influenza virus vaccine

Abstract

The present invention relates inter alia to an immunogenic composition comprising (a) a first hemagglutinin (HA) antigen derived from an influenza virus strain, or a first nucleic acid, preferably mRNA, encoding the first HA antigen, and (b) a second HA antigen derived from an influenza virus strain, or a second nucleic acid, preferably mRNA, encoding the second HA antigen, wherein (a) and (b) are different and the ratio of (a):(b) is comprised between 1.5:1 and 5:1. The present invention also relates to a vaccine and kit or kit-of-parts comprising the same. The immunogenic compositions, vaccines and kits-of-parts provided herein are suitable for use as medicines, in particular for use in the treatment or prevention of infection with influenza viruses, preferably influenza A and / or influenza B viruses.

Description

[Technical Field]

[0001] Technical Field The present invention relates inter alia to immunogenic compositions comprising a hemagglutinin (HA) antigen from an influenza virus strain or a nucleic acid, preferably mRNA, encoding said HA antigen. The present invention also relates to vaccines and kits or kits of parts comprising same. The immunogenic compositions, vaccines and kits of parts provided herein are suitable for pharmaceutical use, in particular for use in the treatment or prevention of infection with influenza viruses, preferably influenza A viruses and / or influenza B viruses. [Background technology]

[0002] background Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family (NCBI classification ID: 11308), subdivided into, for example, alphainfluenza viruses (a genus that includes influenza A viruses) and betainfluenza viruses (a genus that includes influenza B viruses), which are circulating in all regions of the world. Influenza viruses often cause acute respiratory illness during local outbreaks or seasonal epidemics, and sometimes during pandemics. A typical influenza epidemic results in an increased incidence of pneumonia and lower respiratory tract disease, thereby increasing hospitalization and mortality rates. Although the elderly or those with underlying chronic diseases are most likely to suffer from such complications, infants and young children can also suffer from severe illness. Influenza viruses (mainly influenza A viruses and influenza B viruses) have a significant impact on public health worldwide, causing millions of cases of serious illness, thousands of deaths, and considerable economic losses each year.

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

[0004] Currently, there are 18 described HA (H1-H18) subtypes and 11 described NA (N1-N11) subtypes of influenza A viruses, which potentially form 144 combinations of HA and NA. Unlike influenza A viruses, which have a wide host range, influenza B viruses infect humans almost exclusively. 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 have been circulating worldwide since 1983. Influenza B viruses mutate at a rate two to three times slower than influenza A viruses, yet they continue to have a significant impact on children and young adults each year.

[0005] Vaccination is currently the most widely used method for preventing influenza pandemics, especially in high-risk populations. The constant emergence of new strains of influenza virus due to antigenic drift is the virological cause of seasonal epidemics. Due to their constantly evolving nature, regular updates of the viruses contained in influenza vaccines are necessary for the vaccines to be effective. Public health authorities monitor influenza viruses circulating in humans and update the recommended influenza vaccine composition twice a year. The published recommendations (usually three or four different influenza virus strains) are used by national vaccine regulatory agencies and pharmaceutical companies to develop, manufacture, and license influenza vaccines for the next influenza season.

[0006] 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 subtype A and influenza B viruses contained in the vaccine.

[0007] Because HA is the primary 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 tetravalent vaccines based on four HAs derived from each of the four influenza virus strains (typically two influenza A subtype strains and two influenza B strains) designated by health authorities for inclusion in that year's seasonal vaccine, meaning that the vaccine is designed to protect against those four different influenza virus strains. Each of the four HAs is present in the vaccine in an equimolar ratio. The standard dose for one HA (i.e., per strain) is 15 μg / 0.5 ml, and the standard dose for the total (i.e., four HAs) is 60 μg / 0.5 ml. Some available influenza vaccines are approved with even higher doses, such as 45 μg / 0.5 ml HA per strain (FLUBLOK, Sanofi) or 60 μg / 0.7 ml HA per strain (FLUZONE HIGH-DOSE, Sanofi).

[0008] Clinical studies underlying currently licensed influenza vaccines highlight some variability in vaccine efficacy against different influenza virus strains and in immunogenicity associated with the different antigens (e.g., HA) that comprise influenza vaccines. For example, in healthy adults aged 18-49 years, Flublok efficacy was 54.4% against influenza A subtype but only 23.1% against influenza B subtype. Similarly, corresponding immunogenicity studies have shown HI GMTs (hemagglutination inhibition geometric mean titers) that are up to 17-fold higher with HA A antigen than with HA B antigen.

[0009] Therefore, there remains a need to obtain immunogenic compositions that can induce broad, rapid and potent immune responses against influenza viruses. Summary of the Invention

[0010] Overview of the invention In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first hemagglutinin (HA) antigen from a strain of influenza virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; and (b) a second HA antigen from a strain of influenza virus, or a second nucleic acid, preferably mRNA, encoding the second HA antigen. wherein (a) and (b) are different and the ratio of (a):(b) is within the range of 1.5:1 to 5:1.

[0011] Also, (a) a first mRNA encoding the HA of a first strain of influenza B virus; (b) a second mRNA encoding the HA of the first strain of influenza A virus; (c 1 ) a third mRNA encoding the HA of a second strain of influenza A virus; and (c 2 ) A fourth mRNA encoding the HA of a second strain of influenza B virus An immunogenic composition comprising: (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and preferably is 2:1:1:2 or 3:1:1:3.

[0012] In a second aspect, the present invention provides a vaccine comprising an immunogenic composition as defined herein.

[0013] In a third aspect, the present invention provides an antigen or nucleic acid and / or mRNA as defined herein, preferably comprising (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c5 ) and / or (c 6 The present invention provides a kit or kit of parts (kit of parts) comprising mRNA of the present invention, optionally including a liquid vehicle for solubilization, and optionally including technical instructions providing information regarding administration and dosage of the components.

[0014] In a fourth aspect, the present invention relates to an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein for use as a medicament.

[0015] In a fifth aspect, the present invention relates to an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein for use in the treatment or prevention of infection by an influenza virus, preferably an influenza A virus and / or an influenza B virus.

[0016] In a sixth aspect, the present invention relates to a method for the treatment or prevention of a disorder or disease caused by an influenza virus, preferably an influenza A virus and / or an influenza B virus, comprising applying or administering to a subject in need thereof an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein.

[0017] In a seventh aspect, the present invention relates to a method for eliciting an immune response comprising applying or administering to a subject in need thereof an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein.

[0018] A brief description of arrays SEQ ID NO: 1: Amino acid sequence of HA from A / Michigan / 45 / 2015 (H1N1). SEQ ID NO: 2: Amino acid sequence of NA from A / Michigan / 45 / 2015 (H1N1). SEQ ID NO: 3: Amino acid sequence of HA from A / Switzerland / 8060 / 2017 (H3N2). SEQ ID NO: 4: Amino acid sequence of NA from A / Switzerland / 8060 / 2017 (H3N2). SEQ ID NO: 5: Amino acid sequence of HA from B / Colorado / 06 / 2017. SEQ ID NO: 6: Amino acid sequence of NA from B / Colorado / 06 / 2017. SEQ ID NO: 7: Amino acid sequence of HA from B / Phuket / 3073 / 2013. SEQ ID NO: 8: Amino acid sequence of NA from B / Phuket / 3073 / 2013. SEQ ID NO: 9: Amino acid sequence of HA from A / Singapore / INFIMH-16-0019 / 2016 (H3N2). SEQ ID NO: 10: Amino acid sequence of NA from A / Singapore / INFIMH-16-0019 / 2016 (H3N2). SEQ ID NO: 11: Amino acid sequence of HA from A / Brisbane / 02 / 2018 (H1N1). SEQ ID NO: 12: Amino acid sequence of NA from A / Brisbane / 02 / 2018 (H1N1). SEQ ID NO: 13: Amino acid sequence of HA from A / Kansas / 14 / 2017 (H3N2). SEQ ID NO: 14: Amino acid sequence of NA from A / Kansas / 14 / 2017 (H3N2). SEQ ID NO: 15: Amino acid sequence of HA from A / South Australia / 34 / 2019 (H3N2). SEQ ID NO: 16: Amino acid sequence of NA from A / South Australia / 34 / 2019 (H3N2). SEQ ID NO: 17: Amino acid sequence of HA from B / Washington / 02 / 2019. SEQ ID NO: 18: Amino acid sequence of NA from B / Washington / 02 / 2019. SEQ ID NO: 19: Amino acid sequence of HA from A / Guangdong-Maonan / SWL1536 / 2019 (H1N1). SEQ ID NO: 20: Amino acid sequence of NA from A / Guangdong-Maonan / SWL1536 / 2019 (H1N1). SEQ ID NO: 21: Amino acid sequence of HA from A / Hong Kong / 2671 / 2019 (H3N2). SEQ ID NO: 22: Amino acid sequence of NA from A / Hong Kong / 2671 / 2019 (H3N2). SEQ ID NO: 23: Amino acid sequence of HA from A / Hawaii / 70 / 2019 (H1N1). SEQ ID NO: 24: Amino acid sequence of NA from A / Hawaii / 70 / 2019 (H1N1). SEQ ID NO: 25: Amino acid sequence of HA from A / Hong Kong / 45 / 2019 (H3N2). SEQ ID NO: 26: Amino acid sequence of NA from A / Hong Kong / 45 / 2019 (H3N2). SEQ ID NO: 27: Amino acid sequence of HA from A / Victoria / 2570 / 2019 (H1N1). SEQ ID NO: 28: Amino acid sequence of NA from A / Victoria / 2570 / 2019 (H1N1). SEQ ID NO: 29: Amino acid sequence of HA from A / Wisconsin / 588 / 2019 (H1N1). SEQ ID NO: 30: Amino acid sequence of NA from A / Wisconsin / 588 / 2019 (H1N1). SEQ ID NO: 31: Amino acid sequence of HA from A / Cambodia / e0826360 / 2020 (H3N2). SEQ ID NO: 32: Amino acid sequence of NA from A / Cambodia / e0826360 / 2020 (H3N2). SEQ ID NO: 33: Amino acid sequence of HA from A / Darwin / 9 / 2021 (H3N2). SEQ ID NO: 34: Amino acid sequence of NA from A / Darwin / 9 / 2021 (H3N2). SEQ ID NO: 35: Amino acid sequence of HA from B / Austria / 1359417 / 2021. SEQ ID NO: 36: Amino acid sequence of NA from B / Austria / 1359417 / 2021. SEQ ID NO: 37: Amino acid sequence of HA from A / Darwin / 6 / 2021 (H3N2). SEQ ID NO: 38: Amino acid sequence of NA from A / Darwin / 6 / 2021 (H3N2). SEQ ID NO: 39: Amino acid sequence of HA from A / Victoria / 4897 / 2022 (H1N1). SEQ ID NO: 40: Amino acid sequence of NA from A / Victoria / 4897 / 2022 (H1N1). SEQ ID NO: 41: Amino acid sequence of HA from A / Wisconsin / 67 / 2022 (H1N1). SEQ ID NO: 42: Amino acid sequence of NA from A / Wisconsin / 67 / 2022 (H1N1). SEQ ID NO: 43: Amino acid sequence of HA from A / Sydney / 5 / 2021 (H1N1). SEQ ID NO: 44: Amino acid sequence of NA from A / Sydney / 5 / 2021 (H1N1). [Brief explanation of the drawings]

[0019] [Figure 1] Domain structure of influenza A virus (IAV) HA protein. Domains in HA1 include the fusion (F1), vestigial esterase (VE), and receptor binding domain (RBD). Domains in HA2 include the HA2 ectodomain, transmembrane region (TM), and cytoplasmic tail (CT). The HA head contains the receptor binding subdomain and the vestigial esterase subdomain. The stalk (also called the "stem") contains the HA1 fusion domain and the HA2 ectodomain. [Figure 2A]Figures 2A-C: Reactogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine trial. Figure 2A: Solicited adverse events in subjects at the mRNA dose levels indicated at the bottom of the graph. Figure 2B: Solicited adverse events in subjects at the indicated mRNA dose levels split into young adults and elderly. Figures 2A-B: Percentage of grade 0 events above the dose level designation at the bottom of the graph, and events with increasing grades arranged vertically. Figure 2C: Solicited adverse events in subjects at the indicated mRNA dose levels split into young adults and elderly and split into local and systemic events. Grade 0-1 events above the dose level designation at the bottom of the graph. Percentages of grade 0-1 and grade ≥ 2 are shown. [Figure 2B] Figures 2A-C: Reactogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine trial. Figure 2A: Solicited adverse events in subjects at the mRNA dose levels indicated at the bottom of the graph. Figure 2B: Solicited adverse events in subjects at the indicated mRNA dose levels split into young adults and elderly. Figures 2A-B: Percentage of grade 0 events above the dose level designation at the bottom of the graph, and events with increasing grades arranged vertically. Figure 2C: Solicited adverse events in subjects at the indicated mRNA dose levels split into young adults and elderly and split into local and systemic events. Grade 0-1 events above the dose level designation at the bottom of the graph. Percentages of grade 0-1 and grade ≥ 2 are shown. [Figure 2C]Figures 2A-C: Reactogenicity assessment of subjects in the CVSQIV Phase I influenza vaccine trial. Figure 2A: Solicited adverse events in subjects at the mRNA dose levels indicated at the bottom of the graph. Figure 2B: Solicited adverse events in subjects at the indicated mRNA dose levels split into young adults and elderly. Figures 2A-B: Percentage of grade 0 events above the dose level designation at the bottom of the graph, and events with increasing grades arranged vertically. Figure 2C: Solicited adverse events in subjects at the indicated mRNA dose levels split into young adults and elderly and split into local and systemic events. Grade 0-1 events above the dose level designation at the bottom of the graph. Percentages of grade 0-1 and grade ≥ 2 are shown. [Figure 3A] Figures 3A-D: Graphs show hemagglutinin inhibition assay (HAI) geometric mean titers (95% CI) for the Per Protocol Immunogenicity Set. The left panel shows HAI titers for all subjects at days 1, 22, and 183 at the indicated vaccine mRNA dose levels. The data in the right panel are split between young adults (YA) and elderly adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figure 3A), H3N2 (Figure 3B), B / Phuket (Figure 3C), and B / Washington (Figure 3D). [Figure 3B]Figures 3A-D: Graphs show hemagglutinin inhibition assay (HAI) geometric mean titers (95% CI) for the Per Protocol Immunogenicity Set. The left panel shows HAI titers for all subjects at days 1, 22, and 183 at the indicated vaccine mRNA dose levels. The data in the right panel are split between young adults (YA) and elderly adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figure 3A), H3N2 (Figure 3B), B / Phuket (Figure 3C), and B / Washington (Figure 3D). [Figure 3C] Figures 3A-D: Graphs show hemagglutinin inhibition assay (HAI) geometric mean titers (95% CI) for the Per Protocol Immunogenicity Set. The left panel shows HAI titers for all subjects at days 1, 22, and 183 at the indicated vaccine mRNA dose levels. The data in the right panel are split between young adults (YA) and elderly adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figure 3A), H3N2 (Figure 3B), B / Phuket (Figure 3C), and B / Washington (Figure 3D). [Figure 3D]Figures 3A-D: Graphs show hemagglutinin inhibition assay (HAI) geometric mean titers (95% CI) for the Per Protocol Immunogenicity Set. The left panel shows HAI titers for all subjects at days 1, 22, and 183 at the indicated vaccine mRNA dose levels. The data in the right panel are split between young adults (YA) and elderly adults (OA) at the indicated mRNA dose levels. Data are shown separately for each HA component encoded by the vaccine mRNA: H1N1 (Figure 3A), H3N2 (Figure 3B), B / Phuket (Figure 3C), and B / Washington (Figure 3D). [Figure 4] Seroconversion rates (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 ≥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 subjects. 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 subjects. [Figure 5]The percentage of study subjects who showed a 4-fold or greater increase in anti-HA titer by microneutralization (MN) assay is shown. The table in the upper left panel shows the percentage of subjects who showed a 4-fold or greater increase in anti-HA titer by MN assay. Data are 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 4-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 4-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 4-fold increase in anti-HA by MN assay for each coded HA at each dose level in elderly subjects. [Figure 6] The percentage of study subjects who showed a 4-fold or greater increase in anti-NA titers by enzyme-linked lectin assay (ELLA) is shown. The table in the upper left panel shows the percentage of subjects who showed a 4-fold or greater increase in anti-NA titers by the ELLA assay. Data are presented 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 4-fold increase in anti-NA by the ELLA assay for each coded HA at each dose level. The graph in the upper right panel shows the 4-fold increase in anti-NA by the ELLA assay for each coded HA at each dose level in young adults. The graph in the lower right panel shows the 4-fold increase in anti-NA by the ELLA assay for each coded HA at each dose level in elderly subjects. [Figure 7]Figure 1 shows the results of a ferret immunization study using a tetravalent mRNA vaccine encoding HA from two influenza A strains (A / California / 07 / 2009 (H1N1pdm09) and A / Hong Kong / 4801 / 2014 (H3N2)) and two influenza B strains (B / Phuket / 3073 / 2013 and B / Brisbane / 60 / 2008). The vaccines contained equal (1:1) μg amounts of mRNA encoding influenza A and B antigens, or a four-fold excess (1:4) of B antigen-encoding mRNA. Animals were immunized intramuscularly on days 0 and 21. Functional antibody responses were assessed in serum samples collected on days 0, 21, 35, and 49 by MN assay for influenza B antigens (upper panel) and HAI assay for influenza A antigens (lower panel). [Figure 8] Figure 1 shows the results of IFNα levels upon intramuscular (i.m.) immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized i.m. with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of licensed split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the i.m. route on days 0 and 21. IFNα levels were measured using ELISA in serum samples collected 18 hours after the first immunization. [Figure 9A]Figures 9A-D show the HI response upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. HI titers against influenza (A) A / Wisconsin / 588 / 2019 (H1N1pdm09), (B) A / Darwin / 6 / 2021 (H3N2), (C) B / Austria / 1359417 / 2021, and (D) B / Phuket / 3073 / 2013 were measured in sera collected 2 weeks after the second immunization. [Figure 9B] Figures 9A-D show the HI response upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. HI titers against influenza (A) A / Wisconsin / 588 / 2019 (H1N1pdm09), (B) A / Darwin / 6 / 2021 (H3N2), (C) B / Austria / 1359417 / 2021, and (D) B / Phuket / 3073 / 2013 were measured in sera collected 2 weeks after the second immunization. [Figure 9C]Figures 9A-D show the HI response upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. HI titers against influenza (A) A / Wisconsin / 588 / 2019 (H1N1pdm09), (B) A / Darwin / 6 / 2021 (H3N2), (C) B / Austria / 1359417 / 2021, and (D) B / Phuket / 3073 / 2013 were measured in sera collected 2 weeks after the second immunization. [Figure 9D] Figures 9A-D show the HI response upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. HI titers against influenza (A) A / Wisconsin / 588 / 2019 (H1N1pdm09), (B) A / Darwin / 6 / 2021 (H3N2), (C) B / Austria / 1359417 / 2021, and (D) B / Phuket / 3073 / 2013 were measured in sera collected 2 weeks after the second immunization. [Figure 10A]Figures 10A-D show the T cell responses induced upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. T cell immune responses were analyzed by ICS 2 weeks after the second immunization in isolated splenocytes restimulated with a 15-mer overlapping peptide library spanning the full-length HA of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) or the full-length HA of influenza B / Austria / 1359417 / 2021. Influenza A / Wisconsin / 588 / 2019 HA-specific IFNγ+TNF+-producing (A) CD4+ and (B) CD8+ T cells, and influenza B / Austria / 1359417 / 2021 HA-specific IFNγ+TNF+-producing (C) CD4+ and (D) CD8+ T cells were measured. [Figure 10B]Figures 10A-D show the T cell responses induced upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. T cell immune responses were analyzed by ICS 2 weeks after the second immunization in isolated splenocytes restimulated with a 15-mer overlapping peptide library spanning the full-length HA of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) or the full-length HA of influenza B / Austria / 1359417 / 2021. Influenza A / Wisconsin / 588 / 2019 HA-specific IFNγ+TNF+-producing (A) CD4+ and (B) CD8+ T cells, and influenza B / Austria / 1359417 / 2021 HA-specific IFNγ+TNF+-producing (C) CD4+ and (D) CD8+ T cells were measured. [Figure 10C]Figures 10A-D show the T cell responses induced upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. T cell immune responses were analyzed by ICS 2 weeks after the second immunization in isolated splenocytes restimulated with a 15-mer overlapping peptide library spanning the full-length HA of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) or the full-length HA of influenza B / Austria / 1359417 / 2021. Influenza A / Wisconsin / 588 / 2019 HA-specific IFNγ+TNF+-producing (A) CD4+ and (B) CD8+ T cells, and influenza B / Austria / 1359417 / 2021 HA-specific IFNγ+TNF+-producing (C) CD4+ and (D) CD8+ T cells were measured. [Figure 10D]Figures 10A-D show the T cell responses induced upon intramuscular immunization of mice with four- and eight-component seasonal influenza mRNA vaccines containing different ratios of influenza A and B HA components. Female Balb / c mice were immunized intramuscularly with the different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth the human dose of the approved split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the intramuscular route on days 0 and 21. T cell immune responses were analyzed by ICS 2 weeks after the second immunization in isolated splenocytes restimulated with a 15-mer overlapping peptide library spanning the full-length HA of influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) or the full-length HA of influenza B / Austria / 1359417 / 2021. Influenza A / Wisconsin / 588 / 2019 HA-specific IFNγ+TNF+-producing (A) CD4+ and (B) CD8+ T cells, and influenza B / Austria / 1359417 / 2021 HA-specific IFNγ+TNF+-producing (C) CD4+ and (D) CD8+ T cells were measured. [Figure 11A]Figures 11A-D show HI titers induced by four- or seven-component mRNA vaccines containing unmodified or modified (ψ and N1-mψ) nucleosides at equimolar ratios between mRNA sequences. Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the four-component (4HA; unmodified, ψ and N1-mψ) and 1 μg or 2.84 μg of the seven-component (4HA + 3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra NH21-22 or FLUZONE HD NH21-22. 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 2 weeks after the second immunization. [Figure 11B] Figures 11A-D show HI titers induced by four- or seven-component mRNA vaccines containing unmodified or modified (ψ and N1-mψ) nucleosides at equimolar ratios between mRNA sequences. Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the four-component (4HA; unmodified, ψ and N1-mψ) and 1 μg or 2.84 μg of the seven-component (4HA + 3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra NH21-22 or FLUZONE HD NH21-22. 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 2 weeks after the second immunization. [Figure 11C]Figures 11A-D show HI titers induced by four- or seven-component mRNA vaccines containing unmodified or modified (ψ and N1-mψ) nucleosides at equimolar ratios between mRNA sequences. Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the four-component (4HA; unmodified, ψ and N1-mψ) and 1 μg or 2.84 μg of the seven-component (4HA + 3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra NH21-22 or FLUZONE HD NH21-22. 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 2 weeks after the second immunization. [Figure 11D] Figures 11A-D show HI titers induced by four- or seven-component mRNA vaccines containing unmodified or modified (ψ and N1-mψ) nucleosides at equimolar ratios between mRNA sequences. Female Balb / c mice (n = 10 / group) were vaccinated with 0.56 μg or 2.84 μg of the four-component (4HA; unmodified, ψ and N1-mψ) and 1 μg or 2.84 μg of the seven-component (4HA + 3NA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine. Control animals (n = 5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra NH21-22 or FLUZONE HD NH21-22. 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 2 weeks after the second immunization. [Figure 12A]Figures 12A-C show NI titers induced by a seven-component mRNA vaccine containing unmodified or modified (ψ and N1-mψ) nucleosides at an equimolar ratio between mRNA sequences. Female Balb / c mice (n=10 / group) were vaccinated with 1 μg or 2.84 μg of the seven-component (4 HA + 3 NA; unmodified, ψ, and N1-mψ) mRNA vaccine. Control animals (n=5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra (NH21-22) or FLUZONE HD (NH21-22). NI titers against influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) (A), A / Cambodia / e0826360 / 2020 (H3N2) (B), and B / Washington / 02 / 2019 (C) were measured in serum 2 weeks after the second immunization. [Figure 12B] Figures 12A-C show NI titers induced by a seven-component mRNA vaccine containing unmodified or modified (ψ and N1-mψ) nucleosides at an equimolar ratio between mRNA sequences. Female Balb / c mice (n=10 / group) were vaccinated with 1 μg or 2.84 μg of the seven-component (4 HA + 3 NA; unmodified, ψ, and N1-mψ) mRNA vaccine. Control animals (n=5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra (NH21-22) or FLUZONE HD (NH21-22). NI titers against influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) (A), A / Cambodia / e0826360 / 2020 (H3N2) (B), and B / Washington / 02 / 2019 (C) were measured in serum 2 weeks after the second immunization. [Figure 12C]Figures 12A-C show NI titers induced by a seven-component mRNA vaccine containing unmodified or modified (ψ and N1-mψ) nucleosides at an equimolar ratio between mRNA sequences. Female Balb / c mice (n=10 / group) were vaccinated with 1 μg or 2.84 μg of the seven-component (4 HA + 3 NA; unmodified, ψ, and N1-mψ) mRNA vaccine. Control animals (n=5 / group) received saline (NaCl) or one-tenth the human dose of the approved QIV FLUARIX Tetra (NH21-22) or FLUZONE HD (NH21-22). NI titers against influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) (A), A / Cambodia / e0826360 / 2020 (H3N2) (B), and B / Washington / 02 / 2019 (C) were measured in serum 2 weeks after the second immunization. [Figure 13] Figures 13A-D show the HI responses induced upon intramuscular immunization of naive ferrets with four-component and eight-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly with 12.5 μg and 25 μg of the four-component and 25 μg and 50 μg of the eight-component seasonal influenza N1mψ mRNA vaccine on days 0 and 28 (n=6). Control animals received a full human dose of saline (NaCl) (n=6 / group) or the approved split-inactivated QIV FLUARIX Tetra (NH22-23) (n=6 / group) on days 0 and 28. HI titers against 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. [Figure 14]Figures 14A-D show microneutralization (MN) titers induced upon intramuscular immunization of naive ferrets with four-component and eight-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly with 12.5 μg and 25 μg of the four-component and 25 μg and 50 μg of the eight-component seasonal influenza N1mψ mRNA vaccine on days 0 and 28 (n=6). Control animals received a full human dose of saline (NaCl) (n=6 / group) or the approved split-inactivated QIV FLUARIX Tetra (NH22-23) (n=6 / group) on days 0 and 28. MN titers against 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. [Figure 15] Figures 15A-D show neuraminidase inhibition (NI) titers measured using an enzyme-linked lectin assay (ELLA) upon intramuscular immunization of naive ferrets with four-component and eight-component seasonal influenza mRNA vaccine formulations. Female ferrets were immunized intramuscularly with 12.5 μg and 25 μg of the four-component and 25 μg and 50 μg of the eight-component seasonal influenza N1mψ mRNA vaccine on days 0 and 28 (n=6). Control animals received a full human dose of saline (NaCl) (n=6 / group) or the approved split-inactivated QIV FLUARIX Tetra (NH22-23) (n=6 / group) on days 0 and 28. NI titers against 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. [Figure 16A]Figures 16A-D show HI titers induced upon immunization of healthy human adults (ages 18-50) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers against 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 16B] Figures 16A-D show HI titers induced upon immunization of healthy human adults (ages 18-50) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers against 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 16C] Figures 16A-D show HI titers induced upon immunization of healthy human adults (ages 18-50) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers against 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 16D]Figures 16A-D show HI titers induced upon immunization of healthy human adults (ages 18-50) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza (Flu) D-QIV (FLUARIX, NH2022-23). ​​HI titers against 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 17A] Figures 17A-D show NI titers induced upon immunization of healthy human adults (18-50 years of age) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers against 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 17B] Figures 17A-D show NI titers induced upon immunization of healthy human adults (18-50 years of age) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers against 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 17C]Figures 17A-D show NI titers induced upon immunization of healthy human adults (18-50 years of age) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers against 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 17D] Figures 17A-D show NI titers induced upon immunization of healthy human adults (18-50 years of age) with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​NI titers against 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 18A] Figures 18A-D show the percentage of healthy human adults (18-50 years old) who experienced solitary events (A), local events (B), and systemic events (C) within 7 days of immunization with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary by event, including grade 3 events. [Figure 18B] Figures 18A-D show the percentage of healthy human adults (18-50 years old) who experienced solitary events (A), local events (B), and systemic events (C) within 7 days of immunization with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary by event, including grade 3 events. [Figure 18C]Figures 18A-D show the percentage of healthy human adults (18-50 years old) who experienced solitary events (A), local events (B), and systemic events (C) within 7 days of immunization with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary by event, including grade 3 events. [Figure 18D] Figures 18A-D show the percentage of healthy human adults (18-50 years old) who experienced solitary events (A), local events (B), and systemic events (C) within 7 days of immunization with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control was influenza D-QIV (FLUARIX, NH2022-23). ​​(D) shows an overall summary by event, including grade 3 events. [Figure 19] Figure 19 shows the percentage of healthy human adults (18-50 years old) who experienced a related spontaneous event within 7 days of immunization with one-, four-, and eight-component seasonal influenza mRNA vaccine formulations. The control is influenza D-QIV (FLUARIX, NH2022-23).

[0020] Detailed Description of the Invention This application has been submitted with an electronic sequence listing (WIPO Standard ST.26), which is a part of the present specification. The information contained in the sequence listing is incorporated herein by reference in its entirety. When a "SEQ ID NO" is mentioned herein, reference is made to the corresponding nucleic acid (na) sequence or amino acid (aa) sequence in the sequence listing with the respective identifier. For many sequences, the sequence listing also provides further details, such as specific structural features, sequence optimization, GenBank (NCBI) or GISAID (epi) identifiers, or their coding capacity. When a "SEQ ID NO" of another published patent application or patent is mentioned, said sequence, e.g., amino acid sequence or nucleic acid sequence, is expressly incorporated herein by reference. These sequences therefore constitute an essential part of the underlying description.

[0021] Immunogenic composition: The protective immune response induced by vaccination against influenza virus is primarily directed against the viral HA protein, a glycoprotein on the surface of the virus that mediates the interaction of the virus with host cell receptors.

[0022] The HA protein on the viral surface is a homotrimer of HA protein monomers, which is enzymatically cleaved to generate an amino-terminal HA1 polypeptide and a carboxy-terminal HA2 polypeptide. Structurally, the hemagglutinin protein is composed of several domains: a globular head domain, a stalk domain (also called a stem domain), a transmembrane domain, and a cytoplasmic domain (Figure 1, see Russell et al., 2021).

[0023] During influenza virus infection of host cells (e.g., eukaryotic cells, e.g., human cells), it is generally believed that the hemagglutinin protein recognizes and binds to sialic acid of receptors on the surface of the host cells, promoting attachment of the virus to the host cells. After viral endocytosis and endosomal acidification, the hemagglutinin protein undergoes a pH-dependent conformational change that allows it to promote fusion of the viral envelope with the endosomal membrane of the host cell and entry of viral nucleic acid into the host cell.

[0024] The globular head consists exclusively of the major portion of the HA1 polypeptide, while the stem, which anchors the HA protein within the viral lipid envelope, is composed of HA2 and a portion of HA1. The globular head of the HA protein contains two domains: the receptor-binding domain (RBD), which contains the sialic acid-binding site, and the vestigial esterase domain, a smaller region immediately downstream of the RBD. Influenza viruses are generally classified based on the amino acid sequence of the viral hemagglutinin protein and / or the viral neuraminidase (NA). Differences in the amino acid sequence between HA proteins of different subtypes are primarily found within the sequence of the head domain of the protein. The amino acid sequence of the stalk domain is thought to be more conserved between HA subtypes than the sequence of the head domain. The domains of the HA protein can be predicted using routine methods known in the art.

[0025] Many naturally occurring and experimentally derived antibodies that bind to and neutralize the HA protein are thought to bind to epitopes within the head domain of HA and inhibit or reduce the interaction of HA with sialic acid on host cell receptors, thereby preventing or reducing cell infection. Alternatively, or additionally, neutralizing antibodies may prevent or reduce fusion of the viral membrane with the endosomal membrane. Such antibodies may bind to epitopes within the stalk domain, thereby inhibiting conformational changes in the protein. Antibodies against influenza often target variable antigenic sites within the globular head of HA and therefore neutralize only antigenically closely related viruses. The variability of the HA head is due to the constant antigenic drift (i.e., changes in the protein sequence) of influenza viruses and is responsible for seasonal influenza epidemics.

[0026] The present inventors (a) a first hemagglutinin (HA) antigen from a strain of influenza virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; and (b) a second HA antigen from a strain of influenza virus, or a second nucleic acid, preferably mRNA, encoding the second HA antigen. The shortcomings of the prior art are overcome by administering an immunogenic composition comprising: (a) and (b), wherein (a) and (b) are different and the ratio of (a):(b) is comprised between 1.5:1 and 5:1.

[0027] The immunogenic compositions of the present invention have been shown to induce broad, rapid and potent immune responses against influenza viruses, eg, influenza A and / or B.

[0028] In particular, or additionally, it has been found that the efficacy of an immunogenic composition comprising (a) a first HA antigen or a first nucleic acid, preferably mRNA, encoding the first HA antigen, and (b) a second HA antigen or a second nucleic acid, preferably mRNA, encoding the second HA antigen, against different strains of influenza virus is enhanced when the ratio of (a):(b) is comprised between 1.5:1 and 5:1.

[0029] In particular, or in addition, it has been found that the immunogenicity associated with the first and / or second HA antigens constituting the immunogenic composition of the present invention is enhanced when the ratio of (a):(b) is comprised between 1.5:1 and 5:1.

[0030] Preferably, the immunogenic compositions of the present invention have at least some of the following advantageous characteristics: - Translation of nucleic acids, preferably mRNA, encoding the first and second HA antigens at the site of injection / vaccination (e.g. muscle). - Induction of antigen-specific immune responses, preferably at low doses and administration regimens. - Suitability of vaccination for infants and / or newborns or for the elderly, especially the elderly. - Suitability of the composition / vaccine for intramuscular administration. - induction of a specific and functional humoral immune response against influenza viruses, preferably influenza A and / or B viruses. - induction of broad and functional cellular T cell responses against influenza viruses, preferably influenza A and / or B viruses. - Induction of specific B cell memory against influenza viruses, preferably influenza A and / or B viruses. - Induction of functional antibodies capable of effectively neutralizing influenza viruses, preferably influenza A and / or B viruses. - Induction of functional antibodies capable of effectively neutralizing new variants of influenza viruses, preferably influenza A and / or B viruses. - Induction of protective immunity against influenza virus infection, for example against influenza A virus and / or influenza B virus or new variants thereof. - rapid onset of immune protection against influenza viruses, preferably influenza A viruses and / or influenza B viruses. - increasing the duration of the induced immune response against influenza viruses, preferably influenza A viruses and / or influenza B viruses. - 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 excessive induction of systemic cytokine or chemokine responses after application of the composition / vaccine, which may result in undesirably high reactogenicity upon injection / vaccination. - Good tolerability of the composition / vaccine, absence of side effects, absence of toxicity. - Advantageous stability properties of nucleic acid-based compositions / vaccines. - Speed, adaptability, ease and scalability of nucleic acid-based composition / vaccine manufacturing. - Advantageous injection / vaccination regimen requiring only low doses of composition / vaccine for adequate protection.

[0031] Thus, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first hemagglutinin (HA) antigen from a strain of influenza virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; and (b) a second HA antigen from a strain of influenza virus, or a second nucleic acid, preferably mRNA, encoding the second HA antigen. wherein (a) and (b) are different and the ratio of (a):(b) is within the range of 1.5:1 to 5:1.

[0032] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0033] As used herein, "weight / weight ratio" or wt / wt ratio or wt:wt ratio refers to the ratio (proportion) between the weights (masses) of different components. "Molar ratio" refers to the ratio between different components (e.g., the number of mRNAs encoding each antigen).

[0034] The terms "hemagglutinin," "hemagglutinin protein," and "HA" are used interchangeably throughout to refer to the hemagglutinin protein that may be present on the surface of an influenza virus.

[0035] In the context of the present invention, any influenza virus can be selected as a "strain of influenza virus," regardless of the particular genotype, species, strain, isolate, or serotype.

[0036] In some embodiments, the strain of influenza virus may be selected from influenza A virus (NCBI Classification ID: 11320) and / or influenza B virus (NCBI Classification ID: 11520) and / or influenza C virus (NCBI Classification ID: 11552) and / or influenza D virus (NCBI Classification ID: 1511084).

[0037] In some embodiments, the strain of influenza virus is selected from the group consisting of influenza A virus and influenza B virus.

[0038] In some embodiments, the composition is a multivalent composition, and the strain of influenza virus in (a) is different from the strain of influenza virus in (b).

[0039] In some embodiments, the strain of influenza A virus is selected from influenza A viruses characterized by a hemagglutinin (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, more preferably from the group consisting of H1 and H3.

[0040] In some embodiments, the strain of influenza A virus is selected from influenza A viruses characterized by a neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11, preferably the group consisting of N1, N2 and N8, more preferably the group consisting of N1 and N2.

[0041] The terms "neuraminidase," "neuraminidase protein," and "NA" are used interchangeably throughout and refer to the neuraminidase protein that may be present on the surface of an influenza virus.

[0042] In some embodiments, the strain 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, preferably H1N1 and H3N2.

[0043] In some embodiments, the strain of influenza A virus is selected from the group consisting of H1N1 and H3N2.

[0044] In some embodiments, the strain of influenza A virus is 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, A / Solomon Islands / 3 / 2006(H1N1)-like virus, A / Brisbane / 59 / 2007(H1N1)-like virus, A / California / 7 / 2009(H1N1)-like virus, A / California / 7 / 2009(H1N1)pdm09-like virus, A / Michigan / 45 / 2015(H1N1)pdm09-like virus, A / Victoria / 2570 / 2019(H1N1)pdm09 -like virus, A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09-like virus, A / Hawaii / 70 / 2019(H1N1)pdm09-like virus, A / Brisbane / 02 / 2018(H1N1)pdm09-like virus, A / Christchurch / 16 / 2010, A / South Dakota / 6 / 2007, 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 / NewYork / 55 / 2004, A / Wisconsin / 67 / 2005 (H3N2)-like virus, A / Hiroshima / 52 / 2005, A / Brisbane / 10 / 2007 (H3N2)-like virus, A / Uruguay / 716 / 2007, A / Perth / 16 / 2009 (H3N2)-like virus, A / Wisconsin / 15 / 2009, A / Victoria / 210 / 2009, A / Victoria / 361 / 2011 (H3N2)-like virus, A / Ohio / 2 / 2012, A / Maryland / 2 / 2012, A / South Australia / 30 / 2012, A / Brisbane / 1 / 2012, A / Brisbane / 6 / 2012, A(H3N2) viruses antigenically similar to the cytotrophic prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like viruses, A / Darwin / 9 / 2021(H3N2)-like viruses, A / Darwin / 6 / 2021(H3N2)-like viruses, A / Cambodia / e0826360 / 2020(H3N2)-like viruses, A / Hong Kong / 2671 / 2019(H3N2)-like viruses, A / Hong Kong / 45 / 2019(H3N2)-like viruses, A / Switzerland / 9715293 / 2013(H3N2)-like viruses, A / South Australia / 55 / 2014, A / Norway / 466 / 2014, A / Stockholm / 6 / 2014, A / Hong Kong / 4801 / 2014(H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2)-like virus, A / Switzerland / 8060 / 2017(H3N2)-like virus, A / Kansas / 14 / 2017(H3N2)-like virus, and A / South Australia / 34 / 2019(H3N2)-like virus.

[0045] In some embodiments, the strain of influenza A virus is H1N1.

[0046] In some embodiments, the strain of influenza A H1N1 virus is selected from the group consisting of: A / Beijing / 262 / 95(H1N1)-like virus, A / New Caledonia / 20 / 99(H1N1)-like virus, A / Solomon Islands / 3 / 2006(H1N1)-like virus, A / Brisbane / 59 / 2007(H1N1)-like virus, A / California / 7 / 2009(H1N1)-like virus, A / California / 7 / 2009(H1N1)pdm09-like virus, A / Michigan / 45 / 2015(H1N1)pdm09-like virus, and A / Victoria / 2570 / 2019(H1N1)pdm09. -like viruses, A / Wisconsin / 588 / 2019(H1N1)pdm09-like viruses, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09-like viruses, A / Hawaii / 70 / 2019(H1N1)pdm09-like viruses, A / Brisbane / 02 / 2018(H1N1)pdm09-like viruses, A / Christchurch / 16 / 2010 and A / South Dakota / 6 / 2007, A / Victoria / 4897 / 2022(H1N1)pdm09-like viruses, A / Wisconsin / 67 / 2022(H1N1)pdm09-like viruses and A / Sydney / 5 / 2021(H1N1)pdm09-like viruses.

[0047] In some embodiments, the strain of influenza A virus is H3N2.

[0048] In some embodiments, the strain of influenza A H3N2 virus is selected from the group consisting of 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)-like virus, A / Hiroshima / 52 / 2005, A / Brisbane / 10 / 2007 (H3N2)-like virus, A / Uruguay / 716 / 2007, A / Perth / 16 / 2009 (H3N2)-like virus, A / Wisconsin / 15 / 2009, A / Victoria / 210 / 2009, A / Victoria / 361 / 2011 (H3N2)-like virus, A / Ohio / 2 / 2012, A / Maryland / 2 / 2012, A / South Australia / 30 / 2012, A / Brisbane / 1 / 2012, A / Brisbane / 6 / 2012, A(H3N2) viruses antigenically similar to the cytotrophic prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like viruses, A / Darwin / 9 / 2021(H3N2)-like viruses, A / Darwin / 6 / 2021(H3N2)-like viruses, A / Cambodia / e0826360 / 2020(H3N2)-like viruses, A / Hong Kong / 2671 / 2019(H3N2)-like viruses, A / Hong Kong / 45 / 2019(H3N2)-like viruses, A / Switzerland / 9715293 / 2013(H3N2)-like viruses, A / South Australia / 55 / 2014, A / Norway / 466 / 2014, A / Stockholm / 6 / 2014, A / HongKong / 4801 / 2014(H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2)-like virus, A / Switzerland / 8060 / 2017(H3N2)-like virus, A / Kansas / 14 / 2017(H3N2)-like virus, and A / South Australia / 34 / 2019(H3N2)-like virus.

[0049] In some embodiments, the strain of influenza A virus is selected from the influenza A viruses listed in Table 1 and / or Table 2.

[0050] In some embodiments, the strain of influenza A virus is selected from influenza A viruses recommended by the WHO for influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0051] [Table 1] TIFF2025532686000002.tif252167TIFF2025532686000003.tif241166TIFF2025532686000004.tif38166

[0052] [Table 2] TIFF2025532686000006.tif252167TIFF2025532686000007.tif246166TIFF2025532686000008.tif59166

[0053] In some embodiments, the strain of influenza B virus is selected from the group consisting of the B / Victoria lineage and the B / Yamagata lineage.

[0054] In some embodiments, the strain of influenza B virus is selected from the group consisting of B / Beijing / 184 / 93-like virus, B / Harbin / 94-like virus, B / Shangdong / 7 / 97-like virus, B / Yamanashi / 166 / 98-like virus, B / Sichuan / 379 / 99-like virus, B / Guangdong / 120 / 2000, B / Johannesburg / 5 / 99, B / Victoria / 504 / 2000, B / Hong Kong / 330 / 2001-like virus, B / Hong Kong / 1434 / 2002, B / Brisbane / 32 / 2002, B / Shanghai / 361 / 2002-like virus, B / Jiangsu / 10 / 2003, B / Jilin / 20 / 2003, B / Malaysia / 2506 / 2004-like virus, B / Malaysia / 2506 / 2004 virus, B / Ohio / 1 / 2005, B / Florida / 4 / 2006-like virus, B / Brisbane / 3 / 2007, B / Brisbane / 60 / 2008-like virus, B / Brisbane / 33 / 2008, B / Wisconsin / 1 / 2010-like virus, B / Hubei-Wujiagang / 158 / 2009, B / Texas / 6 / 2011, B / Massachusetts / 2 / 2012-like viruses, B / Phuket / 3073 / 2013-like viruses, B / Austria / 1359417 / 2021-like viruses, B / Washington / 02 / 2019-like viruses, and B / Colorado / 06 / 2017-like viruses.

[0055] In some embodiments, the strain of influenza B virus is selected from the influenza B viruses listed in Table 1 and / or Table 2.

[0056] In some embodiments, the strain of influenza B virus is selected from influenza B viruses recommended by the WHO for influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0057] In some embodiments, the strain of influenza virus in (b) is an influenza A virus strain.

[0058] In some embodiments, the strain of influenza virus in (a) is an influenza B virus strain.

[0059] In some embodiments, the strain of influenza virus in (b) is an influenza A virus strain and the strain of influenza virus in (a) is an influenza B virus strain.

[0060] Exemplary HA antigens are known in the art and are published, for example, at 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 / ).

[0061] In some embodiments, the first and / or second HA antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98% or 99% identity to the amino acid sequence set forth 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 or 43, or a fragment or variant thereof.

[0062] In some embodiments, the first and / or second HA antigen comprises or consists of an amino acid sequence set forth 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 or 43, or a fragment or variant thereof.

[0063] In some embodiments, the first HA antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 17 or 35, or a fragment or variant thereof.

[0064] In some embodiments, the first HA antigen comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 17, or 35, or a fragment or variant thereof.

[0065] In some embodiments, the second HA antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 9, 11, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41 or 43, or a fragment or variant thereof.

[0066] In some embodiments, the second HA antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 9, 11, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41 or 43, or a fragment or variant thereof.

[0067] In some embodiments, the second HA antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 19, 23, 27, 29, 39, 41 or 43, or a fragment or variant thereof.

[0068] In some embodiments, the second HA antigen comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 19, 23, 27, 29, 39, 41 or 43, or a fragment or variant thereof.

[0069] In some embodiments, the second HA antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 9, 13, 15, 21, 25, 31, 33 or 37, or a fragment or variant thereof.

[0070] In some embodiments, the second HA antigen comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 3, 9, 13, 15, 21, 25, 31, 33, or 37, or a fragment or variant thereof.

[0071] In some embodiments, the first and / or second HA antigens are polypeptides comprising a full-length influenza HA protein. Suitably, the first and / or second HA antigens are polypeptides consisting of a full-length influenza HA protein.

[0072] In some embodiments, the first and / or second HA antigen is a fragment of a hemagglutinin protein, e.g., a truncated hemagglutinin protein. In some embodiments, the fragment is a head-less hemagglutinin, meaning that the fragment does not include the head domain. In some embodiments, the fragment includes a portion of the head domain. In some embodiments, the fragment is the stalk domain. In some embodiments, the fragment does not include the cytoplasmic domain. In some embodiments, the fragment does not include the transmembrane domain. In such embodiments, the fragment may be referred to as a soluble or secreted hemagglutinin protein or fragment.

[0073] In some embodiments, the ratio of (a):(b) is comprised between 1.5:1 and 5:1, optionally between 2:1 and 5:1, optionally between 3:1 and 5:1, optionally between 4:1 and 5:1, optionally between 1.5:1 and 4:1, optionally between 1.5:1 and 3:1, optionally between 2:1 and 4:1, optionally between 2:1 and 3:1.

[0074] In some embodiments, the ratio of (a):(b) is selected from about 1.5:1, about 2:1, about 2.2:1, about 2.4:1, about 2.6:1, about 2.8:1, about 3:1, about 3.2:1, about 3.4:1, about 3.6:1, about 3.8:1, about 4:1, about 4.2:1, about 4.4:1, about 4.6:1, about 4.8:1, or about 5:1. In some embodiments, the ratio of (a):(b) is selected from about 1.5:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, or 5:1. In some embodiments, the ratio of (a):(b) is comprised between 2:1 and 4:1, preferably between 2:1 and 3:1, and preferably is 2:1 or 3:1.

[0075] In some embodiments, the ratio of (a):(b) is about 2:1, preferably 2.1.

[0076] In some embodiments, the ratio of (a):(b) is about 3:1, preferably 3.1.

[0077] In some embodiments, the immunogenic composition comprises: (a) a first hemagglutinin (HA) antigen from a strain of influenza B virus or a first nucleic acid, preferably mRNA, encoding the first HA antigen; and (b) a second HA antigen from a strain of influenza A virus or a second nucleic acid, preferably mRNA, encoding the second HA antigen. Including, Here, (a) and (b) are different, and the ratio of (a):(b) is comprised between 2:1 and 4:1, preferably between 2:1 and 3:1, and preferably is 2:1 or 3:1.

[0078] In some embodiments, the immunogenic composition comprises: (c) at least one further antigen derived from a strain of influenza virus, or at least one further nucleic acid, preferably mRNA, encoding at least one further antigen Includes.

[0079] In some embodiments, the strain of influenza virus in (c) is selected from the group consisting of influenza A virus and influenza B virus.

[0080] In some embodiments, the strain of influenza virus in (c) is an influenza A virus strain.

[0081] As noted above, in some embodiments, the strain of influenza A virus is selected from influenza A viruses characterized by a hemagglutinin (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, more preferably from the group consisting of H1 and H3.

[0082] In some embodiments, the strain of influenza A virus is selected from influenza A viruses characterized by a neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11, preferably the group consisting of N1, N2 and N8, more preferably the group consisting of N1 and N2.

[0083] In some embodiments, the strain 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, preferably H1N1 and H3N2.

[0084] In some embodiments, the strain of influenza A virus is selected from the group consisting of H1N1 and H3N2.

[0085] In some embodiments, the strain of influenza A virus is selected from the group consisting of: A / Beijing / 262 / 95(H1N1)-like virus, A / New Caledonia / 20 / 99(H1N1)-like virus, A / Solomon Islands / 3 / 2006 (H1N1)-like virus, A / Brisbane / 59 / 2007 (H1N1)-like virus, 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 / California / 7 / 2009 (H1N1)-like virus, A / California / 7 / 2009 (H1N1)pdm09-like virus, A / Michigan / 45 / 2015 (H1N1)pdm09-like virus, A / Victoria / 2570 / 2019 (H1N1)pdm09-like virus, A / Wisconsin / 588 / 2019 (H1N1)pdm09-like virus, A / Guangdong-Maonan / SWL1536 / 2019 (H1N1)pdm09-like virus, A / Hawaii / 70 / 2019 (H1N1)pdm09-like virus, A / Brisbane / 02 / 2018 (H1N1)pdm09-like virus, A / Christchurch / 16 / 2010, A / South Dakota / 6 / 2007, 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)-like virus, A / Hiroshima / 52 / 2005, A / Brisbane / 10 / 2007 (H3N2)-like viruses, A / Uruguay / 716 / 2007, A / Perth / 16 / 2009(H3N2)-like viruses, A / Wisconsin / 15 / 2009, A / Victoria / 210 / 2009, A / Victoria / 361 / 2011 (H3N2)-like viruses, A / Ohio / 2 / 2012, A / Maryland / 2 / 2012, A / South Australia / 30 / 2012, A / Brisbane / 1 / 2012, A / Brisbane / 6 / 2012, A(H3N2) viruses antigenically similar to the cytotrophic prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012 (H3N2)-like viruses, A / Darwin / 9 / 2021 (H3N2)-like viruses, A / Darwin / 6 / 2021 (H3N2)-like viruses, and 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 / Hong Kong / 4801 / 2014 (H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016 (H3N2)-like virus, A / Switzerland / 8060 / 2017 (H3N2)-like virus, A / Kansas / 14 / 2017 (H3N2)-like virus, and A / South Australia / 34 / 2019 (H3N2)-like viruses.

[0086] In some embodiments, the strain of influenza A virus is H1N1.

[0087] In some embodiments, the strain of influenza A H1N1 virus is 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, A / Solomon Islands / 3 / 2006 (H1N1)-like virus, A / Brisbane / 59 / 2007 (H1N1)-like virus, A / California / 7 / 2009 (H1N1)-like virus, A / California / 7 / 2009 (H1N1)pdm09-like viruses, A / Michigan / 45 / 2015 (H1N1)pdm09-like viruses, A / Victoria / 2570 / 2019 (H1N1)pdm09-like viruses, A / Wisconsin / 588 / 2019 (H1N1)pdm09-like viruses, A / Guangdong-Maonan / SWL1536 / 2019 (H1N1)pdm09-like viruses, A / Hawaii / 70 / 2019 (H1N1)pdm09-like viruses, A / Brisbane / 02 / 2018 (H1N1)pdm09-like viruses, A / Christchurch / 16 / 2010, and A / South Dakota / 6 / 2007.

[0088] In some embodiments, the strain of influenza A virus is H3N2.

[0089] In some embodiments, the strain of influenza A H3N2 virus is selected from the group consisting of: 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)-like virus, A / Hiroshima / 52 / 2005, A / Brisbane / 10 / 2007 (H3N2)-like viruses, A / Uruguay / 716 / 2007, A / Perth / 16 / 2009 (H3N2)-like viruses, A / Wisconsin / 15 / 2009, A / Victoria / 210 / 2009,A / Victoria / 361 / 2011 (H3N2)-like viruses, A / Ohio / 2 / 2012, A / Maryland / 2 / 2012, A / South Australia / 30 / 2012, A / Brisbane / 1 / 2012, A / Brisbane / 6 / 2012, A(H3N2) viruses that are antigenically similar to the cytotrophic prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012 (H3N2)-like viruses, A / Darwin / 9 / 2021 (H3N2)-like viruses, A / Darwin / 6 / 2021 (H3N2)-like viruses, A / Cambodia / e0826360 / 2020 (H3N2)-like viruses, A / Hong Kong / 2671 / 2019 (H3N2)-like viruses, 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 / Hong Kong / 4801 / 2014 (H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016 (H3N2)-like virus, A / Switzerland / 8060 / 2017 (H3N2)-like virus, A / Kansas / 14 / 2017 (H3N2)-like virus, and A / South Australia / 34 / 2019 (H3N2)-like virus.

[0090] In some embodiments, the strain of influenza A virus is selected from the influenza A viruses listed in Table 1 and / or Table 2.

[0091] In some embodiments, the strain of influenza A virus is selected from influenza A viruses recommended by the WHO for influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0092] In some embodiments, the strain of influenza virus in (c) is an influenza B virus strain.

[0093] In some embodiments, the strain of influenza B virus is selected from the group consisting of the B / Victoria lineage and the B / Yamagata lineage.

[0094] In some embodiments, the strain of influenza B virus is selected from the group consisting of B / Beijing / 184 / 93-like virus, B / Harbin / 94-like virus, B / Shangdong / 7 / 97-like virus, B / Yamanashi / 166 / 98-like virus, B / Sichuan / 379 / 99-like virus, B / Guangdong / 120 / 2000, B / Johannesburg / 5 / 99, B / Victoria / 504 / 2000, B / Hong Kong / 330 / 2001-like virus, B / Hong Kong / 1434 / 2002, B / Brisbane / 32 / 2002, B / Shanghai / 361 / 2002-like virus, B / Jiangsu / 10 / 2003, B / Jilin / 20 / 2003, B / Malaysia / 2506 / 2004-like virus, B / Malaysia / 2506 / 2004 virus, B / Ohio / 1 / 2005, B / Florida / 4 / 2006-like virus, B / Brisbane / 3 / 2007, B / Brisbane / 60 / 2008-like virus, B / Brisbane / 33 / 2008, B / Wisconsin / 1 / 2010-like virus, B / Hubei-Wujiagang / 158 / 2009, B / Texas / 6 / 2011, B / Massachusetts / 2 / 2012-like viruses, B / Phuket / 3073 / 2013-like viruses, B / Austria / 1359417 / 2021-like viruses, B / Washington / 02 / 2019-like viruses, and B / Colorado / 06 / 2017-like viruses.

[0095] In some embodiments, the strain of influenza B virus is selected from the influenza B viruses listed in Table 1 and / or Table 2.

[0096] In some embodiments, the strain of influenza B virus is selected from influenza B viruses recommended by the WHO for influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0097] In some embodiments, the at least one additional antigen comprises, or consists of, a peptide or protein selected from or derived from influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, and / or polymerase basic protein 2 (PB2), or an immunogenic fragment or immunogenic variant thereof.

[0098] In some embodiments, the at least one further antigen comprises or consists of a peptide or protein selected from or derived from influenza virus hemagglutinin (HA) or neuraminidase (NA), or an immunogenic fragment or immunogenic variant thereof.

[0099] In some embodiments, the immunogenic composition comprises a combination of an HA antigen or a nucleic acid, preferably an mRNA, encoding said HA antigen, and said at least one further antigen comprises or consists of a peptide or protein selected or derived from influenza virus HA or a fragment or variant thereof.

[0100] In some embodiments, the immunogenic composition comprises a combination of an HA antigen and an NA antigen, or nucleic acid, preferably mRNA, encoding said HA antigen and said NA antigen, and said at least one further antigen comprises or consists of a peptide or protein selected or derived from influenza virus NA or a fragment or variant thereof.

[0101] Like HA, neuraminidase (NA) is a major surface glycoprotein of influenza viruses. Naturally acquired or vaccine-induced NA-inhibiting (NAI) antibodies have been shown to contribute to the prevention of influenza disease in naturally occurring influenza and in experimental human challenge studies. Compared with hemagglutinin-inhibiting antibodies, NAI antibodies appear to play an independent role in vaccine efficacy / effectiveness. Antigenic drift between HA and NA has been reported to be independent, suggesting that NA-specific immunity is likely to provide some level of protection in the event of HA drift.

[0102] In some embodiments, the HA antigen is a polypeptide comprising a full-length influenza HA protein. Suitably, the HA antigen is a polypeptide consisting of a full-length influenza HA protein.

[0103] In some embodiments, the HA antigen is a fragment of a hemagglutinin protein, such as a truncated hemagglutinin protein. In some embodiments, the fragment is a head-free hemagglutinin, meaning that the fragment does not include the head domain. In some embodiments, the fragment includes a portion of the head domain. In some embodiments, the fragment is the stalk domain. In some embodiments, the fragment does not include the cytoplasmic domain. In some embodiments, the fragment does not include the transmembrane domain. In such embodiments, the fragment may be referred to as a soluble or secreted hemagglutinin protein or fragment.

[0104] In some embodiments, the NA antigen is a polypeptide comprising a full-length influenza NA protein. Preferably, the NA antigen is a polypeptide consisting of a full-length influenza NA protein.

[0105] In some embodiments, the NA antigen is a fragment of a neuraminidase protein, such as a truncated neuraminidase protein.

[0106] In some embodiments, the HA and NA antigens, or the nucleic acids, preferably mRNAs, encoding said HA and NA antigens, are present in an equimolar ratio.

[0107] In some embodiments, the HA and NA antigens, or the nucleic acids, preferably mRNAs, encoding said HA and NA antigens, are not present in an equimolar ratio.

[0108] In some embodiments, the dose (e.g., weight dose or molar dose, preferably weight dose) of the at least one NA antigen or nucleic acid encoding same, preferably mRNA, is different compared to the dose (e.g., weight dose or molar dose, preferably weight dose) of the HA antigen or nucleic acid encoding said HA antigen, preferably mRNA.

[0109] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is comprised between 4:1 and 1:4, preferably between 3:1 and 1:3, preferably between 2:1 and 2:1.

[0110] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is 4:1 or 1:4.

[0111] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is 3:1 or 1:3.

[0112] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is 2:1 or 1:2.

[0113] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids encoding them, preferably mRNA, is 3:2 or 2:3.

[0114] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is 4:3 or 3:4.

[0115] In some embodiments, the ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is about 1: 1. In some embodiments, the dosage ratio of HA:NA antigens, or the nucleic acids, preferably mRNAs, encoding them, is 1:1.

[0116] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0117] In some embodiments, the HA in the ratio of HA:NA antigen or nucleic acid, preferably mRNA, encoding same is HA from a strain of influenza A virus, preferably H1N1 and / or H3N2.

[0118] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-44, or a fragment thereof.

[0119] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1-44, or a fragment thereof.

[0120] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth 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, or 43, or a fragment or variant thereof.

[0121] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth 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 or 43, or a fragment or variant thereof.

[0122] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 17, or 35, or a fragment or variant thereof.

[0123] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 17, or 35, or a fragment or variant thereof.

[0124] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 9, 11, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, or 43, or a fragment or variant thereof.

[0125] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 9, 11, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41 or 43, or a fragment or variant thereof.

[0126] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 19, 23, 27, 29, 39, 41, or 43, or a fragment or variant thereof.

[0127] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 19, 23, 27, 29, 39, 41, or 43, or a fragment or variant thereof.

[0128] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 9, 13, 15, 21, 25, 31, 33, or 37, or a fragment or variant thereof.

[0129] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 3, 9, 13, 15, 21, 25, 31, 33, or 37, or a fragment or variant thereof.

[0130] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth 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, or 44, or a fragment or variant thereof.

[0131] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth 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, or a fragment or variant thereof.

[0132] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 18, 36, or a fragment or variant thereof.

[0133] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 18, 36, or a fragment or variant thereof.

[0134] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 20, 24, 28, 30, 40, 42, or 44, or a fragment or variant thereof.

[0135] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 20, 24, 28, 30, 40, 42, or 44, or a fragment or variant thereof.

[0136] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 4, 10, 14, 16, 22, 26, 32, 34, 38, or a fragment or variant thereof.

[0137] In some embodiments, the at least one additional antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4, 10, 14, 16, 22, 26, 32, 34, 38, or a fragment or variant thereof.

[0138] In some embodiments, the composition is a multivalent composition, and said strain of influenza virus in (a) and / or said strain of influenza virus in (b) and / or said strain of influenza virus in (c) are different.

[0139] In some embodiments, the strain of influenza virus in (c) is a strain of influenza A virus, and the ratio of (a):(b):(c) is comprised between 1.5:1:1 and 5:1:1, optionally between 2:1:1 and 5:1:1, optionally between 3:1:1 and 5:1:1, optionally between 4:1:1 and 5:1:1, optionally between 1.5:1:1 and 4:1:1, optionally between 1.5:1:1 and 3:1:1, optionally between 2:1:1 and 4:1:1, optionally between 2:1:1 and 3:1:1.

[0140] In some embodiments, the strain of influenza virus in (c) is a strain of influenza A virus, and the ratio of (a):(b):(c) is selected from about 1.5:1:1, about 2:1:1, about 2.2:1:1, about 2.4:1:1, about 2.6:1:1, about 2.8:1:1, about 3:1:1, about 3.2:1:1, about 3.4:1:1, about 3.6:1:1, about 3.8:1:1, about 4:1:1, about 4.2:1:1, about 4.4:1:1, about 4.6:1:1, about 4.8:1:1, or about 5:1:1.

[0141] In some embodiments, the strain of influenza virus in (c) is a strain of influenza A virus, and the ratio of (a):(b):(c) is selected from about 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, or 5:1:1.

[0142] In some embodiments, the strain of influenza virus in (c) is a strain of influenza A virus, and the ratio of (a):(b):(c) is comprised between 2:1:1 and 4:1:1, preferably between 2:1:1 and 3:1:1, and preferably is 2:1:1 or 3:1:1.

[0143] In some embodiments, (c) is a third HA antigen or a third nucleic acid, preferably mRNA, encoding a third HA antigen, wherein the third HA antigen is from a second strain of influenza A virus, preferably H3N2.

[0144] In some embodiments, (c) is a third HA antigen, or a third nucleic acid, preferably mRNA, encoding a third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, preferably H3N2, and the ratio of (a):(b):(c) is comprised between 1.5:1:1 and 5:1:1, optionally between 2:1:1 and 5:1:1, optionally between 3:1:1 and 5:1:1, optionally between 4:1:1 and 5:1:1, optionally between 1.5:1:1 and 4:1:1, optionally between 1.5:1:1 and 3:1:1, 2:1:1 and 4:1:1, preferably between 2:1:1 and 3:1:1, and preferably is 2:1:1 or 3:1:1.

[0145] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0146] In some embodiments, the immunogenic composition comprises: (a) a first hemagglutinin (HA) antigen from a first strain of influenza B virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; (b) a second HA antigen from a first strain of influenza A virus, preferably from H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen; and (c) at least one further antigen derived from a strain of influenza virus, or at least one further nucleic acid, preferably mRNA, encoding at least one further antigen Including, wherein (a), (b) and (c) are different and the ratio of (a):(b):(c) is 1.5:1:1 to 5:1:1, optionally 2:1:1 to 5:1:1, optionally 3:1:1 to 5:1:1, optionally 4:1:1 to 5:1:1, optionally 1.5:1:1 to 4:1:1, optionally 1.5:1:1 to 3:1:1, 2:1:1 to 4:1:1, preferably 2:1:1 to 3:1:1, and preferably 2:1:1 or 3:1:1.

[0147] In some embodiments, the immunogenic composition comprises: (a) a first hemagglutinin (HA) antigen from a first strain of influenza B virus, preferably from the B / Victoria lineage, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; (b) a second HA antigen from a first strain of influenza A virus, preferably from H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen; and (c) a third HA antigen from a second strain of influenza A virus, preferably from H3N2, or a third nucleic acid, preferably mRNA, encoding the third HA antigen. Including, wherein (a), (b) and (c) are different and the ratio of (a):(b):(c) is 1.5:1:1 to 5:1:1, optionally 2:1:1 to 5:1:1, optionally 3:1:1 to 5:1:1, optionally 4:1:1 to 5:1:1, optionally 1.5:1:1 to 4:1:1, optionally 1.5:1:1 to 3:1:1, 2:1:1 to 4:1:1, preferably 2:1:1 to 3:1:1, and preferably 2:1:1 or 3:1:1.

[0148] In some embodiments, the immunogenic composition comprises a plurality of (c), e.g., (c) as defined herein. 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) is included.

[0149] In some embodiments, the composition comprises at least 4, 5, 6, 7 or 8 antigens or nucleic acids encoding them, preferably mRNA, optionally 4 to 10 antigens or nucleic acids encoding them, preferably mRNA, optionally 4, 7 or 8 antigens or nucleic acids encoding them, preferably mRNA.

[0150] In some embodiments, the antigens of (a), (b) and / or (c) are derived from at least two, three, or four strains of influenza virus.

[0151] In some embodiments, the composition comprises four antigens, or nucleic acids encoding them, preferably mRNA.

[0152] In some embodiments, the immunogenic composition comprises a combination of four HA antigens, or a combination of four nucleic acids, preferably mRNAs, encoding said four HA antigens.

[0153] In some embodiments, the immunogenic composition comprises: (a) and (b) as defined herein (preferably, (a) is a first hemagglutinin (HA) antigen from a first strain of influenza B virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen, and / or (b) is a second HA antigen from a first strain of influenza A virus, preferably H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen), (c 1 a third HA antigen from a second strain of influenza A virus, preferably from H3N2, or a third nucleic acid, preferably mRNA, encoding the third HA antigen; (c 2 a fourth HA antigen from a second strain of influenza B virus, or a fourth nucleic acid, preferably mRNA, encoding the fourth HA antigen. Including, wherein the ratio of (a):(b) is comprised between 1.5:1 and 5:1, optionally between 2:1 and 5:1, optionally between 3:1 and 5:1, optionally between 4:1 and 5:1, optionally between 1.5:1 and 4:1, optionally between 1.5:1 and 3:1, 2:1 and 4:1, preferably between 2:1 and 3:1, and is preferably 2:1 or 3:1.

[0154] In some embodiments, the immunogenic composition comprises: (a) and (b) as defined herein (preferably, (a) is a first hemagglutinin (HA) antigen from a first strain of influenza B virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen, and / or (b) is a second HA antigen from a first strain of influenza A virus, preferably H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen), (c 1 a third HA antigen from a second strain of influenza A virus, preferably from H3N2, or a third nucleic acid, preferably mRNA, encoding the third HA antigen; (c 2a fourth HA antigen from a second strain of influenza B virus, or a fourth nucleic acid, preferably mRNA, encoding the fourth HA antigen. Including, where (a):(b):(c 1 ) is comprised between 1.5:1:1 and 5:1:1, optionally between 2:1:1 and 5:1:1, optionally between 3:1:1 and 5:1:1, optionally between 4:1:1 and 5:1:1, optionally between 1.5:1:1 and 4:1:1, optionally between 1.5:1:1 and 3:1:1, 2:1:1 and 4:1:1, preferably between 2:1:1 and 3:1:1, and preferably is 2:1:1 or 3:1:1.

[0155] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0156] In some embodiments, the immunogenic composition comprises: (a) and (b) as defined herein (preferably, (a) is a first hemagglutinin (HA) antigen from a first strain of influenza B virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen, and / or (b) is a second HA antigen from a first strain of influenza A virus, preferably H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen), (c 1 a third HA antigen from a second strain of influenza A virus, preferably from H3N2, or a third nucleic acid, preferably mRNA, encoding the third HA antigen; (c 2 a fourth HA antigen from a second strain of influenza B virus, or a fourth nucleic acid, preferably mRNA, encoding the fourth HA antigen. Including, Here, (a), (b), (c 1 ) and (c 2 ) are different, (a):(b):(c 1 ):(c 2 ) ratio is in the range of 1.5:1:1:1.5 to 5:1:1:5.

[0157] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0158] In some embodiments, the immunogenic composition comprises: (a) a first hemagglutinin (HA) antigen from a first strain of influenza B virus, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; (b) a second HA antigen from a first strain of influenza A virus, preferably from H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen; (c 1 a third HA antigen from a second strain of influenza A virus, preferably from H3N2, or a third nucleic acid, preferably mRNA, encoding the third HA antigen; (c 2 a fourth HA antigen from a second strain of influenza B virus, or a fourth nucleic acid, preferably mRNA, encoding the fourth HA antigen. Including, Here, (a), (b), (c 1 ) and (c 2 ) are different, (a):(b):(c 1 ):(c 2 ) ratio is in the range of 1.5:1:1:1.5 to 5:1:1:5.

[0159] In some embodiments, (a):(b):(c 1 ):(c 2 ) is comprised within the range of 1.5:1:1:1.5 to 5:1:1:5, optionally 2:1:1:2 to 5:1:1:5, optionally 3:1:1:3 to 5:1:1:5, optionally 4:1:1:4 to 5:1:1:5, optionally 1.5:1:1:1.5 to 4:1:1:4, optionally 1.5:1:1:1.5 to 3:1:1:3, optionally 2:1:1:2 to 4:1:1:4, optionally 2:1:1:2 to 3:1:1:3.

[0160] In some embodiments, (a):(b):(c 1 ):(c 2) is about 1.5:1:1:1.5, about 2:1:1:2, about 2.2:1:1:2.2, about 2.4:1:1:2.4, about 2.6:1:1:2.6, about 2.8:1:1:2.8, about 3:1:1:3, about 3.2:1:1:3.2, about 3.4:1:1:3.4, about 3.6:1:1:3.6, about 3.8:1:1:3.8, about 4:1:1:4, about 4.2:1:1:4.2, about 4.4:1:1:4.4, about 4.6:1:1:4.6, about 4.8:1:1:4.8, or about 5:1:1:5. In some embodiments, the ratio of (a):(b):(c 1 ):(c 2 ) is about 1.5:1:1:1.5, 2:1:1:2, 2.2:1:1:2.2, 2.4:1:1:2.4, 2.6:1:1:2.6, 2.8:1:1:2.8, 3:1:1:3, 3.2:1:1:3.2, 3.4:1:1:3.4, 3.6:1:1:3.6, 3.8:1:1:3.8, 4:1:1:4, 4.2:1:1:4.2, 4.4:1:1:4.4, 4.6:1:1:4.6, 4.8:1:1:4.8 or 5:1:1:5.

[0161] In some embodiments, (a):(b):(c 1 ):(c 2 ) is in the range of 2:1:1:2 to 4:1:1:4, preferably in the range of 2:1:1:2 to 3:1:1:3, and more preferably 2:1:1:2 or 3:1:1:3.

[0162] In some embodiments, the composition comprises seven antigens, or nucleic acids encoding them, preferably mRNA.

[0163] In some embodiments, the immunogenic composition comprises a combination of four HA antigens or four nucleic acids, preferably mRNAs, encoding said four HA antigens, and three NA antigens or three nucleic acids, preferably mRNAs, encoding said three NA antigens.

[0164] In some embodiments, the immunogenic composition further comprises: (c 3) a first NA antigen from a first strain of influenza A virus, or a first nucleic acid, preferably mRNA, encoding the first NA antigen; (c 4 ) a second NA antigen from a second strain of influenza A virus, or a second nucleic acid, preferably mRNA, encoding the second NA antigen; and (c 5 a third NA antigen from a first strain of influenza B virus, or a third nucleic acid, preferably mRNA, encoding the third NA antigen; Includes.

[0165] In some embodiments, the immunogenic composition further comprises: (c 3 ) a first NA antigen from a first strain of influenza A virus, or a first nucleic acid, preferably mRNA, encoding the first NA antigen; (c 4 a second NA antigen from a second strain of influenza A virus, or a second nucleic acid, preferably mRNA, encoding the second NA antigen; (c 5 a third NA antigen from a first strain of influenza B virus, or a third nucleic acid, preferably mRNA, encoding the third NA antigen; Including, Here, (a), (b), (c 1 ), (c 2 ), (c 3 ), (c 4 ) and (c 5 ) are different, (a):(b):(c 1 ):(c 2 ) ratio is in the range of 1.5:1:1:1.5 to 5:1:1:5.

[0166] In some embodiments, (a):(b):(c 1 ):(c 2) is comprised within the range of 1.5:1:1:1.5 to 5:1:1:5, optionally 2:1:1:2 to 5:1:1:5, optionally 3:1:1:3 to 5:1:1:5, optionally 4:1:1:4 to 5:1:1:5, optionally 1.5:1:1:1.5 to 4:1:1:4, optionally 1.5:1:1:1.5 to 3:1:1:3, optionally 2:1:1:2 to 4:1:1:4, optionally 2:1:1:2 to 3:1:1:3.

[0167] In some embodiments, (a):(b):(c 1 ):(c 2 ) is about 1.5:1:1:1.5, about 2:1:1:2, about 2.2:1:1:2.2, about 2.4:1:1:2.4, about 2.6:1:1:2.6, about 2.8:1:1:2.8, about 3:1:1:3, about 3.2:1:1:3.2, about 3.4:1:1:3.4, about 3.6:1:1:3.6, about 3.8:1:1:3.8, about 4:1:1:4, about 4.2:1:1:4.2, about 4.4:1:1:4.4, about 4.6:1:1:4.6, about 4.8:1:1:4.8, or about 5:1:1:5. In some embodiments, the ratio of (a):(b):(c 1 ):(c 2 ) is about 1.5:1:1:1.5, 2:1:1:2, 2.2:1:1:2.2, 2.4:1:1:2.4, 2.6:1:1:2.6, 2.8:1:1:2.8, 3:1:1:3, 3.2:1:1:3.2, 3.4:1:1:3.4, 3.6:1:1:3.6, 3.8:1:1:3.8, 4:1:1:4, 4.2:1:1:4.2, 4.4:1:1:4.4, 4.6:1:1:4.6, 4.8:1:1:4.8 or 5:1:1:5.

[0168] In some embodiments, (a):(b):(c 1 ):(c 2 ) is in the range of 2:1:1:2 to 4:1:1:4, preferably in the range of 2:1:1:2 to 3:1:1:3, and more preferably 2:1:1:2 or 3:1:1:3.

[0169] In some embodiments, (a):(b):(c 1 ):(c 2 ):(c 3):(c 4 ):(c 5 ) is comprised between 9:3:3:9:1:1:1 and 3:1:1:3:3:3:3, preferably between 6:2:2:6:1:1:1 and 3:1:1:3:2:2:2, and is preferably 6:2:2:6:1:1:1 or 3:1:1:3:2:2:2.

[0170] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0171] In some embodiments, the composition comprises eight antigens, or nucleic acids, preferably mRNAs, encoding them.

[0172] In some embodiments, the immunogenic composition comprises a combination of four HA antigens or four nucleic acids, preferably mRNAs, encoding said four HA antigens, and four NA antigens or four nucleic acids, preferably mRNAs, encoding said four NA antigens.

[0173] In some embodiments, the composition further comprises: (c 6 a fourth NA antigen from a second strain of influenza B virus, or a fourth nucleic acid, preferably mRNA, encoding the fourth NA antigen; Includes.

[0174] In some embodiments, the composition comprises: (c 6 a fourth NA antigen from a second strain of influenza B virus, or a fourth nucleic acid, preferably mRNA, encoding the fourth NA antigen; Including, Here, (a), (b), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and (c 6 ) are different, (a):(b):(c 1 ):(c 2 ) ratio is in the range of 1.5:1:1:1.5 to 5:1:1:5.

[0175] In some embodiments, (a):(b):(c 1 ):(c 2 ) is in the range of 1.5:1:1:1.5 to 5:1:1:5, optionally 2:1:1:2 to 5:1:1:5, optionally 3:1:1:3 to 5:1:1:5, optionally 4:1:1:4 to 5:1:1:5, optionally 1.5:1:1:1.5 to 4:1:1:4, optionally 1.5:1:1:1.5 to 3:1:1:3, optionally 2:1:1:2 to 4:1:1:4, optionally 2:1:1:2 to 3:1:1:3.

[0176] In some embodiments, (a):(b):(c 1 ):(c 2 ) is about 1.5:1:1:1.5, about 2:1:1:2, about 2.2:1:1:2.2, about 2.4:1:1:2.4, about 2.6:1:1:2.6, about 2.8:1:1:2.8, about 3:1:1:3, about 3.2:1:1:3.2, about 3.4:1:1:3.4, about 3.6:1:1:3.6, about 3.8:1:1:3.8, about 4:1:1:4, about 4.2:1:1:4.2, about 4.4:1:1:4.4, about 4.6:1:1:4.6, about 4.8:1:1:4.8, or about 5:1:1:5. In some embodiments, the ratio of (a):(b):(c 1 ):(c 2 ) is about 1.5:1:1:1.5, 2:1:1:2, 2.2:1:1:2.2, 2.4:1:1:2.4, 2.6:1:1:2.6, 2.8:1:1:2.8, 3:1:1:3, 3.2:1:1:3.2, 3.4:1:1:3.4, 3.6:1:1:3.6, 3.8:1:1:3.8, 4:1:1:4, 4.2:1:1:4.2, 4.4:1:1:4.4, 4.6:1:1:4.6, 4.8:1:1:4.8 or 5:1:1:5.

[0177] In some embodiments, (a):(b):(c 1 ):(c 2 ) is comprised between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and is preferably 2:1:1:2 or 3:1:1:3.

[0178] In some embodiments, (a):(b):(c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ):(c 6 ) is comprised between 9:3:3:9:1:1:1:1 and 3:1:1:3:3:3:3, preferably between 6:2:2:6:1:1:1:1 and 3:1:1:3:2:2:2:2, and more preferably is 6:2:2:6:1:1:1:1 or 3:1:1:3:2:2:2.

[0179] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0180] In some embodiments, the composition comprises six antigens, or the nucleic acids, preferably mRNA, encoding them.

[0181] In some embodiments, the immunogenic composition comprises a combination of three HA antigens or three nucleic acids, preferably mRNAs, encoding said three HA antigens, and three NA antigens or three nucleic acids, preferably mRNAs, encoding said three NA antigens.

[0182] In some embodiments, the immunogenic composition comprises: (a) a first hemagglutinin (HA) antigen from a first strain of influenza B virus, preferably from the B / Victoria lineage, or a first nucleic acid, preferably mRNA, encoding the first HA antigen; (b) a second HA antigen from a first strain of influenza A virus, preferably from H1N1, or a second nucleic acid, preferably mRNA, encoding the second HA antigen; (c 1 a third HA antigen from a second strain of influenza A virus, preferably from H3N2, or a third nucleic acid, preferably mRNA, encoding the third HA antigen; (c 3) a first NA antigen derived from a first strain of influenza A virus, preferably derived from H1N1, or a first nucleic acid, preferably mRNA, encoding the first NA antigen; (c 4 ) a second NA antigen from a second strain of influenza A virus, preferably from H3N2, or a second nucleic acid, preferably mRNA, encoding the second NA antigen; and (c 5 a third NA antigen derived from a first strain of influenza B virus, preferably from the B / Victoria lineage, or a third nucleic acid, preferably mRNA, encoding the third NA antigen; Including, Here, (a), (b), (c 1 ), (c 3 ), (c 4 ) and (c 5 ) are different, (a):(b):(c 1 ) ratio is included in the range of 1.5:1:1 to 5:1:1.

[0183] It should be noted that certain features and embodiments described in the context of the first aspect of the invention, i.e. the immunogenic composition of the invention, are equally applicable to the second aspect (the vaccine of the invention), the third aspect (the kit or kit-of-parts of the invention) or further aspects, including e.g. medical uses (first and second medical uses) and e.g. methods of treatment.

[0184] nucleic acid In some embodiments, at least one nucleic acid of the immunogenic composition, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 In some embodiments, the nucleic acid of at least one of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j ... 1 ), (c 2 ), (c 3 ), (c 4 ), (c5 ) and / or (c 6 ) nucleic acid is DNA.

[0185] In some embodiments, at least one nucleic acid of the immunogenic composition, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The nucleic acid of is an artificial nucleic acid, such as an artificial DNA or an artificial RNA, preferably an mRNA.

[0186] Nucleic acid-based vaccination, including DNA or RNA, preferably mRNA, is one of the promising technologies for novel vaccines against emerging viruses and for providing combination vaccines. Nucleic acids can be genetically manipulated and administered to human subjects. Transfected cells directly produce the encoded antigen (e.g., provided by DNA or RNA, particularly mRNA), which elicits a protective immune response.

[0187] The nucleic acids according to the invention, such as DNA or RNA, preferably mRNA, form the basis of a nucleic acid-based immunogenic composition or a nucleic acid-based vaccine.

[0188] 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.

[0189] In general, protein-based or live-attenuated vaccines are not optimal for use in developing countries due to their high production costs. Protein-based or live-attenuated vaccines also require long development times, making them unsuitable for rapid response to pandemic virus outbreaks, such as influenza virus outbreaks. Indeed, because traditional methods for producing standard inactivated influenza vaccines have long development times, GISRS recommendations are made 6 to 7 months before the start of the influenza season, during which time influenza viruses may continue to evolve.

[0190] In contrast, nucleic acid-based immunogenic compositions and vaccines according to the present invention allow for very rapid and cost-effective production. Thus, compared to known vaccines, nucleic acid-based compositions / vaccines can be produced and manufactured significantly more cheaply and quickly, which is highly advantageous, particularly for use in developing countries or in situations of annual epidemics or global pandemics. Nucleic acid-based compositions / vaccines provide GISRS with additional time to monitor circulating viruses and make GISRS recommendations closer to the influenza season. This extension of the GISRS monitoring timeline should allow GISRS predictions to become more accurate, resulting in more effective vaccines designed to target circulating viruses closer to the influenza season. Furthermore, to ensure or enhance the effectiveness of the immune response against influenza viruses, different nucleic acids encoding different antigens (e.g., antigens of different influenza strains) can be combined in a single immunogenic composition / vaccine.

[0191] The use of RNA, preferably mRNA, in or as a vaccine overcomes the drawbacks of conventional genetic vaccination, which involves the introduction of DNA into cells, in terms of safety, feasibility, applicability, and effectiveness in generating an immune response. RNA molecules, preferably mRNA, are considered significantly safer than DNA vaccines because RNA, preferably mRNA, is more easily degraded. They are rapidly eliminated from the organism and do not integrate into the genome or affect cellular gene expression in an uncontrolled manner. RNA, preferably mRNA vaccines are also less likely to cause serious side effects, such as autoimmune diseases or the production of anti-DNA antibodies. Transfection with RNA, preferably mRNA, simply involves insertion into the cell's cytoplasm, which is more easily achieved than insertion into the nucleus.

[0192] In some embodiments, at least one nucleic acid of the immunogenic composition, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) is RNA.

[0193] Thus, in some embodiments, (a) is a first RNA encoding a first HA antigen, and / or (b) is a second RNA encoding a second HA antigen.

[0194] In some embodiments, (c) is at least one additional RNA encoding at least one additional antigen.

[0195] In some embodiments, the immunogenic composition comprises a plurality of (c) that is RNA.

[0196] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5) and / or (c 6 ) is RNA.

[0197] Messenger RNA (mRNA) is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by ribosomes during the production of proteins. mRNA vaccines can utilize non-replicating mRNA or self-replicating RNA (also called self-amplifying mRNA or SAM). Non-replicating mRNA-based vaccines 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 machinery that allows for intracellular RNA amplification.

[0198] mRNA-based influenza vaccine candidates are currently in clinical trials. For example, mRNA-1010 is an mRNA vaccine candidate encoding the HA glycoproteins of four influenza strains recommended by the WHO for influenza prevention. A Phase I study evaluated mRNA-1010 at equimolar dose levels of 50, 100, and 200 μg in cohorts of young adults and elderly subjects.

[0199] In some embodiments, at least one nucleic acid of the immunogenic composition, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) is mRNA.

[0200] In some embodiments, (a) is a first mRNA encoding a first HA antigen, and / or (b) is a second mRNA encoding a second HA antigen.

[0201] In some embodiments, the dose of each of said first mRNA and / or said second mRNA is 1 to 200 μg, preferably 1 to 60 μg, preferably 2 to 25 μg.

[0202] In some embodiments, the dose of each of the first mRNA and / or the second mRNA 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, optionally 3-6 μg.

[0203] In some embodiments, the dose of each of said first mRNA and / or said second mRNA 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, optionally 2, 3, 6, 9 or 18 μg.

[0204] In some embodiments, the dose of each of said first mRNA and / or said second mRNA is 3, 6, 9, 12, or 18 μg.

[0205] In some embodiments, (c) is at least one additional mRNA encoding at least one additional antigen.

[0206] In some embodiments, the dose of each of said at least one further mRNA is between 1 and 200 μg, preferably between 1 and 60 μg, preferably between 2 and 25 μg.

[0207] In some embodiments, the dose of each of said at least one additional mRNA is between 2 and 25 μg, optionally between 2 and 18 μg, optionally between 2 and 9 μg, optionally between 2 and 6 μg, optionally between 3 and 25 μg, 3 and 18 μg, 3 and 9 μg, optionally between 3 and 6 μg.

[0208] In some embodiments, the dose of each of the at least one additional mRNA 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, optionally 2, 3, 6, 9 or 18 μg.

[0209] In some embodiments, the dose of each of the at least one additional mRNA is 3, 6, 9, 12, or 18 μg.

[0210] In some embodiments, the immunogenic composition comprises a plurality of (c) that is mRNA.

[0211] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) is mRNA.

[0212] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The dose of ) is 1 to 200 μg, preferably 1 to 60 μg, preferably 1 to 25 μg, preferably 2 to 25 μg.

[0213] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) is administered in a dose of 1 to 25 μg, optionally 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, or optionally 3 to 6 μg.

[0214] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6) 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, optionally 1, 2, 3, 6, 9 or 18 μg.

[0215] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) doses are 1, 2, 3, 6, 9, 12 or 18 μg.

[0216] Also, (a) a first mRNA encoding the HA of a first strain of influenza B virus; (b) a second mRNA encoding the HA of a first strain of influenza A virus, preferably H1N1; (c 1 ) a third mRNA encoding the HA of a second strain of influenza A virus, preferably H3N2; and (c 2 ) A fourth mRNA encoding the HA of a second strain of influenza B virus An immunogenic composition comprising: (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and preferably is 2:1:1:2 or 3:1:1:3.

[0217] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Preferably, the ratio is a weight / weight ratio.

[0218] In some embodiments, (a) and (c) 2 ) is administered in a dose of 5 to 50 μg, sometimes 10 to 40 μg, and sometimes 12 to 36 μg.

[0219] In some embodiments, (a) and (c) 2 ) is 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.

[0220] In some embodiments, (b) and (c) 1 ) is administered in a dose of 2 to 20 μg, sometimes 5 to 15 μg, and sometimes 6 to 12 μg.

[0221] In some embodiments, (b) and (c) 1 ) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μg.

[0222] In some embodiments, (b) and (c) 1 ) doses are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μg.

[0223] In some embodiments, (a), (b), (c) 1 ) and (c 2 ) is administered in a dose of 5 to 75 μg, sometimes 10 to 60 μg, and sometimes 12 to 48 μg.

[0224] In some embodiments, (a), (b), (c) 1 ) and (c 2 The dose is 35-75 μg.

[0225] In some embodiments, (a), (b), (c) 1 ) and (c 2 ) doses 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.

[0226] In some embodiments, (a), (b), (c) 1 ) and (c 2) doses are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 45, 46, 47, 48, 49, 50, 55, and 60 μg.

[0227] In some embodiments, (a) and (c) 2 ) is administered at a dose of 5 to 50 μg, optionally 10 to 40 μg, optionally 12 to 36 μg, and (b) and (c) 1 ) is administered in a dose of 2 to 20 μg, sometimes 5 to 15 μg, and sometimes 6 to 12 μg.

[0228] In some embodiments, (a) and (c) 2 ) is 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 (b) and (c 1 ) doses are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μg.

[0229] In some embodiments, the immunogenic composition further comprises: (c 3 ) a first mRNA encoding the NA of a first strain of influenza A virus, preferably H1N1; (c 4 ) a second mRNA encoding the NA of a second strain of influenza A virus, preferably H3N2; and (c 5 ) a third mRNA encoding the NA of the first strain of influenza B virus Including, where (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and preferably is 2:1:1:2 or 3:1:1:3.

[0230] In some embodiments, (a):(b):(c1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ) is in the range of 9:3:3:9:1:1:1 to 3:1:1:3:3:3:3, preferably in the range of 6:2:2:6:1:1:1 to 3:1:1:3:2:2:2, and more preferably 6:2:2:6:1:1:1 or 3:1:1:3:2:2:2.

[0231] In some embodiments, (a), (b), (c) 1 ) and (c 2 ) is administered in a dose of 5 to 75 μg, sometimes 10 to 60 μg, and sometimes 12 to 48 μg.

[0232] In some embodiments, (a), (b), (c) 1 ) and (c 2 The dose is 35-75 μg.

[0233] In some embodiments, (a), (b), (c) 1 ) and (c 2 In some embodiments, the dose of (a), (b), (c) is 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. 1 ) and (c 2 ) doses are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 45, 46, 47, 48, 49, 50, 55, and 60 μg.

[0234] In some embodiments, (c 3 ), (c 4 ) and (c 5 The dose of (c) is 2 to 50 μg, optionally 2 to 30 μg, optionally 5 to 20, and optionally 9 to 18 μg. 3 ), (c 4 ) and (c 5) doses are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0235] In some embodiments, (c 3 ), (c 4 ) and (c 5 ) dose is 9-36μg.

[0236] In some embodiments, (c 3 ), (c 4 ) and (c 5 ) doses are 9, 18, 27 or 36 μg.

[0237] In some embodiments, the immunogenic composition further comprises: (c 6 ) A fourth mRNA encoding the NA of a second strain of influenza B virus Including, where (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and preferably is 2:1:1:2 or 3:1:1:3. In some embodiments, (a):(b):(c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ):(c 6 ) is in the range of 9:3:3:9:1:1:1:1 to 3:1:1:3:3:3:3, preferably 6:2:2:6:1:1:1:1 to 3:1:1:3:2:2:2:2, and more preferably 6:2:2:6:1:1:1:1 or 3:1:1:3:2:2:2.

[0238] In some embodiments, (c 3 ), (c 4 ) and (c 5 ) is administered in a dose of 5 to 50 μg, sometimes 10 to 30 μg, and sometimes 12 to 24 μg.

[0239] In some embodiments, (c 3 ), (c 4 ) and (c 5 ) doses are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 μg.

[0240] In some embodiments, (c 3 ), (c 4 ), (c 5 ) and (c 6 ) is administered in a dose of 5 to 50 μg, sometimes 10 to 50 μg, and sometimes 12 to 48 μg.

[0241] In some embodiments, (c 3 ), (c 4 ), (c 5 ) and (c 6 ) doses 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.

[0242] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The dose of ) is 1 to 200 μg, preferably 1 to 60 μg, preferably 1 to 25 μg, preferably 2 to 25 μg.

[0243] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) is administered in a dose of 1 to 25 μg, optionally 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, optionally 3 to 12 μg, optionally 3 to 9 μg, or optionally 3 to 6 μg.

[0244] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) 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, optionally 1, 2, 3, 6, 9 or 18 μg.

[0245] In some embodiments, each of (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) doses are 1, 2, 3, 6, 9, 12 or 18 μg.

[0246] As used herein, mRNA is preferably provided in a purified or substantially purified form, i.e., substantially free of proteins (e.g., enzymes), other nucleic acids (e.g., DNA and nucleoside phosphate monomers), etc., and generally has a purity of at least about 50% (by weight), usually at least 90% purity, for example, at least 95% or at least 98% purity.

[0247] As used herein, mRNA can be produced in a number of ways, for example, by chemical synthesis in whole or in part, by digesting longer nucleic acids using nucleases (e.g., restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), from genomic or cDNA libraries, etc. In particular, mRNA can be produced enzymatically using a DNA template.

[0248] As used herein, mRNA may be an artificial nucleic acid. As used herein, the term "artificial nucleic acid" is intended to refer to a nucleic acid that does not occur in nature. In other words, an artificial nucleic acid may be understood as a non-natural nucleic acid molecule. Such a nucleic acid molecule may be non-natural due to its individual sequence (e.g., G / C content modified coding sequence, UTR) and / or due to other modifications of nucleotides, such as structural modifications. Typically, an artificial nucleic acid may be designed and / or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, an artificial nucleic acid is a sequence that cannot occur in nature, i.e., a sequence that differs from a wild-type or reference sequence / naturally occurring sequence by at least one nucleotide (e.g., through a codon modification as further specified below). The term "artificial nucleic acid" is not limited to meaning "one single molecule" but is understood to include an ensemble of essentially identical nucleic acid molecules. Thus, it may refer to multiple essentially identical nucleic acid molecules.

[0249] Alternatively, or in addition, the sequence or chemical structure of the nucleic acid may be modified compared to the naturally occurring sequence encoding the antigen. The sequence of the nucleic acid molecule may be modified, for example, to increase the efficiency of expression or replication of the nucleic acid, or to provide greater stability or resistance to degradation.

[0250] Thus, the mRNA used herein may be a modified and / or stabilized nucleic acid, preferably a modified and / or stabilized artificial nucleic acid.

[0251] According to some embodiments, the mRNA used herein may therefore be provided as a "stabilized artificial nucleic acid" or "stabilized coding nucleic acid", i.e. a nucleic acid that exhibits improved resistance to in vivo degradation and / or improved stability in vivo and / or improved translatability in vivo. Below, certain preferred modifications / adaptations in this context that are suitable for "stabilizing" a nucleic acid are described.

[0252] Below, suitable modifications that are capable of "stabilizing" mRNA are described.

[0253] The mRNA used herein may also be codon-optimized. In some embodiments, the mRNA used herein comprises 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.

[0254] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA comprises a coding sequence that is a codon-modified coding sequence, wherein the amino acid sequence encoded by the codon-modified coding sequence is optionally unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0255] In some embodiments, mRNA as used herein may be codon-optimized for expression in human cells. By "codon-optimized" is intended a modification in codon usage that may increase the translation efficiency and / or half-life of a nucleic acid. The term "codon-modified coding sequence" refers to a coding sequence that differs in at least one codon (a triplet of nucleotides encoding an amino acid) compared to a corresponding wild-type or reference coding sequence. Advantageously, in the context of the present invention, the codon-modified coding sequence may exhibit improved resistance to in vivo degradation and / or improved in vivo stability and / or improved in vivo translatability. Codon modification in its broadest sense exploits the degeneracy of the genetic code, where multiple codons may encode the same amino acid and be used interchangeably (see Table 1 in WO2020002525) as outlined herein, to optimize / modify the coding sequence for in vivo applications.

[0256] In some embodiments, the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage adapted coding sequence, a G / C content modified coding sequence, and a G / C optimized coding sequence, or any combination thereof.

[0257] In some embodiments, the codon-modified coding sequence has a G / C content of at least about 45%, 50%, 55%, or 60%. In certain embodiments, at least one coding sequence of an mRNA 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%.

[0258] When transfected into a mammalian host cell, the mRNA used herein that includes a codon-modified coding sequence has a stability of 12 to 18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours, and is capable of being expressed by the mammalian host cell (e.g., muscle cell).

[0259] When transfected into a mammalian host cell, the mRNA used herein comprising a codon-modified coding sequence is translated into a protein, wherein the amount of protein is at least equivalent to, or preferably at least 10% more, or at least 20% more, or at least 30% more, or at least 40% more, or at least 50% more, or at least 100% more, or at least 200% more than, the amount of protein obtained by a naturally occurring or wild-type or reference coding sequence transfected into a mammalian host cell.

[0260] In embodiments, the mRNA used herein may be modified, wherein the C content of at least one coding sequence is increased, preferably maximized, compared to the C content of the corresponding wild-type or reference coding sequence (referred to herein as a "C-maximized coding sequence"). The amino acid sequence encoded by the C-maximized coding sequence of the 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 WO 2015 / 062738. In this context, the disclosure of WO 2015 / 062738 is incorporated herein by reference.

[0261] In some embodiments, the mRNA used herein may be modified, wherein the G / C content of at least one coding sequence may be optimized compared to the G / C content of a corresponding wild-type or reference coding sequence (referred to herein as a "G / C content-optimized coding sequence"). "Optimization" in this context refers to a coding sequence, wherein the G / C content is preferably increased to essentially the maximum G / C content possible. The amino acid sequence encoded by the G / C content-optimized coding sequence of an mRNA is preferably unmodified compared to the amino acid sequence encoded by the respective wild-type or reference coding sequence. The generation of an mRNA sequence with optimized G / C content may be carried out using the method described in WO2002 / 098443. In this context, the disclosure of WO2002 / 098443 is included in its entirety in the present invention.

[0262] In some embodiments, the mRNA used herein may be modified, wherein the codons in at least one coding sequence may be adapted to human codon usage (herein referred to as "human codon usage-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 so that the frequencies of codons encoding the same amino acid correspond to the naturally occurring frequencies of that codon according to human codon usage. For example, for the amino acid Ala, the wild-type or reference coding sequence is preferably adapted in such a way that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" is used at a frequency of 0.28, the codon "GCA" is used at a frequency of 0.22, the codon "GCG" is used at a frequency of 0.10, etc. (see, for example, Table 1 of WO2020002525). Thus, such a technique (exemplified for Ala) is adapted to each amino acid encoded by the coding sequence of the RNA to obtain a sequence adapted to human codon usage.

[0263] In embodiments, the mRNA used herein may be modified, wherein the G / C content of at least one coding sequence may be modified compared to the G / C content of a corresponding wild-type or reference coding sequence (herein referred to as a "G / C content-modified coding sequence"). In this context, the term "G / C optimized" or "G / C content-modified" refers to a nucleic acid that contains a modified, preferably increased, number of guanosine and / or cytosine nucleotides compared to a corresponding wild-type or reference coding sequence. Such an increased number may be generated by replacing codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. Preferably, a nucleic acid sequence with an increased G / C content is more stable or exhibits better expression than a sequence with an increased A / U content. The amino acid sequence encoded by a G / C content-modified coding sequence of an 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 coding sequence of the nucleic acid is increased by at least 10%, 20%, 30%, preferably at least 40% compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence.

[0264] In embodiments, the mRNA used herein may be modified such that the codon compatibility index (CAI) is increased, or preferably maximized, in at least one coding sequence (referred to herein as a "CAI-maximized coding sequence"). In some embodiments, for example, all codons of a wild-type or reference nucleic acid sequence that are relatively rare in humans are replaced with respective codons that are frequent in humans, where the frequent codons encode the same amino acids as the relatively rare codons. Preferably, the most frequent codons are used for each amino acid in 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 compatibility index (CAI) of at least one coding sequence is 1 (CAI=1). For example, for the amino acid Ala, a wild-type or reference coding sequence may be adapted in a way that the most frequent human codon "GCC" is always used for the amino acid. Thus, such an approach (exemplified for Ala) may be adapted to each amino acid encoded by the coding sequence of an mRNA to obtain a CAI-maximized coding sequence.

[0265] In embodiments, mRNAs used herein may be modified by altering the number of A and / or U nucleotides in a nucleic acid sequence relative to the number of A and / or U nucleotides in the original nucleic acid sequence (e.g., a wild-type or reference sequence). In some embodiments, such A-U alterations are made to (i) modify the retention time of individual nucleic acids in a composition to allow for co-purification using HPLC methods and / or 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.

[0266] In some embodiments, at least one coding sequence of an mRNA used herein is a codon-modified coding sequence, wherein the codon-modified coding sequence is selected from a G / C-optimized coding sequence, a human codon usage-compatible coding sequence, or a G / C-modified coding sequence.

[0267] A poly-A tail (eg, about 30 or more adenosine residues) can be attached to the 3' end of the RNA to increase its half-life.

[0268] In some embodiments, an mRNA as used herein comprises at least one poly(N) sequence, e.g., at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or a combination thereof.

[0269] In some embodiments, mRNA as used herein comprises at least one poly(A) sequence.

[0270] The terms "poly(A) sequence," "poly(A) tail," or "3' poly(A) tail" as used herein are recognized and understood by those skilled in the art, and are intended to refer to, for example, a sequence of adenosine nucleotides typically located at the 3' end of a linear RNA (or in a circular RNA) of up to about 1,000 adenosine nucleotides. In some embodiments, the poly(A) sequence is essentially homopolymeric; 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 that is different from adenosine nucleotides; for example, a poly(A) sequence of, for example, 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide or section of nucleotides that is different from adenosine nucleotides).

[0271] The poly(A) sequence may comprise 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 even longer than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides.

[0272] In some embodiments, an mRNA as used herein comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides. In some embodiments, the poly(A) sequence comprises about 64 adenosine nucleotides (A64). In other embodiments, the poly(A) sequence comprises about 100 adenosine nucleotides (A100). In other embodiments, the poly(A) sequence comprises about 150 adenosine nucleotides.

[0273] In a further embodiment, the mRNA used herein comprises at least one poly(A) sequence comprising approximately 100 adenosine nucleotides, the poly(A) sequence being interrupted by non-adenosine nucleotides, preferably by 10 non-adenosine nucleotides (A30-N10-A70).

[0274] The poly(A) sequence 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 to mean located exactly 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.

[0275] In one embodiment, the mRNA used herein comprises a poly(A) sequence of at least 70 adenosine nucleotides, preferably at least 70 consecutive adenosine nucleotides, wherein the 3' terminal nucleotide is an adenosine nucleotide.

[0276] In embodiments, the poly(A) sequence of the nucleic acid is obtained from a DNA template during in vitro RNA 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 generated by enzymatic polyadenylation of the RNA (after in vitro RNA transcription) using a commercially available polyadenylation kit and corresponding protocols known in the art, or alternatively, by using immobilized poly(A) polymerase, for example, using the methods and means described in WO2016174271.

[0277] As used herein, mRNA may contain poly(A) sequences obtained by enzymatic polyadenylation, with the majority of the nucleic acid molecule containing from about 100 (+ / -20) to about 500 (+ / -50), preferably about 250 (+ / -20), adenosine nucleotides.

[0278] In embodiments, mRNA as used herein comprises a poly(A) sequence derived from a template DNA, and optionally further comprises at least one additional poly(A) sequence generated by enzymatic polyadenylation, e.g., as described in WO2016091391.

[0279] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6) mRNA contains at least one poly(A) tail sequence containing 30 to 200 adenosine nucleotides, preferably 100 adenosine nucleotides, wherein the 3'-terminal nucleotide of the RNA is adenosine.

[0280] In embodiments, the mRNA used herein comprises at least one polyadenylation signal.

[0281] In embodiments, mRNA as used herein comprises at least one poly(C) sequence.

[0282] As used herein, the term "poly(C) sequence" is intended to mean a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In embodiments, the poly(C) sequence contains about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In one embodiment, the poly(C) sequence contains about 30 cytosine nucleotides.

[0283] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The mRNA of ) preferably contains 30 to 200 adenosine nucleotides and / or at least one poly(C) sequence, and preferably contains a poly(A) tail sequence containing 10 to 40 cytosine nucleotides.

[0284] In embodiments, an mRNA as used herein comprises at least one histone stem loop (hSL) or histone stem loop structure.

[0285] The term "histone stem loop" (eg, abbreviated as "hSL" in the sequence listing) is intended to refer to nucleic acid sequences that form stem-loop secondary structures found primarily in histone mRNAs.

[0286] The histone stem-loop sequence / structure may preferably be selected from the histone stem-loop sequences disclosed in WO2012019780, the disclosure of which is incorporated herein by reference. The histone stem-loop sequence 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.

[0287] In some embodiments, the first mRNA and / or the second mRNA comprises at least one histone stem loop.

[0288] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA contains at least one histone stem loop.

[0289] In other embodiments, the mRNA used herein does not comprise hsL as defined herein.

[0290] In embodiments, an mRNA as used herein comprises 3' terminal sequence elements, including a poly(A) sequence and optionally a histone stem-loop sequence.

[0291] As used herein, the 5' end of an mRNA may be capped. As used herein, an mRNA may be modified by the addition of a 5' cap structure, which preferably stabilizes the RNA and / or enhances expression of the encoded antigen and / or reduces stimulation of the innate immune system (after administration to a subject).

[0292] For example, the 5' end of an RNA may be capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (Cap 0 structure) or its derivative, which may be incorporated during RNA synthesis or enzymatically generated after RNA transcription (e.g., by using vaccinia virus capping enzyme (VCE), which consists of mRNA triphosphatase, 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 efficiency of an RNA molecule. The 5' cap of an mRNA molecule may be further modified by 2'-O-methyltransferase, resulting in the generation of the Cap 1 structure (m7Gppp[m2'-O]N), which may further increase translational efficiency.

[0293] In some embodiments, the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA comprises a 5' cap structure, preferably an m7G, cap 0, cap 1, cap 2, modified cap 0, or modified cap 1 structure, suitably a 5' cap 1 structure.

[0294] The term "5' cap structure," as used herein, is recognized and understood by those skilled in the art and is intended to refer to a 5'-modified nucleotide, particularly a guanine nucleotide, located at the 5' end of an RNA, e.g., an mRNA. In some embodiments, the 5' cap structure is connected to the RNA via a 5'-5'-triphosphate linkage.

[0295] Suitable 5' cap structures are cap 0 (methylation of the first nucleobase, e.g., m7GpppN), cap 1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), cap 2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap 3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap 4 (additional methylation of the ribose of the fourth nucleotide downstream 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.

[0296] The 5' cap (cap0 or cap1) structure can be formed by chemical RNA synthesis or by RNA in vitro transcription using a cap analog (co-transcriptional capping).

[0297] The term "cap analog," as used herein, will be recognized and understood by those of skill in the art and is intended to refer to a non-polymerizable di- or tri-nucleotide that has a cap functionality, e.g., in that it facilitates translation or localization and / or prevents degradation of a nucleic acid molecule, particularly an RNA molecule, when incorporated at the 5' end of a nucleic acid molecule. By non-polymerizable, we mean that the cap analog is incorporated only at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent polymerase, particularly a template-dependent RNA polymerase. Examples of cap analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G), or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG, and their tetraphosphate derivatives). Additional cap analogs have been previously described (WO2008016473, WO2008157688, WO2009149253, WO2011015347, and WO2013059475). Further suitable cap analogs in this context are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017 / 053297, WO2017066782, WO2018075827, and WO2017066797, the disclosures of which regarding cap analogs are incorporated herein by reference.

[0298] In embodiments, modified cap structure 1 is generated using the tri-nucleotide cap analogs disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, and WO2017066797. In particular, any cap structure derivable from the structures disclosed in claims 1-5 of WO2017053297 may be suitably used to co-transcriptionally generate modified cap structure 1. Furthermore, any cap structure derivable from the structures defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate modified cap structure 1.

[0299] In embodiments, the mRNA used herein comprises a Cap 1 structure.

[0300] In embodiments, a 5' cap structure may be added co-transcriptionally, preferably in an RNA in vitro transcription reaction as defined herein, using a tri-nucleotide cap analog as defined herein.

[0301] In embodiments, the Cap 1 structure of an mRNA is formed using co-transcriptional capping with the tri-nucleotide cap 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.

[0302] In another embodiment, the cap 1 structure of the mRNA is formed using co-transcriptional capping with the tri-nucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0303] In other embodiments, the cap 0 structure of an mRNA used herein is formed using co-transcriptional capping with the cap analog 3'OMe-m7G(5')ppp(5')G.

[0304] In other embodiments, the 5' cap structure is formed via enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or a 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 also be added using immobilized capping enzyme and / or cap-dependent 2'-O methyltransferase using the methods and means disclosed in WO2016193226.

[0305] To determine the presence / absence of cap0 or cap1 structures, one can use the capping assay described in published PCT application WO2015101416, in particular claims 27 to 46 of published PCT application WO2015101416. Other capping assays that can be used to determine the presence / absence of cap0 or cap1 structures in RNA are described in PCT / EP2018 / 08667, or published PCT applications WO2014152673 and WO2014152659.

[0306] In embodiments, an mRNA as used herein comprises an m7G(5')ppp(5')(2'OMeA) cap structure. In such embodiments, the mRNA comprises an m7G cap at the 5' end and an additional methylation of the ribose of the adjacent nucleotide of m7GpppN, in this case a 2'O-methylated adenosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, or 95% of the RNA (species) comprises such a Cap 1 structure as determined using a capping assay.

[0307] In other embodiments, an mRNA as used herein comprises an m7G(5')ppp(5')(2'OMeG) cap structure. In such embodiments, the mRNA comprises an m7G cap at the 5' end and an additional methylation of the ribose of the adjacent nucleotide, in this case, a 2'0-methylated guanosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, or 95% of the coding RNA (species) comprises such a cap structure as determined using a capping assay.

[0308] Thus, the first nucleotide of the mRNA sequence, ie, the nucleotide downstream of the m7G(5')ppp structure, may be a 2'O-methylated guanosine or a 2'O-methylated adenosine.

[0309] In embodiments, the A / U (A / T) content in the environment of the ribosome binding site of an 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. Efficient binding of ribosomes to the ribosome binding site then has the effect of efficient translation of the mRNA.

[0310] Thus, in some embodiments, the mRNA used herein comprises a ribosome binding site, also referred to as a "Kozak sequence."

[0311] In some embodiments, an mRNA as used herein may include at least one heterologous untranslated region (UTR), for example, a 5' UTR and / or a 3' UTR.

[0312] The terms "untranslated region" or "UTR" or "UTR element" are recognized and understood by those skilled in the art and are intended to refer to, for example, a portion of a nucleic acid molecule that is typically located 5' or 3' of a coding sequence. A UTR is not translated into protein. A UTR may be a portion of a nucleic acid, e.g., DNA or RNA. A 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, promoter elements, etc.

[0313] In embodiments, the term "mRNA" as used herein includes a protein-coding region ("coding sequence" or "cds") and a 5'UTR and / or a 3'UTR. In particular, UTRs may have regulatory sequence elements that determine nucleic acid, e.g., RNA, turnover, stability, and localization. Furthermore, UTRs may have sequence elements that enhance translation. In pharmaceutical applications of nucleic acid sequences (including DNA and RNA), translation of the nucleic acid into at least one peptide or protein is paramount to therapeutic efficacy. Certain combinations of 3'UTRs and / or 5'UTRs may enhance expression of an operably linked coding sequence encoding a peptide or protein of the present invention. Nucleic acid molecules having UTR combinations advantageously enable rapid and transient expression of an antigenic peptide or protein after administration to a subject, preferably after intramuscular administration. Therefore, mRNAs comprising certain combinations of 3'UTRs and / or 5'UTRs provided herein are particularly suitable for administration as vaccines, particularly to the muscle, dermis, or epidermis of a subject.

[0314] In some embodiments, an mRNA as 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 a naturally occurring gene or may be synthetically produced. In embodiments, an mRNA comprises at least one coding sequence as defined herein operably linked to at least one (heterologous) 3'UTR and / or at least one (heterologous) 5'UTR.

[0315] In embodiments, the mRNA used herein comprises at least one heterologous 3'UTR.

[0316] In some embodiments, the first mRNA and / or the second mRNA comprises a 3'UTR.

[0317] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA contains the 3'UTR.

[0318] The terms "3' untranslated region" or "3' UTR" or "3' UTR element" are recognized and understood by those of skill in the art and are intended to refer, for example, to a portion of a nucleic acid molecule that is located 3' (i.e., downstream) of a coding sequence and is not translated into protein. A 3' UTR may be a portion of nucleic acid, e.g., DNA or RNA, located between the coding sequence and an (optional) terminal poly(A) sequence. A 3' 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.

[0319] In some embodiments, mRNA as used herein comprises a 3'UTR that may be derivable from a gene for an RNA with an enhanced half-life (ie, resulting in a stable RNA).

[0320] In some embodiments, the 3'UTR comprises one or more of a polyadenylation signal, a binding site for a protein that affects nucleic acid stability at its location in the cell, or one or more miRNAs or binding sites for miRNAs.

[0321] In embodiments, an mRNA as used herein comprises at least one heterologous 3'UTR, wherein the at least one heterologous 3'UTR comprises 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.

[0322] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA comprises a 3'-UTR that comprises 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 variant of any one of these genes.

[0323] Nucleic acid sequences in that context can be derived from published PCT application WO2019077001A1, in particular claim 9 of WO2019077001A1. The corresponding 3'-UTR sequence of claim 9 of WO2019077001A1 is incorporated herein by reference.

[0324] In some embodiments, the mRNA used herein may comprise a 3'UTR as described in WO2016107877, the disclosure of which regarding 3'UTR sequences is incorporated herein by reference. Suitable 3'UTRs are SEQ ID NOS: 1-24 and SEQ ID NOS: 49-318 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA used herein comprises a 3'UTR as described in WO2017036580, the disclosure of which regarding 3'UTR sequences is incorporated herein by reference. Suitable 3'UTRs are SEQ ID NOS: 152-204 of WO2017036580, or fragments or variants of these sequences. In other embodiments, the mRNA used herein comprises a 3'UTR as described in WO2016022914, the disclosure of which regarding 3'UTR sequences is incorporated herein by reference. Particularly suitable 3'UTRs are the nucleic acid sequences set forth in SEQ ID NOs: 20-36 of WO2016022914, or fragments or variants of these sequences.

[0325] In embodiments, the mRNA used herein comprises at least one heterologous 5'UTR.

[0326] In some embodiments, the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA contains a 5' untranslated region (UTR).

[0327] The terms "5' untranslated region" or "5' UTR" or "5' UTR element" are recognized and understood by those skilled in the art and are intended to refer, for example, to a portion of a nucleic acid molecule that is located 5' (i.e., upstream) of a coding sequence and is not translated into protein. A 5' UTR may be a portion of a nucleic acid that is located 5' of a coding sequence. Typically, a 5' UTR begins at the transcription start site and ends before the start codon of the coding sequence. A 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. A 5' UTR may be post-transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5' cap structure (e.g., for mRNA as defined herein).

[0328] In some embodiments, mRNA as used herein comprises a 5'UTR that may be derivable from a gene for an RNA with an enhanced half-life (ie, resulting in a stable RNA).

[0329] In some embodiments, the 5'UTR comprises one or more of a binding site for a protein that affects RNA stability or RNA location in a cell, or one or more miRNAs or binding sites for miRNAs.

[0330] In an embodiment, the mRNA used herein is preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA comprises at least one heterologous 5' UTR, wherein the at least one heterologous 5' UTR comprises 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 a homolog, fragment, or variant of any one of these genes.

[0331] The nucleic acid sequence in that context may be selected from published PCT application WO2019077001A1, in particular claim 9 of WO2019077001A1. The corresponding 5'UTR sequence of claim 9 of WO2019077001A1 is incorporated herein by reference (e.g., SEQ ID NOs: 1-20 of WO2019077001A1, or a fragment or variant thereof).

[0332] In some embodiments, the mRNA used herein may comprise a 5' UTR described in WO2013143700, the disclosure of which regarding 5' UTR sequences is incorporated herein by reference. Particularly suitable 5' UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, 1395, 1421, and 1422 of WO2013143700, or fragments or variants of these sequences. In other embodiments, the mRNA used herein comprises a 5' UTR described in WO2016107877, the disclosure of which regarding 5' UTR sequences is incorporated herein by reference. Particularly suitable 5' UTRs are nucleic acid sequences set forth in SEQ ID NOs: 25-30 and 319-382 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the nucleic acid comprises a 5' UTR as described in WO2017036580, the disclosure of which regarding 5' UTR sequences is incorporated herein by reference. Particularly suitable 5' UTRs are the nucleic acid sequences set forth in SEQ ID NOS: 1-151 of WO2017036580, or fragments or variants of these sequences. In other embodiments, the nucleic acid comprises a 5' UTR as described in WO2016022914, the disclosure of which regarding 5' UTR sequences is incorporated herein by reference. Particularly suitable 5' UTRs are the nucleic acid sequences set forth in SEQ ID NOS: 3-19 of WO2016022914, or fragments or variants of these sequences.

[0333] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA comprises a heterologous 5'-UTR comprising or consisting of a nucleic acid sequence derived from the 5'-UTR from HSD17B4, and at least one heterologous 3'-UTR comprising or consisting of a nucleic acid sequence derived from the 3'-UTR of PSMB3.

[0334] In some embodiments, the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is from 5' to 3': i) 5' Cap 1 structure; ii) a 5'-UTR derived from the 5'-UTR of the HSD17B4 gene; iii) coding sequences; iv) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene; v) optionally, a histone stem-loop sequence, and vi) a poly(A) sequence containing approximately 100 A nucleotides wherein the nucleotide at the 3' end of the RNA is adenosine.

[0335] In embodiments, RNA, preferably mRNA, may be prepared using any method known in the art, including chemical synthesis, such as, for example, solid-phase RNA synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions.

[0336] Thus, in an embodiment, RNA, preferably mRNA as used herein, is in vitro transcribed RNA.

[0337] The term "RNA in vitro transcription" or "in vitro transcription" refers to a process in which RNA is synthesized in a cell-free system (in vitro). RNA may be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which may be a linear plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription may be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5 RNA polymerase. In one embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme, after which it is subjected to RNA in vitro transcription.

[0338] Reagents used in RNA in vitro transcription typically include a DNA template (linear plasmid DNA or PCR product) bearing a promoter sequence with high binding affinity for its respective RNA polymerase, e.g., a bacteriophage-encoded RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); optionally, a cap analog as defined herein; optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5 RNA polymerase) capable of binding to the promoter sequence within the DNA template; optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNases; and optionally, an RNA in MgCl to provide Mg ions as a cofactor for the polymerase; an antioxidant (e.g., DTT), and / or a polyamine, e.g., spermidine at an optimal concentration, e.g., a buffer (TRIS or HEPES) to maintain a suitable pH value, which may also contain a buffer system comprising TRIS citrate as disclosed in WO2017109161.

[0339] In embodiments, the Cap 1 structure of the mRNA used herein is formed using co-transcriptional capping with the tri-nucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A preferred Cap 1 analog that may be used in producing the coding RNA, preferably the coding mRNA, used herein is m7G(5')ppp(5')(2'OMeA)pG.

[0340] In another embodiment, the Cap 1 structure of an RNA, preferably an mRNA, used herein is formed using co-transcriptional capping with the tri-nucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0341] In another embodiment, the cap 0 structure of an RNA, preferably an mRNA, used herein is formed using co-transcriptional capping with the cap analog 3'OMe-m7G(5')ppp(5')G.

[0342] In embodiments, the nucleotide mixture used for RNA in vitro transcription may further comprise modified nucleotides as defined herein. In this context, suitable modified nucleotides may be selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methylcytosine, and 5-methoxyuridine. In embodiments, uracil nucleotides in the nucleotide mixture are replaced (either partially or completely) by pseudouridine (Ψ) and / or N1-methylpseudouridine (m1Ψ) to obtain modified RNA.

[0343] In some other embodiments, the nucleotide mixture used for RNA in vitro transcription does not include modified nucleotides as defined herein, hi embodiments, the nucleotide mixture used for RNA in vitro transcription includes only G, C, A, and U nucleotides, and optionally, cap analogs as defined herein.

[0344] In embodiments, the nucleotide mixture (i.e., the fraction of each nucleotide in the mixture) used in the RNA in vitro transcription reaction may suitably be optimized for a given RNA sequence, as described in WO2015188933.

[0345] In this context, in vitro transcription is carried out in the presence of a sequence-optimized nucleotide mixture and, optionally, a cap analog.

[0346] In this context, a sequence-optimized nucleoside triphosphate (NTP) mix is ​​a mixture of nucleoside triphosphates (NTPs) for use in an in vitro transcription reaction of an RNA molecule of a given sequence, comprising the four nucleoside triphosphates (NTPs) GTP, ATP, CTP, and UTP, where the fractions of each of the four nucleoside triphosphates (NTPs) in the sequence-optimized nucleoside triphosphate (NTP) mix correspond to the fractions of each nucleotide in the RNA molecule. If a ribonucleotide is absent from the RNA molecule, the corresponding nucleoside triphosphate is also absent from the sequence-optimized nucleoside triphosphate (NTP) mix.

[0347] In embodiments where two or more different RNAs, preferably mRNAs as defined herein, have to be produced, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different RNAs have to be produced, the techniques described in WO2017109134 may suitably be used.

[0348] In the context of nucleic acid-based vaccine production, it may be required to provide GMP-grade nucleic acids, such as GMP-grade RNA or DNA. GMP-grade RNA or DNA may be produced using a manufacturing process approved by a regulatory agency. Thus, in some embodiments, RNA production is preferably performed under current Good Manufacturing Practice (GMP) procedures, which include various quality control steps at the DNA and RNA levels, as described in WO2016180430. In embodiments, the mRNA of the present invention is GMP-grade mRNA. Thus, RNA for vaccines is preferably GMP-grade RNA.

[0349] The resulting RNA product can be purified using PUREMESSENGER® (CureVac, Tübingen, Germany; RP-HPLC as described in WO2008077592) and / or tangential flow filtration (as described in WO2016193206) and / or oligo d(T) purification (see WO2016180430).

[0350] In some embodiments, RNA, preferably mRNA, used herein is purified using RP-HPLC, preferably using reversed-phase high-pressure liquid chromatography (RP-HPLC) using a macroporous styrene / divinylbenzene column (e.g., 30 μm particle size, 4000 Å pore size), further using a filter cassette with a cellulose-based membrane with a molecular weight cut-off of approximately 100 kDa.

[0351] In a further embodiment, the RNA, preferably the mRNA, used herein is freeze-dried (e.g. as described in WO2016165831 or WO2011069586) to obtain a temperature-stable dried RNA, preferably the mRNA (powder). The RNA, preferably the mRNA, used herein may also be dried using spray drying or spray freeze drying (e.g. as described in WO2016184575 or WO2016184576) to obtain a temperature-stable RNA, preferably the mRNA (powder) as defined herein. Accordingly, the disclosures of WO2017109161, WO2015188933, WO2016180430, WO2008077592, WO2016193206, WO2016165831, WO2011069586, WO2016184575, and WO2016184576 in the context of RNA production and purification are incorporated herein by reference.

[0352] Thus, in an embodiment, the RNA, preferably mRNA, used herein is dried RNA, preferably mRNA.

[0353] The term "dried RNA (or mRNA)" as used herein should be understood as RNA (or mRNA) that has been freeze-dried or spray-dried or spray-freeze-dried as defined above to obtain a temperature-stable dried mRNA (powder).

[0354] In an embodiment, the RNA, preferably mRNA, used herein is purified RNA, preferably mRNA.

[0355] As used herein, the term "purified RNA (or mRNA)" refers to RNA that has a higher purity than the starting material (e.g., in vitro-transcribed RNA) after a specific purification step (e.g., HPLC, TFF, oligo d(T) purification, precipitation step). Typical impurities that are essentially absent from purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, e.g., RNA polymerases, RNases, pyrophosphatases, restriction endonucleases, DNases), spermidine, BSA, defective RNA sequences, RNA fragments (short double-stranded RNA fragments, defective sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl), etc. Other possible impurities that may be derived, for example, from fermentation procedures include bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (e.g., organic solvents). Therefore, in this context, it is desirable for the "degree of RNA purity" to be as close to 100% as possible. Regarding the degree of RNA purity, it is desirable that the amount of full-length RNA transcripts is as close to 100% as possible. Thus, as used herein, "purified RNA" has a degree of purity greater than 75%, 80%, or 85%, more specifically greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably greater than 99%. The degree of purity may be determined, for example, by analytical HPLC, where the percentages provided above correspond to the ratio of the area of ​​the target RNA peak to the total area of ​​all peaks representing by-products. Alternatively, the degree of purity may be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0356] It should be understood that "dried RNA (or mRNA)" as defined herein and "purified RNA (or mRNA)" as defined herein, or "GMP-grade RNA (or mRNA)" as defined herein, may have superior stability characteristics (in vitro, in vivo) and improved efficacy (e.g., better translatability of mRNA in vivo), and are therefore particularly suitable for medical purposes, e.g., vaccines.

[0357] In an embodiment, the RNA, preferably mRNA, has been purified by RP-HPLC and / or TFF to remove double-stranded RNA, uncapped RNA, and / or RNA fragments.

[0358] For example, the formation of double-stranded RNA as a by-product during RNA in vitro transcription can lead to the induction of innate immune responses, particularly IFN-alpha, a major factor in inducing fever in vaccinated subjects, which is, of course, an undesirable side effect. Current techniques of immunoblotting of dsRNA (such as by dot blot, serological specific electron microscopy (SSEM), or ELISA) are used to detect and size dsRNA species from mixtures of nucleic acids.

[0359] In some embodiments, RNA, preferably mRNA, is purified by RP-HPLC and / or TFF as described herein to reduce the amount of dsRNA.

[0360] In embodiments, the RNA, preferably mRNA, contains about 5%, 10%, or 20% less double-stranded RNA by-products than the double-stranded RNA by-products as the RNA, preferably mRNA, that has not been purified by RP-HPLC and / or TFF.

[0361] In some embodiments, RNA, preferably mRNA, purified by RP-HPLC and / or TFF contains about 5%, 10%, or 20% less double-stranded RNA by-products than RNA, preferably mRNA, purified using oligo-dT purification, precipitation, filtration, and / or AEX.

[0362] In embodiments, the RNA, preferably mRNA, of the composition has an RNA integrity ranging from about 40% to about 100%.

[0363] The term "RNA integrity" generally describes whether a complete RNA sequence is present in a composition.Low RNA integrity can be due to, among other things, RNA degradation, RNA cleavage, incorrect or incomplete chemical synthesis of RNA, incorrect base pairing, incorporation of modified nucleotides or modification of already incorporated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation, or incomplete RNA in vitro transcription.RNA is a fragile molecule that can be easily degraded, which can be caused by, for example, temperature, ribonuclease, pH, or other factors (e.g., nucleophilic attack, hydrolysis, etc.), which can reduce RNA integrity and consequently RNA functionality.

[0364] Those skilled in the art can choose from a variety of different chromatographic or electrophoretic methods for determining RNA integrity. Chromatographic and electrophoretic methods are well known in the art. When chromatography is used (e.g., RP-HPLC), the analysis of RNA integrity can be based on determining the peak area (or "area under the peak") of full-length RNA in the corresponding chromatogram. The peak area can 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 relative to the peak area of ​​the total RNA in each sample. RNA integrity can be expressed as % RNA integrity.

[0365] In the context of embodiments 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 encapsulating RNA may be treated with a detergent (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 Agencourt 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 integrity of the RNA. Typically, to determine RNA integrity, the RNA sample may be diluted to a concentration of 0.1 g / L, for example, using water for injection (WFI). Approximately 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 (0.1 M TEAA, pH 7.0); buffer B (0.1 M TEAA, pH 7.0, containing 25% acetonitrile). Starting with 30% buffer B, the gradient is extended to 32% buffer B in 2 minutes, followed by 55% buffer B over 15 minutes at a flow rate of 1 ml / min. HPLC chromatograms are typically recorded at a wavelength of 260 nm. The resulting chromatograms can be evaluated using software, and relative peak areas can be determined in percentage (%), as 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 areas provides information about the integrity of the RNA. Therefore, for example, if 100ng of RNA is injected in total and 100ng is determined as the relative peak area, the RNA integrity is 100%.For example, if the relative peak area corresponds to 80ng, the RNA integrity is 80%.Therefore, in the context of the present invention, RNA integrity is determined using analytical HPLC, preferably analytical RP-HPLC.

[0366] In embodiments, the RNA, preferably mRNA, of the composition has an RNA integrity in the range of about 40% to about 100%. In embodiments, the RNA, preferably mRNA, has an RNA integrity in the range of about 50% to about 100%. In embodiments, the RNA, preferably mRNA, has an RNA integrity in the range of about 60% to about 100%. In embodiments, the RNA, preferably mRNA, has an RNA integrity in the range of 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.

[0367] In embodiments, the RNA, preferably mRNA, of the composition 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%, preferably as determined using analytical HPLC, more preferably analytical RP-HPLC.

[0368] After co-transcriptional capping as defined herein and after purification as defined herein, the degree of capping of the resulting RNA may be determined using the capping assay described in published PCT application WO2015101416, in particular claims 27 to 46 of published PCT application WO2015101416. Alternatively, the capping assay described in PCT / EP2018 / 08667 may be used.

[0369] In embodiments, automated devices for RNA in vitro transcription may be used to produce and purify mRNA of the present invention. Such devices may also be used to produce compositions or vaccines (described in more detail below). In some embodiments, devices described in WO2020002598, particularly those described in claims 1-59 and / or 68-76 (and Figures 1-18) of WO2020002598, may be suitably used.

[0370] The methods described herein may be applied to methods of producing immunogenic compositions or vaccines, which are described in more detail below.

[0371] In various embodiments, the mRNA used herein suitably contains, in the 5' to 3' direction, the following elements: A) a 5' cap structure, preferably a 5' cap structure as specified herein; B) a 5' terminal initiation element, preferably a 5' terminal initiation element as specified herein; C) optionally a 5'UTR, preferably a 5'UTR as specified herein; D) a ribosome binding site, preferably a ribosome binding site as defined herein; E) at least one coding sequence, preferably at least one coding sequence identified herein; F) a 3'UTR, preferably a 3'UTR as specified herein; G) optionally a poly(A) sequence, preferably a poly(A) sequence as specified herein; H) optionally a poly(C) sequence, preferably a poly(C) sequence as specified herein; I) optionally a histone stem loop, preferably a histone stem loop as defined herein; J) optionally, a 3'-terminal sequence element, preferably a 3'-terminal sequence element as specified herein; Includes.

[0372] In some embodiments, the RNA, preferably mRNA, used herein does not include replicase elements (eg, nucleic acid encoding a replicase).

[0373] In some embodiments, the RNA used herein, preferably the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c4 ), (c 5 ) and / or (c 6 ) mRNA is not self-replicating, in some cases.

[0374] In some embodiments, the RNA used herein, preferably the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNAs are sometimes self-replicating.

[0375] chemical modification In some embodiments, the RNA, preferably mRNA, used herein does not include chemically modified nucleotides.

[0376] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA does not contain chemically modified nucleotides.

[0377] In an embodiment, the RNA, preferably mRNA, used herein comprises a coding sequence consisting only of G, C, A and U nucleotides and therefore does not contain modified nucleotides (except for the 5' terminal cap structure (cap0, cap1, cap2)).

[0378] In some embodiments, RNA, preferably mRNA, as used herein, is modified RNA, preferably mRNA, where modification refers to a chemical modification including a backbone modification and a sugar or base modification.

[0379] 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" refers to a nucleotide containing one or more chemical modifications (e.g., substitutions) in or on the nitrogenous 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 in or on the backbone phosphate. A nucleotide analog may further contain chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, modified ribose, six-membered sugar analog, or open-chain sugar analog) or on the phosphate. The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art, see the following references: U.S. Patent Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642. Many modified nucleosides and nucleotides are commercially available.

[0380] The backbone modifications described herein are modifications in which the phosphate of the backbone of a nucleotide of RNA, preferably mRNA, is chemically modified. The sugar modifications described herein are chemical modifications of the sugar of a nucleotide of RNA, preferably mRNA. Furthermore, the base modifications described herein are chemical modifications of the base portion of a nucleotide of RNA, preferably mRNA. In this context, the nucleotide analogs or modifications are suitably selected from nucleotide analogs applicable to transcription and / or translation.

[0381] In some embodiments, RNA, preferably mRNA, as used herein comprises at least one chemical modification.

[0382] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c4 ), (c 5 ) and / or (c 6 ) mRNA contains at least one chemical modification.

[0383] Modified nucleic acid bases (chemical modifications) that can be incorporated into modified nucleosides and nucleotides and that can be present in 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-isomethyladenosine). 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-hi hydroxynorvalylcarbamoyl adenosine; ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine); 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-acetylcytidine) Cetyl-2-O-methylcytidine; k2C (lycidine); 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); OHyW (hydroxywybutosine); OHyW *(Incompletely modified hydroxywybutosine); imG (wybutosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G *(Archaeosin); 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 derivatives, 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 guanine, 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 (abasic residue), mC, mU, mA, sU, W, or 2'-O-methyl-U. Many of these modified nucleobases and the corresponding ribonucleosides are available from commercial suppliers;

[0384] In some embodiments, the nucleotide analogue / modification that may be incorporated into the modified RNA, preferably mRNA, is selected from the following: 2-amino-6-chloropurine riboside-5'-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-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate 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 base-modifying nucleotides selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleosides,5-Azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-aza ... Cetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 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-methylthio-adenine and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-azaguanosine Inosine, 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-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine, 5 '-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza- Uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0385] In some embodiments, the chemical modification is selected from the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0386] Particularly suitable in this context are pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and 5-methoxyuridine, more preferably pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), and even more preferably N1-methylpseudouridine (m1ψ).

[0387] In some embodiments, essentially all, e.g., essentially 100%, of the uracils in the coding sequence of an RNA, preferably an mRNA, used herein have a chemical modification, preferably the chemical modification is at the 5-position of the uracil.

[0388] In some embodiments, the RNA, preferably the mRNA, used herein comprises chemical modifications that are uridine modifications, preferably 100% of the uridine positions in the mRNA are modified.

[0389] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6) mRNA is uridine-modified, and preferably, 100% of the uridine positions in the mRNA are modified.

[0390] Incorporation of modified nucleotides, such as pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and / or 5-methoxyuridine, into the coding sequence of RNA, preferably mRNA, used herein can be advantageous because unwanted innate immune responses (upon administration of the encoding mRNA or vaccine) can be modulated or reduced (if necessary).

[0391] In an embodiment, the coding sequence of the RNA, preferably the mRNA, used herein comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), preferably in which all uracil nucleotides are substituted by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides, and optionally in which all uracil nucleotides are substituted by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0392] In some embodiments, the RNA, preferably the mRNA, used herein does not contain N1-methylpseudouridine (m1ψ) substituted positions. In further embodiments, the RNA, preferably the mRNA, used herein does not contain pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine substituted positions.

[0393] In some embodiments, the chemical modification is N1-methylpseudouridine and / or pseudouridine. In some embodiments, the chemical modification is N1-methylpseudouridine.

[0394] Carrier Various carrier systems have been described that encapsulate or complex mRNA to enhance mRNA delivery, and consequently expression of the encoded antigen, compared to unencapsulated or uncomplexed mRNA. The present invention may use any suitable carrier system. Specific carrier systems of note are described in further detail below.

[0395] In embodiments, the RNA, preferably mRNA, used herein may be complexed, encapsulated, partially encapsulated or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming a lipid-based carrier, such as a liposome, lipid nanoparticle (LNP), lipoplex and / or nanoliposome, preferably a lipid nanoparticle.

[0396] In some embodiments, (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA, either separately or together, are formulated in lipid nanoparticles (LNPs).

[0397] In some embodiments, the RNA, preferably the mRNA, used herein is formulated separately (in any formulation or complexing agent as defined herein), preferably the RNA, preferably the mRNA, used herein is formulated in separate liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes.

[0398] In some embodiments, the RNA used herein, preferably the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is formulated separately.

[0399] In an embodiment, the RNA, preferably the mRNA, as used herein is co-formulated (in any formulation or complexing agent as defined herein), wherein preferably the RNA, preferably the mRNA, as used herein is formulated in separate liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes.

[0400] In some embodiments, the RNA used herein, preferably the mRNA used herein, preferably (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA are co-formulated, i.e., formulated together.

[0401] LNP The term "lipid nanoparticles," also referred to as "LNPs," is not limited to any particular form and includes any form produced when cationic lipids and, optionally, one or more additional 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).

[0402] Lipid nanoparticles (LNPs) are non-virion liposomal particles that can encapsulate mRNA. The incorporation of nucleic acids into LNPs is also referred to herein as "encapsulation," where the nucleic acid, e.g., RNA, is contained within the interior space of a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome.

[0403] LNP delivery systems and methods for their preparation are known in the art.

[0404] The particles may contain some external RNA, preferably mRNA (e.g., on the surface of the particle), but desirably at least half (and preferably at least 85%, especially at least 95%, e.g., all) of the RNA, preferably mRNA, is encapsulated.

[0405] LNPs are preferably characterized as microscopic vesicles with an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of LNPs is typically formed by amphipathic molecules, e.g., synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains. The bilayer membrane of liposomes can also be formed by amphipathic polymers and surfactants (e.g., polymersomes, niosomes, etc.). In the context of the present invention, LNPs typically function to transport RNA, preferably mRNA, to target tissues.

[0406] Thus, in embodiments, the RNA used herein, preferably mRNA, is complexed with one or more lipids to form a lipid nanoparticle (LNP), liposome, nanoliposome, lipoplex, preferably LNP. In some embodiments, the LNP is suitable for intramuscular and / or intradermal administration.

[0407] In embodiments, at least about 80%, 85%, 90%, 95% of the lipid-based carrier, preferably the LNP, has a spherical morphology, preferably comprising a solid or partially solid core.

[0408] LNPs typically contain 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 encapsulated by the lipid portion of the LNP or a partial or complete lipid portion of the LNP. RNA, preferably mRNA, or portions thereof may also be associated with and complexed with the LNP. LNPs may contain any lipid capable of forming particles to which nucleic acids can be bound or in which one or more nucleic acids are encapsulated. In some embodiments, LNPs containing nucleic acids, preferably RNA, more preferably mRNA, contain one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.

[0409] In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and a cationic lipid.

[0410] LNPs can be formed, for example, from a mixture of (i) PEG-modified lipids, (ii) non-cationic lipids, (iii) sterols, and (iv) ionizable cationic lipids. Alternatively, LNPs can be formed, for example, from a mixture of (i) PEG-modified lipids, (ii) non-cationic lipids, (iii) sterols, and (iv) non-ionizable cationic lipids.

[0411] In some embodiments, the non-cationic lipid is a neutral lipid.

[0412] In some embodiments, the cationic lipid is ionizable.

[0413] The in vivo characteristics and behavior of LNPs can be modified by the addition of hydrophilic polymer coatings, such as polyethylene glycol (PEG), to the LNP surface to confer steric stabilization. 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 attached PEG chains (e.g., via PEGylated lipids or PEGylated cholesterol).

[0414] In one embodiment, RNA, preferably mRNA, is complexed with one or more lipids to form a lipid nanoparticle, wherein the LNP (or liposome, nanoliposome, lipoplex) comprises a polymer-conjugated lipid, preferably a PEGylated lipid / PEG-lipid.

[0415] In some embodiments, the LNP comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid portion 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 comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), etc. The terms "PEGylated lipid" and "PEG-modified lipid" are used interchangeably herein.

[0416] Polymer-conjugated lipids, such as PEG-lipids, as defined herein, can function as aggregation-reducing lipids.

[0417] In certain embodiments, the LNPs comprise a stabilizing 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. Representative 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 comprises a PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate, e.g., ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0418] In some embodiments, the PEG-modified lipid comprises PEG-DMG or PEG-cDMA.

[0419] In embodiments, the PEGylated lipid is preferably derived from formula (IV) of published PCT patent application WO2018078053A1, and the respective disclosures related thereto are hereby incorporated by reference.

[0420] In some embodiments, the PEG-modified lipid has Formula IV: [ka] (In the formula, R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein said alkyl chain may optionally be interrupted by one or more ester bonds; w has an average value of 30 to 60) It has.

[0421] In some embodiments, the PEG-modified lipid R 8 and R 9 is a saturated alkyl chain.

[0422] In some embodiments, RNA, preferably mRNA, is complexed with one or more lipids to form LNPs, wherein the LNPs comprise a polymer-conjugated lipid, preferably a PEGylated lipid, wherein the PEGylated lipid is preferably derived from formula (IVa) in published PCT patent application WO2018078053A1. Accordingly, the PEGylated lipid derived from formula (IVa) in published PCT patent application WO2018078053A1 and each of the disclosures related thereto are incorporated herein by reference.

[0423] In some embodiments, the PEG lipid or PEGylated lipid has formula (IVa): [ka] (In the formula, n has an average value in the range of 30 to 60, e.g., 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 is about 49. In another embodiment, n is 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.

[0424] In some embodiments, the PEG-modified lipid has the formula (IVa):

[0425] [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, and most preferably n has an average value of 49 or 45; or n is an integer selected so that the average molecular weight of the PEG lipid is about 2500 g / mol. It has.

[0426] The lipid of formula IVa suitable for use herein has the chemical term 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referred to as ALC-0159.

[0427] Further examples of suitable PEG-lipids in this context are provided in US20150376115A1 and WO2015199952, each of which is incorporated by reference in its entirety.

[0428] In some embodiments, the LNPs comprise less than about 3, 2, or 1 mole percent PEG or PEG-modified lipids, based on the total moles of lipids in the LNP.

[0429] In further embodiments, the LNP comprises about 0.1% to about 20% on a molar basis, e.g., 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% PEG-modified lipids (based on 100% total moles of lipids in the LNP). In embodiments, the LNP comprises about 1.0% to about 2.0% PEG-modified lipid on a molar basis, e.g., about 1.2% to about 1.9%, about 1.2% to about 1.8%, about 1.3% to about 1.8%, about 1.4% to about 1.8%, about 1.5% to about 1.8%, about 1.6% to about 1.8%, particularly about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, and most preferably 1.7% (based on 100% total moles of lipid in the LNP). In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0430] In some embodiments, the LNP comprises about 0.5-10 mol%, optionally 0.5-5 mol%, or 0.5-3 mol% of a PEG-modified lipid.

[0431] In embodiments, LNPs comprise one or more additional lipids that stabilize particle formation during their formulation or manufacturing process (e.g., a neutral lipid and / or one or more steroids or steroid analogs).

[0432] In embodiments, RNA, preferably mRNA, is complexed with one or more lipids to form a lipid nanoparticle, wherein the LNP comprises one or more neutral lipids and / or one or more steroids or steroid analogs.

[0433] Suitable stabilizing lipids include neutral lipids and anionic lipids.The term "neutral lipid" refers to any one of several lipid species that exist at physiological pH in either uncharged or neutral zwitterionic form.Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0434] In some embodiments, the non-cationic 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); preferably, the neutral lipid is DSPC.

[0435] In embodiments, the LNP (or liposome, nanoliposome, lipoplex) comprises one or more neutral lipids, wherein the neutral lipids are distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl- The phosphatidylethanolamine is selected from the group consisting of phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearyoyl-2-oleoylphosphatidiethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE), or a mixture thereof.

[0436] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipid to neutral lipid ranges from about 2:1 to about 8:1.

[0437] In an embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Preferably, the molar ratio of the cationic lipid to DSPC is in the range of about 2:1 to about 8:1.

[0438] In some embodiments, the steroid is a sterol, preferably cholesterol.

[0439] In embodiments, the steroid is cholesterol. Preferably, the molar ratio of cationic lipid to cholesterol ranges from about 2:1 to about 1:1. In some embodiments, the cholesterol may be pegylated.

[0440] The sterol can be 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 particle. In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol% of the total lipid present in the lipid particle. In another embodiment, the LNP comprises about 5% to about 50% sterol on a molar basis, e.g., 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% on a molar basis (based on 100% total moles of lipid in the lipid nanoparticle).

[0441] The cationic lipid of LNP may be ionizable, i.e., it becomes protonated when the pH is reduced below the pK of the ionizable group of the lipid, but becomes progressively more neutral at higher pH values.Then, at a pH value below the pK, the lipid can associate with negatively charged nucleic acid.In certain embodiments, the cationic lipid comprises a zwitterionic lipid, which becomes positively charged when the pH is reduced.

[0442] 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-dioleoyltrimethylammonium propane chloride (DOTAP) (N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, and 1,2-dioleoyloxy. (also known as cy-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-y-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-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), ICE (imidazole based), 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-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-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-DM), A), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (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-tetrahydro- tetraen-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)piperazin-1-yl)ethylazanediyl)didodecan-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-(undecylamino) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-propylN-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (commercially available cationic liposomes containing N-(1-(2,3 dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and TRANSFECTAM® (Promega Commercially available cationic lipids include dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol from Biosynthetic Chemistry Corp., Madison, Wis., or any combination of any of the foregoing. Additional suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Application Publications WO2010053572 (and in particular CI 2-200 described in paragraph

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

[0443] In embodiments, the cationic lipid of the liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome may be an amino lipid.

[0444] Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 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), 1,2-dilinoleyloxy -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).

[0445] In embodiments, the cationic lipid of the liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome may be an amino alcohol lipidoid.

[0446] Aminoalcohol lipidoids can be prepared by the methods described in U.S. Patent No. 8,450,298, which is incorporated herein by reference in its entirety. Suitable (ionizable) lipids can also be the compounds disclosed in Tables 1, 2, and 3 of WO2017075531A1, which is incorporated herein by reference, and defined in claims 1 to 24.

[0447] In another embodiment, suitable lipids may also be compounds disclosed in WO2015074085A1 (i.e., ATX-001 to ATX-032 or the compounds identified in claims 1 to 26), U.S. Patent Application Nos. 61 / 905,724 and 15 / 614,499, or U.S. Patent Nos. 9,593,077 and 9,567,296, the entireties of which are incorporated herein by reference.

[0448] 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 identified in the claims), the entire contents of which are incorporated herein by reference.

[0449] In some embodiments, the ionizable or cationic lipid 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 disclosure of which is incorporated herein by reference in its entirety. In this 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 of the disclosures related thereto, are incorporated herein by reference.

[0450] In some embodiments, the cationic lipid may be derived from Formula III of published PCT patent application WO2018078053A1, and therefore, Formula III of WO2018078053A1, and certain disclosures relating thereto, are incorporated herein by reference.

[0451] In some embodiments, RNA, preferably mRNA, is complexed with one or more lipids to form an LNP (or liposome, nanoliposome, lipoplex), wherein the cationic lipid of the LNP is selected from structures III-1 through III-36 in Table 9 of published PCT patent application WO2018078053A1. Accordingly, formulas III-1 through III-36 of WO2018078053A1, and the specific disclosures relating thereto, are incorporated herein by reference.

[0452] In some embodiments, the ionizable cationic lipid has Formula III:

[0453] [ka] (In the formula, L 1 or L 2 are each independently —O(C═O)— or (C═O)O—; G 1 and G 2 are each independently an unsubstituted C1-C 12 Alkylene or C1-C 12 Alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R 1 and R 2 are each independently a branched or linear C6-C 24 Alkyl or C6-C 24 is alkenyl; R 3 is 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 is alkyl; R5 is H or C1-C6 alkyl) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0454] In some embodiments, the ionizable cationic lipid has Formula III:

[0455] [ka] (In the formula, L 1 or L 2 are each independently —O(C═O)— or (C═O)O—; G 1 and G 2 are each independently an unsubstituted C1-C 12 is alkylene; G 3 is C1-C 24 is alkylene; R 1 and R 2 are each independently a branched or linear C6-C 24 is alkyl; R 3 is OR 5 and R 5 is H) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0456] In some embodiments, the ionizable cationic lipid has formula III, where R 1 , R 2 or R 1 and R 2 and both have the following structure:

[0457] [ka] It has one of the following.

[0458] In some embodiments, R2 has the following structure:

[0459] [ka] It has.

[0460] In some embodiments, the cationic lipid has the formula:

[0461] [ka] It has.

[0462] In some embodiments, the ionizable cationic lipid has the formula:

[0463] [ka] It has.

[0464] In some embodiments, the ionizable cationic lipid has formula III-3:

[0465] [ka] It has.

[0466] The lipid of formula III-3 suitable for use herein has the chemical name ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also referred to as ALC-0315, i.e., CAS number 2036272-55-4.

[0467] In certain embodiments, a cationic lipid as defined herein, more preferably cationic lipid compound III-3 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), is 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 %, based on the total lipid content of the LNP. When two or more cationic lipids are included in the LNP, such percentages apply to the combined cationic lipids.

[0468] In some embodiments, a cationic lipid as defined herein is present in the LNP in an amount of about 20 mol % to about 60 mol %.

[0469] In some embodiments, the LNP has the following structure:

[0470] [ka] The cationic lipids include those having the formula:

[0471] In embodiments, the cationic lipid is present in the LNP in an amount of about 30 mol% to about 70 mol%. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 mol% to about 60 mol%, e.g., 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 embodiments, the cationic lipid is present in the LNP in an amount of about 47 mol% to about 48 mol%, e.g., 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%, respectively, with 47.4 mol% being particularly preferred.

[0472] In some embodiments, the cationic lipid is present in a ratio of about 20 mol% to about 70 mol% or 75 mol%, or about 45 mol% to about 65 mol%, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol% of the total lipid present in the LNP. In further embodiments, the LNP comprises about 25% to about 75% cationic lipid on a molar basis, e.g., 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% cationic lipid on a molar basis (based on 100% total moles of lipid in the lipid nanoparticle). In some embodiments, the ratio of cationic lipid to nucleic acid, preferably RNA, more preferably mRNA, is about 3 to about 15, e.g., about 5 to about 13 or about 7 to about 11.

[0473] Other suitable (cationic or ionizable) lipids include those described in WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO2013063468, US20110256175, US20120128760, US2013063468 ... 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 and 8,569,256, and U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, US20140039032, and WO2017112865. In that context, WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO2013063468, US20110256175, US20120 128760, US20120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017070620, WO20170 99823, WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012 061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122, and 8,569,256 and the disclosures of U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, and US20140039032, and WO2017112865, all of which are incorporated by reference herein.

[0474] In other embodiments, the cationic or ionizable lipid is

[0475] [ka] TIFF2025532686000021.tif222141TIFF2025532686000022.tif123141.

[0476] In embodiments, amino or cationic lipids as defined herein have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above 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 reference to charged or neutral lipids refers to the nature of the predominant species and does not require that all of the lipids be present in a charged or neutral form. Lipids having two or more protonatable or deprotonatable groups or that are zwitterionic are not excluded and may be similarly suitable in the context of the present invention. In some embodiments, the protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., about 5 to about 7.

[0477] An LNP (or liposome, nanoliposome, lipoplex) can contain two or more (different) cationic lipids as defined herein. The cationic lipids may be selected to contribute different advantageous properties. For example, cationic lipids with different properties, such as amine pKa, chemical stability, circulatory half-life, tissue half-life, net tissue accumulation, or toxicity, can be used in an LNP (or liposome, nanoliposome, lipoplex). In particular, cationic lipids can be selected so that the properties of the mixed LNP are more desirable than those of a single LNP of individual lipids.

[0478] The amount of the constitutive cationic lipid or lipidoid can be selected taking into account the amount of nucleic acid cargo. In one embodiment, these amounts are selected to provide an N / P ratio of the nanoparticle or composition ranging from about 0.1 to about 20, for example. (i) in an amount to achieve an N / P ratio in the range of from about 1 to about 20, preferably from about 2 to about 15, more preferably from about 3 to about 10, even more preferably from about 4 to about 9, and most preferably about 6; (ii) in an amount to achieve an N / P ratio in the range of about 5 to about 20, more preferably about 10 to about 18, even more preferably about 12 to about 16, and most preferably about 14; (iii) in an amount to achieve 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) for lipid nanoparticles according to the invention, in particular lipid nanoparticles comprising cationic lipid III-3, in an amount to achieve an N / P ratio in the range of about 6, be selected.

[0479] In this context, the N / P ratio is defined as the molar ratio of the nitrogen atom ("N") of the basic nitrogen-containing group of the lipid or lipidoid to the phosphate group ("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 constitutive cationic and, if present, cationizable groups. When two or more cationic lipids are present, the N value should be calculated based on all cationic lipids contained in the lipid nanoparticle.

[0480] In some embodiments, the composition has a lipid to RNA molar ratio (N / P ratio) of about 2 to about 12, optionally an N / P ratio of 3 to about 8.

[0481] In one embodiment, the lipid nanoparticles comprise about 40% cationic lipid LKY750, about 10% zwitterionic lipid DSPC, about 48% cholesterol, and about 2% PEGylated lipid DMG (w / w).

[0482] In some embodiments, the LNP is (a) RNA, preferably mRNA, as used herein; (b) cationic lipid; (c) aggregation-reducing agent (e.g., polyethylene glycol (PEG) lipid or PEG-modified lipid); (d) optionally, non-cationic lipid (e.g., neutral lipid); and (e) optionally, sterol. Includes.

[0483] In some embodiments, the cationic lipid (defined above), non-cationic lipid (defined above), cholesterol (defined above), and / or PEG-modified lipid (defined above) may be combined in various relative molar ratios. For example, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid may be between about 30-60:20-35:20-30:1-15, or about 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 about 50:25:20:5, 50:20:25:5, 50:27:20:3, 40:30:20:10, 40:30:25:5, or 40:32:20:8, 40:32:25:3, or 40:33:25:2.

[0484] In some embodiments, the LNP (or liposome, nanoliposome, lipoplex) comprises ALC-0315, RNA as used herein, preferably mRNA, a neutral lipid that is DSPC, a steroid that is cholesterol, and a PEGylated lipid of formula ALC-0159.

[0485] In some embodiments, the LNPs comprise about 0.5-15 mol % PEG-modified lipid, about 5-25 mol % non-cationic lipid, about 25-55 mol % sterol, and about 20-60 mol % ionizable cationic lipid.

[0486] In one embodiment, the LNP consists essentially 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 about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.

[0487] In some embodiments, the RNA, preferably mRNA, is complexed with one or more lipids to form a lipid nanoparticle (LNP), wherein the LNP comprises: 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 analogue 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 PEGylated lipid of or derived from formula (IVa-ALC-0159): Includes.

[0488] In some embodiments, the mRNA is complexed with one or more lipids to form lipid nanoparticles (LNPs), wherein the LNPs comprise (i)-(iv) in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% polymer-conjugated lipid, preferably PEG-lipid.

[0489] In some embodiments, the lipid nanoparticles (or liposomes, nanoliposomes, lipoplexes) comprise a cationic lipid having formula (III-3) and / or a PEG-lipid having formula (IVa), optionally a neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and optionally a steroid, preferably cholesterol, wherein the molar ratio of the cationic lipid to DSPC is optionally in the range of about 2:1 to 8:1, and wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range of about 2:1 to 1:1.

[0490] In one embodiment, the composition comprises lipid nanoparticles (LNPs) having a molar ratio of RNA, preferably mRNA, of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or greater, more preferably 47.4:10:40.9:1.7 (i.e., cationic lipid (preferably a lipid of formula III-3 (ALC-0315)), DSPC, cholesterol, and polymer-conjugated lipid, preferably PEG-lipid (preferably a PEG-lipid of formula (IVa) having n=49, even more preferably a PEG-lipid of formula (IVa) having n=45, ALC-0159); dissolved in ethanol).

[0491] WO2017 / 070620 provides general information regarding LNP compositions and is incorporated herein by reference. 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, WO2016 / 037053, which are also incorporated herein by reference.

[0492] In various embodiments, LNPs suitable for encapsulating mRNA of the present invention have a size of about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm, about 90 nm to about 200 nm, about 90 nm to about 190 nm, about 90 nm to about 180 nm, about 90 nm to about 170 nm, about 90 nm to about 160 nm, about 90 nm to about 150 nm, about 90 nm to about 140 nm, about 90 nm to about 130 nm, about 90 nm to about 120 nm, about 90 nm to about 100 nm, about 70 nm to about The particles have an average diameter of 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 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. As used herein, the average diameter may be expressed in terms of the z-average size determined by dynamic light scattering, as commonly known in the art.

[0493] In some embodiments, the LNPs are 50-200 nm in diameter.

[0494] Suitably, the LNPs have a polydispersity of 0.4 or less, for example 0.3 or less. Typically, the PDI is determined by dynamic light scattering.

[0495] In some embodiments, the compositions have a polydispersity 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.

[0496] Vaccines and combination vaccines The immunogenic compositions described herein are suitable for use as vaccines.

[0497] In a second aspect, the present invention relates to a vaccine comprising the immunogenic composition described herein.

[0498] The vaccine may be a live attenuated vaccine, an inactivated vaccine, a recombinant vaccine or a nucleic acid-based vaccine.

[0499] The vaccine is suitable for active immunization against disease caused by the influenza viruses contained in the vaccine, preferably influenza subtype A viruses and influenza type B viruses.

[0500] In some embodiments, the vaccine is a multivalent vaccine.

[0501] In some embodiments, the vaccine is a trivalent influenza virus vaccine (i.e., contains immunogenic components from three strains of influenza virus) or a quadrivalent influenza virus vaccine (i.e., contains immunogenic components from four strains of influenza virus).

[0502] In some embodiments, the vaccine is a trivalent influenza virus vaccine.

[0503] In some embodiments, the trivalent influenza virus vaccine comprises three HA antigens, or nucleic acids, preferably mRNAs, encoding them.

[0504] In some embodiments, the trivalent influenza virus vaccine comprises two HA antigens or nucleic acids, preferably mRNA, encoding them from a strain of influenza A virus and one HA antigen or nucleic acid, preferably mRNA, encoding it from a strain of influenza B virus.

[0505] In some embodiments, the trivalent influenza virus vaccine comprises three mRNAs encoding three HA antigens.

[0506] In some embodiments, the trivalent influenza virus vaccine comprises two mRNAs encoding two HA antigens from a strain of influenza A virus and one mRNA encoding one HA antigen from a strain of influenza B virus.

[0507] In some embodiments, the trivalent influenza virus vaccine comprises two HA antigens and two NA antigens, or nucleic acids, preferably mRNA, encoding them, from a strain of influenza A virus, and one HA antigen and one NA antigen, or nucleic acids, preferably mRNA, encoding them, from a strain of influenza B virus.

[0508] In some embodiments, the trivalent influenza virus vaccine comprises six mRNAs encoding three HA antigens and three NA antigens.

[0509] In some embodiments, the trivalent influenza virus vaccine comprises four mRNAs encoding two HA antigens and two NA antigens from an influenza A virus strain and two mRNAs encoding one HA antigen and one NA antigen from an influenza B virus strain.

[0510] In some embodiments, the trivalent influenza virus vaccine comprises (a), (b), and (c) as defined herein, wherein the ratio of (a):(b):(c) is comprised between 1.5:1:1 and 5:1:1, preferably between 2:1:1 and 4:1:1, preferably between 2:1:1 and 3:1:1, and preferably is 2:1:1 or 3:1:1. In some embodiments, the vaccine is a quadrivalent influenza virus vaccine.

[0511] In some embodiments, the tetravalent influenza virus vaccine comprises four HA antigens, or nucleic acids, preferably mRNAs, encoding them.

[0512] In some embodiments, the quadrivalent influenza virus vaccine comprises two HA antigens or nucleic acids, preferably mRNA, encoding them from a strain of influenza A virus and two HA antigens or nucleic acids, preferably mRNA, encoding them from a strain of influenza B virus.

[0513] In some embodiments, the tetravalent influenza virus vaccine comprises four mRNAs encoding four HA antigens.

[0514] In some embodiments, the quadrivalent influenza virus vaccine comprises two mRNAs encoding two HA antigens from a strain of influenza A virus and two mRNAs encoding two HA antigens from a strain of influenza B virus.

[0515] In some embodiments, the tetravalent influenza virus vaccine comprises at least one of (a), (b), (c), and (d) as defined herein. 1 ) and (c 2 ), where (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and is preferably 2:1:1:2 or 3:1:1:3.

[0516] In some embodiments, the tetravalent influenza virus vaccine comprises four HA antigens or nucleic acids, preferably mRNAs, encoding them, and three NA antigens or nucleic acids, preferably mRNAs, encoding them (i.e., for example, a seven-component tetravalent influenza virus vaccine).

[0517] In some embodiments, the tetravalent influenza virus vaccine comprises four mRNAs encoding four HA antigens and three mRNAs encoding three NA antigens.

[0518] In some embodiments, the tetravalent influenza virus vaccine comprises at least one of (a), (b), (c), and (d) as defined herein. 1 ), (c 2 ), (c 3 ), (c 4 ) and (c 5 ), where (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and is preferably 2:1:1:2 or 3:1:1:3.

[0519] In some embodiments, the tetravalent influenza virus vaccine comprises four HA antigens or nucleic acids, preferably mRNA, encoding them, and four NA antigens or nucleic acids, preferably mRNA, encoding them (i.e., an eight-component tetravalent influenza virus vaccine).

[0520] In some embodiments, the tetravalent influenza virus vaccine comprises four mRNAs encoding four HA antigens and four mRNAs encoding four NA antigens.

[0521] In some embodiments, the tetravalent influenza virus vaccine comprises at least one of (a), (b), (c), and (d) as defined herein. 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and (c 6 ), where (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, and is preferably 2:1:1:2 or 3:1:1:3.

[0522] In some embodiments, the vaccine further comprises at least one antigen or at least one nucleic acid encoding said at least one antigen, e.g., at least one mRNA encoding an antigen from an additional pathogen, preferably the pathogen is a virus, preferably a respiratory virus.

[0523] In some embodiments, the antigen is derived from an additional virus selected from the group consisting of coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), Pneumoviridae (e.g., respiratory syncytial virus, metapneumovirus), and Paramyxoviridae (e.g., parainfluenza virus, henipavirus), and preferably the antigen from the additional virus is a spike protein or antigenic fragment thereof from the SARS-CoV-2 virus, or an mRNA encoding a spike protein or antigenic fragment thereof 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 set forth in Table 1 of published PCT application WO2021156267A1 or Table 1 of published PCT application WO2022137133A1 (each of which is incorporated herein by reference).

[0524] Kit or parts kit In a third aspect, the present invention provides an antigen or nucleic acid and / or mRNA as defined herein, preferably comprising (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The present invention provides a kit or kit of parts comprising mRNA of a gene encoding a nucleotide sequence ...

[0525] The technical description of the kit may include information regarding administration and dosage and patient population. Such a kit, preferably a kit-of-parts, may for example be applicable to any of the applications or uses described herein, preferably the use of an immunogenic composition or vaccine for the treatment or prevention of infection or disease caused by influenza viruses, preferably influenza A and / or B viruses.

[0526] In some embodiments, the immunogenic composition or vaccine is provided in a separate part of the kit, wherein the immunogenic composition or vaccine is suitably freeze-dried or spray-dried or spray-freeze-dried.

[0527] The kit may further comprise, as part thereof, a vehicle (eg, a buffer solution) for solubilizing the dried or lyophilized nucleic acid composition or vaccine.

[0528] In some embodiments, the antigen or nucleic acid and / or mRNA as defined herein, preferably (a), (b), (c), (d) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is formulated separately.

[0529] In some embodiments, the antigen or nucleic acid and / or mRNA as defined herein, preferably (a), (b), (c), (d) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is provided as part of the kit.

[0530] In some embodiments, the antigen or nucleic acid and / or mRNA as defined herein, preferably (a), (b), (c), (d) 1 ), (c2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) are provided as separate parts of the kit. Preferably, the kit or kit of parts comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight parts, each of which comprises at least one of the nucleic acids and / or mRNAs defined herein, preferably (a), (b), (c), (d), (e), (f), (g), (h), (i), (j ... 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA.

[0531] In some embodiments, the kits or kits of parts provided herein comprise multi-dose (multi-dose) containers and / or administration devices (e.g., syringes for intramuscular and / or intradermal injection) for administration of the compositions / vaccines.

[0532] Formulation and Administration In some embodiments, the antigen or nucleic acid as defined herein, preferably mRNA, is co-formulated. 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA are co-formulated, i.e., formulated together.

[0533] In some embodiments, the antigens or nucleic acids, preferably mRNA, as defined herein of the kit or kit of parts are formulated separately. 1 ), (c 2 ), (c 3 ), (c4 ), (c 5 ) and / or (c 6 ) mRNA is formulated separately.

[0534] In some embodiments, the antigen or nucleic acid as defined herein, preferably mRNA, is co-packaged. 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNAs are co-packaged, i.e., packed together, optionally formulated separately and then packed together.

[0535] In some embodiments, the antigen or nucleic acid as defined herein, preferably the mRNA, is formulated as a bedside admixture. Preferably, the mRNA as defined herein, preferably the (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is formulated as a bedside admixture.

[0536] A "bedside admixture formulation" as used herein should be understood as a formulation in which several (e.g., one or more) of the immunogenic components (e.g., mRNA) are preferably each formulated independently (e.g., in an LNP) before being mixed to produce the bedside admixture formulation.

[0537] In some embodiments, the bedside mixed formulation is obtained by a method comprising: (1) formulating each antigen or nucleic acid, preferably mRNA, independently (e.g., in an LNP); and (2) mixing each (in an LNP) formulated antigen or nucleic acid, preferably mRNA.

[0538] In some embodiments, the bedside admixture is obtained by a method comprising: (1) co-forming (e.g., in a LNP) the antigen or nucleic acid encoding same, preferably mRNA, derived from a strain of influenza A virus; (2) co-forming (e.g., in a LNP) the antigen or nucleic acid encoding same, preferably mRNA, derived from a strain of influenza B virus; and (3) mixing the co-formulated antigen (in a LNP) or nucleic acid encoding same, preferably mRNA, derived from a strain of influenza A virus with the co-formulated antigen (in a LNP) or nucleic acid encoding same, preferably mRNA, derived from a strain of influenza B virus.

[0539] In some embodiments, the bedside admixture formulation is obtained by a method comprising: (1) co-forming (e.g., in a LNP) the antigens or nucleic acids encoding same, preferably mRNA, derived from a strain of influenza A virus; (2) independently formulating each antigen or nucleic acid encoding same, preferably mRNA, derived from a strain of influenza B virus; and (3) mixing the co-formulated antigens or nucleic acids, preferably mRNA, derived from a strain of influenza A virus with each formulated antigen or nucleic acid, preferably mRNA, derived from a strain of influenza B virus.

[0540] The immunogenic compositions may be administered by a variety of suitable routes, including parenteral administration, e.g., intramuscular, intradermal, intranasal, or subcutaneous administration. Preferably, the immunogenic compositions, vaccines, or kits or kits-of-parts described herein are administered intramuscularly and / or intradermally.

[0541] In some embodiments, intramuscular administration of the immunogenic compositions described herein results in expression of the encoded antigen construct in a subject. Administration of the immunogenic compositions described herein results in translation of the mRNA and production of the encoded antigen in a subject.

[0542] The immunogenic compositions described herein may be provided in liquid or dried (eg, lyophilized) form.

[0543] In some embodiments, the immunogenic composition is provided in liquid form.

[0544] In embodiments, the immunogenic compositions may be lyophilized to improve the storage stability of the formulation and / or RNA, preferably mRNA. In embodiments, the immunogenic compositions described herein may be spray dried to improve the storage stability of the formulation and / or RNA, preferably mRNA. The lyoprotectant for lyophilization and / or spray drying may be selected from trehalose, sucrose, mannose, dextran, and inulin.

[0545] Preferably, the immunogenic compositions described herein are freeze-dried (e.g., according to WO2016165831 or WO2011069586) to obtain a thermostable dry RNA (preferably mRNA) (powder) composition as defined herein. Alternatively, the immunogenic compositions can be dried using spray drying or spray freeze drying (e.g., according to WO2016184575 or WO2016184576) to obtain a thermostable composition (powders) as defined herein.

[0546] Thus, in some embodiments, the immunogenic composition is a dry composition.

[0547] The term "dry composition" as used herein should be understood as a composition that has been freeze-dried or spray-dried or spray-freeze-dried as described above to obtain a temperature-stable dry composition (powder) comprising, for example, RNA, preferably mRNA (as described above), complexed with LNPs.

[0548] In embodiments, the freeze-dried or spray-dried composition has a moisture content of less than about 10%.

[0549] In some embodiments, the freeze-dried or spray-dried composition has a moisture content of about 0.5% to 5%.

[0550] In some embodiments, the freeze-dried or spray-dried composition is stable for at least 2 months, preferably at least 3 months, 4 months, 5 months, or 6 months after storage at about 5°C.

[0551] The liquid used for reconstitution is substantially aqueous, such as water for injection, phosphate buffered saline, etc. The need for a buffer and / or tonicity adjuster depends on both the container contents being reconstituted and the subsequent use of the reconstituted contents. The buffer may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate, and TRIS. The buffer may be a phosphate buffer, such as Na / Na2PO4, Na / K2PO4, or K / K2PO4.

[0552] Preferably, the formulations used in the present invention have a dosage volume of 0.05 ml to 1 ml, for example 0.1 ml to 0.6 ml, particularly 0.45 ml to 0.55 ml, for example 0.5 ml. The volume of the composition used may vary depending on the subject, route of administration, and site. Smaller doses may be administered via the intradermal route. A typical human dose for administration via a route such as the intramuscular route is in the range of 200 μl to 750 μl, for example 400 μl to 600 μl, particularly about 500 μl, for example 500 μl.

[0553] The immunogenic compositions described herein can be provided in a variety of physical containers, such as vials or pre-filled syringes.

[0554] In some embodiments, the immunogenic composition is provided in the form of a single dose, hi other embodiments, the immunogenic composition, vaccine, or kit or kit-of-parts is provided in a multi-dose form, e.g., containing 2, 5, or 10 doses.

[0555] When transferring liquids between containers (e.g., from a vial to a syringe), it is common to provide a "surplus" to ensure that the entire amount needed can be conveniently transferred. The level of excess needed will vary depending on the situation, but excessive excess should be avoided to reduce waste, and insufficient excess can cause practical problems. The excess can be on the order of 20-100 μl per dose, e.g., 30 μl or 50 μl.

[0556] Stabilizers may also be present, which may be particularly important when multi-dose containers are provided, as doses of the final formulation may be administered to a subject over a period of time.

[0557] The formulation is preferably sterile.

[0558] Approaches to establishing strong and durable immunity often involve repeated immunizations, i.e., boosting the immune response by administering one or more additional doses. Such boosts can be administered using the same immunogenic composition (allogeneic boost) or a different immunogenic composition (heterogeneous boost). The present invention can be applied as either a priming or boost immunization, as part of an allogeneic or heterogeneous prime / boost regimen.

[0559] Therefore, the administration of the immunogenic composition described herein can be part of a multi-dose administration regimen.For example, the immunogenic composition described herein can be provided as a priming dose in a multi-dose regimen, particularly a 2-dose or 3-dose regimen, particularly a 2-dose regimen.The immunogenic composition described herein can be provided as a boosting dose in a multi-dose regimen, particularly a 2-dose or 3-dose regimen, for example a 2-dose regimen.

[0560] The priming dose and boosting dose can be homologous or heterologous. Thus, the immunogenic compositions described herein can be provided as the priming dose and boosting dose in a homologous multiple-dose regimen, particularly a two-dose or three-dose regimen, particularly a two-dose regimen. Alternatively, the immunogenic compositions described herein can be provided as the priming dose or boosting dose in a heterologous multiple-dose regimen, particularly a two-dose or three-dose regimen, particularly a two-dose regimen, where the boosting doses can be different (e.g., immunogenic compositions described herein; or alternative antigen presentations in the presence or absence of an adjuvant, such as a squalene emulsion adjuvant).

[0561] The interval between multiple administrations can be 2 weeks to 6 months, for example, 3 weeks to 3 months. Periodic, longer-term (e.g., every 2 to 10 years) additional (booster) administrations can also be administered.

[0562] In some embodiments, the immunogenic composition further comprises at least one pharmaceutically acceptable carrier.

[0563] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" preferably includes the liquid or non-liquid basis of a composition for administration. When the composition is provided in liquid form, the carrier may be water, e.g., pyrogen-free water; isotonic saline; or a buffered (aqueous) solution, e.g., a phosphate, citrate, or other buffer solution. Water or a buffer, more preferably an aqueous buffer, containing sodium salts, preferably at least 50 mM sodium salts, calcium salts, preferably at least 0.01 mM calcium salts, and optionally potassium salts, preferably at least 3 mM potassium salts, may be used. According to some embodiments, the sodium, calcium, and optionally potassium salts may occur in the form of their halides, e.g., chlorides, iodides, or bromides; their hydroxides, carbonates, bicarbonates, or sulfates; or the like. Examples of sodium salts include NaCl, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of optional potassium salts include KCl, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include CaCl2, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2.

[0564] Additionally, the aforementioned cationic organic anions may be present in a buffer solution. Thus, in embodiments, the immunogenic composition may include one or more pharmaceutically acceptable carriers or excipients, for example, to increase in vivo stability, increase cell transfection, enable sustained or delayed translation, increase translation of the encoded antigenic peptide or protein, and / or modify the release profile of the encoded antigenic peptide or protein in vivo. In addition to traditional excipients, such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, and preservatives, excipients may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, polynucleotide-transfected cells, hyaluronidase, nanoparticle mimics, and combinations thereof. In embodiments, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may also be used, which are suitable for administration to a subject. As used herein, the term "compatible" means that the components of the composition can be mixed with at least one nucleic acid of component A and / or component B, and optionally multiple nucleic acids of the composition, in a manner that does not result in interactions that substantially reduce the biological activity or pharmaceutical efficacy of the composition under typical use conditions (e.g., intramuscular or intradermal administration). Pharmaceutically acceptable carriers or excipients must have sufficiently high purity and sufficiently low toxicity to be suitable for administration to the subject to be treated.Compounds that can be used as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, trehalose, mannose, and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid lubricants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oils derived from the cacao genus; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; and alginic acid.

[0565] At least one pharmaceutically acceptable carrier or excipient of the immunogenic composition may be selected to be suitable for intramuscular or intradermal delivery / administration of the immunogenic composition. The immunogenic composition is preferably a composition suitable for intramuscular administration to a subject.

[0566] Subjects to which the immunogenic compositions are contemplated include, but are not limited to, humans and / or other primates, commercially relevant mammals, such as mammals including cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats, and / or commercially relevant birds, such as birds including poultry, chickens, ducks, geese, and / or turkeys.

[0567] In various embodiments, the immunogenic composition does not exceed a certain percentage of free RNA, preferably mRNA.

[0568] In this context, "free RNA, preferably mRNA" or "uncomplexed RNA, preferably mRNA" or "unencapsulated RNA, preferably mRNA" includes RNA, preferably mRNA molecules that are not encapsulated in a lipid-based carrier as defined herein. During formulation of a composition (e.g., during encapsulation of RNA, preferably mRNA, in a lipid-based carrier), free RNA, preferably mRNA, may represent a contamination or impurity.

[0569] In embodiments, the immunogenic composition comprises free RNA, preferably mRNA, in the range of about 30% to about 0%. In embodiments, the composition comprises about 20% free RNA, preferably mRNA (and about 80% encapsulated RNA, preferably mRNA), about 15% free RNA, preferably mRNA (and about 85% encapsulated RNA, preferably mRNA), about 10% free RNA, preferably mRNA (and about 90% encapsulated RNA, preferably mRNA), or about 5% free RNA, preferably mRNA (and about 95% encapsulated RNA, preferably mRNA). In some embodiments, the composition comprises less than about 20% free RNA, preferably mRNA, preferably less than about 15% free RNA, preferably mRNA, more preferably less than about 10% free RNA, preferably mRNA, and most preferably less than about 5% free RNA, preferably mRNA.

[0570] The term "encapsulated RNA, preferably mRNA" includes RNA, preferably mRNA molecules encapsulated in a lipid-based carrier as defined herein. The percentage of encapsulated RNA, preferably mRNA, in the context of the present invention is typically determined using a RiboGreen assay.

[0571] Medical Uses (Primary and Secondary / Further Medical Uses) and Methods of Treatment In a fourth aspect, the present invention relates to an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein for use as a medicament.

[0572] Also described herein is the use of an immunogenic composition, vaccine or kit or kit of parts described herein as a medicine.

[0573] In a fifth aspect, the present invention relates to an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein for use in the treatment or prevention of infection by an influenza virus, preferably influenza A and / or influenza B.

[0574] Also described herein is the use of the immunogenic compositions, vaccines or kits or kits of parts described herein in the treatment or prevention of infection by influenza viruses, preferably influenza A and / or influenza B.

[0575] In some embodiments, a single dose of the immunogenic composition is 0.1 to 1000 μg, particularly 1 to 500 μg, particularly 2 to 500 μg, particularly 10 to 250 μg, and preferably 25 to 150 μg, in terms of total mRNA.

[0576] In a further embodiment, a single dose of the immunogenic composition comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different mRNAs, of which each mRNA is 1 to 200 μg, preferably 1 to 60 μg, preferably 1 to 25 μg, preferably 2 to 25 μg, preferably 3 to 18 μg.

[0577] In some embodiments, a single dose of the composition is 2 to 500 μg, particularly 10 to 250 μg, for example 10 to 75 μg, of total mRNA.

[0578] In some embodiments, a single dose of the immunogenic composition is 10-100 μg.

[0579] In some embodiments, a single dose of the composition is 6, 12, 15, 16, 18, 24, 32, 36, 48, 54, 60, 72, 84, 96, or 120 μg of total mRNA.

[0580] In some embodiments, a single dose of the composition is 1-10 μg of each mRNA for a young adult, eg, 18-64 years old.

[0581] In some embodiments, a single dose of the composition is 1, 2, 3, 6, or 9 μg of each mRNA for a young adult, eg, 18-64 years of age.

[0582] In some embodiments, a single dose of the composition is 15-50 μg of total mRNA for a young adult, eg, 18-64 years old.

[0583] In some embodiments, a single dose of the composition is 16, 32, or 48 μg of total mRNA for a young adult, eg, 18-64 years of age.

[0584] In some embodiments, a single dose of the composition is 2-20 μg of each mRNA for elderly people, eg, people 65 years of age or older.

[0585] In some embodiments, a single dose of the composition is 2, 3, 6, 9, or 18 μg of each mRNA for an elderly person, eg, 65 years or older.

[0586] In some embodiments, a single dose of the composition is 30 to 100 μg of total mRNA for elderly people, eg, people 65 years of age or older.

[0587] In some embodiments, a single dose of the composition is 32, 48, or 96 μg of total mRNA for an elderly person, eg, 65 years or older.

[0588] In some embodiments, the use is for intramuscular and / or intradermal administration, preferably intramuscular administration.

[0589] In some embodiments, the antigen or nucleic acid and / or mRNA described herein, preferably (a), (b), (c), (c 1 ), (c2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is administered at different injection sites.

[0590] In some embodiments, the antigen or nucleic acid and / or mRNA derived from a strain of influenza A virus is administered at an injection site that is different from the injection site at which the antigen or nucleic acid and / or mRNA derived from a strain of influenza B virus is administered.

[0591] In some embodiments, the antigens or nucleic acids and / or mRNA derived from strains of influenza B virus are administered separately, preferably at different injection sites.

[0592] In some embodiments, an immune response, preferably an adaptive immune response, more preferably a protective adaptive immune response against influenza virus, preferably influenza A and / or influenza B, is elicited.

[0593] In some embodiments, an immune response is elicited.

[0594] In some embodiments, an adaptive immune response is elicited.

[0595] In some embodiments, a protective adaptive immune response against influenza virus is elicited.

[0596] In some embodiments, a protective adaptive immune response against influenza A and / or B viruses is elicited.

[0597] In some embodiments, a protective adaptive immune response is elicited against one or more influenza A virus subtypes and / or influenza B virus lineages, preferably influenza A H1N1, influenza A H3N2, influenza B / Yamagata lineage, and influenza B / Victoria lineage.

[0598] In some embodiments, the immune response induced comprises neutralizing antibody titers against influenza virus, preferably influenza A and / or B virus, more preferably one or more influenza A virus subtypes and / or influenza B virus strains, more preferably influenza A H1N1, influenza A H3N2, influenza B / Yamagata strain, and influenza B / Victoria strain.

[0599] In some embodiments, the immune response elicited comprises functional antibodies capable of effectively neutralizing the respective viruses.

[0600] In some embodiments, the immune response elicited is a cross-reactive immune response, where functional antibodies that can effectively neutralize each virus also neutralize viruses belonging to the same and / or other influenza A subtypes and / or influenza B lineages.

[0601] In some embodiments, the cross-reactive immune response is allogeneic, xenogeneic and / or heterosubtypic.

[0602] The term "homologous" in the context of the immune response to be elicited will be recognized and understood by those skilled in the art. The term refers, for example, to an immune response elicited against the same strain, e.g., the same influenza A strain or the same influenza B strain. For example, an immunogenic composition can include an HA antigen (or a nucleic acid encoding it, preferably RNA, preferably mRNA) from A / Michigan / 45 / 2015 (H1N1pdm9) that can elicit an immune response against the A / Michigan / 45 / 2015 (H1N1pdm9) strain.

[0603] The term "heterologous" in the context of the immune response elicited will be recognized and understood by those skilled in the art. The term refers to, for example, an immune response elicited against a different strain within a subtype (in the case of influenza A virus) or lineage (in the case of influenza B virus), e.g., a different influenza A strain within a subtype, such as an H1 or H3 subtype. For example, an immunogenic composition can include an HA antigen (or a nucleic acid encoding it, preferably RNA, preferably mRNA) from A / Michigan / 45 / 2015 (H1N1pdm9) that can elicit an immune response against the A / New Caledonia / 20 / 1999 (H1N1) strain.

[0604] The term "heterosubtypic" in the context of an elicited immune response will be recognized and understood by those skilled in the art. The term refers to an immune response elicited against, for example, one or more different subtypes (in the case of influenza A viruses) or different strains within a lineage (in the case of influenza B viruses). For example, an immunogenic composition can include an HA antigen (or a nucleic acid encoding it, preferably RNA, preferably mRNA) from A / Michigan / 45 / 2015 (H1N1pdm9) that can elicit an immune response against Hong Kong / 4801 / 2014 (H3N2).

[0605] In a further embodiment, the elicited immune response comprises a broad and functional cellular T cell response against the respective virus, in particular, the elicited immune response comprises a CD4+ T cell immune response and / or a CD8+ T cell immune response.

[0606] In a further embodiment, the immune response elicited comprises a well-balanced B cell and T cell response against each virus.

[0607] In some embodiments, the immune response elicited comprises an antigen-specific immune response.

[0608] In some embodiments, the immune response that is elicited partially or completely reduces the severity of one or more symptoms and / or the length of time that one or more symptoms of influenza virus infection are experienced by the subject.

[0609] In some embodiments, the immune response elicited reduces the likelihood of developing an established influenza virus infection after challenge.

[0610] In some particular embodiments, the immune response elicited slows the progression of influenza, preferably influenza A and / or B.

[0611] In a sixth aspect, the present invention relates to a method for the treatment or prevention of a disorder caused by an influenza virus, preferably influenza A and / or influenza B, comprising applying or administering to a subject in need thereof an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein.

[0612] Prevention (suppression) or treatment of a disease, particularly a viral infection, refers to the suppression of the full development of a disease or condition in a subject at risk of the disease, e.g., a viral infection. "Treatment" refers to a therapeutic intervention that ameliorates signs or symptoms of a disease or condition after they have begun to develop. The term "amelioration" with respect to a disease or condition refers to any observable beneficial effect of treatment. Suppression of a disease can include preventing or reducing the risk of a disease, for example, preventing or reducing the risk of a viral infection. A beneficial effect can be demonstrated, for example, by a delay in the onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease, a delay in the progression of the disease, a reduction in viral load, an improvement in the overall health or well-being of the subject, or other parameters specific to a particular disease. "Prophylactic" treatment is treatment administered to a subject who does not show signs of the disease or who shows only early signs, with the aim of reducing the risk of developing a condition.

[0613] In some embodiments, the composition, vaccine, or kit or kit-of-parts is administered in a therapeutically effective amount.

[0614] In some embodiments, the disorder is infection with an influenza virus, preferably an influenza A and / or B virus.

[0615] In some embodiments, the required subject is a mammalian subject, preferably a human subject.

[0616] In a seventh aspect, the present invention relates to a method for eliciting an immune response comprising applying or administering to a subject in need thereof an immunogenic composition, a vaccine or a kit or kit-of-parts as described herein.

[0617] In some embodiments, the immune response is an adaptive immune response, preferably a protective adaptive immune response against influenza virus, preferably influenza A virus and / or influenza B virus.

[0618] In some embodiments, an immune response is elicited.

[0619] In some embodiments, an adaptive immune response is elicited.

[0620] In some embodiments, a protective adaptive immune response against influenza virus is elicited.

[0621] In some embodiments, a protective adaptive immune response against influenza A and / or B viruses is elicited.

[0622] In some embodiments, a protective adaptive immune response is elicited against one or more influenza A virus subtypes and / or influenza B virus lineages, preferably influenza A H1N1, influenza A H3N2, influenza B / Yamagata lineage, and influenza B / Victoria lineage.

[0623] In some embodiments, the immune response induced comprises neutralizing antibody titers against influenza virus, preferably influenza A and / or B virus, more preferably one or more influenza A virus subtypes and / or influenza B virus strains, more preferably influenza A H1N1, influenza A H3N2, influenza B / Yamagata strain, and influenza B / Victoria strain.

[0624] In some embodiments, the immune response elicited comprises functional antibodies capable of effectively neutralizing the respective viruses.

[0625] In some embodiments, the immune response elicited is a cross-reactive immune response, where functional antibodies that can effectively neutralize each virus also neutralize viruses belonging to the same and / or other influenza A subtypes and / or influenza B lineages.

[0626] In some embodiments, the cross-reactive immune response is allogeneic, xenogeneic and / or heterosubtypic.

[0627] In a further embodiment, the elicited immune response comprises a broad and functional cellular T cell response against the respective virus, in particular, the elicited immune response comprises a CD4+ T cell immune response and / or a CD8+ T cell immune response.

[0628] In a further embodiment, the immune response elicited comprises a well-balanced B cell and T cell response against each virus.

[0629] In some embodiments, the immune response elicited comprises an antigen-specific immune response.

[0630] In some embodiments, the immune response that is elicited partially or completely reduces the severity of one or more symptoms and / or the length of time that one or more symptoms of influenza virus infection are experienced by the subject.

[0631] In some embodiments, the immune response elicited reduces the likelihood of developing an established influenza virus infection after challenge.

[0632] In some particular embodiments, the immune response elicited slows the progression of influenza, preferably influenza A and / or B.

[0633] In some embodiments, the required subject is a mammalian subject, preferably a human subject.

[0634] In some embodiments, the compositions, vaccines, or kits or kits of parts described herein are administered in an amount effective to induce a T cell response against influenza A H1N1, influenza A H3N2, influenza B / Yamagata strain, and influenza B / Victoria strain.

[0635] In some embodiments, the described compositions, vaccines, or kits or kits of parts are administered in an amount effective to induce a neutralizing antibody response against influenza A H1N1, influenza A H3N2, influenza B / Yamagata strain, and influenza B / Victoria strain.

[0636] In embodiments, administration of the immunogenic composition, vaccine, or kit to a subject elicits neutralizing antibodies and does not elicit disease-enhancing antibodies. In particular, administration of the immunogenic composition, vaccine, or kit to a subject does not elicit immunopathological effects, such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0637] Further definitions For clarity and readability, the following definitions are provided. The technical features cited with respect to these definitions can be interpreted in each embodiment of the present invention. Additional definitions and explanations can be specifically provided in the context of these embodiments.

[0638] Throughout this specification, including the claims, where the context permits, the word "comprise" and variations thereof, such as "comprises," should be construed to include the indicated element (e.g., a singular integer) or elements (e.g., multiple integers) without necessarily excluding any other elements (e.g., integers). Thus, a composition "comprising" X can consist of only X, or it can include something additional, such as X + Y.

[0639] The term "substantially" does not exclude "completely." For example, a composition that is "substantially free" of Y can be completely free of Y. If desired, the term "substantially" may be omitted from the definition of the present invention.

[0640] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0641] Unless otherwise indicated, a process that includes mixing two or more components does not require any particular mixing order. Thus, the components can be mixed in any order. If three components are present, two components can be combined with each other, and then that combination can be combined with the third component, etc.

[0642] The term "immunogenic fragment" or "immunogenic variant" should be understood as any fragment / variant of the corresponding influenza antigen that is capable of eliciting an immune response in a subject.

[0643] Percentages in numerical contexts should be understood as relative values ​​to the total number of the respective item. In other cases, and unless the context indicates otherwise, percentages should be understood as percentages by weight (wt.-%).

[0644] About: The term "about" is used when a determinant or value does not necessarily have to be identical (i.e., 100% identical). Thus, "about" means that a determinant or value may deviate by 1% to 20%, e.g., 1% to 10%, particularly 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. For example, those skilled in the art will recognize that certain parameters or determinants may vary slightly depending on how the parameter is determined. For example, if a particular determinant or value is defined herein as having a length of, e.g., "about 100 nucleotides," that length may deviate by 1% to 20%. Thus, those skilled in the art will recognize that, in that particular example, the length may deviate by 1 to 20 nucleotides. Thus, a length of "about 100 nucleotides" may encompass sequences ranging from 80 to 120 nucleotides.

[0645] Adaptive immune response: The term "adaptive immune response" as used herein will be recognized and understood by those skilled in the art. The term is intended to refer to, for example, an antigen-specific response of the immune system (adaptive immune system). Antigen specificity allows for the generation of a response tailored to a particular pathogen or pathogen-infected cell. The ability to mount these tailored responses is usually maintained in the body by "memory cells" (B cells).

[0646] Antigen: The term "antigen" as used herein will be recognized and understood by those of skill in the art. The term is intended to mean a substance that can be recognized by an immune system, such as the adaptive immune system, and that can elicit an antigen-specific immune response (e.g., by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response). Typically, an antigen can be or include a peptide or protein that is presented to T cells by MHC. Also understood as antigens are fragments, variants, and derivatives of peptides or proteins that contain at least one epitope.

[0647] Antigenic peptide, polypeptide, or protein: The term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by those skilled in the art. The term is intended to mean, for example, a peptide, protein derived from a protein (antigenic or immunogenic) that stimulates the body's adaptive immune system to produce an adaptive immune response. Thus, an antigenic / immunogenic peptide or protein comprises at least one epitope (as defined herein) or antigen (as defined herein) of the protein from which it is derived.

[0648] Cationic: Unless otherwise clear from the specific context, the term "cationic" means that the respective structure carries a positive charge, either permanently or non-permanently depending on specific conditions, such as pH. Thus, the term "cationic" includes both "permanently cationic" and "cationizable." The term "permanently cationic," for example, means that the respective compound, group, or atom is positively charged at any pH value or hydrogen ion activity in its environment. Typically, the positive charge is caused by the presence of a quaternary nitrogen atom.

[0649] Cationizable: As used herein, the term "cationizable" means that a compound, group, or atom is positively charged at a lower pH of the environment and uncharged at a higher pH. Also, in non-aqueous environments where the pH value cannot be determined, a cationizable compound, group, or atom is positively charged at a high hydrogen ion concentration and uncharged at a low hydrogen ion concentration or activity. This depends on the individual properties of the cationizable or polycationizable compound, particularly the pKa of each cationizable group or atom (at what pH or hydrogen ion concentration it is charged or uncharged). In a dilute aqueous environment, the proportion of positively charged cationizable compounds, groups, or atoms can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, it is suitably positively charged at pH values ​​of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, e.g., pH values ​​of 9 or less, 8 or less, or 7 or less, e.g., physiological pH values, e.g., about 7.3 to 7.4, i.e., under physiological conditions, particularly the physiological salt conditions of cells in vivo. In other embodiments, the cationizable compound or moiety is suitably primarily neutral at physiological pH values, e.g., about 7.0 to 7.4, but positively charged at lower pH values. In some embodiments, the pKa range of the cationizable compound or moiety is about 5 to about 7.

[0650] Coding sequence / coding region: As used herein, the terms "coding sequence" or "coding region" and the corresponding abbreviation "cds" will be recognized and understood by those skilled in the art. The term is intended to mean a sequence of several nucleotide triplets that can be translated into, for example, a peptide or protein. A coding sequence in the context of the present invention can be an RNA sequence consisting of several nucleotides divisible by three, which begins with a start codon and ends, for example, with a stop codon.

[0651] Derived: The term "derived" as used throughout this specification in the context of nucleic acids [i.e., with respect to a nucleic acid that is "derived" from (another) nucleic acid] means that a nucleic acid derived from (another) nucleic acid shares, for example, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid from which it is derived. Those skilled in the art will recognize that sequence identity is typically calculated with respect to nucleic acids of the same type, i.e., with respect to DNA sequences or with respect to RNA sequences. Thus, when DNA is "derived" from RNA or RNA is "derived" from DNA, it is understood that in a first step the RNA sequence is converted to the corresponding DNA sequence [particularly by substituting uracil (U) with thymine (T) throughout the sequence], or vice versa, the DNA sequence is converted to the corresponding RNA sequence [particularly by substituting T with U throughout the sequence]. The sequence identity of the DNA sequence or the sequence identity of the RNA sequence is then determined. For example, a nucleic acid "derived" from a nucleic acid also refers to a nucleic acid that has been modified compared to the nucleic acid from which it is derived, for example to further enhance RNA stability and / or to extend and / or increase protein production. In the context of amino acid sequences (e.g., antigenic peptides or proteins), the term "derived from" means that an amino acid sequence derived from (another) amino acid sequence shares, for example, at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence from which it is derived.

[0652] Epitope: As used herein, "epitope" (also referred to in the art as "antigenic determinant") will be recognized and understood by those of skill in the art. The term is intended to refer to, for example, T cell epitopes and B cell epitopes. T cell epitopes or portions of antigenic peptides or proteins can include fragments preferably having a length of about 6 to about 20 or more amino acids, such as fragments processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g., 8, 9, or 10 (or even 11 or 12) amino acids, or fragments processed and presented by MHC class II molecules, preferably having a length of about 13 to about 20 or more amino acids. These fragments are typically recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule. That is, the fragments are typically not recognized in their native form. A B-cell epitope is typically a fragment located on the outer surface of a (natural) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, and even more preferably having 6 to 9 amino acids, which can be recognized by an antibody, i.e., in its native form. Such an epitope of a protein or peptide may further be selected from any of the variants of such proteins or peptides listed herein. In this context, an epitope may be a conformational epitope or a discontinuous epitope (which is discontinuous in the amino acid sequence of the protein or peptide defined herein but is held together in the three-dimensional structure) composed of segments of the protein or peptide defined herein, or a continuous or linear epitope composed of a single polypeptide chain.

[0653] Fragment: The term "fragment" as used throughout the specification in the context of a nucleic acid sequence (e.g., RNA or DNA) or an amino acid sequence may typically refer to a shorter portion of the full-length sequence, e.g., of a nucleic acid or amino acid sequence. Thus, a fragment typically consists of a sequence that is identical to a corresponding stretch within the full-length sequence. A specific fragment of a sequence in the context of the present invention consists of a contiguous stretch of an entity, such as nucleotides or amino acids, that corresponds to a contiguous portion of the entity in the molecule from which the fragment is derived, representing at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the entire (i.e., full-length) molecule (e.g., a viral protein) from which the fragment is derived. The term "fragment" as used throughout the specification in the context of a protein or peptide may typically include a protein or peptide sequence as defined herein that, with respect to its amino acid sequence, is truncated at the N-terminus and / or C-terminus compared to the amino acid sequence of the original protein. The term "fragment" as used throughout the specification in the context of an RNA sequence may typically include an RNA sequence that is truncated at the 5' and / or 3' end compared to a reference RNA sequence. Such truncation may therefore occur at the amino acid level or, correspondingly, at the nucleic acid level. Accordingly, the sequence identity with respect to such fragments as defined herein may, for example, be with respect to the entire protein or peptide as defined herein, or the entire (encoding) nucleic acid molecule of such protein or peptide. A fragment of a protein or peptide may comprise at least one epitope of those proteins or peptides.

[0654] Heterologous: The term "heterologous" or "heterologous sequence" as used throughout this specification in the context of a nucleic acid sequence or an amino acid sequence should be understood as a sequence (e.g., RNA, DNA, amino acid) that is derived from another gene, another allele, or, for example, another species or virus. Typically, two sequences are understood to be "heterologous" when they are not derivable from the same gene or the same allele. That is, heterologous sequences may be derivable from the same organism or virus, but in nature they are not found in the same nucleic acid or protein.

[0655] Humoral immune response: The term "humoral immunity" or "humoral immune response" will be recognized and understood by those skilled in the art. The term is intended to refer, for example, to B cell-mediated antibody production and, in some cases, the auxiliary processes associated with antibody production. A humoral immune response can typically be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell generation. Humoral immunity can also refer to the effector functions of antibodies, including neutralization of pathogens and toxins, classical complement activation, and opsonization to promote phagocytosis and pathogen elimination.

[0656] Identity (of a sequence): The term "identity" used throughout this specification in the context of a nucleic acid sequence or an amino acid sequence will be recognized and understood by those skilled in the art. The term is intended to mean, for example, the percentage of two sequences that are identical. To determine the percentage of identity between two sequences, such as the nucleic acid sequences or amino acid (aa) sequences defined herein, for example, the aa sequences encoded by the nucleic acid sequences defined herein or the aa sequences themselves, the sequences can be aligned so that they are subsequently compared to each other. Thus, for example, a position in a first sequence can be compared to the corresponding position in a second sequence. If a position in the first sequence is occupied by the same residue as in the second sequence, the two sequences are identical at this position. Otherwise, the sequences differ at this position. If there is an insertion in the second sequence compared to the first sequence, a gap can be inserted in the first sequence to allow for further alignment. If there is a deletion in the second sequence compared to the first sequence, a gap can be inserted in the second sequence to allow for further alignment. Thus, the percent identity of two sequences is a function of the number of identical positions divided by the total number of positions (including positions occupied in only one sequence). The percent identity of two sequences can be determined using an algorithm (e.g., the algorithm integrated in the BLAST program).Use the EMBOSS Water sequence alignment tool at the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_water / with parameters gap open = 12, gap extend = 1, matrix = BLOSUM62 (for protein sequences) or the EMBOSS Needle sequence alignment tool at the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / with default parameters [e.g. gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10 and end gap extend = 0.5 and matrix = BLOSUM62 (for protein sequences) or matrix = full DNA (in the case of DNA / RNA sequences)] to determine sequence identity. Unless otherwise indicated, when this application refers to sequence identity to a particular reference sequence, it is intended that identity be calculated over the entire length of that reference sequence.

[0657] Immunogen, immunogenic: The terms "immunogen" or "immunogenic" will be recognized and understood by those skilled in the art. The terms are intended to relate to a compound that can, for example, stimulate / elicit an (adaptive) immune response. An immunogen can be a peptide, polypeptide, or protein.

[0658] Immune response: The term "immune response" will be recognized and understood by those skilled in the art. The term is intended to mean, for example, a specific reaction of the adaptive immune system (a so-called specific or adaptive immune response) or a non-specific reaction of the innate immune system (a so-called non-specific or innate immune response) to a particular antigen, or a combination thereof.

[0659] Innate immune system: The term "innate immune system" (also referred to as non-specific or non-specific immune system) will be recognized and understood by those skilled in the art. The term is intended to mean a system that typically includes cells and mechanisms that non-specifically defend the host, for example, from infection by other organisms. This means that cells of the innate immune system can universally recognize and respond to pathogens, but unlike the adaptive immune system, it does not provide the host with long-lasting or protective immunity. The innate immune system can be activated by ligands of pattern recognition receptors, such as Toll-like receptors, NOD-like receptors, or RIG-I-like receptors.

[0660] Lipidoid compounds: Lipidoid compounds, also simply called lipidoids, are lipid-like compounds, i.e., amphiphilic compounds with lipid-like physical properties. In the context of the present invention, the term lipid is considered to encompass lipidoid compounds.

[0661] Nucleic Acid, Nucleic Acid Molecule: As used herein, the terms "nucleic acid" or "nucleic acid molecule" will be recognized and understood by those of skill in the art. The terms "nucleic acid" or "nucleic acid molecule" refer, inter alia, to DNA (molecule) or RNA (molecule). The terms are used interchangeably with the term polynucleotide. For example, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by sugar / phosphate backbone phosphodiester bonds. The terms "nucleic acid" or "nucleic acid molecule" also encompass modified nucleic acids (molecules), such as, for example, base-, sugar-, or backbone-modified DNA or RNA (molecules) as defined herein.

[0662] Nucleic acid sequence, DNA sequence, RNA sequence: The terms "nucleic acid sequence," "DNA sequence," and "RNA sequence" will be recognized and understood by those of skill in the art. The terms refer, for example, to a sequence of nucleotides in a particular, specific order.

[0663] Permanently cationic: The term "permanently cationic" as used herein will be recognized and understood by those skilled in the art. The term means, for example, that the respective compound or group or atom is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge is caused by the presence of a quaternary nitrogen atom. When a compound bears multiple such positive charges, it can be referred to as permanently polycationic.

[0664] Stabilized RNA: The term "stabilized RNA" refers to RNA that has been modified to be more stable, for example, against degradation or degradation due to environmental factors or enzymatic digestion (e.g., exonuclease or endonuclease degradation), compared to RNA that does not have such a modification. Preferably, the stabilized RNA in the context of the present invention is stabilized in a cell, for example, a prokaryotic or eukaryotic cell, preferably a mammalian cell, for example, a human cell. For storage of a composition comprising the stabilized RNA, the stabilizing effect can be exerted outside the cell, for example, in a buffer solution.

[0665] T cell response: The terms "cellular immunity" or "cellular immune response" or "cellular T cell response" as used herein will be recognized and understood by those skilled in the art. The term is intended to mean, for example, the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to an antigen. In more general terms, cellular immunity is not based on antibodies but on the activation of cells of the immune system. Typically, a cellular immune response can be characterized by the activation of antigen-specific cytotoxic T lymphocytes, which can induce apoptosis in cells (e.g., specific immune cells such as dendritic cells or other cells) that present epitopes of foreign antigens on their surface.

[0666] RNA: The term "RNA" is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer composed 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 a so-called backbone. The backbone is typically formed by a phosphodiester bond between the sugar (i.e., ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific order of the monomers, i.e., the order of the bases linked to the sugar / phosphate backbone, is referred to as the RNA sequence. Generally, RNA can be obtained by transcription of a DNA sequence, for example, in a cell or in vitro. In the context of the present invention, RNA can be obtained by RNA in vitro transcription. Alternatively, RNA can be obtained by chemical synthesis.

[0667] RNA in vitro transcription: The term "RNA in vitro transcription" or "in vitro transcription" refers to a 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 is typically a linear DNA template (e.g., linearized plasmid DNA or a PCR product). 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 an appropriate restriction enzyme before being subjected to RNA in vitro transcription. Reagents typically used in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has high binding affinity for the respective RNA polymerase, e.g., a bacteriophage-encoded RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); optionally, a cap analog as defined herein; optionally, modified nucleotides as defined herein; and a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5) that can bind to the promoter sequence within the DNA template. RNA polymerase; optionally, a ribonuclease (RNase) inhibitor to inactivate potentially contaminating RNases; optionally, pyrophosphatase; MgCl2; a buffer (TRIS or HEPES) to maintain an appropriate pH value [which may also contain an antioxidant (e.g., DTT), and / or a polyamine, such as spermidine].

[0668] Variant (of a sequence): The term "variant" as used throughout this specification in the context of a nucleic acid sequence will be recognized and understood by those of skill in the art. The term is intended to refer to a variant of a nucleic acid sequence derived, for example, from another nucleic acid sequence. For example, a variant of a nucleic acid sequence may exhibit a deletion, insertion, addition, and / or substitution of one or more nucleotides compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may be at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence from which the variant is derived. A variant is a functional variant in the sense that it retains at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence from which the variant is derived. A "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotides to such nucleic acid sequence.

[0669] The term "variant" as used throughout the present specification in the context of a protein or peptide is intended to mean a protein or peptide variant having an amino acid sequence that differs from the original sequence, for example, in one or more mutations / substitutions, such as one or more substituted, inserted and / or deleted amino acids. Preferably, these fragments and / or variants have the same or equivalent specific antigenic properties (immunogenic variants, antigenic variants). Insertions and substitutions are possible, in particular, at sequence positions that do not result in modifications to the three-dimensional structure or that do not affect binding regions. Modifications of the three-dimensional structure due to insertions or deletions can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy). A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids with such protein or peptide. Alternatively, a "variant" of a protein or polypeptide may have 1 to 20, such as 1 to 10, single amino acid mutations, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19 or 20 single amino acid mutations, compared to such protein or peptide. Mutations refer to or include substitutions, insertions or deletions. In one embodiment, a mutant (variant) of a protein includes a functional mutant (variant) of a protein, which in the context of the present invention means that the mutant (variant) expresses essentially the same immunogenicity or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity of the protein from which it is derived.

[0670] Multivalent Vaccines / Compositions: Multivalent vaccines or combinations of the invention provide multiple valencies (e.g., antigens) derived from multiple viruses (e.g., at least one influenza virus as defined herein and at least one additional influenza virus as defined herein).

[0671] Example Below are given specific examples illustrating various embodiments and aspects of the present invention. However, the scope of the present invention is not limited by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. However, the scope of the present invention is not limited by the exemplified embodiments, which are intended merely as illustrations of single aspects of the invention, and functionally equivalent methods are within the scope of the invention. Indeed, various modifications of the present invention in addition to those described herein will be readily apparent to those skilled in the art from the foregoing description, the accompanying drawings, and the following examples. All such modifications are within the scope of the appended claims.

[0672] Example 1: Phase 1 Quadrivalent Influenza Vaccine Trial - Unmodified mRNA The Phase 1 trial was designed to evaluate the safety, reactogenicity, and immunogenicity of CVSQIV when administered as a single dose at various dose levels using an adaptive dose-finding design.

[0673] 1. Clinical trial objectives and endpoints Main purpose Evaluate the safety and reactogenicity profile of CVSQIV at various dose levels. Secondary Objectives To evaluate the humoral immune response to CVSQIV at various dose levels in terms of hemagglutination inhibition (HAI) antibody titers.

[0674] Purpose of exploration To evaluate humoral immune responses to CVSQIV at various dose levels in terms of microneutralizing (MN) and NA-inhibiting (NI) antibody titers. Innate immune responses to CVSQIV will be evaluated at various dose levels in all sentinel subjects. · Evaluate cross-reactivity to influenza antigens not included in the vaccine.

[0675] endpoint major The frequency of Grade 3 adverse reactions (ARs) and serious adverse reactions (SARs) within at least 20 hours after administration of the investigational vaccine by dose level for the purpose of determining subsequent vaccination of additional sentinel subjects at the same dose level. The frequency of Grade 3 ARs and SARs within at least 60 hours after administration of the investigational vaccine by dose level for decisions regarding dose escalation and continued enrollment at the same dose level. Frequency, intensity, and duration of solicited local AR by dose level on the day of vaccination and for the following 7 days to characterize the safety and reactogenicity profile. The frequency, intensity, duration, and relationship to study vaccination of unsolicited systemic adverse events (AEs) by dose level on the day of vaccination and for the following 7 days to characterize the safety and reactogenicity profile. The occurrence, intensity, and relationship to study vaccination of unsolicited AEs by dose level on the day of vaccination and for the following 28 days to characterize the safety and reactogenicity profile. - The occurrence of serious adverse events (SAEs) and adverse events of special interest (AESIs) throughout the trial and their relationship to study vaccination to characterize the safety and reactogenicity profile.

[0676] secondary Anti-HA antibody titers measured by HAI assay on days 22 and 183 Geometric mean titers (GMT) of antigen-specific anti-HA antibody titers. Percentage of subjects who showed antigen-specific seroconversion * . *Seroconversion of the HA antigen, as measured by the HAI assay, is defined as a post-vaccination titer of 1:40 or greater (≧1:40) for subjects with a baseline titer of 1:10 or less (≦1:10), and at least a four-fold increase in post-vaccination titer compared to baseline for subjects with a baseline titer of 1:10 or greater (≧1:10). The proportion of subjects who demonstrated a two-fold increase in antigen-specific post-vaccination anti-HA antibody titers compared to baseline. The proportion of subjects who demonstrated a four-fold increase in antigen-specific post-vaccination anti-HA antibody titers compared to baseline. The proportion of subjects with antigen-specific post-vaccination anti-HA antibody titers of 1:40 or greater (≥1:40) and 1:80 or greater (≥1:80).

[0677] exploratory Anti-HA antibody titers measured by microneutralization assay on days 22 and 183 GMT of antigen-specific anti-HA antibody titers. The proportion of subjects who demonstrated a two-fold increase in antigen-specific post-vaccination anti-HA antibody titers compared to baseline. The proportion of subjects who demonstrated a four-fold increase in antigen-specific post-vaccination anti-HA antibody titers compared to baseline.

[0678] Anti-neuraminidase (NA) antibody titers measured by enzyme-linked lectin assay (ELLA) on days 22 and 183 · GMT of antigen-specific anti-NA titers. The proportion of subjects who demonstrated a two-fold increase in antigen-specific post-vaccination anti-NA titers compared to baseline. The proportion of subjects who demonstrated a 4-fold increase in antigen-specific post-vaccination anti-NA titers compared to baseline. · Proportion of subjects with post-vaccination anti-NA titers of 1:40 or greater (≥1:40) and 1:80 or greater (≥1:80).

[0679] Cross-reactivity to antigens not included in the vaccine · Anti-NA antibody titer against B-Phuket. Innate immune response (only in sentinel subjects) Serum cytokine concentrations, including but not limited to, IFN-α, IFN-γ, IL-6, chemokine ligand (CCL)2, and IFN-γ-inducible protein 10 (IP-10), on days 2 and 22.

[0680] 2. Clinical trial design In the Phase 1 trial, subjects were enrolled in a rolling pattern at five dose levels (3, 6, 12, 20, and 28 μg). All subjects received a...

Claims

1. (a) a first hemagglutinin (HA) antigen from a strain of influenza virus, or a first nucleic acid encoding the first HA antigen; and (b) a second HA antigen from a strain of influenza virus, or a second nucleic acid encoding the second HA antigen. wherein (a) and (b) are different and the ratio of (a):(b) is comprised between 1.5:1 and 5:

1.

2. The immunogenic composition of claim 1, wherein (a) is a first RNA encoding a first HA antigen, and / or (b) is a second RNA encoding a second HA antigen.

3. The immunogenic composition of claim 1 or 2, wherein (a) is a first mRNA encoding a first HA antigen, and / or (b) is a second mRNA encoding a second HA antigen.

4. The immunogenic composition of any one of claims 1 to 3, wherein said strain of influenza virus is selected from the group consisting of influenza A virus and influenza B virus.

5. The immunogenic composition of any one of claims 1 to 4, wherein the composition is a multivalent composition and the strain of influenza virus in (a) is different from the strain of influenza virus in (b).

6. The immunogenic composition of any one of claims 1 to 5, wherein the first HA antigen is derived from an influenza B virus strain and the second HA antigen is derived from an influenza A virus strain.

7. 7. The immunogenic composition of any one of claims 1 to 6, wherein the ratio of (a):(b) is 1.5:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1 or 5:

1.

8. The immunogenic composition according to any one of claims 1 to 7, wherein the ratio of (a):(b) is comprised between 2:1 and 4:1, preferably between 2:1 and 3:1, preferably 2:1 or 3:

1.

9. The immunogenic composition according to any one of claims 1 to 8, wherein the dose of each of said first mRNA and / or said second mRNA is 1 to 200 μg, preferably 1 to 60 μg, preferably 2 to 25 μg.

10. (c) at least one additional antigen derived from a strain of influenza virus, or at least one additional nucleic acid encoding said at least one additional antigen. The immunogenic composition of any one of claims 1 to 9, further comprising:

11. (c) is at least one additional RNA encoding said at least one additional antigen. The immunogenic composition of claim 10.

12. 12. The immunogenic composition of claim 10 or 11, wherein (c) is at least one additional mRNA encoding said at least one additional antigen.

13. The immunogenic composition of any one of claims 10 to 12, wherein said strain of influenza virus is selected from the group consisting of influenza A virus and influenza B virus.

14. 14. The immunogenic composition of any one of claims 10 to 13, wherein the at least one additional antigen comprises or consists of a peptide or protein selected from or derived from influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2 and / or polymerase basic protein 2 (PB2), or an immunogenic fragment or immunogenic variant thereof.

15. 15. The immunogenic composition of any one of claims 10 to 14, wherein the at least one additional antigen comprises or consists of a peptide or protein selected from or derived from influenza virus hemagglutinin (HA) or neuraminidase (NA) or an immunogenic fragment or immunogenic variant thereof.

16. The immunogenic composition of any one of claims 10 to 15, wherein the composition comprises a plurality of (c).

17. 17. The immunogenic composition of claim 16, wherein the composition comprises at least 4, 5, 6, 7 or 8 antigens or nucleic acids encoding them, optionally 4 to 10 antigens or nucleic acids encoding them, optionally 4, 7 or 8 antigens or nucleic acids encoding them.

18. 18. The immunogenic composition of claim 16 or 17, comprising at least 4, 5, 6, 7 or 8 mRNAs, optionally 4 to 10 mRNAs, optionally 4, 7 or 8 mRNAs.

19. 19. The immunogenic composition of any one of claims 16 to 18, wherein the composition is a multivalent composition and the strains of influenza virus in (a) and / or the strains of influenza virus in (b) and / or the strains of influenza virus in (c) are different.

20. 20. The immunogenic composition of any one of claims 16 to 19, wherein the antigens of (a), (b) and / or (c) are derived from at least two, three or four strains of influenza virus.

21. 21. The immunogenic composition according to any one of claims 4 to 20, wherein said strain of influenza A virus is selected from influenza A viruses characterized by a hemagglutinin (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 selected from the group consisting of H1, H3, H5, H7, H9 and H10, more preferably selected from the group consisting of H1 and H3.

22. 22. The immunogenic composition according to any one of claims 4 to 21, wherein said strain of influenza A virus is selected from influenza A viruses characterized by a 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, more preferably from the group consisting of N1 and N2.

23. 23. The immunogenic composition according to any one of claims 4 to 22, wherein said strain 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, preferably H1N1 and H3N2.

24. The strain of influenza A virus is 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 / Victoria / 2570 / 2019 (H1N1)pdm09-like virus, A / Darwin / 9 / 2021 (H3N2)-like virus, A / Wisconsin / 588 / 2019 (H1N1)pdm09-like virus, A / Darwin / 6 / 2021 (H3N2)-like virus, A / Cambodia / e0826360 / 2020 (H3N2)-like virus, and A / Guangdong-Maonan / SWL1536 / 2019 (H1N1)pdm09-like virus, A / Hong Kong / 2671 / 2019 (H3N2)-like virus, A / Hawaii / 70 / 2019 (H1N1)pdm09-like virus, A / Hong Kong / 45 / 2019 (H3N2)-like virus, A / Brisbane / 02 / 2018 (H1N1)pdm09-like virus, A / Kansas / 14 / 2017 (H3N2)-like virus, A / California / 7 / 2009 (H1N1)pdm09-like virus, A / Switzerland / 97]5293 / 2013 (H3N2)-like virus, A / Hong Kong / 4801 / 2014 (H3N2)-like virus, A / Michigan / 45 / 2015 (H1N1)pdm09-like virus, A / Singapore / INFIMH-16-0019 / 2016 (H3N2)-like virus, A / Switzerland / 8060 / 2017 (H3N2)-like virus, A / Brisbane / 02 / 2018 (H1N1)pdm09-like virus, A / Kansas / 14 / 2017 (H3N2)-like virus, A / South Australia / 34 / 2019 (H3N2)-like virus, A / Idaho / 07 / 2018 (H1N1)pdm09-like virus, A / Maine / 38 / 2018 (H1N1)pdm09-like virus, A / Nebraska / IS / 2018 (H1N1)pdm09-like virus, A / Nebraska / 14 / 2019 (H1N1)pdm09-like virus, A / Iowa / 33 / 2019H1N1)pdm09-like virus, A / Arkansas / 28 / 2019 H1N1)pdm09-like virus, A / Virginia / 41 / 2019 H1N1)pdm09-like virus, A / Minnesota / 60 / 2019 H1N1)pdm09-like virus, A / Alabama / 27 / 2019 H1N1)pdm09-like virus, A / Iowa / 60 / 2018 (H3N2)-like virus, A / Jamaica / 60361 / 2019 (H3N2)-like virus, A / Florida / 130 / 2019 (H3N2)-like virus, A / Laos / 1789 / 2019 (H3N2)-like virus, A / Vermont / 25 / 2019 (H3N2)-like virus, A / New 24. The immunogenic composition of any one of claims 4 to 23, wherein the virus is selected from the group consisting of Jersey / 34 / 2019 (H3N2)-like virus, A / California / 176 / 2019 (H3N2)-like virus, A / Pennsylvania / 1026 / 2019 (H3N2)-like virus, A / Togo / 634 / 2019 (H3N2)-like virus, A / Kenya / 130 / 2019 (H3N2)-like virus, A / Togo / 1307 / 2019 (H3N2)-like virus, A / Ohio / 30 / 2019 (H3N2)-like virus, A / Guatemala / 93 / 2019 (H3N2)-like virus, A / Guatemala / 10 / 2019 (H3N2)-like virus, and A / Hong Kong / 4801 / 2014 (H3N2)-like virus.

25. The immunogenic composition according to any one of claims 4 to 24, wherein said strain of influenza B virus is selected from the group consisting of the B / Victoria lineage and the B / Yamagata lineage.

26. 26. The immunogenic composition of any one of claims 4 to 25, wherein the strain of influenza B virus is selected from the group consisting of B / Austria / 1359417 / 2021 (B / Victoria lineage)-like viruses, B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like viruses, B / Washington / 02 / 2019 (B / Victoria lineage)-like viruses, B / Colorado / 06 / 2017-like viruses (B / Victoria / 2 / 87 lineage), B / Brisbane / 60 / 2008-like viruses, and B / Colorado / 06 / 2019 (B / Victoria lineage)-like viruses.

27. (a) a first mRNA encoding the HA of a first strain of influenza B virus; (b) a second mRNA encoding the HA of the first strain of influenza A virus; (c 1 ) a third mRNA encoding the HA of a second strain of influenza A virus; and (c 2 ) a fourth mRNA encoding the HA of a second strain of influenza B virus An immunogenic composition comprising: (a):(b):(c 1 ):(c 2 ) is comprised between 1.5:1:1:1.5 and 5:1:1:5, preferably between 2:1:1:2 and 4:1:1:4, preferably between 2:1:1:2 and 3:1:1:3, preferably 2:1:1:2 or 3:1:1:

3.

28. (c 3 ) a first mRNA encoding the NA of a first strain of influenza A virus; (c 4 ) a second mRNA encoding the NA of a second strain of influenza A virus; and (c 5 ) a third mRNA encoding the NA of the first strain of influenza B virus further comprising where (a):(b):(c 1 ):(c 2 28. The immunogenic composition of claim 27, wherein the ratio of:

29. (c 6 ) The eighth mRNA encoding the NA of a second strain of influenza B virus further comprising where (a):(b):(c 1 ):(c 2 29. The immunogenic composition of claim 28, wherein the ratio of:

30. (a):(b):(c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ):(c 6 29. The immunogenic composition of claim 28, wherein the ratio of:

31. (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 30. The immunogenic composition of any one of claims 27 to 29, wherein the mRNAs of said antibodies are formulated, separately or not, in lipid nanoparticles (LNPs).

32. 32. The immunogenic composition of claim 31, wherein the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and a cationic lipid.

33. 33. The immunogenic composition of claim 32, wherein the cationic lipid is ionizable.

34. The ionizable cationic lipid has the formula III: 【Chemical 1】 [In the formula, L 1 or L 2 are each independently —O(C═O)— or (C═O)O—; G 1 and G 2 are each independently an unsubstituted C 1 -C 12 Alkylene or C 1 -C 12 alkenylene; G 3 is C 1 -C 24 Alkylene, C 1 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene or C 3 -C 8 is cycloalkenylene; R 1 and R 2 are each independently a branched or linear C 6 -C 24 Alkyl or C 6 -C 24 alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or NR 5 C(=O)R 4 is; R 4 is C 1 -C 12 is alkyl; R 5 is H or C 1 -C 6 alkyl] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

35. The ionizable cationic lipid has the formula III: 【Chemistry 2】 [In the formula, L 1 or L 2 are each independently —O(C═O)— or (C═O)O—; G 1 and G 2 are each independently an unsubstituted C 1 -C 12 is alkylene; G 3 is C 1 -C 24 is alkylene; R 1 and R 2 are each independently a branched or linear C 6 -C 24 is alkyl; R 3 is OR 5 and R 5 is H] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

36. The ionizable cationic lipid has formula III, where R 1 , R 2 or R 1 and R 2 and both have the following structure: 【Chemistry 3】 33. The immunogenic composition of claim 32, comprising one of:

37. R 2 But the structure: 【Chemistry 4】 37. The immunogenic composition of claim 36, comprising:

38. The cationic lipid has the formula: 【Chemistry 5】 33. The immunogenic composition of claim 32, comprising:

39. The cationic lipid has the formula: 【Chemistry 6】 33. The immunogenic composition of claim 32, comprising:

40. The ionizable cationic lipid has the formula: 【Chemistry 7】 34. The immunogenic composition of claim 33, comprising:

41. The immunogenic composition of any one of claims 32 to 40, wherein the PEG-modified lipid comprises PEG-DMG or PEG-cDMA.

42. The PEG-modified lipid has the formula IV: 【Chemistry 8】 (In the formula, R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain may optionally be interrupted by one or more ester linkages; w has an average value in the range of 30 to 60. The immunogenic composition of any one of claims 32 to 40, comprising:

43. In PEG-modified lipids, R 8 and R 9 43. The immunogenic composition of claim 42, wherein is a saturated alkyl chain.

44. The PEG-modified lipid may be represented by Formula IVa: 【Chemistry 9】 wherein 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, and 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 lipid is about 2500 g / mol.

33. The immunogenic composition of claim 32, comprising:

45. 45. The immunogenic composition of any one of claims 32 to 44, wherein the non-cationic 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), preferably the neutral lipid is DSPC.

46. The immunogenic composition of any one of claims 32 to 45, wherein the sterol is cholesterol.

47. 47. The immunogenic composition of any one of claims 32-46, wherein the LNP comprises about 0.5-15 mol% PEG-modified lipid, about 5-25 mol% non-cationic lipid, about 25-55 mol% sterol, and about 20-60 mol% ionizable cationic lipid.

48. 33. The immunogenic composition of claim 32, wherein the LNP comprises about 0.5 to 10 mol%, optionally 0.5 to 5 mol% or 0.5 to 3 mol% of PEG-modified lipids.

49. 49. The immunogenic composition of any one of claims 32 to 48, wherein the composition has a lipid to RNA molar ratio (N / P ratio) of from about 2 to about 12, optionally an N / P ratio of from 3 to about 8.

50. The immunogenic composition of any one of claims 32 to 49, wherein the LNP has a diameter of 50 to 200 nm.

51. (a), (b), (c), (c 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 51. The immunogenic composition of any one of claims 27 to 50, wherein the mRNA of each of the following is optionally non-replicating:

52. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 52. The immunogenic composition of any one of claims 27 to 51, wherein the mRNA of said polypeptide comprises a coding sequence that is a codon-modified coding sequence, wherein the amino acid sequence encoded by the codon-modified coding sequence is optionally unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

53. The immunogenic composition of claim 52, wherein the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage-adapted coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof.

54. 54. The immunogenic composition of claim 53, wherein the codon-modified coding sequence has a G / C content of at least about 45%, 50%, 55%, or 60%.

55. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 55. The immunogenic composition of any one of claims 27 to 54, wherein the mRNA of said antibody comprises a 5' cap, preferably an m7G, cap 0, cap 1, cap 2, modified cap 0 or modified cap 1 structure, preferably a 5'-cap 1 structure.

56. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 56. The immunogenic composition according to any one of claims 27 to 55, wherein the mRNA of said antibody comprises a poly(A) tail sequence, preferably a poly(A) tail sequence comprising 30 to 200 adenosine nucleotides, and / or at least one poly(C) sequence, preferably at least one poly(C) sequence comprising 10 to 40 cytosine nucleotides.

57. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The immunogenic composition of any one of claims 27 to 56, wherein the mRNA of said polypeptide comprises at least one histone stem-loop.

58. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 58. The immunogenic composition of any one of claims 27 to 57, wherein the mRNA of said RNA comprises at least one poly(A) tail sequence comprising 30 to 200 adenosine nucleotides, preferably 100 adenosine nucleotides, and the 3'-terminal nucleotide of said RNA is adenosine.

59. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The immunogenic composition of any one of claims 27 to 58, wherein the mRNA of said antibody comprises a 5' untranslated region (UTR).

60. 60. The immunogenic composition of claim 59, wherein the 5'UTR comprises or consists of 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 homolog, fragment or variant of any of these genes.

61. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The immunogenic composition of any one of claims 27 to 60, wherein the mRNA of said antibody comprises a 3'UTR.

62. The immunogenic composition of claim 61, wherein the 3'UTR comprises or consists of a nucleic acid sequence that is the 3'UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homolog, fragment or variant of any one of these genes.

63. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 63. The immunogenic composition of any one of claims 27 to 62, wherein the mRNA of HSD17B4 comprises a heterologous 5'-UTR comprising or consisting of a nucleic acid sequence derived from the 5'-UTR from HSD17B4, and at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence derived from the 3'-UTR of PSMB3.

64. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA in the 5' to 3' direction, i) 5'-Cap 1 structure, ii) a 5′-UTR derived from the 5′-UTR of the HSD17B4 gene; iii) coding sequences; iv) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene; v) an optional histone stem-loop sequence; and vi) a poly(A) sequence containing about 100 A nucleotides, the 3' terminal nucleotide of the RNA being an adenosine; The immunogenic composition of any one of claims 27 to 63, comprising:

65. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The immunogenic composition of any one of claims 27 to 64, wherein the mRNA of said antibody is free of chemically modified nucleotides.

66. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 The immunogenic composition of any one of claims 27 to 64, wherein the mRNA of comprises at least one chemical modification.

67. 67. The immunogenic composition of claim 66, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

68. 68. The immunogenic composition according to claim 66 or 67, wherein the chemical modification is N1-methylpseudouridine and / or pseudouridine, preferably N1-methylpseudouridine.

69. Chemical modifications (a), (b), and (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA is uridine-modified, and preferably 100% of the uridine positions in the mRNA are modified. The immunogenic composition of any one of claims 66 to 68.

70. The immunogenic composition of any one of claims 1 to 69, wherein the ratio is a mass ratio (weight / weight ratio).

71. 71. The immunogenic composition of any one of claims 1 to 70, further comprising at least one pharmaceutically acceptable carrier.

72. 72. A vaccine comprising the immunogenic composition of any one of claims 1 to 71.

73. 73. The vaccine of claim 72, further comprising at least one antigen or at least one nucleic acid encoding said at least one antigen, such as at least one mRNA encoding an antigen from an additional pathogen, preferably wherein the pathogen is a virus.

74. 74. The vaccine of claim 73, wherein the antigen is derived from an additional virus 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 an antigenic fragment thereof from the SARS-CoV-2 virus, or an mRNA encoding the spike protein or an antigenic fragment thereof from the SARS-CoV-2 virus.

75. An antigen or nucleic acid according to any one of claims 1 to 26, or an mRNA according to any one of claims 3 to 71, preferably (a), (b), (c) according to any one of claims 27 to 71. 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 ) mRNA, optionally including a liquid vehicle for solubilization, and optionally including technical instructions providing information regarding administration and dosage of the components.

76. Antigen or nucleic acid or mRNA, preferably (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 76. The kit or kit-of-parts of claim 75, wherein the mRNA of

77. Antigen or nucleic acid or mRNA, preferably (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 77. The immunogenic composition of any one of claims 1 to 71, the vaccine of any one of claims 72 to 74 or the kit or kit-of-parts of claim 75 or 76, wherein the mRNA of said antibody or antibody fragment is formulated as a bedside admixture.

78. (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 77. The immunogenic composition of any one of claims 1 to 71, the vaccine of any one of claims 72 to 74 or the kit or kit of parts of claim 75 or 76, wherein an antigen or nucleic acid and / or mRNA of the

79. An immunogenic composition according to any one of claims 1 to 71, a vaccine according to any one of claims 72 to 74 or a kit or kit of parts according to claim 75 or 76 for use as a medicament.

80. 77. An immunogenic composition according to any one of claims 1 to 71, a vaccine according to any one of claims 72 to 74 or a kit or kit of parts according to claim 75 or 76 for use in the treatment or prevention of infection by an influenza virus, preferably an influenza A virus and / or an influenza B virus.

81. 81. The immunogenic composition, vaccine, kit or kit-of-parts for use according to claim 80, wherein a single dose of the composition is from 2 to 500 μg of total mRNA, in particular from 10 to 250 μg, such as from 10 to 75 μg of total mRNA.

82. 82. The immunogenic composition, vaccine, kit or kit-of-parts for use according to claim 80 or 81, for intramuscular administration.

83. Antigen or nucleic acid or mRNA, preferably (a), (b), (c) 1 ), (c 2 ), (c 3 ), (c 4 ), (c 5 ) and / or (c 6 83. The kit or kit of parts for use according to any one of claims 80 to 82, wherein the mRNAs of said medicament are administered at different injection sites.

84. 84. The immunogenic composition, vaccine, kit for use according to any one of claims 80 to 83, wherein an immune response, preferably an adaptive immune response, more preferably a protective adaptive immune response against influenza viruses, preferably influenza A viruses and / or influenza B viruses, is elicited.

85. 76. A method for the treatment or prevention of disorders caused by influenza viruses, preferably influenza A viruses and / or influenza B viruses, comprising applying or administering to a subject in need thereof an immunogenic composition according to any one of claims 1 to 71, a vaccine according to any one of claims 72 to 74 or a kit or kit of parts according to claim 75 or 76.

86. 77. A method for inducing an immune response comprising applying or administering to a subject in need thereof an immunogenic composition according to any one of claims 1 to 71, a vaccine according to any one of claims 72 to 74 or a kit or kit of parts according to claim 75 or 76.

87. 87. The method of claim 86, wherein the immune response is an adaptive immune response, preferably a protective adaptive immune response against influenza virus, preferably against influenza A virus and / or influenza B virus.

88. 88. The method of claim 85 or the method of claim 86 or 87, wherein the required subject is a mammalian subject, preferably a human subject.

89. 88. The method of claim 85 or the method of claim 86 or 87, wherein the immunogenic composition of any one of claims 1 to 71, the vaccine of any one of claims 72 to 74 or the kit or kit of parts of claim 75 or 76 is administered in an amount effective to induce a T cell response against influenza A H1N1, influenza A H3N2, influenza B / Yamagata strain and influenza B / Victoria strain.

90. 88. The method of claim 85 or the method of claim 86 or 87, wherein the immunogenic composition of any one of claims 1 to 71, the vaccine of any one of claims 72 to 74 or the kit or kit of parts of claim 75 or 76 is administered in an amount effective to induce a neutralising antibody response against influenza A H1N1, influenza A H3N2, influenza B / Yamagata lineage and influenza B / Victoria lineage.

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