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / South Australia / 55 / 2014, A / Norway / 466 / 2014, A / Stockholm / 6 / 2014, A / HongViruses such as Kong / 4801 / 2014 (H3N2), A / Singapore / INFIMH-16-0019 / 2016 (H3N2), A / Switzerland / 8060 / 2017 (H3N2), A / Kansas / 14 / 2017 (H3N2), and A / South Australia / 34 / 2019 (H3N2).

[0055] In some embodiments, the strains of the first and / or second subtypes of the influenza A virus are selected from the influenza A viruses listed in Table 1 and / or Table 2.

[0056] In some embodiments, the strains of the first and / or second subtypes of the influenza A virus are selected from the influenza A viruses recommended by the WHO for influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0057] [Table 1] TIFF20265184110000O2.tif2A9163TIFF20265184110000O3.tif199163

[0058] [[ID=2l]] [Table 2] [[ID=Z4]] TIFF20265184110000O5.tif247163TIFF20265184110000O6.tif220163

[0059] In some embodiments, the first strain of the influenza B virus is selected from the group consisting of the B / Victoria lineage and the B / Yamagata lineage.

[0060] As is well known in the art, influenza B viruses are classified into two distinct lineages: B / Victoria / 2 / 1987-like (B / Victoria lineage) viruses, which have been spreading globally since 1983, and B / Yamagata / 16 / 1988-like (B / Yamagata lineage) viruses. In some embodiments, the first and / or second strains of influenza B viruses are selected from the group consisting of: B / Beijing / 184 / 93-like viruses, B / Harbin / 94-like viruses, B / Shangdong / 7 / 97-like viruses, B / Yamanashi / 166 / 98-like viruses, B / Sichuan / 379 / 99-like viruses, B / Guangdong / 120 / 2000, B / Johannesburg / 5 / 99, B / Victoria / 504 / 2000, B / Hong Kong / 330 / 2001-like viruses, and B / Hong Viruses such as Kong / 1434 / 2002, B / Brisbane / 32 / 2002, B / Shanghai / 361 / 2002, B / Jiangsu / 10 / 2003, B / Jilin / 20 / 2003, B / Malaysia / 2506 / 2004, B / Malaysia / 2506 / 2004 virus, B / Ohio / 1 / 2005, B / Florida / 4 / 2006, B / Brisbane / 3 / 2007, B / Brisbane / 60 / 2008, B / Brisbane / 33 / 2008, B / Wisconsin / 1 / 2010, B / Hubei-Wujiagang / 158 / Viruses such as 2009, B / Texas / 6 / 2011, B / Massachusetts / 2 / 2012, B / Phuket / 3073 / 2013, B / Austria / 1359417 / 2021, B / Washington / 02 / 2019, and B / Colorado / 06 / 2017.

[0061] In some embodiments, the first strain of influenza B virus is selected from the influenza B viruses listed in Table 1 and / or Table 2.

[0062] In some embodiments, the first strain of influenza B virus is selected from influenza B viruses recommended by the WHO for influenza virus vaccine composition (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0063] In some embodiments, the first and second strains of influenza B are strains of the B / Victoria lineage.

[0064] In some embodiments, the aforementioned first strain of influenza B is a strain of the B / Victoria lineage.

[0065] In some embodiments, the second strain of influenza B is a strain of the B / Yamagata lineage.

[0066] In some embodiments, the first strain of influenza B virus is selected from the group consisting of B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus, B / Washington / 02 / 2019 (B / Victoria lineage)-like virus, B / Colorado / 06 / 2017-like virus (B / Victoria / 2 / 87 lineage), B / Brisbane / 60 / 2008-like virus, and B / Colorado / 06 / 2019 (B / Victoria lineage)-like virus.

[0067] In some embodiments, the first subtype of influenza A virus is influenza A H1N1, the second subtype of influenza A virus is influenza A H3N2, and the first strain of influenza B virus is influenza B / Victoria lineage.

[0068] In some embodiments, the immunogenic composition is further: (d) The fourth nucleic acid encoding the HA antigen of the second strain of influenza B virus This includes, where the immune response is further induced against the HA antigen of the second strain of influenza B virus.

[0069] In some embodiments, the first strain of influenza B virus in (c) and the second strain of influenza B virus in (d) are identical.

[0070] In some embodiments, the aforementioned first and second strains of influenza B are strains of the B / Victoria lineage.

[0071] In some embodiments, (c) and (d) are identical.

[0072] In some embodiments, the first strain of influenza B virus in (c) is different from the second strain of influenza B virus in (d).

[0073] In some embodiments, the aforementioned first strain of influenza B is a strain of the B / Victoria lineage.

[0074] In some embodiments, the second strain of influenza B is a strain of the B / Yamagata lineage.

[0075] In some embodiments, (c) and (d) are different.

[0076] In some embodiments, the aforementioned second strain of influenza B virus is a B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus.

[0077] In some embodiments, the first subtype of influenza A virus is influenza A H1N1, the second subtype of influenza A virus is influenza A H3N2, the first strain of influenza B virus is of the influenza B / Victoria lineage, and the second strain of influenza B virus is of the influenza B / Yamagata lineage.

[0078] Exemplary HA antigens are publicly known in the art and are available, for example, in the NCBI Influenza Virus Resource (https: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / nph-select.cgi?go=database) and GISRS (https: / / gisaid.org / resources / human-Influenza-vaccine-composition / ).

[0079] In some embodiments, the HA antigen encoded by the first, second, third and / or fourth nucleic acid comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47.

[0080] In some embodiments, the HA antigen encoded by the first, second, third and / or fourth nucleic acid comprises or consists of an amino acid sequence or fragment or variant thereof described in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47.

[0081] In some embodiments, the HA antigen encoded by the third and / or fourth nucleic acid comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs. 5, 7, 17, or 35.

[0082] In some embodiments, the HA antigen encoded by the third and / or fourth nucleic acid comprises or consists of the amino acid sequence, fragment thereof, or variant thereof, described in any one of SEQ ID NOs. 5, 7, 17, or 35.

[0083] In some embodiments, the HA antigen encoded by the first and / or second nucleic acid comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 1, 3, 9, 11, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, or 47.

[0084] In some embodiments, the HA antigen encoded by the first and / or second nucleic acid comprises or consists of an amino acid sequence or fragment or variant thereof described in any one of SEQ ID NOs: 1, 3, 9, 11, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, or 47.

[0085] In some embodiments, the HA antigen encoded by the first nucleic acid comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 1, 11, 19, 23, 27, 29, 39, 41, or 43.

[0086] In some embodiments, the HA antigen encoded by the first nucleic acid comprises or consists of an amino acid sequence, fragment thereof, or a variant thereof, as described in any one of SEQ ID NOs: 1, 11, 19, 23, 27, 29, 39, 41, or 43.

[0087] In some embodiments, the HA antigen encoded by the second nucleic acid comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 3, 9, 13, 15, 21, 25, 31, 33, 37, 45, or 47.

[0088] In some embodiments, the HA antigen encoded by the second nucleic acid comprises or consists of an amino acid sequence, fragment thereof, or a variant thereof, as described in any one of SEQ ID NOs: 3, 9, 13, 15, 21, 25, 31, 33, 37, 45, or 47.

[0089] In some embodiments, the HA antigen encoded by the first, second, third, and / or fourth nucleic acids is a polypeptide comprising a full-length influenza HA protein. Preferably, the HA antigen encoded by the first, second, third, and / or fourth nucleic acids is a polypeptide consisting of a full-length influenza HA protein.

[0090] In some embodiments, the HA antigen encoded by the first, second, third, and / or fourth nucleic acids is a fragment of hemagglutinin protein, for example, a truncated hemagglutinin protein. In some embodiments, the fragment is headless hemagglutinin, meaning that the fragment does not contain the head domain. In some embodiments, the fragment contains a portion of the head domain. In some embodiments, the fragment is the stalk domain. In some embodiments, the fragment does not contain the cytoplasmic domain. In some embodiments, the fragment does not contain the transmembrane domain. In such embodiments, the fragment may be referred to as a soluble or secreted hemagglutinin protein or fragment.

[0091] In some embodiments, the composition does not contain nucleic acids encoding HA antigens from influenza strains not recommended by the WHO.

[0092] In some embodiments, the composition does not contain nucleic acids encoding HA antigens identified or designed by machine learning.

[0093] In some embodiments, the induced immune response is homogeneous, heterosubtype, and optionally heterogeneous or intrasubtype.

[0094] In some embodiments, the induced immune response is further heterogeneous or intraspecific.

[0095] In some embodiments, the immune response is induced against an HA antigen that is antigenically distinct from any of the HA antigens encoded by the nucleic acids present in the composition.

[0096] Influenza virus subtypes and lineages can be further classified into distinct genetic "clades" (also called "groups") and "subclades" (also called "subgroups") based on the similarity of their HA gene sequences. Clades and subclades are shown on the phylogenetic tree as groups and subgroups of viruses that typically have similar genetic variations (i.e., nucleotide or amino acid changes) and share a single common ancestor.

[0097] Genetically distinct clades and subclades are not necessarily antigenically distinct. Viruses from a particular clade or subclade may not have mutations that affect host immunity compared to those from other clades and subclades.

[0098] The term "antigenic properties" is used to describe the immune response triggered by antigens on a particular virus, such as HA and / or NA.

[0099] In some embodiments, the aforementioned at least one further HA antigen subtype of influenza A virus is selected from influenza A viruses characterized by HA selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18, preferably from the group consisting of H1, H2, H3, H5, H7, and H10, and more preferably from the group consisting of H2, H5, H7, and H10.

[0100] In some embodiments, the aforementioned at least one further HA antigenic subtype of influenza A virus is selected from influenza A viruses characterized by neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11, and preferably from the group consisting of N1, N2, N8, and N9.

[0101] In some embodiments, the aforementioned at least one further HA antigen subtype of influenza A virus is selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, and H10N8, and preferably from the group consisting of H1N1, H2N2, H3N2, H5N1, H7N9, and H10N8.

[0102] In some embodiments, the aforementioned at least one further HA antigen subtype of the influenza A virus is from influenza A group 1, preferably from influenza A subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18, more preferably from H1, H2, or H5.

[0103] In some embodiments, at least one further HA antigen subtype of the influenza A virus is H2N2 or H5N1.

[0104] In some embodiments, the aforementioned at least one further HA antigen subtype of influenza A virus is a subtype of influenza A group 2, preferably from influenza A subtypes H3, H4, H7, H10, H14, or H15, more preferably from H3, H7, or H10.

[0105] In some embodiments, the aforementioned at least one further HA antigen subtype of the influenza A virus is H7N9 or H10N8.

[0106] In some embodiments, the strain of the influenza A virus of which the aforementioned at least one further HA antigen subtype is selected from the group consisting of H2 / Singapore 1957, H5 / Vietnam / 2004, H7 / Shanghai / 2013 and H10 / Jiangxi Donghu / 2013.

[0107] In some embodiments, an immune response is induced against the HA antigens of influenza A subtypes H1 and H3, and against at least one, preferably all, of influenza A subtypes H2, H5, H7, or H10.

[0108] In some embodiments, the immune response is further induced to at least one additional HA antigen of the influenza B virus strain, which is different from any of the HA antigens of the influenza B virus strain encoded by the nucleic acids present in the composition.

[0109] In some embodiments, the aforementioned at least one further HA antigen of the influenza B virus strain is derived from a strain selected from the group consisting of B / Victoria and B / Yamagata lineages.

[0110] In some embodiments, the aforementioned at least one further HA antigen of the influenza B virus strain is a B / Victoria lineage strain.

[0111] In some embodiments, the aforementioned at least one further HA antigen of the influenza B virus strain is a B / Yamagata strain.

[0112] In some embodiments, the aforementioned at least one further HA antigen of the influenza B virus strain is B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus, B / Washington / 02 / 2019 (B / Victoria lineage)-like virus, B / Colorado / 06 / 2017-like virus (B / Victoria / 2 / 87 lineage), B / Brisbane / 60 / 2008-like virus, B / Colorado / 06 / 2019 (B / Victoria lineage)-like virus , B / Illinois / NHRC_FDX51486 / 2015, B / Oman / 4241 / 2019, B / Illinois / NHRC_18512 / 2017, B / California / BRD12452N / 2017, B / India / Pun-192 2338 / 2019, B / Japan / 8858 / 2019, B / Washington / 02 / 2019, B / Stockholm / 7 / 2019, B / Phuket / 3073 / 2013 and B / Quebec / 70 / 2015.

[0113] In some embodiments, the aforementioned at least one further HA antigen of the influenza B virus strain is B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus, B / Washington / 02 / 2019 (B / Victoria lineage)-like virus, B / Colorado / 06 / 2017-like virus (B / Victoria / 2 / 87 lineage), B / Brisbane / 60 / 2008-like virus, B / Colorado / 06 / 2019 (B / Victoria lineage)-like virus, B / Illinois / NHRC_FDX51486 / 2015, B / Oman / 4241 / 2019, B / Illinois / NHRC_18512 / 2017, B / California / BRD12452N / 2017 The group is selected from B / India / Pun-1922338 / 2019, B / Japan / 8858 / 2019, B / Washington / 02 / 2019, and B / Stockholm / 7 / 2019.

[0114] In some embodiments, the aforementioned at least one further HA antigen of the influenza B virus strain is selected from the group consisting of B / Phuket / 3073 / 2013 and B / Quebec / 70 / 2015.

[0115] In some embodiments, the immune response is further induced against at least one HA antigen of a heterologous or intra-subtype strain of influenza A virus.

[0116] In some embodiments, the heterogeneous or intrasubtype strain of influenza A virus is selected from the group consisting of H1 / Michigan / 2015, H1 / Hawaii / 2019, H1 / Christchurch / 2010, H1 / California / 2009, H3 / Finland / 2004, H3 / Hong Kong 2019, H3 / Perth / 2009, H3Bejing / 1992, H3 / Philippines / 1982, and H3 / Hong Kong / 1968.

[0117] In some embodiments, the heterogeneous or intraspecific strain of influenza A virus is selected from the group consisting of H1 / Michigan / 2015, H1 / Hawaii / 2019, H1 / Christchurch / 2010, and H1 / California / 2009.

[0118] In some embodiments, the aforementioned heterogeneous or intrasubtype strain of influenza A virus is selected from the group consisting of H3 / Finland / 2004, H3 / Hong Kong2019, H3 / Perth / 2009, H3Bejing / 1992, H3 / Philippines / 1982, and H3 / Hong Kong / 1968.

[0119] In some embodiments, the immune response is induced to at least 3, 4, 5, 6, 7, 8, 9, 10 HA antigens of at least 3, 4, 5, 6, 7, 8, 9, 10 subtypes of influenza A virus, including the aforementioned first and second subtypes of influenza A virus, and / or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 HA antigens of influenza B virus, including the aforementioned first strain of influenza B virus.

[0120] In some embodiments, the immune response is induced to at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 HA antigens of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 HA antigens of influenza B virus, including.

[0121] In some embodiments, the immune response is induced to at least one HA antigen of influenza A from subtypes H1, H2, H3, H5, H7, and H10, respectively, and to at least one HA antigen of influenza B from the B / Victoria lineage.

[0122] In some embodiments, the immune response is induced against at least one HA antigen of influenza A from subtypes H1, H2, H3, H5, H7, and H10, at least one HA antigen of influenza B from the B / Victoria lineage, and at least one strain of influenza B from the B / Yamagata lineage.

[0123] In some embodiments, the nucleic acid encoding the HA antigen, preferably mRNA, is present in equimolar ratios.

[0124] In some embodiments, the ratio of (a):(b):(c) is 1:1:1.

[0125] In some embodiments, the ratio of (a):(b):(c):(d) is 1:1:1:1.

[0126] In some embodiments, the nucleic acid encoding the HA antigen, preferably mRNA, is not present in equimolar ratios.

[0127] In some embodiments, the doses of (c) and / or (d) (e.g., weight dose or molar dose, preferably weight dose) differ from the doses of (a) and / or (b) (e.g., weight dose or molar dose, preferably weight dose).

[0128] In some embodiments, the ratio of (a):(b):(c) falls within the range of 1:1:1.5 to 1:1:20.

[0129] In some embodiments, the ratio of (a):(b):(c) is included in 1:1:1.5 to 1:1:5, optionally 1:1:2 to 1:1:5, optionally 1:1:3 to 1:1:5, optionally 1:1:4 to 1:1:5, optionally 1:1:1.5 to 1:1:4, optionally 1:1:1.5 to 1:1:3, optionally 1:1:2 to 1:1:4, and optionally 1:1:2 to 1:1:3.

[0130] In some embodiments, the ratio of (a):(b):(c) is greater than 1:1:5 and less than or equal to 1:1:20, preferably greater than 1:1:5 and less than or equal to 1:1:15, preferably greater than 1:1:5 and less than or equal to 1:1:12, preferably greater than 1:1:5 and less than or equal to 1:1:10, preferably greater than 1:1:5 and less than or equal to 1:1:8. In some embodiments, the ratio of (a):(b):(c) is 1:1:5.5 to 1:1:20, optionally 1:1:5.5 to 1:1:15, optionally 1:1:5.5 to 1:1:12, optionally 1:1:5.5 to 1:1:10, optionally 1:1:5.5 to 1:1:8, optionally 1:1:6 to 1:1:20, optionally , included in 1:1:6~1:1:15, 1:1:6~1:1:12 (upon request), 1:1:6~1:1:10 (upon request), 1:1:6~1:1:8 (upon request), 1:1:8~1:1:20 (upon request), 1:1:8~1:1:15 (upon request), 1:1:8~1:1:12 (upon request), and 1:1:8~1:1:10 (upon request).

[0131] In some embodiments, the ratio of (a):(b):(c) is approximately 1:1:1.5, approximately 1:1:2, approximately 1:1:2.2, approximately 1:1:2.4:2.4, approximately 1:1:2.6, approximately 1:1:2.8, approximately 1:1:3, approximately 1:1:3.2, approximately 1:1:3.4, approximately 1:1:3.6, approximately 1:1:3.8, approximately 1:1:4, approximately 1:1:4.2, approximately 1:1:4.4, approximately 1:1:4.6, approximately 1:1:4.8, approximately 1:1:5, approximately 1:1:5.5, approximately 1:1:6, approximately 1:1:6.5, approximately 1:1:7, approximately 1:1:7.5, approximately 1:1:8. Selected from approximately 1:1:8.5, 1:1:9, 1:1:9.5, 1:1:10, 1:1:10.5, 1:1:11, 1:1:11.5, 1:1:12, 1:1:12.5, 1:1:13, 1:1:13.5, 1:1:14, 1:1:14.5, 1:1:15, 1:1:15.5, 1:1:16, 1:1:16.5, 1:1:17, 1:1:17.5, 1:1:18, 1:1:18.5, 1:1:19, 1:1:19.5, and / or approximately 1:1:20.

[0132] In some embodiments, the ratio of (a):(b):(c) is 1:1:1.5, 1:1:2, 1:1:2.2, 1:1:2.4, 1:1:2.6, 1:1:2.8, 1:1:3, 1:1:3.2, 1:1:3.4, 1:1:3.6, 1:1:3.8, 1:1:4, 1:1:4.2, 1:1:4.4, 1:1:4.6, 1:1:4.8, 1:1:5, 1:1:5.5, 1:1:6, 1:1:6.5, 1:1:7, 1:1:7.5, 1:1 :8, 1:1:8.5, 1:1:9, 1:1:9.5, 1:1:10, 1:1:10.5, 1:1:11, 1:1:11.5, 1:1:12, 1:1:12.5, 1:1:13, 1:1:13.5, 1:1:14, 1:1:14.5, 1:1:15, 1:1:15.5, 1:1:16, 1:1:16.5, 1:1:17, 1:1:17.5, 1:1:18, 1:1:18.5, 1:1:19, 1:1:19.5, or 1:1:20.

[0133] In some embodiments, the ratio of (a):(b):(c) falls within the range of 1:1:2 to 1:1:4, and is appropriately about 1:1:2, about 1:1:3, or about 1:1:4, and is appropriately 1:1:2, 1:1:3, or 1:1:4.

[0134] In some embodiments, the ratio of (a):(b):(c) is within the range of 1:1:5.5 to 1:1:20, appropriately 1:1:6 to 1:1:12, appropriately 1:1:6 to 1:1:10, appropriately about 1:1:6, about 1:1:8 or about 1:1:10, appropriately 1:1:6, 1:1:8 or 1:1:10.

[0135] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is 1:1.5:1 to 1:20:1, preferably 1:1.5:1 to 1:5:1.

[0136] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is 1:1.5:1 to 1:5:1, preferably 1:2:1 to 1:5:1, preferably 1:3:1 to 1:5:1, preferably 1:4:1 to 1:5:1, preferably 1:1.5:1 to 1:4:1, preferably 1:1.5:1 to 1:3:1, preferably 1:2:1 to 1:4:1, preferably 1:2:1 to 1:3:1.

[0137] In some embodiments, the aforementioned first subtype of influenza A virus (a) is H1, the aforementioned second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is approximately 1:1.5:1, approximately 1:2:1, approximately 1:2.2:1, approximately 1:2.4:1, approximately 1:2.6:1, approximately 1:2.8:1, approximately 1:3:1, approximately 1:3.2:1, approximately 1:3.4:1, approximately 1:3.6:1, approximately 1:3.8:1, approximately 1:4:1, approximately 1:4.2:1, approximately 1:4.4:1, approximately 1:4.6:1, approximately 1:4.8:1, or approximately 1:5:1. In some embodiments, the ratio of (a):(b):(c) is 1:1.5:1, 1:2:1, 1:2.2:1, 1:2.4:1, 1:2.6:1, 1:2.8:1, 1:3:1, 1:3.2:1, 1:3.4:1, 1:3.6:1, 1:3.8:1, 1:4:1, 1:4.2:1, 1:4.4:1, 1:4.6:1, 1:4.8:1, or 1:5:1.

[0138] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is within 1:2:1 to 1:4:1, preferably 1:2:1 to 1:3:1, and preferably 1:2:1 or 1:3:1.

[0139] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is greater than 1:3:1 and less than or equal to 1:20:1.

[0140] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is greater than 1:3:1 and less than or equal to 1:5:1.

[0141] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is 1:1.5:1.5 to 1:20:5, preferably 1:1.5:1.5 to 1:5:5, preferably 1:2:2 to 1:5:5, preferably 1:3:3 to 1:5:5, preferably 1:4:4 to 1:5:5, preferably 1:1.5:1.5 to 1:4:4, preferably 1:1.5:1.5 to 1:3:3, preferably 1:2:2 to 1:4:4, preferably 1:2:2 to 1:3:3, appropriately 1:2:2, 1:2:3, 1:3:2 or 1:3:3.

[0142] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c) is 1:1.5:5.5 to 1:20:20, preferably 1:1.5:5.5 to 1:5:20, preferably 1:2:5.5 to 1:5:20, preferably 1:3:5. The ratios are 5-1:5:20, 1:4:5.5-1:5:20 (as desired), 1:1.5:5.5-1:4:20 (as desired), 1:1.5:5.5-1:3:20 (as desired), 1:2:5.5-1:4:20 (as desired), and 1:2:5.5-1:3:20 (as desired). Appropriately, the ratios are 1:2:6, 1:2:8, 1:2:10, 1:3:6, 1:3:8, or 1:3:10.

[0143] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0144] In some embodiments, the ratio of (a):(b):(c):(d) falls within the range of 1:1:1.5:1.5 to 1:1:20:20.

[0145] In some embodiments, the ratio of (a):(b):(c):(d) falls within the range of 1:1:1.5:1.5 to 1:1:5:5.

[0146] In some embodiments, the ratio of (a):(b):(c):(d) is included in 1:1:1.5:1.5 to 1:1:5:5, optionally 1:1:2:2 to 1:1:5:5, optionally 1:1:3:3 to 1:1:5:5, optionally 1:1:4:4 to 1:1:5:5, optionally 1:1:1.5:1.5 to 1:1:4:4, optionally 1:1:1.5:1.5 to 1:1:3:3, optionally 1:1:2:2 to 1:1:4:4, and optionally 1:1:2:2 to 1:1:3:3.

[0147] In some embodiments, the ratio of (a):(b):(c):(d) is greater than 1:1:5:5 and less than or equal to 1:1:20:20, preferably greater than 1:1:5:5 and less than or equal to 1:1:15:15, preferably greater than 1:1:5:5 and less than or equal to 1:1:12:12, preferably greater than 1:1:5:5 and less than or equal to 1:1:10:10, preferably greater than 1:1:5:5 and less than or equal to 1:1:8:8.

[0148] In some embodiments, the ratio of (a):(b):(c):(d) is 1:1:5.5:5.5 to 1:1:20:20, optionally 1:1:5.5:5.5 to 1:1:15:15, optionally 1:1:5.5:5.5 to 1:1:12:12, optionally 1:1:5.5:5.5 to 1:1:10:10, optionally 1:1:5.5:5.5 to 1:1:8:8, optionally 1:1:6:6 to 1:1:20:20, optionally It is included in 1:1:6:6~1:1:15:15, 1:1:6:6~1:1:12:12 (as requested), 1:1:6:6~1:1:10:10 (as requested), 1:1:6:6~1:1:8:8 (as requested), 1:1:8:8~1:1:20:20 (as requested), 1:1:8:8~1:1:15:15 (as requested), 1:1:8:8~1:1:12:12 (as requested), and 1:1:8:8~1:1:10:10 (as requested).

[0149] In some embodiments, the ratios of (a):(b):(c):(d) are approximately 1:1:1.5:1.5, approximately 1:1:2:2, approximately 1:1:2.2:2.2, approximately 1:1:2.4:2.4, approximately 1:1:2.6:2.6, approximately 1:1:2.8:2.8, approximately 1:1:3:3, approximately 1:1:3.2:3.2, approximately 1:1:3.4:3.4, and approximately 1:1:3.6:3.6 Approximately 1:1:3.8:3.8, approximately 1:1:4:4, approximately 1:1:4.2:4.2, approximately 1:1:4.4:4.4, approximately 1:1:4.6:4.6, approximately 1:1:4.8:4.8, approximately 1:1:5:5, approximately 1:1:5.5:5.5, approximately 1:1:6:6, approximately 1:1:6.5:6.5, approximately 1:1:7:7, approximately 1:1:7.5:7.5, approximately 1:1:8:8, approximately 1:1: 8.5:8.5, approximately 1:1:9:9, approximately 1:1:9.5:9.5, approximately 1:1:10:10, approximately 1:1:10.5:10.5, approximately 1:1:11:11, approximately 1:1:11.5:11.5, approximately 1:1:12:12, approximately 1:1:12.5:12.5, approximately 1:1:13:13, approximately 1:1:13.5:13.5, approximately 1:1:14:14, approximately 1:1:14.5: Select from 14.5, approximately 1:1:15:15, approximately 1:1:15.5:15.5, approximately 1:1:16:16, approximately 1:1:16.5:16.5, approximately 1:1:17:17, approximately 1:1:17.5:17.5, approximately 1:1:18:18, approximately 1:1:18.5:18.5, approximately 1:1:19:19, approximately 1:1:19.5:19.5, or approximately 1:1:20:20.

[0150] In some embodiments, the ratios of (a):(b):(c):(d) are 1:1:1.5:1.5, 1:1:2:2, 1:1:2.2:2.2, 1:1:2.4:2.4, 1:1:2.6:2.6, 1:1:2.8:2.8, 1:1:3:3, 1:1:3.2:3.2, 1:1:3.4:3.4, 1:1:3.6:3 .6, 1:1:3.8:3.8, 1:1:4:4, 1:1:4.2:4.2, 1:1:4.4:4.4, 1:1:4.6:4.6, 1:1:4.8:4.8, 1:1:5:5, 1:1:5.5:5.5, 1:1:6:6, 1:1:6.5:6.5, 1:1:7:7, 1:1:7.5:7.5, 1:1:8:8, 1:1 :8.5:8.5, 1:1:9:9, 1:1:9.5:9.5, 1:1:10:10, 1:1:10.5:10.5, 1:1:11:11, 1:1:11.5:11.5, 1:1:12:12, 1:1:12.5:12.5, 1:1:13:13, 1:1:13.5:13.5, 1:1:14:14, 1:1:14. The times are 5:14.5, 1:1:15:15, 1:1:15.5:15.5, 1:1:16:16, 1:1:16.5:16.5, 1:1:17:17, 1:1:17.5:17.5, 1:1:18:18, 1:1:18.5:18.5, 1:1:19:19, 1:1:19.5:19.5, or 1:1:20:20.

[0151] In some embodiments, the ratio of (a):(b):(c):(d) is within the range of 1:1:2:2 to 1:1:4:4, preferably 1:1:2:2 to 1:1:3:3, and preferably 1:1:2:2 or 1:1:3:3.

[0152] In some embodiments, the ratio of (a):(b):(c):(d) falls within the range of 1:1:2:2 to 1:1:4:4, and is appropriately about 1:1:4:4, and appropriately 1:1:4:4.

[0153] In some embodiments, the ratio of (a):(b):(c):(d) is within the range of 1:1:5.5:5.5 to 1:1:20:20, appropriately 1:1:6:6 to 1:1:12:12, appropriately 1:1:6:6 to 1:1:10:10, and appropriately 1:1:6:6 or 1:1:8:8 or 1:1:10:10.

[0154] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is within 1:1.5:1:1 to 1:20:1:1, and optionally within 1:1.5:1:1 to 1:5:1:1.

[0155] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is 1:1.5:1:1 to 1:20:1:1, preferably 1:1.5:1:1 to 1:5:1:1, preferably 1:2: It is included in 1:1~1:5:1:1, 1:3:1:1~1:5:1:1, 1:4:1:1~1:5:1:1, 1:1.5:1:1~1:4:1:1, 1:1.5:1:1~1:3:1:1, 1:2:1:1~1:4:1:1, and 1:2:1:1~1:3:1:1, as requested.

[0156] In some embodiments, the aforementioned first subtype of influenza A virus (a) is H1, the aforementioned second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is approximately 1:1.5:1:1, approximately 1:2:1:1, approximately 1:2.2:1:1, approximately 1:2.4:1:1, approximately 1:2.6:1:1, approximately 1:2.8:1:1, approximately 1:3:1:1, approximately 1:3.2:1:1, approximately 1:3.4:1:1, approximately 1:3.6:1:1, approximately 1:3.8:1:1, approximately 1:4:1:1, approximately 1:4.2:1:1, approximately 1:4.4:1:1, approximately 1:4.6:1:1, approximately 1:4.8:1:1, or approximately 1:5:1:1. In some embodiments, the ratio of (a):(b):(c):(d) is 1:1.5:1:1, 1:2:1:1, 1:2.2:1:1, 1:2.4:1:1, 1:2.6:1:1, 1:2.8:1:1, 1:3:1:1, 1:3.2:1:1, 1:3.4:1:1, 1:3.6:1:1, 1:3.8:1:1, 1:4:1:1, 1:4.2:1:1, 1:4.4:1:1, 1:4.6:1:1, 1:4.8:1:1, or 1:5:1:1.

[0157] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is within the range of 1:2:1:1 to 1:4:1:1, preferably 1:2:1:1 to 1:3:1:1, and preferably 1:2:1:1 or 1:3:1:1.

[0158] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is greater than 1:3:1:1 and less than or equal to 1:20:1:1.

[0159] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is greater than 1:3:1:1 and less than or equal to 1:5:1:1.

[0160] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is 1:1.5:1.5:1.5 to 1:20:5:5, optionally 1:1.5:1.5:1.5 to 1:5:5:5, optionally 1:2:2:2 to 1:5:5:5, optionally 1:3 :3:3~1:5:5:5, as desired, 1:4:4:4~1:5:5:5, as desired, 1:1.5:1.5:1.5~1:4:4:4, as desired, 1:1.5:1.5:1.5~1:3:3:3, as desired, 1:2:2:2~1:4:4:4, as desired, 1:2:2:2~1:3:3:3, and appropriately, 1:2:2:2, 1:2:3:3, 1:3:2:2 or 1:3:3:3.

[0161] In some embodiments, the first subtype of influenza A virus (a) is H1, the second subtype of influenza A virus (b) is H3, and the ratio of (a):(b):(c):(d) is 1:1.5:5.5:5.5 to 1:20:20:20, preferably 1:1.5:5.5:5.5 to 1:5:20:20, preferably 1:2:5.5:5.5 to 1:5:20:20, preferably 1:3:5.5:5.5 to 1:5:20:20 , as desired, included in 1:4:5.5:5.5~1:5:20:20, as desired, 1:1.5:5.5:5.5~1:4:20:20, as desired, 1:1.5:5.5:5.5~1:3:20:20, as desired, 1:2:5.5:5.5~1:4:20:20, as desired, 1:2:5.5:5.5~1:3:20:20, and appropriately, 1:2:6:6, 1:2:8:8, 1:2:10:10, 1:3:6:6, 1:3:8:8 or 1:3:10:10.

[0162] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0163] In some embodiments, the immunogenic composition is further: (e) at least one further nucleic acid encoding at least one further antigen derived from a strain of influenza virus Includes.

[0164] In some embodiments, the strain of influenza virus from which the at least one further antigen(e) is derived is selected from the group consisting of influenza A viruses and influenza B viruses.

[0165] In some embodiments, the strain of influenza virus from which the at least one further antigen(e) is derived is selected from the group consisting of the first subtype of influenza A virus, the second subtype of influenza A virus, the first strain of influenza B virus, and the second strain of influenza B virus.

[0166] In some embodiments, the immunogenic composition is a trivalent composition.

[0167] In some embodiments, the immunogenic composition is a tetravalent composition.

[0168] In some embodiments, the at least one further antigen comprises or consists of hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acid protein (PA), polymerase basic protein PB1, PB1-F2 and / or polymerase basic protein 2 (PB2), or peptides or proteins selected or derived from immunogenic fragments or immunogenic variants thereof.

[0169] In some embodiments, the at least one further antigen comprises or consists of peptides or proteins selected from or derived from influenza virus NA or immunogenic fragments or immunogenic variants thereof.

[0170] In some embodiments, the immunogenic composition comprises a combination of HA nucleic acids and NA nucleic acids encoding the HA antigen and NA antigen, wherein the at least one further antigen comprises or consists of peptides or proteins selected or derived from influenza virus NA or fragments or variants thereof.

[0171] Similar to hemagglutinin inhibitors (HA), neuraminidase (NA) is a major surface glycoprotein of the influenza virus. Naturally acquired or vaccine-induced NA inhibitor (NAI) antibodies have been shown to contribute to the prevention of influenza disease in naturally occurring influenza and in experimental human challenge studies. NAI antibodies appear to play an independent role in vaccine efficacy / effectiveness compared to hemagglutinin inhibitors.

[0172] In some embodiments, the NA antigen is a polypeptide containing full-length influenza NA protein. Preferably, the NA antigen is a polypeptide consisting of full-length influenza NA protein.

[0173] In some embodiments, the NA antigen is a fragment of a neuraminidase protein, such as a truncated neuraminidase protein.

[0174] In some embodiments, the composition does not contain nucleic acids encoding NA antigens derived from influenza strains not recommended by the WHO.

[0175] In some embodiments, the composition does not contain nucleic acids encoding NA antigens identified or designed by machine learning.

[0176] In some embodiments, the nucleic acids encoding the HA antigen and the NA antigen, preferably mRNA, are present in equimolar ratios.

[0177] In some embodiments, the nucleic acids encoding the HA antigen and the NA antigen, preferably mRNA, are not present in equimolar ratios.

[0178] In some embodiments, the dose (e.g., weight dose or molar dose, preferably weight dose) of the at least one further nucleic acid encoding the NA antigen, preferably mRNA, is different from the dose (e.g., weight dose or molar dose, preferably weight dose) of the nucleic acid encoding the HA antigen, preferably mRNA.

[0179] In some embodiments, the HA:NA antigen ratio encoded by nucleic acids, preferably mRNA, is within the range of 4:1 to 1:4, preferably 3:1 to 1:3, and preferably 2:1 to 2:1.

[0180] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is 4:1 or 1:4.

[0181] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is 3:1 or 1:3.

[0182] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is 2:1 or 1:2.

[0183] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is 3:2 or 2:3.

[0184] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is 4:3 or 3:4.

[0185] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is approximately 1:1.

[0186] In some embodiments, the ratio of HA:NA antigens encoded by nucleic acids, preferably mRNA, is 1:1.

[0187] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0188] In some embodiments, the at least one further antigen comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

[0189] In some embodiments, the at least one further antigen comprises or consists of an amino acid sequence, fragment thereof, or a variant thereof, as described in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

[0190] In some embodiments, the at least one further antigen comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 6, 8, 18, or 36.

[0191] In some embodiments, the at least one further antigen comprises or consists of an amino acid sequence, fragment thereof, or a variant thereof, as described in any one of SEQ ID NOs: 6, 8, 18, or 36.

[0192] In some embodiments, the at least one further antigen comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 2, 4, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, or 48.

[0193] In some embodiments, the at least one further antigen comprises or consists of an amino acid sequence, fragment thereof, or a variant thereof, as described in any one of SEQ ID NOs: 2, 4, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, or 48.

[0194] In some embodiments, the at least one further antigen comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 2, 12, 20, 24, 28, 30, 40, 42, or 44.

[0195] In some embodiments, the at least one further antigen comprises or consists of an amino acid sequence, fragment thereof, or a variant thereof, as described in any one of SEQ ID NOs: 2, 12, 20, 24, 28, 30, 40, 42, or 44.

[0196] In some embodiments, the at least one further antigen comprises, or consists of, an amino acid sequence or fragment or variant thereof having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence described in any one of SEQ ID NOs: 4, 10, 14, 16, 22, 26, 32, 34, 38, 46, or 48.

[0197] In some embodiments, the at least one further antigen comprises or consists of an amino acid sequence or fragment or variant thereof described in any one of SEQ ID NOs: 4, 10, 14, 16, 22, 26, 32, 34, 38, 46, or 48.

[0198] In some embodiments, the immunogenic composition is as defined herein (e 1 ), (e 2 ), (e 3 ) and / or (e 4 This includes multiple (e) such as ).

[0199] In some embodiments, the composition comprises at least 3, 4, 5, 6, 7, or 8 nucleic acids, preferably mRNA, encoding at least 3, 4, 5, 6, 7, or 8 antigens; optionally, 3 to 8 nucleic acids, preferably mRNA, encoding 3 to 8 antigens; optionally, 5 to 10 nucleic acids, preferably mRNA, encoding 5 to 10 antigens; and optionally, 7 or 8 nucleic acids, preferably mRNA, encoding 7 or 8 antigens.

[0200] In some embodiments, the composition is a polyvalent composition, where the antigens (a), (b), (c), (d) and / or (e) are derived from at least three strains of influenza virus.

[0201] In some embodiments, the composition comprises three nucleic acids, preferably mRNA, that encode three antigens.

[0202] In some embodiments, the composition comprises six nucleic acids encoding six antigens, suitably mRNA.

[0203] In some embodiments, the immunogenic composition comprises a combination of the first, second, and third nucleic acids, suitably mRNA, encoding the three HA antigens described above, and three nucleic acids, suitably mRNA, encoding three NA antigens.

[0204] In some embodiments, the composition is a multivalent composition, wherein the antigens of (a), (b), (c), (d) and / or (e) are derived from at least four strains of influenza virus.

[0205] In some embodiments, the composition comprises seven nucleic acids encoding seven antigens, suitably mRNA.

[0206] In some embodiments, the immunogenic composition comprises a combination of the first, second, third, and fourth nucleic acids, suitably mRNA, encoding the four HA antigens described above, and three nucleic acids, suitably mRNA, encoding three NA antigens.

[0207] In some embodiments, the immunogenic composition further comprises (e 1 ) a fifth nucleic acid encoding the NA of the first subtype of influenza A virus, (e 2 ) a sixth nucleic acid encoding the NA of the second subtype of influenza A virus, and (e 3 ) a seventh nucleic acid encoding the NA of the first strain of influenza B virus comprising.

[0208] In some embodiments, (a):(b):(c):(e 1 ):(e 2 ):(e 3The ratio of ) is included in 3:3:9:1:1:1 to 1:1:3:3:3, appropriately in 2:2:6:1:1:1 to 1:1:3:2:2:2, appropriately in 2:2:6:1:1:1 or 1:1:3:2:2:2 or 3:3:6:1:1:1 or 1:1:3:3:3.

[0209] In some embodiments, (a):(b):(c):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:3:24:1:1:1~1:1:8:3:3:3, appropriately in 2:2:16:1:1:1~1:1:8:2:2:2, appropriately in 2:2:16:1:1:1 or 1:1:8:2:2:2 or 3:3:24:1:1:1 or 1:1:8:3:3:3.

[0210] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:9:3:1:1:1~1:3:1:3:3, appropriately in 2:6:2:1:1:1~1:3:1:2:2:2, appropriately in 2:6:2:1:1:1 or 1:3:1:2:2:2 or 3:9:3:1:1:1 or 1:3:1:3:3.

[0211] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:15:3:1:1:1~1 :5:1:3:3:3, and is appropriately 2:10:2:1:1:1 or 3:15:3:1:1:1 or 2:8:2:1:1:1 or 3:12:3:1:1:1.

[0212] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:9:9:1:1:1 to 1:3:3:3:3:3, appropriately in 2:6:6:1:1:1 to 1:3:3:2:2:2, appropriately in 2:6:6:1:1:1 or 1:3:3:2:2:2 or 3:9:9:1:1:1 or 1:3:3:3:3.

[0213] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(e 1 ):(e 2 ):(e 3 The ratio of ) falls within 3:15:15:1:1:1 to 1:5:5:3:3:3, and is appropriately 2:10:10:1:1:1 or 3:15:15:1:1:1 or 2:8:8:1:1:1 or 3:12:12:1:1:1.

[0214] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(e 1 ):(e 2 ):(e 3 The ratio of ) is 3:9:24:1:1:1~1:3:8:3:3:3, appropriately 2:6:16:1:1:1~1:3:8:2:2:2, appropriately 2:6:16:1:1:1 or 1:3:8:2:2:2 or 3:9:24:1:1:1 or 1:3:8:3:3:3.

[0215] In some embodiments, (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3The ratio of ) is included in 3:3:9:9:1:1:1~1:1:3:3:3:3:3, appropriately in 2:2:6:6:1:1:1~1:1:3:3:2:2:2, appropriately in 2:2:6:6:1:1:1 or 1:1:3:3:2:2:2 or 3:3:9:9:1:1:1 or 1:1:3:3:3:3.

[0216] In some embodiments, (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:3:24:24:1:1:1~1:1:8:8:3:3:3, appropriately in 2:2:16:16:1:1:1~1:1:8:8:2:2:2, appropriately in 2:2:16::161:1:1 or 1:1:8:8:2:2:2 or 3:3:24:24:1:1:1 or 1:1:8:8:3:3.

[0217] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:9:3:3:1:1:1~1:3:1:1:3:3, appropriately in 2:6:2:2:1:1:1~1:3:1:1:2:2:2, appropriately in 2:6:2:2:1:1:1 or 1:3:1:1:2:2:2 or 3:9:3:3:1:1:1 or 1:3:1:1:3:3.

[0218] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3The ratio of ) falls within 3:15:3:3:1:1:1~1:5:1:1:3:3:3, and is appropriately 2:10:2:2:1:1:1 or 3:15:3:3:1:1:1 or 2:8:2:2:1:1:1 or 3:12:3:3:1:1:1.

[0219] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 The ratio of ) is included in 3:9:9:9:1:1:1~1:3:3:3:3:3:3, and appropriately in 2:6:6:6:1:1:1~1:3:3:3:2:2:2, and appropriately in 2:6:6:6:1:1:1 or 1:3:3:3:2:2 or 3:9:9:9:1:1:1 or 1:3:3:3:3:3.

[0220] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 The ratio of ) falls within 3:15:15:15:1:1:1~1:5:5:5:3:3:3, and is appropriately 2:10:10:10:1:1:1 or 3:15:15:15:1:1:1 or 2:8:8:8:1:1:1 or 3:12:12:12:1:1:1.

[0221] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3The ratio of ) is included in 3:9:24:24:1:1:1~1:3:8:8:3:3, appropriately in 2:6:16:16:1:1:1~1:3:8:8:2:2:2, appropriately in 2:6:16:16:1:1:1 or 1:3:8:8:2:2:2 or 3:9:24:24:1:1:1 or 1:3:8:8:3:3:3.

[0222] In some embodiments, the composition comprises eight nucleic acids, preferably mRNA, that encode eight antigens.

[0223] In some embodiments, the immunogenic composition comprises a combination of four HA antigens or four nucleic acids, appropriately mRNA, encoding the four HA antigens, and four NA antigens or four nucleic acids, appropriately mRNA, encoding the four NA antigens.

[0224] In some embodiments, the composition is further, (e 4 ) The eighth nucleic acid encoding NA of the second strain of influenza B virus Includes.

[0225] In some embodiments, (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 ):(e 4 The ratio of ) is included in 3:3:9:9:1:1:1:1~1:1:3:3:3:3:3:3, appropriately in 2:2:6:6:1:1:1:1~1:1:3:3:2:2:2:2, appropriately in 2:2:6:6:1:1:1:1 or 1:1:3:3:2:2:2:2 or 3:3:9:9:1:1:1:1 or 1:1:3:3:3:3:3:3.

[0226] In some embodiments, (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 ):(e 4The ratio of ) is included in 3:3:24:24:1:1:1:1~1:1:8:8:3:3:3:3, appropriately in 2:2:16:16:1:1:1:1~1:1:8:8:2:2:2:2, appropriately in 2:2:16:16:1:1:1:1 or 1:1:8:8:2:2:2:2 or 3:3:24:24:1:1:1:1 or 1:1:8:8:3:3:3:3.

[0227] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 ):(e 4 The ratio of ) is included in 3:9:3:3:1:1:1:1~1:3:1:1:3:3:3, appropriately in 2:6:2:2:1:1:1:1~1:3:1:1:2:2:2, appropriately in 2:6:2:2:1:1:1:1 or 1:3:1:1:2:2:2:2 or 3:9:3:3:1:1:1:1 or 1:3:1:1:3:3:3.

[0228] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 ):(e 4 The ratio of ) falls within 3:15:3:3:1:1:1:1~1:5:1:1:3:3:3, and is appropriately 2:10:2:2:1:1:1:1 or 3:15:3:3:1:1:1:1 or 2:8:2:2:1:1:1:1 or 3:12:3:3:1:1:1:1.

[0229] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e2 ):(e 3 ):(e 4 The ratio of ) is included in 3:9:9:9:1:1:1:1~1:3:3:3:3:3:3:3, appropriately in 2:6:6:6:1:1:1:1~1:3:3:3:2:2:2:2, appropriately in 2:6:6:6:1:1:1:1 or 1:3:3:3:2:2:2:2 or 3:9:9:9:1:1:1:1 or 1:3:3:3:3:3:3:1.

[0230] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 ):(e 4 The ratio of ) is 3:15:15:15:1:1:1:1~1:5:5:5:3:3:3:3, appropriately 2:10:10:10:1:1:1:1 or 3:15:15:15:1:1:1:1 or 2:8:8:8:1:1:1:1 or 3:12:12:12:1:1:1:1.

[0231] In some embodiments, the first subtype of influenza A virus in (a) is H1, the second subtype of influenza A virus in (b) is H3, and (a):(b):(c):(d):(e 1 ):(e 2 ):(e 3 ):(e 4 The ratio of ) is included in 3:9:24:24:1:1:1:1~1:3:8:8:3:3:3, appropriately in 2:6:16:16:1:1:1:1~1:3:8:8:2:2:2:2, appropriately in 2:6:16:16:1:1:1:1 or 1:3:8:8:2:2:2:2 or 3:9:24:24:1:1:1:1 or 1:3:8:8:3:3:3:3.

[0232] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0233] In some embodiments, the immune response is further induced to at least one further NA antigen of the influenza A virus and / or influenza B virus strains, which is different from the NA antigens of the aforementioned first and second subtypes of influenza A virus and the aforementioned first strain of influenza B virus, and optionally, the NA antigen encoded by nucleic acids present in the composition.

[0234] In some embodiments, the immune response is further induced to at least one further NA antigen of influenza A virus and / or influenza B virus strains that is different from any of the aforementioned first and second subtype NA antigens of influenza A virus, the aforementioned first and second strains of influenza B virus, and optionally, any NA antigen encoded by nucleic acids present in the composition.

[0235] In some embodiments, the immune response is induced against an NA antigen that is antigenically distinct from any of the NA antigens encoded by the nucleic acids present in the composition.

[0236] It should be noted that certain features and embodiments described in the first aspect of the present invention, i.e., in the context of immunogenic compositions for use according to the present invention, are equally applicable to a second aspect (vaccines for use according to the present invention), a third aspect (kits or parts kits for use according to the present invention), or further aspects including, for example, therapeutic methods.

[0237] nucleic acid In some embodiments, the immunogenic composition comprises at least one nucleic acid, preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The nucleic acid is DNA or RNA, preferably mRNA.

[0238] In some embodiments, at least one nucleic acid of the immunogenic composition, preferably the nucleic acid of (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 ) is DNA.

[0239] In some embodiments, at least one nucleic acid of the immunogenic composition, preferably the nucleic acid of (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 ) is an artificial nucleic acid, such as artificial DNA or artificial RNA, preferably mRNA.

[0240] Nucleic acid-based vaccination, including DNA or RNA, preferably mRNA, is one of the promising technologies for new vaccines against emerging viruses and for the provision of combination vaccines. The nucleic acid is genetically operable and administrable to a human subject. The transfected cells directly produce the encoded antigen (provided, for example, by DNA or RNA, particularly mRNA), which elicits a protective immune response.

[0241] The nucleic acids according to the invention, such as DNA or RNA, preferably mRNA, form the basis of nucleic acid-based immunogenic compositions or nucleic acid-based vaccines.

[0242] The nucleic acid-based immunogenic compositions (first aspect) or nucleic acid-based vaccines (second aspect) provided herein have advantages when compared to classical vaccine approaches.

[0243] Generally, protein-based vaccines or live attenuated vaccines are not ideal for use in developing countries due to their high production costs. Furthermore, protein-based vaccines or live attenuated vaccines require long development periods and are not suitable for rapid responses to outbreaks of epidemic viruses, such as influenza outbreaks. In fact, traditional methods for producing standard inactivated influenza vaccines are lengthy, meaning GISRS recommendations are made 6-7 months before the start of the influenza season, during which time the influenza virus can continue to evolve.

[0244] In contrast, nucleic acid-based immunogenic compositions and vaccines according to the present invention enable very rapid and cost-effective production. Therefore, compared to known vaccines, nucleic acid-based compositions / vaccines can be produced and manufactured significantly less cheaply and quickly, which is highly advantageous, especially for use in developing countries or in situations of annual epidemics or global pandemics. Nucleic acid-based compositions / vaccines provide GISRS with more time to monitor circulating viruses and to make GISRS recommendations closer to the influenza epidemic. This extension of the GISRS monitoring timeline should lead to more effective vaccines, designated to target circulating viruses closer to the influenza epidemic, allowing GISRS predictions to become more accurate. Furthermore, different nucleic acids encoding different antigens (e.g., antigens of different influenza strains) can be combined in a single immunogenic composition / vaccine to ensure or enhance the effectiveness of the immune response against the influenza virus.

[0245] The use of RNA, preferably mRNA, in or as a vaccine overcomes the drawbacks of conventional genetic vaccination that involves integrating DNA into cells with respect to safety, feasibility, applicability, and effectiveness in generating an immune response. RNA molecules, preferably mRNA, are considered to be significantly safer than DNA vaccines because RNA, preferably mRNA, is more readily degraded. They are rapidly removed from organisms and do not integrate into the genome or affect cellular gene expression in an uncontrolled manner. Also, RNA, preferably mRNA vaccines, are less likely to cause severe side effects such as autoimmune diseases or the production of anti-DNA antibodies. Transfection with RNA, preferably mRNA, only requires insertion into the cytoplasm of cells, which is more easily achieved than insertion into the nucleus.

[0246] In some embodiments, at least one nucleic acid of the immunogenic composition, preferably the nucleic acid of (a), (b), (c), (d), (e), (e 1 )、(e 2 )、(e 3 )、and / or (e 4 ) is RNA.

[0247] The term "RNA" is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and cytidine monophosphate (CMP) monomers or their analogs, which are linked to each other along the so-called backbone. The backbone is typically formed by a phosphodiester bond between the sugar of the first monomer, i.e., ribose, and the phosphate moiety of the second adjacent monomer. The specific sequential arrangement of the monomers, i.e., the sequential arrangement of the bases attached to the sugar / phosphate-backbone, is referred to as the RNA sequence.

[0248] Appropriately, the RNA molecule is selected from antisense RNAs, such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and RNAs that constitute part of the single guide RNA (sgRNA)-mediated CRISPR-Cas system.

[0249] In some embodiments, (a) is the first RNA, (b) is the second RNA, (c) is the first RNA, and / or (d) is the fourth RNA.

[0250] In some embodiments, (e) is at least one further RNA encoding at least one further antigen.

[0251] In some embodiments, the immunogenic composition comprises a plurality of (e) which are RNA.

[0252] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 ) is RNA.

[0253] Messenger RNA (mRNA) is a single-stranded RNA molecule that corresponds to the gene sequence of a gene and is read by ribosomes during the process of protein production. mRNA vaccines can utilize non-replicating mRNA or self-replicating RNA (also called self-amplifying mRNA or SAM). Vaccines based on non-replicating mRNA typically encode the antigen of interest and contain 5' and 3' untranslated regions (UTRs), a 5' cap, and a poly(A) tail, while self-amplifying RNA also encodes the viral replication mechanism that enables intracellular RNA amplification.

[0254] In some embodiments, the immunogenic composition comprises at least one nucleic acid, appropriately (a), (b), (c), (d), (e), (e 1 ), (e2 ), (e 3 ) and / or (e 4 ) is mRNA.

[0255] In some embodiments, the first, second, third, and / or fourth nucleic acids are mRNA.

[0256] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 1 to 200 μg, preferably 1 to 60 μg, and preferably 2 to 25 μg.

[0257] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 2 to 25 μg, optionally 2 to 18 μg, optionally 2 to 9 μg, optionally 2 to 6 μg, optionally 3 to 25 μg, 3 to 18 μg, 3 to 9 μg, and optionally 3 to 6 μg.

[0258] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μg, and optionally 2, 3, 6, 9, or 18 μg.

[0259] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 3, 6, 9, 12, or 18 μg.

[0260] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 0.5 to 200 μg, optionally 2 to 25 μg, optionally 2 to 30 μg, optionally 2 to 40 μg, optionally 2 to 45 μg, optionally 2 to 50 μg, and optionally 2 to 75 μg.

[0261] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 71, 72, 73, 74, or 75 μg, and optionally 3, 6, 9, 24, 36, 48, or 72 μg.

[0262] In some embodiments, the respective doses of the first, second, third, and / or fourth mRNAs are 3, 6, 9, 24, 36, 48, or 72 μg.

[0263] In some embodiments, the dose of the first mRNA is 0.5 to 15 μg, and optionally 2 to 10 μg.

[0264] In some embodiments, the dose of the first mRNA is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg.

[0265] In some embodiments, the dose of the first mRNA is 3, 6, or 9 μg.

[0266] In some embodiments, the dose of the second mRNA is contained in 0.5 to 15 μg, and optionally 2 to 10 μg.

[0267] In some embodiments, the dose of the second mRNA is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg.

[0268] In some embodiments, the dose of the second mRNA is 3, 6, or 9 μg.

[0269] In some embodiments, the dose of the third mRNA is contained in 15-100 μg, and optionally 20-75 μg.

[0270] In some embodiments, the dose of the third mRNA is 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 71, 72, 73, 74, or 75 μg.

[0271] In some embodiments, the dose of the third mRNA is 24, 36, 48, or 72 μg.

[0272] In some embodiments, the dose of the fourth mRNA is 15–100 μg, and optionally 20–75 μg.

[0273] In some embodiments, the dose of the fourth mRNA is 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 71, 72, 73, 74, or 75 μg.

[0274] In some embodiments, the dose of the fourth mRNA is 24, 36, 48, or 72 μg.

[0275] In some embodiments, the at least one further nucleic acid(e) is mRNA.

[0276] In some embodiments, the dose of each of the at least one additional mRNAs is 1 to 200 μg, preferably 1 to 60 μg, and preferably 2 to 25 μg.

[0277] In some embodiments, the dose of each of the at least one further mRNAs is 2–25 μg, optionally 2–18 μg, optionally 2–9 μg, optionally 2–6 μg, optionally 3–25 μg, 3–18 μg, 3–9 μg, and optionally 3–6 μg.

[0278] In some embodiments, the dose of each of the at least one further mRNAs is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μg, and optionally 2, 3, 6, 9, or 18 μg.

[0279] In some embodiments, the dose of each of the at least one additional mRNAs is 3, 6, 9, 12, or 18 μg.

[0280] In some embodiments, the dose of each of the at least one further mRNAs is 0.5 to 200 μg, optionally 2 to 10 μg, 2 to 25 μg, optionally 2 to 30 μg, optionally 2 to 40 μg, optionally 2 to 45 μg, optionally 2 to 50 μg, and optionally 2 to 75 μg.

[0281] In some embodiments, the dose of each of the at least one further mRNAs is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 71, 72, 73, 74, or 75 μg, and optionally 3, 6, 9, 24, 36, 48, or 72 μg.

[0282] In some embodiments, the respective doses of at least one additional mRNA are 3, 6, 9, 24, 36, 48, or 72 μg.

[0283] In some embodiments, the dose of at least one additional mRNA is contained in 2 to 10 μg, and preferably 3 μg.

[0284] In some embodiments, the immunogenic composition comprises a plurality of (e) which are mRNAs.

[0285] In some embodiments, the composition comprises at least 3, 4, 5, 6, 7, or 8 mRNAs, optionally 3 to 8 mRNAs, optionally 5 to 10 mRNAs, and optionally 7 or 8 mRNAs.

[0286] In some embodiments, the third, fourth, fifth, sixth, seventh and / or eighth nucleic acid is mRNA.

[0287] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 ) is mRNA.

[0288] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 1-200 μg, appropriately 1-60 μg, appropriately 1-25 μg, and appropriately 2-25 μg.

[0289] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 1-25 μg, 2-25 μg (optional), 2-18 μg (optional), 2-9 μg (optional), 2-6 μg (optional), 3-25 μg (optional), 3-18 μg (optional), 3-9 μg (optional), and 3-6 μg (optional).

[0290] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4The respective doses are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μg, and optionally 1, 2, 3, 6, 9, or 18 μg.

[0291] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 1, 2, 3, 6, 9, 12, or 18 μg.

[0292] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 0.5-200 μg, 2-10 μg, 2-25 μg, 2-30 μg, 2-40 μg, 2-45 μg, 2-50 μg, and 2-75 μg, depending on preference.

[0293] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 71, 72, 73, 74, or 75 μg, and optionally 3, 6, 9, 24, 36, 48, or 72 μg.

[0294] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e4 The respective doses are 3, 6, 9, 24, 36, 48, or 72 μg.

[0295] Furthermore, in this specification, (a) The first mRNA encoding the HA antigen of the first subtype strain of influenza A virus, (b) Second mRNA encoding the HA antigen of a second subtype strain of influenza A virus, and (c) Third mRNA encoding the HA antigen of the first strain of influenza B virus The present invention provides an immunogenic composition for use in the treatment or prevention of infection by influenza virus, wherein the immune response is induced against the first and second subtypes of influenza A virus, the first strain of influenza B virus, and the HA antigen of at least one further HA antigen subtype of influenza A virus (different from any of the HA antigen subtypes of influenza A virus encoded by mRNA present in the composition).

[0296] Furthermore, in this specification, (a) The first mRNA encoding the HA antigen of the first subtype strain of influenza A virus, (b) Second mRNA encoding the HA antigen of a second subtype strain of influenza A virus, (c) Third mRNA encoding the HA antigen of the first strain of influenza B virus, and (d) The fourth mRNA encoding the HA antigen of the second strain of influenza B virus. The present invention provides an immunogenic composition for use in the treatment or prevention of infection by influenza virus, wherein the immune response is induced against the HA antigen of the first and second subtypes of influenza A virus, the first and second subtypes of influenza B virus, and at least one further HA antigen subtype of influenza A virus (different from any of the HA antigen subtypes of influenza A virus encoded by mRNA present in the composition).

[0297] In some embodiments, the first strain of influenza B virus in (c) and the second strain of influenza B virus in (d) are identical.

[0298] In some embodiments, (c) and (d) are identical.

[0299] In some embodiments, the first strain of influenza B virus in (c) is different from the second strain of influenza B virus in (d).

[0300] In some embodiments, (c) and (d) are different.

[0301] In some embodiments, the respective doses of (c) and (d) are 15 to 100 μg, and optionally 20 to 75 μg.

[0302] In some embodiments, the respective doses of (c) and (d) are 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 1, 72, 73, 74, or 75 μg.

[0303] In some embodiments, the doses of (c) and (d) are 24, 36, 48, or 72 μg, respectively.

[0304] In some embodiments, the doses of (c) and (d) are 5 to 50 μg, optionally 10 to 40 μg, and optionally 12 to 36 μg.

[0305] In some embodiments, the doses of (c) and (d) are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 μg.

[0306] In some embodiments, the doses of (c) and (d) are contained in 20-200 μg, and optionally in 40-150 μg.

[0307] In some embodiments, the doses of (c) and (d) are 45, 46, 47, 48, 49, 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 141, 142, 143, 144, 145, or 150 μg.

[0308] In some embodiments, the doses of (c) and (d) are 48, 72, 96, or 144 μg.

[0309] In some embodiments, the respective doses of (a) and (b) are 0.5 to 15 μg, and optionally 2 to 10 μg.

[0310] In some embodiments, the respective doses of (a) and (b) are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg.

[0311] In some embodiments, the doses of (a) and (b) are 3, 6, or 9 μg, respectively.

[0312] In some embodiments, the doses of (a) and (b) are 2 to 20 μg, optionally 5 to 15 μg, and optionally 6 to 12 μg.

[0313] In some embodiments, the doses of (a) and (b) are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg.

[0314] In some embodiments, the doses of (a) and (b) are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg.

[0315] In some embodiments, the doses of (a) and (b) are 2 to 30 μg, and optionally 5 to 20 μg.

[0316] In some embodiments, the doses of (a) and (b) are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg.

[0317] In some embodiments, the doses of (a) and (b) are 6, 12, or 18 μg.

[0318] In some embodiments, the doses of (a), (b), (c), and (d) are 5 to 75 μg, optionally 10 to 60 μg, and optionally 12 to 48 μg.

[0319] In some embodiments, the doses of (a), (b), (c), and (d) are 35 to 75 μg.

[0320] In some embodiments, the doses of (a), (b), (c), and (d) are 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 70, 71, 72, 73, 74, or 75 μg.

[0321] In some embodiments, the doses of (a), (b), (c), and (d) are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 24, 25, 26, 27, 28, 29, 30, 45, 46, 47, 48, 49, 50, 55, and 60 μg.

[0322] In some embodiments, the doses of (a), (b), and (c) are 25 to 150 μg, optionally 25 to 100 μg, and optionally 30 to 90 μg.

[0323] In some embodiments, the doses of (a), (b), and (c) are 25 to 100 μg.

[0324] In some embodiments, the doses of (a), (b), and (c) are 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 100 μg.

[0325] In some embodiments, the doses of (a), (b), and (c) are 30, 36, 54, 60, or 90 μg.

[0326] In some embodiments, the doses of (c) and (d) are 5 to 50 μg, optionally 10 to 40 μg, optionally 12 to 36 μg, and the doses of (a) and (b) are 2 to 20 μg, optionally 5 to 15 μg, optionally 6 to 12 μg.

[0327] In some embodiments, the doses of (c) and (d) are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 μg, and the doses of (a) and (b) are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg.

[0328] In some embodiments, the dose of (c) is contained in 15-100 μg, optionally 20-75 μg, and the doses of (a) and (b) are contained in 2-30 μg, optionally 5-20 μg.

[0329] In some embodiments, the dose of (c) is 24, 36, 48, or 72 μg, and the doses of (a) and (b) are 6, 12, or 18 μg.

[0330] In some embodiments, the immunogenic composition is further: (e 1 ) The fifth mRNA encoding the NA of the first subtype of influenza A virus, (e 2 ) The sixth mRNA encoding the NA of the second subtype of influenza A virus, and (e 3 ) The seventh mRNA encoding the NA of the first strain of influenza B virus Includes.

[0331] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosage is 2-50 μg, 2-30 μg if desired, 5-20 μg if desired, and 9-18 μg if desired.

[0332] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosages are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 μg.

[0333] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosage is 9-36 μg.

[0334] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosage is 9, 18, 27, or 36 μg.

[0335] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dose of ) is 0.5 to 50 μg, optionally 2 to 20 μg, optionally 5 to 15 μg. In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosage is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg.

[0336] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosage is 5-15 μg.

[0337] In some embodiments, (e 1 ), (e 2 ) and (e 3 The dosage is 9 μg.

[0338] In some embodiments, (e 1 ), (e 2 ) and (e 3 The respective doses of ) are 0.5 to 20 μg, optionally 0.5 to 10 μg, optionally 0.5 to 5 μg, optionally 1 to 5 μg, optionally 2 to 5 μg. In some embodiments, (e 1 ), (e 2 ) and (e 3 The respective doses are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg.

[0339] In some embodiments, (e 1 ), (e 2 ) and (e 3 The respective doses are 1-5 μg.

[0340] In some embodiments, (e 1 ), (e 2 ) and (e 3 Each dose of ) is 3 μg.

[0341] In some embodiments, the immunogenic composition is further: (e 4 ) The eighth mRNA encoding the NA of the second strain of influenza B virus mentioned above. Includes.

[0342] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4The dosage is 5-50 μg, 10-50 μg if desired, and 12-48 μg if desired.

[0343] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 The dosages are 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, and 48 μg.

[0344] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 The dosage of ) is included in 0.5-50 μg, 2-25 μg if desired, and 5-20 μg if desired.

[0345] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 The dosage is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg.

[0346] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 The dosage is 12 μg.

[0347] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 The respective doses of ) are 0.5 to 20 μg, optionally 0.5 to 10 μg, optionally 0.5 to 5 μg, optionally 1 to 5 μg, optionally 2 to 5 μg. In some embodiments, (e 1 ), (e 2 ) and (e 3The respective doses are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg.

[0348] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 The respective doses are 1-5 μg.

[0349] In some embodiments, (e 1 ), (e 2 ), (e 3 ) and (e 4 Each dose of ) is 3 μg.

[0350] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 1-200 μg, appropriately 1-60 μg, appropriately 1-25 μg, and appropriately 2-25 μg.

[0351] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 1-25 μg, 2-25 μg (optional), 2-18 μg (optional), 2-9 μg (optional), 2-6 μg (optional), 3-25 μg (optional), 3-18 μg (optional), 3-12 μg (optional), 3-9 μg (optional), and 3-6 μg (optional).

[0352] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4The respective doses are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μg, and optionally 1, 2, 3, 6, 9, or 18 μg.

[0353] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 1, 2, 3, 6, 9, 12, or 18 μg.

[0354] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 0.5-200 μg, 2-10 μg, 2-25 μg, 2-30 μg, 2-40 μg, 2-45 μg, 2-50 μg, and 2-75 μg.

[0355] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The respective doses are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 71, 72, 73, 74, or 75 μg, and 3, 6, 9, 24, 36, 48, or 72 μg, as desired.

[0356] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ) and / or (e4 The respective doses are 3, 6, 9, 24, 36, 48, or 72 μg.

[0357] The mRNA used herein is preferably provided in a purified or substantially purified form, i.e., a form substantially free of proteins (e.g., enzymes), other nucleic acids (e.g., DNA and nucleoside phosphate monomers), etc., and generally having a purity of at least about 50% (by weight), usually at least 90%, for example, at least 95% or at least 98%.

[0358] The mRNA used herein can be produced by a number of methods, such as by whole or partial chemical synthesis, by digesting longer nucleic acids using nucleases (e.g., restriction enzymes), by ligating shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), or from genomes or cDNA libraries. In particular, mRNA can be produced enzymatically using DNA templates.

[0359] The mRNA used herein may be synthetic nucleic acids. The term “synthetic nucleic acid” as used herein is intended to refer to nucleic acids that do not occur naturally. In other words, synthetic nucleic acids can be understood as non-natural nucleic acid molecules. Such nucleic acid molecules may be non-natural due to their individual sequences (e.g., G / C content modified coding sequences, UTRs) and / or due to other modifications of nucleotides, e.g., structural modifications. Typically, synthetic nucleic acids can be designed and / or produced by genetic engineering to correspond to desired synthetic sequences of nucleotides. In this context, synthetic nucleic acids are sequences that do not occur naturally, i.e., sequences that differ from the wild-type or reference sequence / naturally occurring sequence by at least one nucleotide (e.g., via codon modifications, as further specified below). The term “synthetic nucleic acid” is not limited to meaning “a single molecule” but is understood to include ensembles of essentially identical nucleic acid molecules. Thus, it may refer to multiple essentially identical nucleic acid molecules.

[0360] In some embodiments, the mRNA used herein may be modified and / or stabilized nucleic acids, preferably modified and / or stabilized artificial mRNA.

[0361] According to some embodiments, mRNA used herein may therefore be provided as a “stabilized artificial nucleic acid” or “stabilized coding nucleic acid,” i.e., a nucleic acid exhibiting improved resistance to in vivo degradation, and / or improved stability in vivo, and / or improved translatability in vivo. Specific preferred modifications / fittings in this context that are suitable for “stabilizing” nucleic acids are described below.

[0362] The mRNA used herein may also be codon-optimized. In some embodiments, the mRNA used herein includes at least one codon-modified coding sequence. In some embodiments, the coding sequence of the mRNA used herein is a codon-modified coding sequence. Preferably, the amino acid sequence encoded by the codon-modified coding sequence is unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0363] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) contains a coding sequence which is a codon-modified coding sequence, where the amino acid sequence encoded by the codon-modified coding sequence is, in some cases, unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0364] In some embodiments, the mRNA used herein may be codon-optimized for expression in human cells. “Codon-optimized” means that modifications relating to codon usage frequency are intended to increase the translation efficiency and / or half-life of the nucleic acid. The term “codon-modified coding sequence” refers to a coding sequence in which at least one codon (a triplet of nucleotides encoding one amino acid) differs from the corresponding wild-type or reference coding sequence. Preferably, in the context of the present invention, a codon-modified coding sequence may exhibit improved resistance to degradation in vivo and / or improved stability in vivo and / or improved translatability in vivo. Codon modification in its broadest sense utilizes the degeneracy of the gene code, where multiple codons may encode the same amino acid and be used interchangeably, as outlined herein (see Table 1 of WO2020002525), so that the coding sequence may be optimized / modified for in vivo application.

[0365] In embodiments, the mRNA used herein may be modified, where the C content of at least one coding sequence is increased, and preferably maximized, compared to the C content of the corresponding wild-type or reference coding sequence (referred to herein as the "C-maximized coding sequence"). The amino acid sequence encoded by the C-maximized coding sequence of mRNA is preferably unmodified compared to the amino acid sequence encoded by the respective wild-type or reference coding sequence. The generation of the C-maximized nucleic acid sequence may preferably be carried out using the modification method described in WO2015 / 062738. In this context, the disclosure of WO 2015 / 062738 is incorporated herein by reference.

[0366] In some embodiments, the mRNA used herein may be modified, where at least one codon in the coding sequence may be adapted to human codon usage frequencies (referred to herein as the "human codon usage frequency adapted coding sequence"). Codons encoding the same amino acid occur at different frequencies in humans. Therefore, the coding sequence of the mRNA used herein is preferably modified such that the frequency of codons encoding the same amino acid corresponds to the naturally occurring frequency of those codons according to human codon usage frequencies. For example, in the case of the amino acid Ala, the wild-type or reference coding sequence is preferably adapted such that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" at a frequency of 0.28, the codon "GCA" at a frequency of 0.22, and the codon "GCG" at a frequency of 0.10 (see, for example, Table 1 of WO2020002525). Therefore, by adapting such a method (exemplified for Ala) to each amino acid encoded by the RNA coding sequence, a sequence that matches the frequency of human codon use can be obtained.

[0367] In embodiments, the mRNA used herein may be modified, where the codon compatibility index (CAI) may be increased or preferably maximized in at least one coding sequence (referred to herein as the “CAI-maximized coding sequence”). In some embodiments, for example, all codons in a wild-type or reference nucleic acid sequence that are relatively rare in humans may be replaced, for example, with each of the codons that are frequent in humans, where the frequent codons code for the same amino acid as the relatively rare codon. Preferably, the most frequent codons are used for each amino acid of the encoded protein (see Table 1 of WO2020002525, where the most frequent human codons are marked with an asterisk). Preferably, the mRNA used herein comprises at least one coding sequence, where the codon compatibility index (CAI) of at least one coding sequence is at least 0.5, at least 0.8, at least 0.9, or at least 0.95. In some embodiments, the codon fit index (CAI) of at least one coding sequence is 1 (CAI=1). For example, in the case of the amino acid Ala, the wild-type or reference coding sequence may be fitted in the same way as the most frequent human codon "GCC" is always fitted to the amino acid. Thus, such a method (exemplified for Ala) may be fitted to each amino acid encoded by the mRNA coding sequence to obtain a CAI-maximizing coding sequence.

[0368] In embodiments, the mRNA used herein may be modified, where the G / C content of at least one coding sequence is modified compared to the G / C content of the corresponding wild-type or reference coding sequence (referred to herein as the "G / C content modified coding sequence"). In this context, the terms "G / C optimization" or "G / C content modification" refer to nucleic acids containing a modified, appropriately increased number of guanosine and / or cytosine nucleotides compared to the corresponding wild-type or reference coding sequence. Such an increased number can be obtained by substituting codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. Preferably, nucleic acid sequences with increased G / C content are more stable or exhibit better expression than sequences with increased A / U. The amino acid sequence encoded by the G / C content modified coding sequence of mRNA is preferably unmodified compared to the amino acid sequence encoded by the respective wild-type or reference sequence. In some embodiments, the G / C content of the nucleic acid coding sequence is increased by at least 10%, 20%, 30%, and preferably at least 40%, compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence. The preparation of G / C content optimized mRNA sequences may be carried out using methods according to WO2002 / 098443. In this context, the entire scope of the disclosure of WO2002 / 098443 is included in the present invention.

[0369] In embodiments, mRNA used herein may be modified by altering the number of A and / or U nucleotides in the nucleic acid sequence compared to the number of A and / or U nucleotides in the original nucleic acid sequence (e.g., wild-type or reference sequence). In some embodiments, such AU modification is performed to alter the retention time of individual nucleic acids in the composition, and / or to enable co-purification using HPLC, and / or to enable analysis of the resulting nucleic acid composition. Such methods are described in detail in published PCT application WO2019092153A1. Claims 1 to 70 of WO2019092153A1 are incorporated herein by reference.

[0370] In some embodiments, the modified RNA sequence is selected from C-maximizing coding sequences, CAI-maximizing coding sequences, human codon frequency-adapted coding sequences, G / C content modification (or optimization) sequences, A / U modification, or any combination thereof.

[0371] In some embodiments, the RNA sequence has a G / C content of at least about 45%, 50%, 55%, or 60%. In certain embodiments, the RNA sequence has a G / C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

[0372] Ideally, mRNA containing modified sequences, when transfected into mammalian host cells, should have a stability of 12–18 hours or longer, e.g., 24, 36, 48, 60, 72 hours or longer, and be expressible by mammalian host cells (e.g., muscle cells).

[0373] Appropriately, when mRNA containing a modified RNA sequence is transfected into a mammalian host cell, it is translated into a protein in which the amount of protein is at least equivalent to the amount of protein obtained by a naturally occurring or wild-type or reference coding sequence transfected into a mammalian host cell, or appropriately, at least 10%, 20%, 30%, 40%, 50%, 100%, or 200% greater than the amount of such protein.

[0374] In some embodiments, the mRNA used herein comprises at least one poly(N) sequence, for example, at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or a combination thereof.

[0375] In some embodiments, the mRNA used herein includes at least one poly(A) sequence. Preferably, a poly(A) tail (e.g., a poly(A) tail of about 30 or more adenosine residues) can be attached to the 3' end of the RNA to increase its half-life.

[0376] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” or “3' poly(A) tail” are intended to be recognized and understood by those skilled in the art, and are, for example, typically a sequence of adenosine nucleotides located at the 3' end of a linear (or circular) RNA (or cyclic RNA) of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is homopolymerized in nature, for example, a poly(A) sequence of, for example, 100 adenosine nucleotides has a length of essentially 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from the adenosine nucleotides, for example, a poly(A) sequence of, for example, 100 adenosine nucleotides may have a nucleotide length longer than 100 (including 100 adenosine nucleotides and further including at least one nucleotide or nucleotide compartment different from the adenosine nucleotides).

[0377] The poly(A) sequence may contain about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. In some embodiments, the length of the poly(A) sequence may be at least about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides, or further longer than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides.

[0378] In some embodiments, the mRNA used herein includes at least one poly(A) sequence containing about 30 to about 200 adenosine nucleotides. In some embodiments, the poly(A) sequence contains about 64 adenosine nucleotides (A64). In other embodiments, the poly(A) sequence contains about 100 adenosine nucleotides (A100). In other embodiments, the poly(A) sequence contains about 150 adenosine nucleotides.

[0379] In further embodiments, the mRNA used herein comprises at least one poly(A) sequence containing about 100 adenosine nucleotides, the poly(A) sequence being interrupted by non-adenosine nucleotides, preferably 10 non-adenosine nucleotides (A30-N10-A70).

[0380] The poly(A) sequences as defined herein may be located directly at the 3' end of mRNA. In some embodiments, the 3' terminal nucleotide (the last 3' terminal nucleotide in the polynucleotide chain) is the 3' terminal A nucleotide of at least one poly(A) sequence. The term “located directly at the 3' end” should be understood as being precisely located at the 3' end; in other words, the 3' end of the nucleic acid consists of a poly(A) sequence and terminates with an A nucleotide.

[0381] In one embodiment, the mRNA used herein comprises at least 70 adenosine nucleotides, preferably a poly(A) sequence of at least 70 consecutive adenosine nucleotides, where the 3' terminal nucleotide is an adenosine nucleotide.

[0382] In some embodiments, the poly(A) sequence of nucleic acid is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly(A) sequence is not necessarily transcribed from a DNA template but is obtained in vitro by common chemical synthesis methods. In other embodiments, the poly(A) sequence is produced by enzymatic polyadenylation of RNA (after RNA in vitro transcription) using a commercially available polyadenylation kit and corresponding protocols known in the art, or alternatively, by using an immobilized poly(A) polymerase using, for example, the methods and means described in WO2016174271.

[0383] In embodiments, the mRNA used herein comprises at least one poly(C) sequence.

[0384] As used herein, the term “poly(C) sequence” is intended to mean a sequence of cytosine nucleotides of up to approximately 200 cytosine nucleotides. In embodiments, the poly(C) sequence may contain approximately 10 to approximately 200 cytosine nucleotides, approximately 10 to approximately 100 cytosine nucleotides, approximately 20 to approximately 70 cytosine nucleotides, approximately 20 to approximately 60 cytosine nucleotides, or approximately 10 to approximately 40 cytosine nucleotides. In one embodiment, the poly(C) sequence may contain approximately 30 cytosine nucleotides.

[0385] In some embodiments, the mRNA used herein includes at least one histone stem-loop (hSL) or histone stem-loop structure. In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) contains at least one histone stem loop.

[0386] The term "histone stem-loop" (for example, abbreviated as "hSL" in sequence listings) is intended to refer to the nucleic acid sequence that forms the stem-loop secondary structure primarily found in histone mRNA.

[0387] The histone stem-loop sequence / structure may preferably be selected from the histone stem-loop sequences disclosed in WO2012019780, and disclosures relating to histone stem-loop sequences / structures are incorporated herein by reference. The histone stem-loop sequences that may be used may be derived from formula (I) or (II) of WO2012019780. According to a further embodiment, the mRNA comprises at least one histone stem-loop sequence derived from at least one of the specific formulas (Ia) or (IIa) of patent application WO2012019780.

[0388] In other embodiments, the mRNA used herein does not contain hsL as defined herein.

[0389] The mRNA used herein may be modified by the addition of a 5' cap structure, which preferably stabilizes the RNA and / or enhances the expression of the encoded antigen and / or reduces stimulation of the innate immune system (after administration to the subject).

[0390] As used herein, the term “5' cap structure” is intended to be recognized and understood by those skilled in the art, and to refer, for example, to a 5' modified nucleotide, particularly a guanine nucleotide located at the 5' end of RNA, such as mRNA.

[0391] For example, the 5' end of RNA may be capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis or enzymatically produced after RNA transcription (e.g., by using a vaccinia virus capping enzyme (VCE) consisting of mRNA triphothphatase, guanylyl-transferase, and guanine-7-methyltransferase to catalyze the construction of the N7-monomethylated cap 0 structure). The cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the mRNA molecule may be further modified by a 2'-O-methyltransferase, resulting in the generation of a cap 1 structure (m7Gppp[m2'-O]N) which can further increase translational efficacy.

[0392] In some embodiments, the 5' cap structure is connected to RNA via a 5'-5'-triphosphate bond.

[0393] Possible suitable 5' cap structures include cap 0 (methylation of the first nucleic acid base, e.g., m7GpppN), cap 1 (further methylation of the ribose of the nucleotide adjacent to m7GpppN), cap 2 (further methylation of the ribose of the second downstream nucleotide of m7GpppN), cap 3 (further methylation of the ribose of the third downstream nucleotide of m7GpppN), cap 4 (further methylation of the ribose of the fourth downstream nucleotide of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0394] In some embodiments, mRNA used herein is preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) includes a 5' cap, preferably m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structure, and preferably a 5' cap1 structure.

[0395] The 5' cap (cap 0 or cap 1) structure can be formed by chemical RNA synthesis or by RNA in vitro transcription (simultaneous transcription capping) using a cap analog.

[0396] As used herein, the term “cap analog” is intended to refer to a non-polymerizable dinucleotide or trinucleotide that has cap functionality in that it promotes translation or localization and / or prevents the degradation of nucleic acid molecules, particularly RNA molecules, when incorporated at the 5' end of a nucleic acid molecule, as recognized and understood by those skilled in the art. Non-polymerizable means that the cap analog lacks a 5' triphosphate and therefore cannot be extended in the 3' direction by template-dependent polymerases, particularly by template-dependent RNA polymerases, and is therefore incorporated only at the 5' end. Examples of capped analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; non-methylated capped analogs (e.g., GpppG); dimethylated capped analogs (e.g., m2,7GpppG); trimethylated capped analogs (e.g., m2,2,7GpppG); dimethylated symmetrical capped analogs (e.g., m7Gpppm7G); or anti-reverse capped analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). Further capped analogs have been previously described (WO2008016473, WO2008157688, WO2009149253, WO2011015347, and WO2013059475). Further preferred capping analogs in that context are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017 / 053297, WO2017066782, WO2018075827, and WO2017066797, and disclosures relating to capping analogs are incorporated herein by reference.

[0397] In embodiments, the modified cap 1 structure is generated using the tri-nucleotide cap analogs disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, and WO2017066797. In particular, the modified cap structure 1 may be generated co-transcribed using any cap structure derivable from the structures disclosed in claims 1 to 5 of WO2017053297. Furthermore, the modified cap structure 1 may be generated co-transcribed using any cap structure derivable from the structures defined in claim 1 or claim 21 of WO2018075827.

[0398] In the embodiments described herein, the mRNA includes a cap 1 structure.

[0399] In embodiments, the 5' cap structure may be added co-transcribed, preferably in an RNA in vitro transcription reaction as defined herein, using a tri-nucleotide cap analog as defined herein.

[0400] In the embodiment, the mRNA cap 1 structure is formed by co-transcriptional capping using the tri-nucleotide capping analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A preferred cap 1 analog in that context is m7G(5')ppp(5')(2'OMeA)pG.

[0401] In other embodiments, the mRNA cap 1 structure is formed using co-transcriptional capping with the tri-nucleotide capping analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0402] In other embodiments, the cap 0 structure of the mRNA used herein is formed by co-transcriptional capping using the cap analog 3'OMe-m7G(5')ppp(5')G.

[0403] In other embodiments, the 5' cap structure is formed via enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase) to generate a cap 0, cap 1, or cap 2 structure. The 5' cap structure (cap 0 or cap 1) may be added using an immobilized capping enzyme and / or cap-dependent 2'-O methyltransferase using the methods and means disclosed in WO2016193226.

[0404] To determine the presence or absence of a cap 0 or cap 1 structure, a capping assay described in published PCT application WO2015101416, particularly claims 27-46 of published PCT application WO2015101416, can be used. Other capping assays that may be used to determine the presence or absence of a cap 0 or cap 1 structure in RNA are described in PCT / EP2018 / 08667, or published PCT applications WO2014152673 and WO2014152659.

[0405] In embodiments, the mRNA used herein contains an m7G(5')ppp(5')(2'OMeA) cap structure. In such embodiments, the mRNA contains an m7G cap at the 5' end and further methylation of the ribose of the nucleotides adjacent to m7GpppN, in that case, 2'O methylated adenosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, and 95% of the RNA(species) contain such cap structures as determined by capping assays.

[0406] In other embodiments, the mRNA used herein contains an m7G(5')ppp(5')(2'OMeG) cap structure. In such embodiments, the mRNA contains an m7G cap at the 5' end and further methylation of the ribose of the adjacent nucleotide, in that case, 2'O methylated guanosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, and 95% of the coding RNA (species) contain such cap structures as determined by a capping assay.

[0407] Therefore, the first nucleotide of the mRNA sequence, i.e., the downstream nucleotide of the m7G(5')ppp structure, may be 2'O-methylated guanosine or 2'O-methylated adenosine.

[0408] Preferably, the mRNA used herein includes a ribosome-binding site also known as a "Kozak sequence." In embodiments, the A / U (A / T) content in the environment of the ribosome-binding site of the mRNA used herein may be increased compared to the A / U (A / T) content in the environment of the ribosome-binding site of its respective wild-type or reference nucleic acid. This modification (increased A / U (A / T) content around the ribosome-binding site) increases the efficiency of ribosome binding to the mRNA. Effective binding of ribosomes to the ribosome-binding site then has the effect of efficient translation of the mRNA.

[0409] In some embodiments, the mRNA used herein may include at least one heterologous untranslated region (UTR), for example, a 5' UTR and / or a 3' UTR.

[0410] The terms “untranslated region,” “UTR,” or “UTR element” are as recognized and understood by those skilled in the art and are intended to refer to a portion of a nucleic acid molecule typically located at 5' or 3' of a coding sequence. UTRs are not translated into proteins. UTRs may be portions of nucleic acids, such as DNA or RNA. UTRs may also contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, promoter elements, and the like.

[0411] In embodiments, the mRNA used herein comprises a protein-coding region ("coding sequence" or "cds"), as well as a 5'UTR and / or 3'UTR. In particular, the UTR may have regulatory sequence elements that determine the nucleic acid, e.g., RNA turnover, stability, and localization. Furthermore, the UTR may have sequence elements that enhance translation. In the pharmaceutical application of nucleic acid sequences (including DNA and RNA), the translation of the nucleic acid into at least one peptide or protein is of paramount importance to therapeutic efficacy. Certain combinations of 3'UTR and / or 5'UTR can enhance the expression of the operably linked coding sequence encoding the peptide or protein of the present invention. Nucleic acid molecules having a combination of UTRs advantageously allow for rapid and transient expression of the antigenic peptide or protein after administration to a subject, preferably after intramuscular administration. Therefore, mRNAs comprising certain combinations of 3'UTR and / or 5'UTR provided herein are particularly suitable for administration as a vaccine, and especially suitable for administration into the muscle, dermis, or epidermis of a subject.

[0412] In some embodiments, the mRNA used herein comprises at least one heterologous 5'UTR and / or at least one heterologous 3'UTR. The heterologous 5'UTR or 3'UTR may be derived from naturally occurring genes or synthesized. In embodiments, the mRNA comprises at least one coding sequence as defined herein, operably ligated to at least one (heterologous) 3'UTR and / or at least one (heterologous) 5'UTR.

[0413] In embodiments, the mRNA used herein includes at least one heterologous 3'UTR.

[0414] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) includes the 3'UTR.

[0415] The terms “3' untranslated region,” “3'UTR,” or “3'UTR element” are intended to be recognized and understood by those skilled in the art and refer to, for example, a portion of a nucleic acid molecule located 3' (i.e., downstream) of a coding sequence that is not translated into a protein. The 3'UTR may be a portion of nucleic acid located between the coding sequence and (any) terminal poly(A) sequence, such as DNA or RNA. The 3'UTR may also include elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, and the like.

[0416] In some embodiments, the mRNA used herein may include a 3'UTR that can be derived from a gene relating to RNA with an enhanced half-life (i.e., resulting in stable RNA).

[0417] In some embodiments, the 3'UTR includes one or more of the following: a polyadenylation signal, a protein binding site that affects the nucleic acid stability of the cell location, or one or more miRNAs or miRNA binding sites.

[0418] In embodiments, the mRNA used herein comprises at least one heterologous 3'UTR, the at least one heterologous 3'UTR comprising a nucleic acid sequence derived from or selected from the 3'UTR of a gene selected from PSMB3, ALB7, alpha-globin (referred to as "muag"), CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a homolog, fragment, or variant of any one of these genes.

[0419] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) contains a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or from the 3'-UTR of a homolog, fragment, or variant of any one of these genes, or contains a 3'-UTR consisting of such sequences.

[0420] The nucleic acid sequence in that context may be derived from the published PCT application WO2019077001A1, in particular from claim 9 of WO2019077001A1. The corresponding 3'-UTR sequence of claim 9 of WO2019077001A1 is incorporated herein by reference.

[0421] In some embodiments, the mRNA used herein may include 3'UTRs described in WO2016107877, the disclosures of WO2016107877 relating to 3'UTR sequences are incorporated herein by reference. Preferred 3'UTRs are SEQ ID NOs. 1-24 and 49-318 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA used herein may include 3'UTRs described in WO2017036580, the disclosures of WO2017036580 relating to 3'UTR sequences are incorporated herein by reference. Preferred 3'UTRs are SEQ ID NOs. 152-204 of WO2017036580, or fragments or variants of these sequences. In other embodiments, the mRNA used herein includes the 3'UTR described in WO2016022914, and the disclosure of WO2016022914 relating to the 3'UTR sequences is incorporated herein by reference. Particularly preferred 3'UTRs are the nucleic acid sequences described in SEQ ID NOs. 20-36 of WO2016022914, or fragments or variants thereof.

[0422] In embodiments, the mRNA used herein includes at least one heterologous 5'UTR.

[0423] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) includes the 5' untranslated region (UTR).

[0424] The terms “5' untranslated region,” “5'UTR,” or “5'UTR element” are intended to be recognized and understood by those skilled in the art and refer, for example, to a portion of a nucleic acid molecule located at 5' (i.e., upstream) of a coding sequence that is not translated into a protein. The 5'UTR may also be a portion of nucleic acid located at 5' of a coding sequence. Typically, the 5'UTR begins at the transcription start site and ends before the start codon of the coding sequence. The 5'UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc. The 5'UTR may be post-transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5' cap structure (for example, for mRNA as defined herein).

[0425] In some embodiments, the mRNA used herein may include a 5'UTR that can be derived from a gene relating to RNA with an enhanced half-life (i.e., resulting in stable RNA).

[0426] In some embodiments, the 5'UTR includes one or more protein binding sites that affect RNA stability or RNA position in a cell, or one or more miRNAs or miRNA binding sites.

[0427] In embodiments, mRNA used herein is preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) contains at least one heterologous 5'UTR, and at least one heterologous 5'UTR contains a nucleic acid sequence derived from or selected from the 5'UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or from the 5'UTR of a homolog, fragment, or variant of any one of these genes.

[0428] The nucleic acid sequence in that context may be selected from the published PCT application WO2019077001A1, in particular from claim 9 of WO2019077001A1. The corresponding 5'UTR sequence of claim 9 of WO2019077001A1 is incorporated herein by reference (e.g., sequence numbers 1-20 of WO2019077001A1, or fragments or variants thereof).

[0429] In some embodiments, the mRNA used herein may include 5'UTRs described in WO2013143700, and the disclosures of WO2013143700 relating to 5'UTR sequences are incorporated herein by reference. Particularly preferred 5'UTRs are the sequence numbers 1-1363, 1395, 1421, and 1422 of WO2013143700, or nucleic acid sequences derived from fragments or variants of these sequences. In other embodiments, the mRNA used herein may include 5'UTRs described in WO2016107877, and the disclosures of WO2016107877 relating to 5'UTR sequences are incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in sequence numbers 25-30 and 319-382 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the nucleic acid comprises a 5'UTR described in WO2017036580, the disclosure of WO2017036580 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in SEQ ID NOs. 1 to 151 of WO2017036580, or fragments or variants thereof. In other embodiments, the nucleic acid comprises a 5'UTR described in WO2016022914, the disclosure of WO2016022914 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in SEQ ID NOs. 3 to 19 of WO2016022914, or fragments or variants thereof.

[0430] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) includes a heterologous 5'-UTR containing or consisting of a nucleic acid sequence derived from the 5'-UTR of HSD17B4, and at least one heterologous 3'-UTR containing or consisting of a nucleic acid sequence derived from the 3'-UTR of PSMB3.

[0431] In some embodiments, mRNA used herein is preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is located from 5' to 3': i) 5' cap 1 structure; ii) The 5'-UTR derived from the 5'-UTR of the HSD17B4 gene, iii) Code array, iv) Derived from the 3'-UTR of the PSMB3 gene, 3'-UTR, v) Depending on the case, the histone stem-loop sequence, and vi) A poly(A) sequence containing approximately 100 A nucleotides It contains, and the nucleotide at the 3' end of the RNA is adenosine.

[0432] In the embodiment, the RNA of the composition, preferably mRNA, has an RNA integrity in the range of about 40% to about 100%.

[0433] The term "RNA integrity" generally describes whether a complete RNA sequence is present in a composition. Low RNA integrity can result from, among other things, RNA degradation, RNA cleavage, inaccurate or incomplete chemosynthesis of RNA, inaccurate base pairing, incorporation of modified nucleotides or modification of already incorporated nucleotides, lack or incomplete capping, lack or incomplete polyadenylation, or incomplete RNA in vitro transcription. RNA is a fragile molecule that can be easily degraded, which can be caused, for example, by temperature, ribonucleases, pH, or other factors (e.g., nucleophilic attack, hydrolysis, etc.), which can reduce RNA integrity and consequently the functionality of the RNA.

[0434] Those skilled in the art can select from a variety of different chromatography or electrophoresis methods for determining RNA integrity. Chromatography and electrophoresis are well known in the art. When chromatography is used (e.g., RP-HPLC), the analysis of RNA integrity may be based on determining the peak area (or "sub-peak area") of full-length RNA in the corresponding chromatogram. The peak area may be determined by any suitable software that evaluates the signal of the detector system. The process of determining the peak area is also called integration. The peak area representing full-length RNA is typically set in relation to the peak area of ​​total RNA in each sample. RNA integrity may be expressed as %RNA integrity.

[0435] In the context of aspects of the present invention, RNA integrity may be determined using analytical (RP)HPLC. Typically, a test sample of a composition containing a lipid-based carrier for encapsulating RNA may be treated with a surfactant (e.g., about 2% Triton X100) to dissociate the lipid-based carrier and release the encapsulated RNA. The released RNA may be captured using a suitable binding compound, such as Agentcourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), essentially according to the manufacturer's instructions. After preparation of the RNA sample, analytical (RP)HPLC may be performed to determine the RNA integrity. Typically, to determine RNA integrity, the RNA sample may be diluted to a concentration of 0.1 g / l using, for example, water for injection (WFI). About 10 μl of the diluted RNA sample may be injected into an HPLC column (e.g., an integrated poly(styrene-divinylbenzene) matrix). Analytical (RP)HPLC may be performed using standard conditions, for example, gradient 1: buffer A (0.1M TEAA (pH 7.0)); buffer B (0.1M TEAA (pH 7.0), containing 25% acetonitrile). Starting with 30% buffer B, the gradient is expanded to 32% buffer B over 2 minutes, followed by expansion to 55% buffer B over 15 minutes at a flow rate of 1 ml / min. The HPLC chromatogram is typically recorded at a wavelength of 260 nm. The resulting chromatogram can be evaluated using software, and the relative peak area can be determined as a percentage (%) commonly known in the art. The relative peak area indicates the amount of RNA with 100% RNA integrity. Since the amount of RNA injected into the HPLC is typically known, analysis of the relative peak area provides information about the integrity of the RNA. Therefore, for example, if a total of 100 ng of RNA is injected and 100 ng is determined as the relative peak area, the RNA integrity is 100%. For example, if the relative peak area corresponds to 80 ng, the RNA integrity is 80%. Therefore, RNA integrity is determined in the context of this invention using analytical HPLC, preferably analytical RP-HPLC.

[0436] In embodiments, the RNA, preferably mRNA, of the composition has RNA integrity ranging from about 40% to about 100%. In embodiments, the RNA, preferably mRNA, has RNA integrity ranging from about 50% to about 100%. In embodiments, the RNA, preferably mRNA, has RNA integrity ranging from about 60% to about 100%. In embodiments, the RNA, preferably mRNA, has RNA integrity ranging from about 70% to about 100%. In embodiments, the RNA, preferably mRNA integrity is, for example, about 50%, about 60%, about 70%, about 80%, or about 90%. RNA integrity is preferably determined using analytical HPLC, preferably analytical RP-HPLC.

[0437] In embodiments, the RNA of the composition, preferably mRNA, has an RNA integrity of at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 80% or about 90%. RNA integrity is preferably determined using analytical HPLC, more preferably analytical RP-HPLC.

[0438] In some embodiments, the RNA, preferably mRNA, used herein does not contain a replicase element (e.g., a nucleic acid encoding a replicase).

[0439] In some embodiments, RNA used herein, preferably mRNA used herein, preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of each of these organisms does not self-replicate in all cases.

[0440] In some embodiments, RNA used herein, preferably mRNA used herein, preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4The mRNA of each of these organisms may self-replicate depending on the circumstances.

[0441] chemical modification In some embodiments, the RNA, preferably mRNA, contains a coding sequence consisting only of G, C, A, and U nucleotides, and therefore does not contain modified nucleotides (excluding the 5' terminal cap structures (cap0, cap1, cap2)).

[0442] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) does not contain chemically modified nucleotides.

[0443] In some embodiments, the RNA, preferably mRNA, used herein is modified RNA, preferably mRNA, where modification means chemical modification including skeletal modification and sugar modification or base modification.

[0444] Modified RNA, preferably mRNA, may contain one or more nucleotide analogs or modified nucleotides (nucleotide analogs / modifications, e.g., backbone modifications, sugar modifications, or base modifications). As used herein, "nucleotide analog" or "modified nucleotide" means a nucleotide containing one or more chemical modifications (e.g., substitution) within or on the nitrogen base of the nucleoside (e.g., cytosine (C), thymine (T), or uracil (U), adenine (A), or guanine (G)), and / or one or more chemical modifications within or on the phosphate of the backbone. Nucleotide analogs may contain further chemical modifications within or on the sugar portion of the nucleoside (e.g., ribose, modified ribose, six-membered sugar analog, or open-chain sugar analog) or on the phosphate. The production of nucleotides, modified nucleotides, and nucleosides is well known in the art; see the following references: U.S. Patents 4,373,071, 445,8066, 4500,707, 466,8777, 4973,679, 5047,524, 5132,418, 5153,319, 5262,530, and 5700,642. Numerous modified nucleosides and modified nucleotides are commercially available.

[0445] The skeletal modifications described herein are modifications in which the phosphate of the nucleotide backbone of RNA, preferably mRNA, is chemically modified. The sugar modifications described herein are chemical modifications of the sugars of RNA, preferably mRNA, nucleotides. Furthermore, the base modifications described herein are chemical modifications of the base portion of RNA, preferably mRNA, nucleotides. In this context, nucleotide analogs or modifications are appropriately selected from nucleotide analogs applicable to transcription and / or translation.

[0446] In some embodiments, the RNA, preferably mRNA, used herein includes at least one chemical modification.

[0447] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3), and / or (e 4 The mRNA of ) contains at least one chemical modification.

[0448] Modified nucleic acid bases (chemical modifiers) that can be incorporated into modified nucleosides and nucleotides and may be present within RNA, preferably mRNA molecules, include the following: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-iso Pentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-H droxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p)(2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2'-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-A Cetyl-2-O-methylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHaW (hydroxywybutosine); OHaW *(Incompletely modified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queosine); oQ (epoxyqueosine); galQ (galtactosylqueosine); manQ (mannosylqueosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G *(Archeosin); D(Dihydrouridine); m5Um(5,2'-O-dimethyluridine); s4U(4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um(2-thio-2'-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U(5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U(uridine 5-oxyacetic acid); mcmo5U(uridine 5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U(5-methoxycarbonylmethyluridine); mcm5Um(S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U(5-methylaminomethyluridine); mnm5s2U(5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine) ;ncm5U(5-carbamoylmethyluridine);ncm5Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm5U(5-carboxymethylaminomethyluridine);cnmm5Um(5-carboxymethylaminomethyl-2-LO-methyluridine);cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine);m62A(N6,N6-dimethyladenosine);Tm(2'-O-methylinosine);m4C(N4-methylcytidine);m4Cm(N4,2-O-dimethylcytidine);hm5C(5 -Hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,2'-O-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2'-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); mlGm (1,2'-O-dimethylguanosine);m'Am(1,2-O-dimethyladenosine)irinomethyluridine); tm5s2U(S-taurinomethyl-2-thiouridine)); iniG-14(4-demethylguanosine); imG2(isoguanosine); ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxoadenine, its 7-substituted derivative, dihydrouracil, pseudouracil, 2- Thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkyl Cytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine Nin, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (debasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Many of these modified nucleic acid bases and their corresponding ribonucleosides are available from commercial suppliers.

[0449] In some embodiments, the modified RNA, preferably the nucleotide analog / modification that can be incorporated into mRNA, is selected from the following: 2-amino-6-chloropurine riboside-5'-triphosphate (triphosphate), 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine- 5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5 '-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-tri Phosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particularly preferred are the base modification nucleotides selected from the group of base modification nucleotides consisting of the following: 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, pyridine-4-oneribonucleoside,5-Azauridine, 2-Thio-5-Azauridine, 2-Thiouridine, 4-Thio-Pseudouridine, 2-Thio-Pseudouridine, 5-Hydroxyuridine, 3-Methyluridine, 5-Carboxymethyluridine, 1-Carboxymethyl-Pseudouridine, 5-Propynyluridine, 1-Propynyl-Pseudouridine, 5-Taurinomethyluridine, 1-Taurinomethyl-Pseudouridine, 5-Taurinomethyl-2-Thiouridine, 1-Taurinomethyl-4-Thiouridine, 5-Methyluridine, 1-Methyl-Pseudouridine N, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine and 4-methoxy-2-thio-pseuduridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-a Cetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolocytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methylzebralin, 5-aza-2-thio-zebralin, 2-thio-zebraline, 2-methoxy-cytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine and 2-methoxyadenine, inosine, 1-methylinosine, waiosin, waibtosin, 7-deaza-guanosine, 7-deaza-8-aza-guanosine Anosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine and N2,N2-dimethyl-6-thioguanosine, 5 '-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseudruridine, 6-aza-cytidine, 2-thiocytidine, alpha-thiocytidine, pseudo-isocytidine, 5-aminoallyl-uridine, 5-iod-uridine, N1-methyl-pseudruridine, 5,6-dihydrouridine, alpha-thiouridine, 4-thiouridine, 6-aza- Uridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methylcytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudoisocytidine, 6-chloropurine, N6-methyladenosine, alpha-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0450] In some embodiments, the chemical modification is selected from the following: pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0451] Particularly suitable in that context are pseudouridine(ψ), N1-methylpseudridine(m1ψ), 5-methylcytosine, and 5-methoxyuridine, more preferably pseudouridine(ψ) and N1-methylpseudridine(m1ψ), and even more preferably N1-methylpseudridine(m1ψ).

[0452] In some embodiments, the RNA used herein, preferably mRNA, has essentially all, for example, essentially 100%, of the uracil in its coding sequence, and preferably the chemical modification is located at position 5 of the uracil.

[0453] In some embodiments, the RNA used herein, preferably mRNA, includes a chemical modification which is a uridine modification, preferably in which 100% of the uridine positions in the mRNA are modified.

[0454] It may be advantageous to incorporate modified nucleotides, such as pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and / or 5-methoxyuridine, into the coding sequence of the RNA, and appropriately mRNA, used herein, because undesirable innate immune responses (in response to the administration of coding mRNA or vaccine) may be regulated or reduced (as needed).

[0455] In embodiments, the coding sequence of RNA, preferably mRNA, used herein comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudridine (m1ψ), wherein preferably all uracil nucleotides are substituted with pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides, and optionally, wherein all uracil nucleotides are substituted with pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0456] In some embodiments, the RNA, and preferably the mRNA, used herein does not contain the N1-methylpseudridine (m1ψ) substitution site. In further embodiments, the RNA, and preferably the mRNA, used herein does not contain the pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine substitution sites.

[0457] In some embodiments, the chemical modification is N1-methylpseudridine and / or pseudouridine.

[0458] In the context of manufacturing nucleic acid-based vaccines or therapeutic substances, it may be required to provide GMP-grade nucleic acids, such as GMP-grade RNA or DNA. GMP-grade RNA or DNA can be manufactured using manufacturing processes approved by regulatory authorities. Therefore, in some embodiments, RNA is manufactured in accordance with current Good Manufacturing Practices (GMP), and various quality control steps at the DNA and RNA levels are implemented, appropriately in accordance with WO2016180430. In embodiments, the RNA, appropriately mRNA, of the present invention is GMP-grade RNA.

[0459] RNA synthesis In some embodiments, RNA, preferably mRNA, may be produced using any method known in the Art, including chemical synthesis, such as solid-phase RNA synthesis, and in vitro methods, such as RNA in vitro transcription reactions.

[0460] The RNA, and more appropriately the mRNA, used herein is in vitro transcription RNA.

[0461] The terms "RNA in vitro transcription" or "in vitro transcription" refer to the process by which RNA is synthesized in a cell-free system (in vitro). RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template [which may be a linearized plasmid DNA template or a PCR-amplified DNA template]. The promoter for controlling RNA in vitro transcription can be any promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases include T7, T3, SP6, or Syn5 RNA polymerase. In one embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme before being subjected to RNA in vitro transcription. Reagents typically used in RNA in vitro transcription typically include: a DNA template (linear plasmid DNA or PCR product) having a promoter sequence with high binding affinity to its respective corresponding RNA polymerase, e.g., bacteriophage-coding RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) relating to four bases (adenine, cytosine, guanine, and uracil); optionally, cap analogues as defined herein; optionally, further modified nucleotides as defined herein; and DNA-dependent RNA polymerases (e.g., T7, T3, SP6, or Syn5) capable of binding to the promoter sequence within the DNA template. RNA polymerase; optionally, a ribonuclease (RNase) inhibitor to inactivate potentially contaminating RNases; optionally, a pyrophosphatase to degrade pyrophosphatases that may inhibit RNA in vitro transcription; MgCl2 to supply Mg2+ ions as a cofactor for polymerase; a buffer (TRIS or HEPES) to maintain an appropriate pH value [which may also contain antioxidants (e.g., DTT) and / or polyamines, e.g., spermidine, at optimal concentrations], e.g., a buffer system containing TRIS-citric acid as disclosed in WO2017109161.

[0462] In embodiments, the nucleotide mixture used in RNA in vitro transcription may further comprise modified nucleotides as defined herein. In this context, suitable modified nucleotides may be selected from, in particular, pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. In embodiments, uracil nucleotides in the nucleotide mixture may be (partially or completely) substituted with pseudouridine (ψ) and / or N1-methylpseudridine (m1ψ) to obtain modified RNA.

[0463] In other embodiments, the nucleotide mixture used for RNA in vitro transcription does not contain the modified nucleotides defined herein. In embodiments, the nucleotide mixture used in RNA in vitro transcription contains only G, C, A, and U nucleotides and may optionally contain the cap analogs defined herein.

[0464] In some embodiments, the nucleotide mixture used in the RNA in vitro transcription reaction (i.e., fractions of each nucleotide in the mixture) can be optimized with respect to a given RNA sequence, as appropriately described in WO2015188933.

[0465] In this context, in vitro transcription was performed in the presence of a sequence-optimized nucleotide mixture and optionally a cap analog.

[0466] RNA purification In embodiments, the RNA, and more preferably the mRNA, used herein is purified RNA, and more preferably the mRNA.

[0467] As used herein, “purified RNA (or mRNA)” should be understood as RNA that has a higher purity than the starting material (e.g., in vitro transcription RNA) after a specific purification step (e.g., HPLC, TFF, oligo-d(T) purification, precipitation step). Typical impurities that are not inherently present in purified RNA include: peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, e.g., RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, incomplete RNA sequences, RNA fragments (short double-stranded RNA fragments, incomplete sequences, etc.), free nucleotides (modified nucleotides, normal NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other potential impurities that may originate from methods such as fermentation include bacterial impurities (bioburden, bacterial DNA) or impurities from the purification method (organic solvents, etc.). Therefore, in this regard, it is desirable that the “RNA purity” be as close to 100% as possible. Furthermore, with respect to RNA purity, it is desirable that the amount of full-length RNA transcript be as close to 100% as possible. Therefore, the "purified RNA (or mRNA)" used herein has a purity of 75%, 80%, 85%, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably 99% or higher. Purity can be measured, for example, by analytical HPLC, in which case the percentage corresponds to the ratio of the peak area of ​​the target RNA to the total area of ​​all peaks representing byproducts. Alternatively, purity can be measured, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0468] In some embodiments, RNA is purified using RP-HPLC, and preferably using reversed-phase high-pressure liquid chromatography (RP-HPLC) with additional use of a macroporous styrene / divinylbenzene column (e.g., particle size 30 μm, pore size 4000 angstroms) and a filter cassette having a cellulose membrane with a molecular weight cutoff of approximately 100 kDa. RNA may be purified in particular using PUREMESSENGER (RP-HPLC according to CureVac, Tubingen, Germany; WO2008077592) and / or tangent flow filtration (described in WO2016193206) and / or oligo-d(T) purification (see WO2016180430).

[0469] In some embodiments, RNA, preferably mRNA, is purified by RP-HPLC and / or TFF to remove double-stranded RNA, uncapped RNA, and / or RNA fragments.

[0470] For example, the formation of double-stranded RNA as a byproduct during RNA in vitro transcription can lead to the induction of an innate immune response, particularly IFN-alpha (which is a major cause of fever in vaccinated subjects), which is, of course, an undesirable side effect. Current techniques for immunoblotting of dsRNAs (e.g., dot blotting, serologically specific electron microscopy (SSEM), ELISA, etc.) are used to detect dsRNA species from nucleic acid mixtures and to determine their size.

[0471] In embodiments, the RNA, preferably mRNA, contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts compared to the RNA, preferably mRNA, that has not been purified by RP-HPLC and / or TFF.

[0472] In some embodiments, the RNA purified by RP-HPLC and / or TFF, preferably mRNA, contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts compared to the RNA purified by oligo-dT purification, precipitation, filtration, and / or AEX, preferably mRNA.

[0473] Carrier Various carrier systems have been described that can be used to protect RNA, preferably mRNA, and to encapsulate or complex RNA, preferably mRNA, in order to facilitate its delivery to target cells. The present invention can utilize any suitable carrier system. Notable specific carrier systems are described in further detail below.

[0474] In embodiments, RNA, preferably mRNA, used herein can be complexed, encapsulated, partially encapsulated, or bound using one or more lipids (e.g., cationic lipids and / or neutral lipids) to form lipid-based carriers, such as liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes, preferably lipid nanoparticles.

[0475] In some embodiments, (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is formulated (combined, formulated) separately or together in lipid nanoparticles (LNPs).

[0476] In some embodiments, the RNA, preferably mRNA, used herein is formulated separately (in any formulation or complexing agent as defined herein), preferably the RNA, preferably mRNA, used herein is formulated in separate liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes.

[0477] In some embodiments, RNA used herein, preferably mRNA used herein, preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is formulated separately.

[0478] In embodiments, the RNA, preferably mRNA, used herein is co-formulated (in any formulation or complexing agent as defined herein), where preferably the RNA, preferably mRNA, used herein is formulated in separate liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes.

[0479] In some embodiments, RNA used herein, preferably mRNA used herein, preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is co-formulated, that is, formulated together.

[0480] LNP The term “lipid nanoparticles” (or “LNPs”) refers to non-viral particles in which nucleic acid molecules, such as RNA, can be encapsulated. LNPs are not limited to any particular form and include any form produced when ionizable (or cationic) lipids and optionally one or more further lipids are combined, for example, in an aqueous environment and / or in the presence of nucleic acids, such as RNA. For example, liposomes, lipid complexes, lipoplexes, etc., fall within the scope of lipid nanoparticles (LNPs). LNP delivery systems and methods for their preparation are known in the art.

[0481] The particles may contain some external RNA, preferably mRNA (e.g., on the surface of the particle), but preferably RNA, preferably at least half of the mRNA (and preferably at least 85%, particularly at least 95%, e.g., all of it), is encapsulated.

[0482] In some embodiments, LNP is suitable for intramuscular and / or intradermal administration.

[0483] In embodiments, a lipid-based carrier, preferably one in which at least about 80%, 85%, 90%, and 95% of the LNPs have a spherical shape.

[0484] LNPs typically comprise cationic lipids and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., pegylated lipids). RNA, preferably mRNA, may be encapsulated in an aqueous space enclosed by the lipid portion of the LNP or by part or all of the lipid portion of the LNP. RNA, preferably mRNA, or its portion may also associate with and complex with the LNP. LNPs may comprise any lipid capable of binding nucleic acids or forming particles in which one or more nucleic acids are encapsulated. In some embodiments, an LNP comprising nucleic acids, preferably RNA, more preferably mRNA, comprises one or more cationic lipids and one or more stabilizing lipids. The stabilizing lipids include neutral lipids and pegylated lipids.

[0485] In some embodiments, the LNP includes PEG-modified lipids, noncationic lipids, sterols, and cationic lipids.

[0486] LNPs can be formed from, for example, a mixture of (i) PEG-modified lipids, (ii) non-cationic lipids, (iii) sterols, and (iv) ionizable cationic lipids. Alternatively, LNPs can be formed from, for example, a mixture of (i) PEG-modified lipids, (ii) non-cationic lipids, (iii) sterols, and (iv) non-ionizable cationic lipids.

[0487] In some embodiments, the noncationic lipid is a neutral lipid.

[0488] In some embodiments, cationic lipids are ionizable.

[0489] The in vivo characteristics and behavior of LNPs can be modified to impart steric stabilization by adding a hydrophilic polymer coating, such as polyethylene glycol (PEG), to the LNP surface. Furthermore, LNPs (or liposomes, nanoliposomes, lipoplexes) can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the attached PEG chains (e.g., via pegylated lipids or pegylated cholesterol).

[0490] In one embodiment, RNA, preferably mRNA, is complexed with one or more lipids to form lipid nanoparticles, where LNP (or liposome, nanoliposome, lipoplex) includes polymer-conjugated lipids, preferably PEGylated lipids / PEG-lipids.

[0491] In some embodiments, the LNP includes polymer-conjugated lipids. The term “polymer-conjugated lipid” refers to a molecule containing both a lipid and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” or “PEG-modified lipid” refers to a molecule containing both a lipid and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), among others. The terms “PEGylated lipid” and “PEG-modified lipid” are used interchangeably herein.

[0492] Polymer-conjugated lipids as defined herein, such as PEG-lipids, can function as lipids that reduce aggregation.

[0493] In certain embodiments, the LNP includes a stabilized lipid that is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Typical polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP includes pegylated diacylglycerol (PEG-DAG), for example, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), for example, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, for example, ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0494] In some embodiments, the PEG-modified lipid includes PEG-DMG or PEG-cDMA.

[0495] In embodiments, the pegylated lipid is preferably derived from formula (IV) of the published PCT patent application WO2018078053A1. Accordingly, the pegylated lipid derived from formula (IV) of the published PCT patent application WO2018078053A1 and their respective related disclosures are incorporated herein by reference.

[0496] In some embodiments, PEG-modified lipids are expressed in formula IV:

[0497] [ka] (In the formula, R 8 and R 9 Each of these is independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain may be interrupted by one or more ester bonds; w has an average value of 30-60. It has.

[0498] In some embodiments, PEG-modified lipids R 8 and R 9 This is a saturated alkyl chain.

[0499] In some embodiments, RNA, preferably mRNA, is complexed with one or more lipids to form an LNP, where the LNP comprises a polymer-conjugated lipid, preferably a PEG lipid, where the PEG lipid is preferably derived from formula (IVa) of the published PCT patent application WO2018078053A1. Accordingly, the PEG lipids derived from formula (IVa) of the published PCT patent application WO2018078053A1 and their respective related disclosures are incorporated herein by reference.

[0500] In some embodiments, the PEG lipid or PEGylated lipid is defined by formula (IVa):

[0501] [ka] (In the formula, n has an average value in the range of 30 to 60, for example, about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2, or 60±2. In one embodiment, n has about 49. In another embodiment, n has about 45. In a further embodiment, the PEG lipid has formula (IVa), where n is an integer selected such that the average molecular weight of the PEG lipid is about 2000 g / mol to about 3000 g / mol or about 2300 g / mol to about 2700 g / mol, preferably about 2500 g / mol.

[0502] In some embodiments, the PEG-modified lipid is of formula (IVa):

[0503] [ka] (In the formula, n has an average value in the range of 30 to 60, preferably n has an average value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most preferably n has an average value of 49 or 45; or n is an integer selected such that the average molecular weight of the PEG lipids is approximately 2500 g / mol. It has.

[0504] The lipid of formula IVa, which is preferably used in this specification, has the chemical term 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide and is also referred to as ALC-0159.

[0505] Further examples of suitable PEG-lipids in that context are provided in US20150376115A1 and WO2015199952, each of which is incorporated in whole by reference.

[0506] In some embodiments, the LNP contains about 3, 2, or less than 1 mole percent of PEG or PEG-modified lipids, based on the total moles of lipids in the LNP.

[0507] In further embodiments, the LNP contains PEG-modified lipids in molar terms of about 0.1% to about 20%, for example, about 0.5 to about 15%, about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.5%, or about 0.3% (based on 100% total molars of lipids in the LNP). In embodiments, the LNP contains approximately 1.0% to approximately 2.0% on a molar basis, for example, approximately 1.2 to approximately 1.9%, approximately 1.2 to approximately 1.8%, approximately 1.3 to approximately 1.8%, approximately 1.4 to approximately 1.8%, approximately 1.5 to approximately 1.8%, approximately 1.6 to approximately 1.8%, particularly approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, most preferably 1.7% (based on 100% total molars of lipids in the LNP) of PEG-modified lipids. In various embodiments, the molar ratio of cationic lipids to PEGylated lipids is in the range of approximately 100:1 to approximately 25:1.

[0508] In some embodiments, the LNP contains about 0.5 to 10 mol%, optionally 0.5 to 5 mol%, or 0.5 to 3 mol%, of PEG-modified lipids.

[0509] In the embodiment, the LNP comprises one or more further lipids that stabilize particle formation during their formulation or manufacturing process (e.g., neutral lipids and / or one or more steroids or steroid analogs).

[0510] Suitable stabilizing lipids include neutral lipids and anionic lipids. The term "neutral lipid" refers to one of several lipid species that exist either uncharged or in a neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0511] In some embodiments, the noncationic lipid is a neutral lipid, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or sphingomyelin (SM), and preferably the neutral lipid is DSPC.

[0512] In embodiments, LNP (or liposome, nanoliposome, lipoplex) comprises one or more neutral lipids, where the neutral lipids are distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and dioleoyl - Selected from the group comprising phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearyl-2-oleoylphosphatidiethanolamine (SOPE), and 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (transDOPE), or mixtures thereof.

[0513] In some embodiments, the LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipids to neutral lipids is in the range of about 2:1 to about 8:1.

[0514] In the embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Preferably, the molar ratio of the cationic lipid to DSPC can be in the range of about 2:1 to about 8:1.

[0515] In some embodiments, the steroid is a sterol, preferably cholesterol.

[0516] In some embodiments, the steroid is cholesterol. Preferably, the molar ratio of cationic lipids to cholesterol can be in the range of about 2:1 to about 1:1. In some embodiments, the cholesterol may be pegylated.

[0517] Sterols may constitute about 10 mol% to about 60 mol%, or about 25 mol% to about 55 mol%, or about 25 mol% to about 40 mol% of the lipid particles. In one embodiment, sterols are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol% of the total lipids present in the lipid particles. In another embodiment, the LNP contains sterols in a molar basis of about 5% to about 50%, for example, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5%, or about 31% in a molar basis (based on 100% total moles of lipids in the lipid nanoparticles).

[0518] The cationic lipids of LNPs may be ionizable, meaning they are protonated when the pH drops below the pK of the lipid's ionizable group, but gradually become more neutral at higher pH values. Then, at pH values ​​below the pK, the lipids can associate with negatively charged nucleic acids. In certain embodiments, the cationic lipids include zwitterionic lipids that become positively charged as the pH decreases.

[0519] Such cationic lipids (in the case of liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes) include, but are not limited to, DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP)(N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, and 1,2-dioleyloxypropyl (Also known as c-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane Pan(γ-DLenDMA), 98N12-5, 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3 -Dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), ICE (imidazole base), HGT5000, HGT5001, DMDMA, ClinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC(2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) HGT4003, 1,2-Dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A) 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analog, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-te Traen-19-yl-4-(dimethylamino)butanoate (MC3), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl )amino)ethyl)piperazine-1-yl)ethylazanejyl)didodecane-2-ol(C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), NC98-5(4,7,13-tris(3-oxo-3-(undecylamine (N)propyl)-N,N16-diundecyl-4,7,10,13-tetraazahexadecane-l,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropane-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutane-1-amine (MC4 ether), LIPOFECTIN® (commercial cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (commercial cationic liposomes containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and TRANSFECTAM® (Promega The composition includes commercially available cationic lipids (including dioctadecylamideglycylcarboxyspermine (DOGS) in ethanol from Corp., Madison, Wis.) or any combination thereof. Further suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Application Publication WO2010053572 (and in particular CI 2-200 as described in paragraph

[0225] ) and WO2012170930, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1), all of which are incorporated herein by reference.

[0520] In embodiments, the cationic lipids of liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be aminolipids.

[0521] Representative aminolipids, though not limited to them, include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), and 1,2-dilinoleyloxy This includes -3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120).

[0522] In embodiments, the cationic lipids of liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be amino alcohol lipidoids.

[0523] Amino alcohol lipidoids can be prepared by the method described in U.S. Patent No. 8,450,298, which is incorporated herein by reference in its entirety. Suitable (ionizable) lipids may also be compounds as defined in claims 1 to 24, which are disclosed in Tables 1, 2 and 3 of WO2017075531A1, which is incorporated herein by reference.

[0524] In another embodiment, preferred lipids may also be compounds disclosed in WO2015074085A1 (i.e., ATX-001 to ATX-032 or compounds specified in claims 1 to 26), U.S. Patent Applications No. 61 / 905,724 and 15 / 614,499, or U.S. Patents No. 9,593,077 and 9,567,296, which are incorporated in whole by reference herein.

[0525] In other embodiments, suitable cationic lipids may also be compounds disclosed in WO2017117530A1 (i.e., lipids 13, 14, 15, 16, 17, 18, 19, 20, or compounds specified in claims), which are incorporated in whole by reference herein.

[0526] In some embodiments, ionizable or cationic lipids may also be selected from the lipids disclosed in WO2018078053A1 (i.e., lipids derived from formulas I, II, and III of WO2018078053A1, or lipids specified in claims 1 to 12 of WO2018078053A1), the disclosures of WO2018078053A1 being incorporated in their entirety herein by reference. In that context, the lipids disclosed in Table 7 of WO2018078053A1 (e.g., lipids derived from formulas I-1 to I-41) and the lipids disclosed in Table 8 of WO2018078053A1 (e.g., lipids derived from formulas II-1 to II-36) may be suitably used in the context of the present invention. Accordingly, formulas I-1 to I-41 and II-1 to II-36 of WO2018078053A1, and certain related disclosures, are incorporated herein by reference.

[0527] In some embodiments, the cationic lipid may be derived from Formula III of the published PCT patent application WO2018078053A1. Accordingly, Formula III of WO2018078053A1 and certain related disclosures are incorporated herein by reference.

[0528] In some embodiments, RNA, preferably mRNA, is complexed with one or more lipids to form LNPs (or liposomes, nanoliposomes, lipoplexes), where the cationic lipids of the LNPs are selected from structures III-1 to III-36 in Table 9 of the published PCT patent application WO2018078053A1. Thus, formulas III-1 to III-36 of WO2018078053A1 and certain related disclosures are incorporated herein by reference.

[0529] In some embodiments, the ionizable cationic lipid is given by formula III:

[0530] [ka] (In the formula, L 1 or L 2 These are independently -O(C=O)- or (C=O)O-; G 1 and G 2 These are, independently, unsubstituted C1-C 12 Alkylene or C1-C 12 It is an alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 These are alkenylenes, C3-C8 cycloalkylenes, or C3-C8 cycloalkenylenes; R 1 and R 2 Each of these is independently a branched or linear C6-C 24 Alkyl or C6-C 24 It is Alkenil; R 3 H, OR 5 , CN, -C(=O)OR 4 -OC(=O)R 4 or NR 5 C(=O)R 4 is; R 4 is C1-C 12 It is alkyl; R5 (It is H or C1-C6 alkyl) A compound having [the specified characteristic] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0531] In some embodiments, the ionizable cationic lipid is given by formula III:

[0532] [ka] (In the formula, L 1 or L 2 These are independently -O(C=O)- or (C=O)O-; G 1 and G 2 These are, independently, unsubstituted C1-C 12 It is alkylene; G 3 is C1-C 24 It is alkylene; R 1 and R 2 Each of these is independently a branched or linear C6-C 24 It is alkyl; R 3 is OR 5 is; and R 5 (is H) A compound having [the specified characteristic] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0533] In some embodiments, the ionizable cationic lipid has formula III, where R 1 , R 2 or R 1 and R 2 Both have the following structure:

[0534] [ka] Take one of them.

[0535] In some embodiments, R2 The structure is as follows:

[0536] [ka] It has.

[0537] In some embodiments, cationic lipids are expressed by formula:

[0538] [ka] It has.

[0539] In some embodiments, the ionizable cationic lipid is given by formula:

[0540] [ka] It has TIFF2026518411000016.tif116161.

[0541] In some embodiments, the ionizable cationic lipid is given by formula III-3:

[0542] [ka] It has.

[0543] The lipid of formula III-3 as appropriately used herein has the chemical name ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) and is also known as ALC-0315, or CAS number 2036272-55-4.

[0544] In certain embodiments, the cationic lipids defined herein, more preferably the cationic lipid compound III-3((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), are present in the LNP in an amount of about 30 mol% to about 80 mol%, preferably about 30 mol% to about 60 mol%, more preferably about 40 mol% to about 55 mol%, and more preferably about 47.4 mol%, relative to the total lipid content of the LNP. If the LNP contains two or more cationic lipids, such proportions apply to the combined cationic lipids.

[0545] In some embodiments, cationic lipids as defined herein are present in the LNP in an amount of about 20 mol% to about 60 mol%.

[0546] In some embodiments, the LNP has the following structure:

[0547] [ka] Contains cationic lipids.

[0548] In one embodiment, cationic lipids are present in the LNP in amounts of about 30 mol% to about 70 mol%. In another embodiment, cationic lipids are present in the LNP in amounts of about 40 mol% to about 60 mol%, for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%, respectively. In yet another embodiment, cationic lipids are present in the LNP in amounts of about 47 mol% to about 48 mol%, for example, about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 50.0 mol%, where 47.4 mol% is particularly preferred.

[0549] In some embodiments, cationic lipids are present in a ratio of about 20 mol% to about 70 mol% or 75 mol% of the total lipids present in the LNP, or about 45 mol% to about 65 mol%, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol%. In further embodiments, the LNP contains about 25% to about 75% on a molar basis, for example, about 20% to about 70%, about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 57.1%, about 50%, or about 40% on a molar basis (based on 100% total moles of lipids in the lipid nanoparticles) of cationic lipids. In some embodiments, the ratio of cationic lipids to nucleic acids, preferably RNA, more preferably mRNA, is about 3% to about 15%, for example, about 5% to about 13% or about 7% to about 11%.

[0550] Other suitable (cationic or ionizable) lipids include WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO2013063468, US20110256175, US20120128760, US2 0120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017070620, WO2017 099823, WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO201102 2460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 Disclosed in , and Patent No. 8,569,256, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, US20140039032, and WO2017112865.In that context, the following (cationic) lipids are particularly suitable for LNPs (or liposomes, nanoliposomes, lipoplexes): WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO2013063468, US2011025617 5, US20120128760, US20120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017 070620, WO2017099823, WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, W O2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122, and 8 The disclosures of Patent No. 569,256, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, and US20140039032, as well as WO2017112865, are incorporated herein by reference.

[0551] In other embodiments, cationic or ionizable lipids are

[0552] [ka] The filename is TIFF2026518411000020.tif233161.

[0553] In embodiments, amino or cationic lipids as defined herein have at least one protonable or deprotonable group such that the lipid is positively charged at a pH below the physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above the physiological pH. It will be understood, of course, that the addition or removal of protons as a function of pH is an equilibration process, and that references to charged or neutral lipids refer to the properties of the dominant species and do not require that all lipids exist in charged or neutral forms. Lipids having two or more protonable or deprotonable groups, or being zwitterionic, are not excluded and may be equally suitable in the context of the present invention. In some embodiments, protonable lipids have a pKa of protonable groups in the range of about 4 to about 11, for example, about 5 to about 7.

[0554] LNPs (or liposomes, nanoliposomes, lipoplexes) may contain two or more (different) cationic lipids as defined herein. The cationic lipids may be selected to contribute to different advantageous properties. For example, cationic lipids with different properties, such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity, can be used in LNPs (or liposomes, nanoliposomes, lipoplexes). In particular, cationic lipids can be selected such that the properties of the mixed LNP are more desirable than the properties of a single LNP of the individual lipids.

[0555] The amount of constitutive cationic lipids or lipidoids can be selected considering the amount of nucleic acid cargo. In one embodiment, these amounts are, for example, such as resulting in an N / P ratio of nanoparticles or composition in the range of about 0.1 to about 20, or (i) an amount that achieves an N / P ratio in the range of approximately 1 to approximately 20, preferably approximately 2 to approximately 15, more preferably approximately 3 to approximately 10, even more preferably approximately 4 to approximately 9, and most preferably approximately 6. (ii) an amount that achieves an N / P ratio in the range of approximately 5 to approximately 20, more preferably approximately 10 to approximately 18, even more preferably approximately 12 to approximately 16, and most preferably approximately 14. (iii) an amount that achieves a lipid:mRNA weight ratio in the range of 20 to 60, preferably about 3 to about 15, 5 to about 13, about 4 to about 8, or about 7 to about 11, or (iv) With respect to lipid nanoparticles according to the present invention, particularly lipid nanoparticles containing cationic lipid III-3, in an amount that achieves an N / P ratio in the range of about 6, Selected.

[0556] In this context, the N / P ratio is defined as the molar ratio of nitrogen atoms ("N") in the basic nitrogen-containing groups of a lipid or lipidoid to phosphate groups ("P") of the nucleic acid used as cargo. The N / P ratio can be calculated, for example, based on the fact that 1 μg of RNA typically contains about 3 nmol of phosphate residues, although RNA exhibits a statistical distribution of bases. The "N" value of a cationic lipid or lipidoid can be calculated based on its molecular weight and the relative content of constitutively cationic and, if present, cationizable groups. If two or more cationic lipids are present, the N value should be calculated based on all cationic lipids contained in the lipid nanoparticles.

[0557] In some embodiments, the composition has a lipid-to-RNA molar ratio (N / P ratio) of about 2 to about 12, and optionally an N / P ratio of 3 to about 8.

[0558] In one embodiment, the lipid nanoparticles contain approximately 40% cationic lipid LKY750, approximately 10% zwitterionic lipid DSPC, approximately 48% cholesterol, and approximately 2% pegylated lipid DMG (w / w).

[0559] In some embodiments, the LNP comprises (a) RNA as used herein, preferably mRNA, (b) a cationic lipid, (c) an anti-aggregation agent (e.g., polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (e.g., a neutral lipid), and (e) optionally a sterol.

[0560] In some embodiments, cationic lipids (as defined above), non-cationic lipids (as defined above), cholesterol (as defined above), and / or PEG-modified lipids (as defined above) may be combined in various relative molar ratios. For example, the ratio of cationic lipids, non-cationic lipids, cholesterol-based lipids, and pegylated lipids may be between approximately 30-60:20-35:20-30:1-15, or between approximately 40:30:25:5, 50:25:20:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:32:25:3, or 40:33:25:2, or between approximately 50:25:20:5, 50:20:25:5, 50:27:20:3, 40:30:20:10, 40:30:25:5, or between 40:32:20:8, 40:32:25:3, or 40:33:25:2.

[0561] In some embodiments, the LNP (or liposome, nanoliposome, lipoplex) comprises ALC-0315, RNA as used herein, preferably mRNA, a neutral lipid which is DSPC, a steroid which is cholesterol, and a pegylated lipid which is formula ALC-0159.

[0562] In some embodiments, the LNP comprises about 0.5 to 15 mol% of PEG-modified lipids, about 5 to 25 mol% of noncationic lipids, about 25 to 55 mol% of sterols, and about 20 to 60 mol% of ionizable cationic lipids.

[0563] In one embodiment, the LNP essentially consists of (i) at least one cationic lipid, (ii) a neutral lipid, (iii) a sterol, e.g., cholesterol, and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of approximately 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.

[0564] In some embodiments, RNA, preferably mRNA, is complexed with one or more lipids to form lipid nanoparticles (LNPs), where the LNPs are I. At least one cationic lipid as defined herein, preferably a lipid of formula III-3 (ALC-0315), II. At least one neutral lipid as defined herein, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), III. At least one steroid or steroid analog as defined herein, preferably cholesterol, and IV. At least one polymer-conjugated lipid as defined herein, preferably a PEG-lipid, such as PEG-DMG or PEG-cDMA, preferably a PEG-conjugated lipid of formula (IVa-ALC-0159) or derived therefrom. Includes.

[0565] In some embodiments, mRNA is complexed with one or more lipids to form lipid nanoparticles (LNPs), where the LNPs contain approximately 20-60% cationic lipids, 5-25% neutral lipids, 25-55% sterols, and 0.5-15% polymer-conjugated lipids, preferably in the molar ratio of PEG-lipids, (i)-(iv).

[0566] In some embodiments, the lipid nanoparticles (or liposomes, nanoliposomes, lipoplexes) include cationic lipids having formula (III-3) and / or PEG lipids having formula (IVa), optionally neutral lipids, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and optionally steroids, preferably cholesterol, where the molar ratio of cationic lipids to DSPC is optionally in the range of about 2:1 to 8:1, and where the molar ratio of cationic lipids to cholesterol is optionally in the range of about 2:1 to 1:1.

[0567] In one embodiment, the composition comprises lipid nanoparticles (LNPs) having a molar ratio of RNA, preferably mRNA, about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or higher, more preferably 47.4:10:40.9:1.7 (i.e., cationic lipids (preferably lipids of formula III-3 (ALC-0315)), DSPC, cholesterol, and polymer-conjugated lipids, preferably PEG-lipids (preferably PEG-lipids of formula (IVa) having n=49, more preferably PEG-lipids of formula (IVa) having n=45, ALC-0159) (mol%); soluble in ethanol).

[0568] Other useful LNPs are described in the following references: WO2012 / 006376, WO2012 / 030901, WO2012 / 031046, WO2012 / 031043, WO2012 / 006378, WO2011 / 076807, WO2013 / 033563, WO2013 / 006825, WO2014 / 136086, WO2015 / 095340, WO2015 / 095346, and WO2016 / 037053, which are also incorporated herein by reference.

[0569] Suitably, the LNPs are about 50nm to about 200nm, about 60nm to about 200nm, about 70nm to about 200nm, about 80nm to about 200nm, about 90nm to about 200nm, about 90nm to about 190nm, about 90nm to about 180nm, about 90nm m ~ about 170nm, about 90nm - about 160nm, about 90nm - about 150nm, about 90nm - about 140nm, about 90nm - about 130nm, about 90nm - about 120nm, about 90nm - about 100nm, about 70nm - about 90nm, about 80nm They have an average diameter of approximately 90 nm, approximately 70 nm to approximately 80 nm, or approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, and are substantially non-toxic. The average diameter used herein may be expressed by the z-average size determined by dynamic light scattering, which is generally known in the art.

[0570] Preferably, the LNP has a polydispersity of 0.4 or less, for example, 0.3 or less. Typically, the PDI is determined by dynamic light scattering.

[0571] In some embodiments, the composition has a polyvariance index (PDI) value of less than about 0.4, preferably less than about 0.3, more preferably less than about 0.2, and most preferably less than about 0.1.

[0572] Appropriately, at least 50%, and more appropriately, at least 60%, 70%, 80%, 85%, 90%, or 95% of the RNA is encapsulated within the LNP. In this context, “encapsulated RNA” is understood as RNA (appropriately mRNA) that forms a complex with the lipids that make up the LNP and / or is contained within the internal space of the LNP. The percentage of encapsulated RNA can typically be measured using the RiboGreeN assay.

[0573] The composition appropriately contains less than approximately 30%, and appropriately less than 20%, less than 15%, less than 10%, or less than 5% of unencapsulated RNA (or free RNA). In this context, the terms “free RNA” or “unencapsulated RNA” are understood to mean RNA (appropriately mRNA) that is not encapsulated in an LNP as defined herein. In therapeutic compositions, free RNA may correspond to contaminants or impurities.

[0574] Furthermore, in this specification, (a) The first nucleic acid encoding the hemagglutinin (HA) antigen of the first subtype strain of influenza A virus, (b) The second nucleic acid encoding the HA antigen of the second subtype strain of influenza A virus, (c) The third nucleic acid encoding the HA antigen of the first strain of influenza B virus, and (d) A fourth nucleic acid encoding the HA antigen of a second strain of influenza B virus, which may be included as desired. The present invention provides an immunogenic composition containing the following:

[0575] In some embodiments, the immunogenic composition further comprises (d).

[0576] In some embodiments, the first subtype of the influenza A virus is a subtype of influenza A group 1, which is appropriately influenza A subtype H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18, and more appropriately H1.

[0577] In some embodiments, the aforementioned first subtype of influenza A virus is the influenza A H1N1 subtype.

[0578] In some embodiments, the second subtype of the influenza A virus is a subtype of influenza A group 2, preferably influenza A subtypes H3, H4, H7, H10, H14, and H15, and more preferably H3.

[0579] In some embodiments, the aforementioned second strain subtype of influenza A virus is the influenza A H3N2 subtype.

[0580] In some embodiments, the aforementioned first strain of influenza B is a strain of the B / Victoria lineage.

[0581] In some embodiments, the second strain of influenza B is a strain of the B / Yamagata lineage.

[0582] In some embodiments, the first, second, third, and / or fourth nucleic acids are messenger ribonucleic acid (mRNA).

[0583] In some embodiments, the immunogenic composition is further: (e) at least one further nucleic acid, preferably mRNA, that codes for at least one further antigen. The antigen comprises, wherein the at least one further antigen is derived from a strain of influenza virus, and is preferably selected from the group consisting of influenza A virus and influenza B virus, and more preferably selected from the group consisting of the strain of the first subtype of influenza A virus, the strain of the second subtype of influenza A virus, the first strain of influenza B virus, and the second strain of influenza B virus, which may optionally be included.

[0584] In some embodiments, the at least one further antigen comprises, or consists of, peptides or proteins selected from or derived from influenza virus NA or its immunogenic fragments or immunogenic variants.

[0585] In some embodiments, the composition comprises a plurality of (e).

[0586] In some embodiments, the immunogenic composition is further: (e 1 ) The fifth nucleic acid, appropriately mRNA, that codes for the NA of the first subtype of influenza A virus. (e 2 ) The sixth nucleic acid, appropriately mRNA, that codes for the NA of the aforementioned second subtype of influenza A virus, (e 3 ) The seventh nucleic acid encoding the NA of the first strain of influenza B virus, preferably mRNA, and may optionally be included, (e 4 ) The eighth nucleic acid encoding the NA of the second strain of influenza B virus, properly known as mRNA Includes.

[0587] In some embodiments, (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The mRNAs of each molecule are incorporated into lipid nanoparticles (LNPs), either separately or together.

[0588] In some embodiments, the LNP appropriately comprises about 0.5 to 15 mol% of PEG-modified lipids, appropriately about 0.5 to 25 mol% of non-cationic lipids, appropriately about 25 to 55 mol% of sterols, and cationic lipids, appropriately about 20 to 60 mol% of ionizable cationic lipids.

[0589] In some embodiments, (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The mRNAs of these organisms are, if desired, not self-replicating.

[0590] In some embodiments, (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4The mRNA of ) includes a 5' untranslated region (UTR), which, appropriately, contains or consists of nucleic acid sequences derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or a homolog, fragment, or mutant of any one of these genes.

[0591] In some embodiments, (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The mRNA of ) includes a 3'UTR, and appropriately, the 3'UTR contains, or consists of, a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a homolog, fragment, or mutant of any one of these genes.

[0592] In some embodiments, (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 The mRNA of ) contains at least one chemical modification, which is preferably N1-methylpseudridine and / or pseudouridine, and preferably N1-methylpseudridine.

[0593] Vaccines and combination vaccines In a second embodiment, the present invention relates to a vaccine for use in the treatment or prevention of infection by influenza virus, comprising an immunogenic composition as defined herein, wherein the immune response is induced as defined herein.

[0594] The vaccine is a nucleic acid-based vaccine, more precisely, an mRNA-based vaccine.

[0595] In some embodiments, the vaccine is a polyvalent vaccine.

[0596] In some embodiments, the vaccine is a trivalent influenza virus vaccine (i.e., one containing immunogenic components derived from three strains of the influenza virus).

[0597] In some embodiments, the trivalent influenza virus vaccine comprises three different nucleic acids, appropriately mRNAs, that encode three HA antigens.

[0598] In some embodiments, the trivalent influenza virus vaccine comprises two distinct nucleic acids, appropriately mRNA, encoding two HA antigens derived from a strain of influenza A virus, preferably H1N1 and / or H3N2, and one nucleic acid, appropriately mRNA, encoding one HA antigen derived from a strain of influenza B virus, preferably the B / Victoria lineage.

[0599] In some embodiments, the trivalent influenza virus vaccine comprises six different nucleic acids, preferably mRNA, encoding three HA antigens and three NA antigens.

[0600] In some embodiments, the trivalent influenza virus vaccine comprises four different nucleic acids, appropriately mRNA, encoding two HA antigens and two NA antigens derived from a strain of influenza A virus, preferably H1N1 and / or H3N2, and two different nucleic acids, appropriately mRNA, encoding one HA antigen and one NA antigen derived from a strain of influenza B virus, preferably B / Victoria lineage.

[0601] In some embodiments, the trivalent influenza virus vaccine comprises (a), (b), and (c) as defined herein.

[0602] In some embodiments, the trivalent influenza virus vaccine is (a), (b), (c), (e) as defined herein. 1 ), (e 2 ) and (e 3 ) includes.

[0603] In some embodiments, the vaccine is a quadrivalent influenza virus vaccine (i.e., one containing immunogenic components derived from four strains of the influenza virus).

[0604] In some embodiments, the quadrivalent influenza virus vaccine contains four mRNAs encoding four HA antigens.

[0605] In some embodiments, the quadrivalent influenza virus vaccine is (a) The first mRNA encoding the hemagglutinin (HA) antigen of the first subtype strain of influenza A virus, (b) Second mRNA encoding the HA antigen of a second subtype strain of influenza A virus, (c) Third mRNA encoding the HA antigen of the first strain of influenza B virus, and (d) The fourth mRNA encoding the HA antigen of the second strain of influenza B virus. Includes.

[0606] In some embodiments, the quadrivalent influenza virus vaccine comprises four mRNAs encoding four HA antigens and three mRNAs encoding three NA antigens (i.e., a seven-component quadrivalent influenza virus vaccine).

[0607] In some embodiments, the quadrivalent influenza virus vaccine is further, (e 1 ) The fifth mRNA encoding the NA of the first subtype of influenza A virus, (e 2) The sixth mRNA encoding the NA of the second subtype of influenza A virus, and (e 3 ) The seventh mRNA encoding the NA of the first strain of influenza B virus Includes.

[0608] In some embodiments, the quadrivalent influenza virus vaccine comprises four mRNAs encoding four HA antigens and four mRNAs encoding four NA antigens (i.e., an eight-component quadrivalent influenza virus vaccine).

[0609] In some embodiments, the quadrivalent influenza virus vaccine is further, (e 4 ) The eighth mRNA encoding the NA of the second strain of influenza B virus Includes.

[0610] In some embodiments, the vaccine further comprises at least one antigen or at least one nucleic acid encoding the said at least one antigen, for example, at least one mRNA encoding an antigen derived from an additional pathogen, preferably a virus, preferably a respiratory virus.

[0611] In some embodiments, the antigen is derived from an additional virus, which is selected from the group consisting of coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), Pneumoviridae viruses (e.g., respiratory syncytial virus, metapneumovirus), and Paramyxoviridae viruses (e.g., parainfluenza virus, henipavirus), and preferably, the antigen derived from the additional virus is a spike protein or antigenic fragment thereof derived from the SARS-CoV-2 virus, or mRNA encoding a spike protein or antigenic fragment thereof derived from the SARS-CoV-2 virus. For example, the antigen may be a SARS-CoV-2 virus spike protein or antigenic fragment thereof selected from those listed in Table 1 of published PCT application WO2021156267A1 or Table 1 of published PCT application WO2022137133A1 (each of which is incorporated herein by reference).

[0612] This specification also provides vaccines comprising immunogenic compositions as defined herein.

[0613] Kit or parts kit In a third embodiment, the present invention relates to a kit or kit of parts for use in the treatment or prevention of infection by influenza virus, wherein the kit or kit of parts comprises nucleic acids, preferably mRNA as defined herein, preferably (a), (b), (c), (d), (e) as defined herein. 1 ), (e 2 ), (e 3 ), and / or (e 4 The product contains mRNA of ) and may optionally include a liquid vehicle for solubilization, and may optionally include a technical description providing information on the administration and dosage of the immune response-inducing component as defined herein.

[0614] The technical description of the kit may include information regarding administration and dosage and patient groups. Such a kit, preferably a parts kit, is applicable, for example, to any of the uses or applications described herein, and is preferably applicable to the use of immunogenic compositions or vaccines for the treatment or prevention of infection or disease caused by influenza virus, preferably influenza A and / or B virus.

[0615] In some embodiments, the immunogenic composition or vaccine is provided in a separate part of a kit, where the immunogenic composition or vaccine is preferably freeze-dried, spray-dried, or spray-freeze-dried.

[0616] The kit may further contain, as part of a vehicle (e.g., a buffer solution) for solubilizing a dried or freeze-dried nucleic acid composition or vaccine.

[0617] In some embodiments, nucleic acids and / or mRNA as defined herein, preferably (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is formulated separately.

[0618] In some embodiments, nucleic acids and / or mRNA as defined herein, preferably (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA for ) is provided as part of the kit.

[0619] In some embodiments, nucleic acids and / or mRNA as defined herein, preferably (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ), and / or (e 4Each of the mRNAs of (a), (b), (c), (d), (e) is provided as an individual part of the kit. Preferably, the kit or parts kit comprises at least two, at least three, at least four, at least five, at least six, at least seven, and at least eight parts, each comprising at least one nucleic acid and / or mRNA as defined herein, preferably (a), (b), (c), (d), (e) 1 ), (e 2 ), (e 3 ), and / or (e 4 It contains at least one mRNA of ).

[0620] In some embodiments, a kit or parts kit as defined herein includes multiple-dose (multiple-dose) containers and / or administration devices (e.g., syringes for intramuscular and / or intradermal injection) for administering a composition / vaccine.

[0621] In this specification, nucleic acids as defined herein, preferably mRNA, preferably (a), (b), (c), (d), (e) as defined herein. 1 ), (e 2 ), (e 3 ), and / or (e 4 Kits or parts kits are also provided that contain mRNA of ), optionally include a liquid vehicle for solubilization, and optionally include technical instructions providing information on the administration and dosage of components that induce an immune response as defined herein.

[0622] Formulation and administration In some embodiments, nucleic acids as defined herein, preferably mRNA, are co-formulated. Preferably, (a), (b), (c), (d), (e) as defined herein. 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is co-formulated, that is, formulated together.

[0623] In some embodiments, the nucleic acids, preferably mRNA, as defined herein, are formulated separately in a kit or parts kit. In some embodiments, (a), (b), (c), (d), (e) as defined herein 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is formulated separately.

[0624] In some embodiments, the antigen or nucleic acid, preferably mRNA, as defined herein, is co-packed. Preferably, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The mRNAs of the ) are co-packed, that is, packed together, and in some cases, packed together after being formulated separately.

[0625] In some embodiments, nucleic acids as defined herein, preferably mRNA, are formulated as bedside mixed formulations. Preferably mRNA as defined herein, preferably (a), (b), (c), (d), (e), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 The mRNA of ) is formulated as a bedside mixed formulation.

[0626] The “bedside mixed formulations” as described herein should be understood as formulations in which several (e.g., one or more) immunogenic components (e.g., mRNA) are preferably each formulated independently (e.g., in LNPs) before being mixed to produce the bedside mixed formulation.

[0627] In some embodiments, a bedside mixed formulation is obtained by a method comprising (1) independently formulation each nucleic acid, preferably mRNA, (for example, in LNPs), and (2) mixing the respective (in LNP) formulated nucleic acids, preferably mRNA.

[0628] In some embodiments, the bedside compound formulation is obtained by a method comprising: (1) co-formulating (e.g., in an LNP) the nucleic acid, preferably mRNA, encoding a strain of influenza A virus; (2) co-formulating (e.g., in an LNP) the nucleic acid, preferably mRNA, encoding a strain of influenza B virus; and (3) mixing (in an LNP) the co-formulated (in an LNP) nucleic acid, preferably mRNA, encoding a strain of influenza A virus with (in an LNP) the co-formulated (in an LNP) nucleic acid, preferably mRNA, encoding a strain of influenza B virus.

[0629] In some embodiments, the bedside compound formulation is obtained by a method comprising: (1) co-formulating the nucleic acid, preferably mRNA, encoding a strain of influenza A virus (for example, in an LNP); (2) independently formulating each nucleic acid, preferably mRNA, encoding a strain of influenza B virus; and (3) mixing the co-formulated nucleic acid, preferably mRNA, encoding a strain of influenza A virus (in an LNP) with each formulated nucleic acid, preferably mRNA, encoding a strain of influenza B virus (in an LNP).

[0630] Immunogenic compositions can be administered by various suitable routes, including parenteral administration, such as intramuscular, intradermal, intranasal, or subcutaneous administration. Preferably, the immunogenic compositions, vaccines, or kits or parts kits described herein are administered intramuscularly and / or intradermally.

[0631] In some embodiments, intramuscular administration of the immunogenic compositions described herein results in the expression of an encoding antigen construct in a subject. Administration of the immunogenic compositions described herein results in mRNA translation and the production of an encoding antigen in a subject.

[0632] The immunogenic compositions described herein may be provided in liquid or dry (e.g., lyophilized) form.

[0633] In some embodiments, the immunogenic composition is provided in liquid form.

[0634] In some embodiments, the immunogenic composition is a dry composition.

[0635] As used herein, the term "dried composition" may refer to a freeze-dried composition (e.g., a composition according to WO2016165831, WO2011069586, WO2022 / 232585, WO2022 / 101461, WO2022 / 076562, WO2012 / 170889, or WO2022 / 036170), or a dried composition in powder form, preferably a spray-dried or freeze-dried composition for obtaining a temperature-stable composition (e.g., a composition according to WO2016184575, WO2016184576, or WO2021 / 216541).

[0636] In one embodiment, the composition according to the present invention is a lyophilized, freeze-dried, or spray-dried dry composition comprising one or more further excipients selected from cryoprotectants, plasticizers, and polymers. Preferably, before administration to a patient, the lyophilized, freeze-dried, or spray-dried dry composition is mixed with a liquid, preferably an aqueous liquid such as sterile water or physiological saline, to form a "reconstituted liquid formulation."

[0637] In this embodiment, the freeze-dried or spray-dried composition has a moisture content of less than about 10%.

[0638] In some embodiments, the lyophilized, freeze-dried, or spray-dried composition has a moisture content of approximately 0.5% to 5%.

[0639] In some embodiments, the lyophilized, freeze-dried, or spray-dried composition is stable for at least two months, preferably at least three, four, five, or six months, after storage at approximately 5°C.

[0640] The liquid used for reconstitution (reduction) is substantially aqueous, such as sterile water for injection or phosphate-buffered saline. The need for buffers and / or tonic modifiers depends on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. Buffers can be selected from acetates, citrates, histidines, maleates, phosphates, succinates, tartrates, and TRIS. The buffer may be a phosphate buffer, such as Na / Na2PO4, Na / K2PO4, or K / K2PO4.

[0641] Preferably, the formulations used in the present invention have a dose volume of 0.05 ml to 1 ml, for example 0.1 ml to 0.6 ml, and especially 0.45 ml to 0.55 ml, for example 0.5 ml. The volume of the composition used may vary depending on the target, route of administration, and site. Smaller doses may be administered via the intradermal route. Typical human doses for administration via routes such as the intramuscular route are in the range of 200 μl to 750 μl, for example 400 μl to 600 μl, and especially about 500 μl, for example 500 μl.

[0642] The immunogenic compositions described herein may be provided in various physical containers, such as vials or pre-filled syringes.

[0643] In some embodiments, the immunogenic composition is provided in a single-dose form. In other embodiments, the immunogenic composition, vaccine, or kit or parts kit is provided in a multiple-dose form containing, for example, 2, 5, or 10 doses.

[0644] When transferring liquids between containers (for example, from a vial to a syringe), it is common practice to include a "surplus" to ensure that the entire required volume can be easily transferred. The required level of surplus varies depending on the situation, but excessive surplus should be avoided to reduce waste, while insufficient surplus can cause practical problems. The surplus may be around 20-100 μl per dose, for example, 30 μl or 50 μl.

[0645] A stabilizer may be present. Stabilizers can be particularly important when multiple doses are provided, because the final dosage of the formulation may be administered to the subject over a period of time.

[0646] The formulation is preferably sterile.

[0647] Approaches to establishing robust and sustained immunity often involve repeated immunization, i.e., boosting the immune response by administering one or more additional doses.

[0648] Therefore, the administration of immunogenic compositions described herein may be part of a multi-dose regimen. For example, the immunogenic compositions described herein may be provided as a priming dose in a multi-dose regimen, particularly a two-dose or three-dose regimen, especially a two-dose regimen. The immunogenic compositions described herein may be provided as a boost dose in a multi-dose regimen, particularly a two-dose or three-dose regimen, for example, a two-dose regimen.

[0649] The interval between multiple doses may be 2 weeks to 6 months, for example, 3 weeks to 3 months. Regular, longer-term (e.g., every 2 to 10 years) additional (booster) doses may also be administered.

[0650] In some embodiments, the immunogenic composition further comprises at least one pharmaceutically acceptable carrier.

[0651] As used herein, the terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” preferably...

Claims

1. An immunogenic composition for use in the treatment or prevention of infection by influenza virus, wherein the immunogenic composition is (a) The first nucleic acid encoding the hemagglutinin (HA) antigen of the first subtype strain of influenza A virus, (b) The second nucleic acid encoding the HA antigen of the second subtype strain of influenza A virus, (c) The third nucleic acid encoding the HA antigen of the first strain of influenza B virus, and (d) A fourth nucleic acid encoding the HA antigen of a second strain of influenza B virus, which may be included as desired. The immunogenic composition comprising, wherein the immune response is induced against the aforementioned strains of the first and second subtypes of influenza A virus, the aforementioned first strain and optionally a second strain of influenza B virus, and the HA antigen of at least one further HA antigen subtype of influenza A virus (which is different from any of the HA antigen subtypes of influenza A virus encoded by the nucleic acids present in the composition).

2. The immunogenic composition for use according to claim 1, wherein the composition further comprises (d) the fourth nucleic acid encoding the HA antigen of a second strain of influenza B virus, and the immune response is further induced against the HA antigen of the second strain of influenza B virus.

3. An immunogenic composition for use according to claim 1 or 2, wherein the first subtype of influenza A virus is a subtype of influenza A group 1, preferably influenza A subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18, more preferably H1.

4. An immunogenic composition for use according to any one of claims 1 to 3, wherein the first subtype of influenza A virus is the H1N1 subtype of influenza A virus.

5. An immunogenic composition for use according to any one of claims 1 to 4, wherein the second subtype of influenza A virus is a subtype of influenza A group 2, preferably influenza A subtypes H3, H4, H7, H10, H14 and H15, more preferably H3.

6. An immunogenic composition for use according to any one of claims 1 to 5, wherein the second strain subtype of influenza A virus is influenza A H3N2 subtype.

7. An immunogenic composition for use according to any one of claims 1 to 6, wherein the first strain of influenza type B is a strain of the B / Victoria lineage.

8. An immunogenic composition for use according to any one of claims 2 to 7, wherein the second strain of influenza B is a strain of the B / Yamagata lineage.

9. An immunogenic composition for use according to any one of claims 1 to 8, wherein the induced immune response is homogeneous, hetero-subtype, and optionally heterogeneous or intra-subtype.

10. An immunogenic composition for use according to any one of claims 1 to 9, wherein the aforementioned at least one further HA antigen subtype of influenza A virus is from influenza A group 1, appropriately influenza A subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18, more appropriately H1, H2, or H5.

11. An immunogenic composition for use according to any one of claims 1 to 10, wherein the aforementioned at least one further HA antigen subtype of influenza A virus is derived from a strain of influenza A group 2, and is appropriately influenza A subtype H3, H4, H7, H10, H14, or H15, more appropriately H3, H7, or H10.

12. An immunogenic composition for use according to any one of claims 1 to 11, wherein an immune response is induced against at least one, appropriately all, of the HA antigens of influenza A subtypes H1, H3, and influenza A subtypes H2, H5, H7, or H10.

13. An immunogenic composition for use according to any one of claims 1 to 12, wherein the immune response is further induced to at least one further HA antigen of an influenza B virus strain, which is different from any of the HA antigens of the influenza B virus strain encoded by the nucleic acid present in the composition.

14. The immunogenic composition for use according to claim 13, wherein the at least one further HA antigen of the influenza B virus strain is derived from a strain selected from the group consisting of B / Victoria and B / Yamagata lineages.

15. An immunogenic composition for use according to any one of claims 1 to 14, wherein the first, second, third, and / or fourth nucleic acid is messenger ribonucleic acid (mRNA).

16. An immunogenic composition for use according to any one of claims 1 to 15, further comprising (e) at least one further nucleic acid, preferably mRNA, encoding at least one further antigen, wherein the at least one further antigen is derived from a strain of influenza virus, preferably selected from the group consisting of influenza A virus and influenza B virus, and more preferably selected from the group consisting of the strain of the first subtype of influenza A virus, the strain of the second subtype of influenza A virus, the first strain of influenza B virus, and optionally comprising the second strain of influenza B virus.

17. The immunogenic composition for use according to claim 16, wherein the at least one further antigen comprises, or consists of, a peptide or protein selected from or derived from influenza virus NA or its immunogenic fragment or immunogenic variant.

18. An immunogenic composition for use according to claim 16 or 17, wherein the composition comprises a plurality of (e).

19. (e 1 ) The fifth nucleic acid, appropriately mRNA, that codes for the NA of the first subtype of influenza A virus. (e 2 ) The sixth nucleic acid, appropriately mRNA, that codes for the NA of the second subtype of influenza A virus. (e 3 ) The seventh nucleic acid encoding the NA of the first strain of influenza B virus, preferably mRNA, and may optionally be included, (e 4 ) The eighth nucleic acid, appropriately mRNA, that codes for the NA of the second strain of influenza B virus. An immunogenic composition for use according to any one of claims 16 to 18, further comprising:

20. An immunogenic composition for use according to any one of claims 1 to 19, wherein the immune response is further induced to the NA antigens of the strains of the first and second subtypes of influenza A virus, the first strain and optionally a second strain of influenza B virus, and optionally at least one further NA antigen (different from any of the NA antigens encoded by nucleic acids present in the composition) of the strains of influenza A virus and / or influenza B virus.

21. (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 15 to 20, wherein the mRNAs of ) are each incorporated separately or together in lipid nanoparticles (LNPs).

22. The immunogenic composition for use according to claim 21, wherein the LNP comprises PEG-modified lipids, preferably about 0.5 to 15 mol% of PEG-modified lipids, non-cationic lipids, preferably about 5 to 25 mol% of non-cationic lipids, sterols, preferably about 25 to 55 mol% of sterols, and cationic lipids, preferably about 20 to 60 mol% of ionizable cationic lipids.

23. (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ), and / or (e 4 ), the mRNA of which does not self-replicate, if desired, respectively, an immunogenic composition for use according to any one of claims 15 to 22.

24. (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 15 to 23, wherein the mRNA of ) comprises a 5' untranslated region (UTR), and preferably the 5'UTR comprises a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or a nucleic acid sequence derived from a homolog, fragment, or variant of any one of these genes.

25. (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 15 to 24, wherein the mRNA of ) comprises a 3'UTR, and preferably the 3'UTR comprises a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a nucleic acid sequence derived from a homolog, fragment, or variant of any one of these genes.

26. (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 15 to 25, wherein the mRNA of ) comprises at least one chemical modification, preferably the chemical modification being N1-methylpseudridine and / or pseudouridine, preferably N1-methylpseudridine.

27. A vaccine for use in the treatment or prevention of infection by influenza virus, comprising an immunogenic composition according to any one of claims 1 to 26, wherein the immune response is induced as described in any one of claims 1 to 26.

28. The vaccine for use according to claim 27, wherein the vaccine further comprises at least one antigen, or at least one nucleic acid encoding the said at least one antigen, for example, at least one mRNA encoding an antigen from a further pathogen, wherein the pathogen is preferably a virus, for example, a coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), a Pneumoviridae virus (e.g., respiratory syncytial virus, metapneumovirus), and / or a Paramyxoviridae virus (e.g., parainfluenza virus, henipavirus).

29. A kit or parts kit for use in the treatment or prevention of infection by influenza virus, wherein the kit or parts kit comprises a nucleic acid, preferably mRNA, as described in any one of claims 1 to 26, preferably (a), (b), (c), (d), (e) as described in any one of claims 15 to 26. 1 ), (e 2 ), (e 3 ) and / or (e 4 The kit or parts kit comprises mRNA of ), optionally comprising a liquid vehicle for solubilization, optionally comprising a technical description providing information regarding the administration and dosage of the components, wherein an immune response is induced as described in any one of claims 1 to 26.

30. Nucleic acid or mRNA, appropriately (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 A kit or parts kit for use according to claim 29, wherein the mRNAs of ) are formulated separately.

31. Nucleic acid or mRNA, appropriately (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 1 to 26, or a vaccine for use according to any one of claims 27 to 28, or a kit or parts kit for use according to claim 29 or 30, wherein the mRNA of ) is formulated as a bedside mixed formulation.

32. Antigen or nucleic acid or mRNA, appropriately (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 1 to 26, or a vaccine for use according to any one of claims 27 to 28, or a kit or parts kit for use according to claim 29 or 30, comprising co-combination of mRNA of ).

33. (a), (b), (c), (d), (e 1 ), (e 2 ), (e 3 ) and / or (e 4 An immunogenic composition for use according to any one of claims 1 to 26, or a vaccine for use according to any one of claims 27 to 28, or a kit or parts kit for use according to claim 29 or 30, wherein the dose of each mRNA is 1 to 200 μg, preferably 1 to 60 μg, preferably 2 to 25 μg.

34. An immunogenic composition for use according to any one of claims 1 to 26, or a vaccine for use according to any one of claims 27 to 28, or a kit or parts kit for use according to claim 29 or 30, wherein the single dose of the composition is 2 to 500 μg, particularly 10 to 250 μg of total mRNA, for example 10 to 150 μg of total mRNA.

35. A method for inducing an immune response to an influenza virus, comprising applying or administering an immunogenic composition according to any one of claims 1 to 26 to a subject requiring such composition, wherein the immune response is induced as described in any one of claims 1 to 26.

36. A method for treating or preventing a disorder caused by an influenza virus, comprising applying or administering an immunogenic composition according to any one of claims 1 to 26 to a subject requiring such treatment, wherein an immune response is induced as described in any one of claims 1 to 26.

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