Influenza virus vaccine

Abstract

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

Description

Technical Field

[0001] The present invention particularly relates to immunogenic compositions comprising hemagglutinin (HA) antigens or nucleic acids encoding these HA antigens (suitably mRNA), wherein these HA antigens are derived from strains of influenza virus. The present invention also relates to vaccines and kits or kits comprising the same. The immunogenic compositions, vaccines and kits provided herein are suitable for use as medicaments, in particular for the treatment or prevention of influenza virus infection, suitably influenza A and / or influenza B. Background Art

[0002] Influenza virus is an RNA virus (NCBI classification system number: 11308) belonging to the family Orthomyxoviridae, further divided into, for example, influenza A virus genus (including the genus of influenza A virus) and influenza B virus genus (including the genus of influenza B virus), which spreads throughout the world. Influenza virus usually causes acute respiratory diseases during local outbreaks or seasonal epidemics and occasionally during pandemics. Typical influenza epidemics lead to increased incidence of pneumonia and lower respiratory tract diseases, which are shown by increased hospitalization rates or mortality rates. Elderly people or those with underlying chronic diseases are most likely to experience such complications, but infants and young children may also suffer from severe illness. Influenza viruses (mainly influenza A and influenza B viruses) have a significant impact on global public health, causing millions of serious illnesses, thousands of deaths and huge economic losses every year.

[0003] Influenza viruses (such as influenza A and influenza B) are enveloped viruses that contain eight segmented negative-sense RNAs encoding 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The best characterized of these viral proteins are hemagglutinin (HA) and neuraminidase (NA), two large glycoproteins present 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 viral binding to target cells and entry of the viral genome into the target cell.

[0004] Currently, there are 18 described HA (H1-H18) subtypes and 11 described NA (N1-N11) subtypes of influenza A virus, which potentially form 144 HA and NA combinations. Unlike influenza A virus, which has a wide host range, influenza B virus infects humans almost exclusively. Influenza B virus is divided into two different lineages: B / Victoria / 2 / 1987-like (B / Victoria lineage) and B / Yamagata / 16 / 1988-like (B / Yamagata lineage), which have circulated worldwide since 1983. The mutation rate of influenza B virus is 2 to 3 times slower than that of influenza A; however, it significantly affects children and young adults every year.

[0005] Vaccination is currently the most widely used method to prevent influenza outbreaks, especially among high-risk groups. The continuous emergence of new strains of influenza viruses through antigenic drift is the virological basis of seasonal epidemics. Due to its evolving nature, regular updates of the viruses contained in influenza (influenza) vaccines are necessary for the vaccine to be effective. Public health authorities monitor influenza viruses circulating in humans and update the recommended composition of influenza vaccines twice a year. National vaccine regulatory agencies and pharmaceutical companies use the published recommendations (usually three or four different strains of influenza viruses) to develop, produce 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 available on the market for active immunization against diseases caused by the influenza A and B viruses contained in these vaccines.

[0007] Since HA is the main influenza virus antigen recognized by neutralizing antibodies, this glycoprotein has always been the focus of currently approved inactivated influenza vaccines and recombinant influenza vaccines. Most of these influenza vaccines are quadrivalent vaccines, based on 4 HAs from each of the four influenza virus strains (usually two strains of influenza A virus and two strains of influenza B virus) specified by the health department to be included in the annual seasonal vaccine, meaning that it is designed to provide protection for these four different influenza virus strains. Each of these 4 HAs is present in an equimolar ratio. The standard dose of 1 HA (i.e., each strain) is 15 μg / 0.5 ml, resulting in a total (i.e., for 4 HAs) standard dose of 60 μg / 0.5 ml. Some available influenza vaccines are further approved for higher doses, such as 45 μg / 0.5 ml HA (FLUBLOK, Sanofi) per strain or 60 μg / 0.7 ml HA (FLUZONE high dose, Sanofi) per strain.

[0008] Clinical studies that are the basis for currently approved influenza vaccines have highlighted some variation in vaccine efficacy against different strains of influenza virus and in the immunogenicity associated with the different antigens (e.g., HA) that form the influenza vaccine. For example, in healthy adults aged 18-49, FLUBLOK was 54.4% effective against influenza A, but only 23.1% against influenza B. Similarly, corresponding immunogenicity studies revealed that the HI GMT (geometric mean titer of hemagglutination inhibition) of the HA A antigen was up to 17 times higher than that of the HA B antigen.

[0009] Therefore, there remains a need to provide immunogenic compositions that are capable of eliciting a broad, rapid and potent immune response against influenza virus. Summary of the invention

[0010] In a first aspect, the present invention provides an immunogenic composition comprising:

[0011] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a strain of influenza virus; and

[0012] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a strain of influenza virus,

[0013] wherein (a) and (b) are different, and wherein the ratio of (a):(b) is between 1.5:1 and 5:1.

[0014] The present invention also provides an immunogenic composition comprising:

[0015] (a) a first mRNA encoding HA of a first strain of influenza B virus;

[0016] (b) a second mRNA encoding HA of a first strain of influenza A virus;

[0017] (c 1 ) a third mRNA encoding HA of a second strain of influenza A virus; and

[0018] (c 2 ) a fourth mRNA encoding HA of a second strain of influenza B virus,

[0019] Where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0020] In a second aspect, the invention provides a vaccine comprising the immunogenic composition as defined herein.

[0021] In a third aspect, the invention provides a kit or set of parts comprising an antigen or nucleic acid and / or mRNA as defined herein (suitably (a), (b), (c) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )), optionally containing a liquid carrier for dissolution, and optionally technical instructions providing information about the administration and dosage of the components.

[0022] In a fourth aspect, the invention relates to an immunogenic composition, vaccine or kit or set of parts as described herein for use as a medicament.

[0023] In a fifth aspect, the invention relates to an immunogenic composition, vaccine or kit or set of parts as described herein for use in treating or preventing influenza virus infection, suitably influenza A and / or influenza B.

[0024] In a sixth aspect, the present invention relates to a method of treating or preventing a disorder or disease caused by influenza virus (suitably influenza A and / or influenza B), wherein the method comprises applying or administering to a subject in need thereof an immunogenic composition, vaccine or kit or set of parts as described herein.

[0025] In a seventh aspect, the present invention relates to a method of eliciting an immune response, wherein the method comprises applying or administering to a subject in need thereof an immunogenic composition, vaccine or kit or set of parts as described herein.

[0026] A brief description of the sequence

[0027] SEQ ID NO: 1 Amino acid sequence of HA from A / Michigan / 45 / 2015 (H1N1)

[0028] SEQ ID NO:2 Amino acid sequence of NA from A / Michigan / 45 / 2015 (H1N1)

[0029] SEQ ID NO:3 Amino acid sequence of HA from A / Switzerland / 8060 / 2017 (H3N2)

[0030] SEQ ID NO:4 Amino acid sequence of NA from A / Switzerland / 8060 / 2017 (H3N2)

[0031] SEQ ID NO:5 Amino acid sequence of HA from B / Colorado / 06 / 2017

[0032] SEQ ID NO:6 Amino acid sequence of NA from B / Colorado / 06 / 2017

[0033] SEQ ID NO:7 Amino acid sequence of HA from B / Phuket / 3073 / 2013

[0034] SEQ ID NO:8 Amino acid sequence of NA from B / Phuket / 3073 / 2013

[0035] SEQ ID NO:9 Amino acid sequence of HA from A / Singapore / INFIMH-16-0019 / 2016 (H3N2)

[0036] SEQ ID NO: 10 Amino acid sequence of NA from A / Singapore / INFIMH-16-0019 / 2016 (H3N2)

[0037] SEQ ID NO: 11 Amino acid sequence of HA from A / Brisbane / 02 / 2018 (H1N1)

[0038] SEQ ID NO: 12 Amino acid sequence of NA from A / Brisbane / 02 / 2018 (H1N1)

[0039] SEQ ID NO: 13 Amino acid sequence of HA from A / Kansas / 14 / 2017 (H3N2)

[0040] SEQ ID NO: 14 Amino acid sequence of NA from A / Kansas / 14 / 2017 (H3N2)

[0041] SEQ ID NO: 15 Amino acid sequence of HA from A / South Australia / 34 / 2019 (H3N2)

[0042] SEQ ID NO: 16 Amino acid sequence of NA from A / South Australia / 34 / 2019 (H3N2)

[0043] SEQ ID NO: 17 Amino acid sequence of HA from B / Washington / 02 / 2019

[0044] SEQ ID NO: 18 Amino acid sequence of NA from B / Washington / 02 / 2019

[0045] SEQ ID NO: 19 Amino acid sequence of HA from A / Guangdong-Maonan / SWL1536 / 2019 (H1N1)

[0046] SEQ ID NO: 20 Amino acid sequence of NA from A / Guangdong-Maonan / SWL1536 / 2019 (H1N1)

[0047] SEQ ID NO:21 Amino acid sequence of HA from A / Hong Kong / 2671 / 2019 (H3N2)

[0048] SEQ ID NO:22 Amino acid sequence of NA from A / Hong Kong / 2671 / 2019 (H3N2)

[0049] SEQ ID NO:23 Amino acid sequence of HA from A / Hawaii / 70 / 2019 (H1N1)

[0050] SEQ ID NO:24 Amino acid sequence of NA from A / Hawaii / 70 / 2019 (H1N1)

[0051] SEQ ID NO:25 Amino acid sequence of HA from A / Hong Kong / 45 / 2019 (H3N2)

[0052] SEQ ID NO:26 Amino acid sequence of NA from A / Hong Kong / 45 / 2019 (H3N2)

[0053] SEQ ID NO: 27 Amino acid sequence of HA from A / Victoria / 2570 / 2019 (H1N1)

[0054] SEQ ID NO:28 Amino acid sequence of NA from A / Victoria / 2570 / 2019 (H1N1)

[0055] SEQ ID NO: 29 Amino acid sequence of HA from A / Wisconsin / 588 / 2019 (H1N1)

[0056] SEQ ID NO:30 Amino acid sequence of NA from A / Wisconsin / 588 / 2019 (H1N1)

[0057] SEQ ID NO:31 Amino acid sequence of HA from A / Cambodia / e0826360 / 2020 (H3N2)

[0058] SEQ ID NO:32 Amino acid sequence of NA from A / Cambodia / e0826360 / 2020 (H3N2)

[0059] SEQ ID NO:33 Amino acid sequence of HA from A / Darwin / 9 / 2021 (H3N2)

[0060] SEQ ID NO:34 Amino acid sequence of NA from A / Darwin / 9 / 2021 (H3N2)

[0061] SEQ ID NO:35 Amino acid sequence of HA from B / Austria / 1359417 / 2021

[0062] SEQ ID NO:36 Amino acid sequence of NA from B / Austria / 1359417 / 2021

[0063] SEQ ID NO:37 Amino acid sequence of HA from A / Darwin / 6 / 2021 (H3N2)

[0064] SEQ ID NO:38 Amino acid sequence of NA from A / Darwin / 6 / 2021 (H3N2)

[0065] SEQ ID NO:39 Amino acid sequence of HA from A / Victoria / 4897 / 2022 (H1N1)

[0066] SEQ ID NO:40 Amino acid sequence of NA from A / Victoria / 4897 / 2022 (H1N1)

[0067] SEQ ID NO:41 Amino acid sequence of HA from A / Wisconsin / 67 / 2022 (H1N1)

[0068] SEQ ID NO:42 Amino acid sequence of NA from A / Wisconsin / 67 / 2022 (H1N1)

[0069] 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 DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 :The domain structure of influenza A virus (IAV) HA protein. The domains in HA1 include the fusion domain (F1), the residual (vestigial) esterase domain (VE) and the receptor binding domain (RBD). The domains in HA2 include the HA2 extracellular domain, the transmembrane region (TM) and the cytoplasmic tail (CT). The HA head contains the receptor binding subdomain and the residual esterase subdomain. The stem (also called the "stem") contains the HA1 fusion domain and the HA2 extracellular domain.

[0071] Figure 2A-2C : Reactogenicity assessment in subjects in the CVSQIV Phase I influenza vaccine trial. Figure 2A , solicited adverse events for subjects at the specified mRNA dose level shown at the bottom of the graph. Figure 2B , subjects actively enrolled for adverse events at a given mRNA dose level, distinguishing between younger and older adults. Figure 2A-2B , grade 0 events are at the bottom of the graph, above the dose level indication, and the percentages of increasing grade events are arranged vertically. Figure 2C , actively solicited adverse events by subject at the specified mRNA dose level, distinguished for younger and older adults, and distinguished between local and systemic events. Grade 0-1 events are at the bottom of the chart, above the dose level indication. The percentages of Grade 0-1 vs. Grade ≥2 are shown.

[0072] Figure 3A-3D: Graph showing the geometric mean titers (95% CI) of the hemagglutinin inhibition assay (HAI) in each regimen immunogenicity set. The left column shows the HAI titers for all subjects at the specified vaccine mRNA dose level at Day 1, Day 22, and Day 183. The data in the right column distinguish between young adults (YA) and older adults (OA) at the specified mRNA dose level. For each HA component encoded by the vaccine mRNA, the data are shown separately: H1N1 ( Figure 3A ); H3N2( Figure 3B ); B / Phuket( Figure 3C ); and B / Washington( Figure 3D ).

[0073] Figure 4 :Seroconversion rate (SCR) from HAI determination. The table in the upper left column shows SCR (defined as pre-vaccination titer <1:10, post-vaccination titer should be ≥1:40; if pre-vaccination titer ≥1:10, post-vaccination titer should be increased ≥fourfold from baseline). Data for each coded HA at each dose level and for all subjects or for distinction between young adults and elderly adults are shown. The chart in the lower left column shows the overall SCR of each coded HA at each dose level. The chart in the upper right column shows the SCR of each coded HA in young adults at each dose level. The chart in the lower right column shows the SCR of each coded HA in elderly adults at each dose level.

[0074] Figure 5 : Shows the percentage of study subjects who showed an increase of ≥ four times in anti-HA titer by microneutralization (MN) assay. The table in the upper left column shows the percentage of subjects who showed an increase of ≥ four times in anti-HA titer by MN assay. Shows data for each encoded HA at each dose level and for all subjects or for differentiation between young adults and elderly adults. The chart in the lower left column shows a 4-fold increase in anti-HA overall demonstrated by MN assay for each encoded HA at each dose level. The chart in the upper right column shows a 4-fold increase in anti-HA demonstrated by MN assay in young adults for each encoded HA at each dose level. The chart in the lower right column shows a 4-fold increase in anti-HA demonstrated by MN assay in elderly adults for each encoded HA at each dose level.

[0075] Figure 6: The percentage of study subjects who exhibited a ≥ four-fold increase in anti-NA titer by enzyme-linked lectin assay (ELLA) is shown. The table in the upper left column shows the percentage of subjects who exhibited a ≥ four-fold increase in anti-NA titer by ELLA assay. Data are shown for each encoded NA at each dose level and for all subjects or for differentiation between young and older adults. The chart in the lower left column shows the overall 4-fold increase in anti-NA exhibited by each encoded NA by ELLA assay at each dose level. The chart in the upper right column shows the 4-fold increase in anti-NA exhibited by each encoded HA by ELLA assay in young adults at each dose level. The chart in the lower right column shows the 4-fold increase in anti-NA exhibited by each encoded NA by ELLA assay in older adults at each dose level.

[0076] Figure 7 : Shown are the results of a ferret immunization study using a quadrivalent mRNA vaccine encoding HA from two influenza A strains (A / California / 07 / 2009 (H1N1pdm09) and A / HongKong / 4801 / 2014 (H3N2)) and two influenza B strains (B / Phuket / 3073 / 2013 and B / Brisbane / 60 / 2008). The mRNA encoding influenza A and influenza B antigens was included in equal (1:1) μg amounts, or four times more mRNA encoding B antigens (1:4). Animals were immunized IM on days 0 and 21. Functional antibody responses were assessed by MN assay (upper column) for influenza B antigens or by HAI assay (lower column) for influenza A antigens in serum samples collected on days 0, 21, 35, and 49.

[0077] Figure 8 : Shows the results of IFNα levels when mice were immunized im with 4-component and 8-component influenza seasonal mRNA vaccines with different ratios of influenza A and influenza B HA components. Female Balb / c mice were immunized im with different influenza seasonal mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth of the human dose of licensed split-inactivated QIV FLUARIX TetraNH22-23 (n=8 / group) by the im route on days 0 and 21, respectively. IFNα levels were determined using ELISA in serum samples collected 18h ​​after the first immunization.

[0078] Figure 9A-9D: HI responses when mice were immunized with 4-component and 8-component influenza seasonal mRNA vaccines with different ratios of influenza A and influenza B HA components are shown. Female Balb / c mice were immunized with different influenza seasonal mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (NaCl) (n=5 / group) or one-tenth of the human dose of licensed split-inactivated QIV FLUARIX Tetra NH22-23 (n=8 / group) by the im route on days 0 and 21. HI titers against the following influenza were measured in sera collected two weeks after the secondary immunization: (A) A / Wisconsin / 588 / 2019 (H1N1pdm09), (B) A / Darwin / 6 / 2021 (H3N2), (C) B / Austria / 1359417 / 2021 and (D) B / Phuket / 3073 / 2013.

[0079] Figures 10A-10D :T cell responses induced when mice were immunized with 4-component and 8-component influenza seasonal mRNA vaccines with different ratios of influenza A and influenza B HA components were shown. Female Balb / c mice were immunized with different seasonal influenza mRNA vaccines on days 0 and 21 (n=8 / group). Control animals received saline (n=5 / group) or one-tenth of the human dose of licensed split-inactivated QIV FLUARIX TetraNH22-23 (n=8 / group) by the im route on days 0 and 21. Two weeks after the secondary immunization, T cell immune responses were analyzed by ICS in isolated splenocytes, which were re-stimulated 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 (A) CD4+ and (B) CD8+ T cells producing IFNγ+TNF+; Influenza B / Austria / 1359417 / 2021 HA-specific (C) CD4+ and (D) CD8+ T cells producing IFNγ+TNF+ were measured.

[0080] Figures 11A-11D: Shown are HI titers induced by 4-component or 7-component mRNA vaccines containing unmodified or modified (ψ and N1-mψ) nucleosides with equimolar ratios between mRNA sequences. Female Balb / c mice (n=10 / group) were vaccinated with 0.56 μg or 2.84 μg of 4-component (4HA; unmodified, ψ and N1-mψ) mRNA-LNP vaccine and 1 μg or 2.84 μg of 7-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 licensed QIV FLUARIX Tetra NH21-22 or FLUZONE HD NH21-22. Two weeks after the secondary immunization, 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 the sera of mice.

[0081] Figure 12A-12C : NI titers induced by 7-component mRNA vaccines containing unmodified or modified (ψ and N1-mψ) nucleosides and equimolar ratios between mRNA sequences are shown. Female Balb / c mice (n=10 / group) were vaccinated with 1 μg or 2.84 μg of 7-component (4HA+3NA; unmodified, ψ and N1-mψ) mRNA vaccines. Control animals (n=5 / group) received saline (NaCl) or one-tenth of the human dose of licensed QIV FLUARIX Tetra (NH21-22) or FLUZONE HD (NH21-22). Two weeks after the secondary immunization, 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.

[0082] Fig.13AD: shows the HI response induced when naive ferrets were immunized im with 4-component and 8-component influenza seasonal mRNA vaccine formulations. Female ferrets were immunized by the im route on days 0 and 28 with 12.5 μg and 25 μg of the 4-component influenza seasonal N1mψmRNA vaccine and 25 μg and 50 μg of the 8-component influenza seasonal N1mψmRNA vaccine (n=6). Control animals received saline (NaCl) (n=6 / group) or a complete human dose of a licensed split-inactivated QIV FLUARIX Tetra (NH22-23) by the im route on days 0 and 28 (n=6 / group). 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 sera from vaccinated animals collected on day 55.

[0083] Fig.14 AD: shows microneutralization (MN) titers induced when naive ferrets were immunized im with 4-component and 8-component influenza seasonal mRNA vaccine formulations. Female ferrets were immunized by the im route on days 0 and 28 with 12.5 μg and 25 μg of the 4-component influenza seasonal N1mψmRNA vaccine and 25 μg and 50 μg of the 8-component influenza seasonal N1mψmRNA vaccine (n=6). Control animals received saline (NaCl) (n=6 / group) or a full human dose of a licensed split-inactivated QIV FLUARIX Tetra (NH22-23) by the im route on days 0 and 28 (n=6 / group). 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 sera from vaccinated animals collected on day 55.

[0084] Fig.15AD: shows the neuraminidase inhibition (NI) titers induced when naive ferrets were immunized im with 4-component and 8-component influenza seasonal mRNA vaccine formulations. Female ferrets were immunized by the im route on days 0 and 28 with 12.5 μg and 25 μg of the 4-component influenza seasonal N1mψmRNA vaccine and 25 μg and 50 μg of the 8-component influenza seasonal N1mψmRNA vaccine (n=6). Control animals received saline (NaCl) (n=6 / group) or a full human dose of a licensed split-inactivated QIV FLUARIX Tetra (NH22-23) by the im route on days 0 and 28 (n=6 / group). 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 sera from vaccinated animals collected on day 55.

[0085] Figures 16A-16D : Shown are the HI titers induced when immunizing healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component influenza seasonal mRNA vaccine formulations. The control is influenza D-QIV (FLUARIX, NH 2022-23). ​​HI titers were measured on day 29 for influenza A / Victoria / 2570 / 2019 (H1N1pdm09) (A), A / Darwin / 6 / 2021 (H3N2) (B), B / Connecticut / 01 / 2021 (C), and B / Phuket / 3073 / 2013 (D).

[0086] Figures 17A-17D : Shown are the NI titers induced when immunizing healthy human adults (18-50 years old) with 1-component, 4-component, and 8-component influenza seasonal mRNA vaccine formulations. The control is influenza D-QIV (FLUARIX, NH 2022-23). ​​The NI titers for influenza A / Wisconsin / 588 / 2019 (H1N1pdm09) (A), influenza A / Cambodia / e0826360 / 2020 (H3N2) (B), influenza B / Austria / 1359417 / 2021 (C), and B / Phuket / 3073 / 2013 (D) were measured on day 29.

[0087] Figures 18A-18D: Shows the percentage of human healthy adults (18-50 years) experiencing active solicited events (any; A), local events (B), and systemic events (C) within 7 days of immunization with 1-component, 4-component, and 8-component influenza seasonal mRNA vaccine formulations. The control is influenza D-QIV (FLUARIX, NH 2022-23). ​​(D) shows an overall overview by event, including grade 3 events.

[0088] Fig.19 : Shows the percentage of human healthy adults (18-50 years) with relevant non-active solicited events within 7 days after immunization with 1-component, 4-component, and 8-component influenza seasonal mRNA vaccine formulations. The control is influenza D-QIV (FLUARIX, NH 2022-23). DETAILED DESCRIPTION

[0089] The present application is submitted together with a sequence table (WIPO standard ST.26) in electronic format as a part of the specification. The information contained in the sequence table is incorporated herein by reference in its entirety. When "SEQ ID NO" is mentioned in this article, it refers to the corresponding nucleic acid (na) sequence or amino acid (aa) sequence in the sequence table with respective identifiers. For many sequences, the sequence table also provides additional detailed information, such as about certain structural features, sequence optimization, GenBank (NCBI) or GISAID (epi) identifiers, or about the additional detailed information of its encoding ability. When referring to the "SEQ ID NO" of other published patent applications or patents, the sequence (such as amino acid sequence or nucleic acid sequence) is clearly incorporated herein by reference. Therefore, these sequences constitute the components of the basic description.

[0090] Immunogenic composition:

[0091] 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 responsible for the interaction of the virus with host cell receptors.

[0092] The HA protein on the surface of influenza virus is a homotrimer of HA protein monomers, which are enzymatically cleaved to produce an amino-terminal HA1 polypeptide and a carboxyl-terminal HA2 polypeptide. Structurally, the hemagglutinin protein consists of several domains: a globular head domain, a stalk domain (also called a stalk domain), a transmembrane domain, and a cytoplasmic domain (see Figure 1 , Russell et al., 2021).

[0093] It is generally believed that during infection of a host cell (e.g., a eukaryotic cell, such as a human cell) with influenza virus, the hemagglutinin protein recognizes and binds to the sialic acid of a receptor on the surface of the host cell, promoting the attachment of the virus to the host cell. After endocytosis of the virus and endosomal acidification, the hemagglutinin protein undergoes a pH-dependent conformational change that allows the hemagglutinin protein 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.

[0094] The spherical head is composed of only the main part of the HA1 polypeptide, while the stem that anchors the HA protein to the viral lipid envelope is composed of HA2 and part of HA1. The spherical head of the HA protein includes two domains: a receptor binding domain (RBD), i.e., a domain containing a sialic acid binding site, and a residual esterase domain, i.e., a smaller area located just below the RBD. In general, influenza viruses are classified based on the amino acid sequence of the viral hemagglutinin protein and / or the amino acid sequence of the viral neuraminidase (NA). The amino acid sequence differences between the HA proteins of different subtypes are mainly present in the sequence of the head domain of the protein. Compared with the sequence of the head domain, the amino acid sequence of the stem domain is considered to be more conservative between HA subtypes. The domains of the HA protein can be predicted using conventional methods known in the art.

[0095] Many naturally occurring antibodies and experimentally derived antibodies that bind and neutralize HA proteins are thought to bind to epitopes within the head domain of HA and prevent or reduce the interaction of HA with sialic acid on the receptors of host cells, thereby preventing or reducing infection of cells. Alternatively, or in addition, neutralizing antibodies can prevent or reduce membrane fusion of viral membranes with endosomes. Such antibodies can bind to epitopes within the stem domain, thereby inhibiting conformational changes of the protein. Antibodies against influenza typically target variable antigenic sites in the globular head of HA, and therefore only neutralize closely related viruses on the antigen. The variability of the HA head is due to the continuous antigenic drift (i.e., changes in the protein sequence) of influenza viruses, and is the cause of seasonal prevalence of influenza.

[0096] The present inventors have overcome the disadvantages of the prior art by administering an immunogenic composition comprising:

[0097] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a strain of influenza virus; and

[0098] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a strain of influenza virus,

[0099] wherein (a) and (b) are different, and wherein the ratio of (a):(b) is between 1.5:1 and 5:1.

[0100] It has been found that the immunogenic compositions of the present invention induce a broad, rapid and strong immune response against influenza viruses (eg, influenza A and / or influenza B).

[0101] In particular, or in addition, it has been found that the efficacy of an immunogenic composition comprising (a) a first HA antigen or a first nucleic acid encoding the first HA antigen (suitably mRNA) and (b) a second HA antigen or a second nucleic acid encoding the second HA antigen (suitably mRNA) against different strains of influenza virus is enhanced when the ratio of (a):(b) is between 1.5:1 and 5:1.

[0102] In particular, or in addition, it has been found that the immunogenicity associated with the first HA antigen and / or the second HA antigen forming the immunogenic composition of the invention is enhanced when the ratio of (a):(b) is between 1.5:1 and 5:1.

[0103] Suitably, the immunogenic compositions of the invention have at least some of the following advantageous characteristics:

[0104] - translating the nucleic acid (suitably mRNA) encoding the first HA antigen and the second HA antigen at the injection / vaccination site (e.g. muscle);

[0105] - Induce antigen-specific immune responses, suitably at low doses and dosing schedules;

[0106] - Vaccinations for infants and / or newborns or the elderly (particularly the elderly);

[0107] - the composition / vaccine is suitable for intramuscular administration;

[0108] - inducing a specific and functional humoral immune response against influenza virus (suitably influenza A virus and / or influenza B virus);

[0109] - inducing a broadly functional cellular T cell response against influenza virus (suitably influenza A virus and / or influenza B virus);

[0110] - inducing specific B cell memory against influenza virus (suitably influenza A virus and / or influenza B virus);

[0111] - inducing functional antibodies that are capable of effectively neutralizing influenza viruses (suitably influenza A virus and / or influenza B virus);

[0112] - inducing functional antibodies that are able to effectively neutralize emerging variants of influenza viruses (suitably influenza A and / or influenza B viruses);

[0113] - Inducing protective immunity against infection with influenza virus (e.g., against influenza A virus and / or influenza B virus) or emerging variants thereof;

[0114] - rapid onset of immune protection against influenza virus (suitably influenza A virus and / or influenza B virus);

[0115] - the persistence of the induced immune response against influenza virus (suitably influenza A virus and / or influenza B virus);

[0116] - No enhancement of viral infection (e.g. influenza virus infection) due to vaccination or immunopathological effects;

[0117] - No antibody-dependent enhancement (ADE) caused by the nucleic acid-based composition / vaccine;

[0118] - after administration of the composition / vaccine, there is no excessive induction of systemic cytokine or chemokine responses, which could lead to undesirable high reactogenicity upon injection / vaccination;

[0119] - The composition / vaccine is well tolerated, without side effects and toxicity;

[0120] - favorable stability characteristics of the nucleic acid-based composition / vaccine;

[0121] - The speed, adaptability, simplicity and scalability of production of the nucleic acid-based composition / vaccine;

[0122] - Favorable injection / vaccination regimens, which require only low doses of the composition / vaccine to provide adequate protection.

[0123] Thus, in a first aspect, the present invention relates to an immunogenic composition comprising:

[0124] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a strain of influenza virus; and

[0125] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a strain of influenza virus,

[0126] wherein (a) and (b) are different, and wherein the ratio of (a):(b) is between 1.5:1 and 5:1.

[0127] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0128] As used herein, "weight / weight ratio" or wt / wt ratio or wt:wt ratio refers to the ratio between the weights (masses) of the different components. "Molar ratio" refers to the ratio between the different components (e.g., the number of mRNAs encoding each antigen).

[0129] The terms "hemagglutinin," "hemagglutinin protein," and "HA" may be used interchangeably throughout and refer to the hemagglutinin protein that may be present on the surface of an influenza virus.

[0130] In the context of the present invention, any influenza virus, regardless of the specific genotype, species, strain, isolate or serotype, may be selected as a "strain of influenza virus".

[0131] In some embodiments, the influenza virus strain can be selected from influenza A virus (NCBI classification system number: 11320) and / or influenza B virus (NCBI classification system number: 11520) and / or influenza C virus (NCBI classification system number: 11552) and / or influenza D virus (NCBI classification system number: 1511084).

[0132] In some embodiments, the strain of influenza virus is selected from the group consisting of influenza A virus and influenza B virus.

[0133] In some embodiments, the composition is a multivalent composition, and the strain of the influenza virus of (a) and the strain of the influenza virus of (b) are different.

[0134] In some embodiments, the strain of influenza A virus is selected from an influenza A virus characterized by hemagglutinin (HA), which is 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, suitably selected from the group consisting of H1, H3, H5, H7, H9 and H10, and more suitably selected from the group consisting of H1 and H3.

[0135] In some embodiments, the strain of influenza A virus is selected from influenza A virus characterized by neuraminidase (NA), which is selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11, suitably selected from the group consisting of N1, N2 and N8, and more suitably selected from the group consisting of N1 and N2.

[0136] The terms "neuraminidase," "neuraminidase protein," and "NA" may be used interchangeably throughout and refer to the neuraminidase protein that may be present on the surface of an influenza virus.

[0137] 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, suitably H1N1 and H3N2.

[0138] In some embodiments, the strain of influenza A virus is selected from the group consisting of H1N1 and H3N2.

[0139] 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, influenza A(H3N2) viruses antigenically similar to the cell-propagated prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like virus, A / Darwin / 9 / 2021(H3N2)-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, A / Cambodia / e0826360 / 2020(H3N2)-like virus, A / Hong Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus 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.

[0140] In some embodiments, the strain of influenza A virus is H1N1.

[0141] 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, A / Victoria / 257 0 / 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, and A / South Dakota / 6 / 2007, A / Victoria / 4897 / 2022(H1N1)pdm09-like virus, A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus, and A / Sydney / 5 / 2021(H1N1)pdm09-like virus.

[0142] In some embodiments, the strain of influenza A virus is H3N2.

[0143] 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, influenza A(H3N2) viruses antigenically similar to the cell-propagated prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like virus, A / Darwin / 9 / 2021(H3N2)-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, A / Cambodia / e0826360 / 2020(H3N2)-like virus, A / Hong Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus 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.

[0144] In some embodiments, the strain of influenza A virus is selected from the influenza A viruses listed in Table 1 and / or Table 2.

[0145] In some embodiments, the strain of influenza A virus is selected from the influenza A virus recommended by WHO for use in influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0146] Table 1: Recommended composition of influenza virus vaccines for use in the 1998-2024 northern hemisphere influenza seasons

[0147]

[0148]

[0149]

[0150]

[0151] Table 2: Recommended composition of influenza virus vaccines for use in the 1999-2023 southern hemisphere influenza season

[0152]

[0153]

[0154]

[0155]

[0156] In some embodiments, the strain of influenza B virus is selected from the group consisting of B / Victoria lineage and B / Yamagata lineage.

[0157] 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 / 2004virus, B / Ohio / 1 / 2005, B / Florida / 4 / 2006-like virus, B / Brisbane / 3 / 2007, B / Brisbane / 60 / 20 08-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 virus, B / Phuket / 3073 / 2013-like virus, B / Austria / 1359417 / 2021-like virus, B / Washington / 02 / 2019-like virus and B / Colorado / 06 / 2017-like virus.

[0158] In some embodiments, the strain of influenza B virus is selected from the influenza B virus listed in Table 1 and / or Table 2.

[0159] In some embodiments, the strain of influenza B virus is selected from the influenza B virus recommended by WHO for use in influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0160] In some embodiments, the strain of influenza virus of (b) is a strain of influenza A virus.

[0161] In some embodiments, the strain of influenza virus of (a) is a strain of influenza B virus.

[0162] In some embodiments, the strain of influenza virus of (b) is a strain of influenza A virus, and the strain of influenza virus of (a) is a strain of influenza B virus.

[0163] Exemplary HA antigens are known in the art and are publicly available, for example, at NCBI's Influenza Virus Resource (https: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / nph-select.cgi?go=database) and GISRS (https: / / gisaid.org / resources / human-influency-vaccine-composition / ).

[0164] In some embodiments, the first HA antigen and / or the second HA antigen comprises, or consists of, an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 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.

[0165] In some embodiments, the first HA antigen and / or the second HA antigen comprises or consists of the amino acid sequence of any one of SEQ ID NO: 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.

[0166] In some embodiments, the first HA antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 5, 7, 17 or 35, or a fragment or variant thereof.

[0167] In some embodiments, the first HA antigen comprises or consists of the amino acid sequence of any one of SEQ ID NO: 5, 7, 17 or 35, or a fragment or variant thereof.

[0168] In some embodiments, the second HA antigen comprises, or consists of, an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 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.

[0169] In some embodiments, the second HA antigen comprises or consists of the amino acid sequence of any one of SEQ ID NO: 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.

[0170] In some embodiments, the second HA antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 1, 11, 19, 23, 27, 29, 39, 41 or 43, or a fragment or variant thereof.

[0171] In some embodiments, the second HA antigen comprises or consists of the amino acid sequence of any one of SEQ ID NO: 1, 11, 19, 23, 27, 29, 39, 41 or 43, or a fragment or variant thereof.

[0172] In some embodiments, the second HA antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 3, 9, 13, 15, 21, 25, 31, 33 or 37, or a fragment or variant thereof.

[0173] In some embodiments, the second HA antigen comprises or consists of the amino acid sequence of any one of SEQ ID NO: 3, 9, 13, 15, 21, 25, 31, 33 or 37, or a fragment or variant thereof.

[0174] In some embodiments, the first HA antigen and / or the second HA antigen is a polypeptide comprising a full-length influenza HA protein. Suitably, the first HA antigen and / or the second HA antigen is a polypeptide consisting of a full-length influenza HA protein.

[0175] In some embodiments, the first HA antigen and / or the second HA antigen is a fragment of a hemagglutinin protein, such as a truncated hemagglutinin protein. In some embodiments, the fragment is a headless hemagglutinin, meaning that the fragment does not include a head domain. In some embodiments, the fragment includes a portion of a head domain. In some embodiments, the fragment is a stem domain. In some embodiments, the fragment does not include a cytoplasmic domain. In some embodiments, the fragment does not include a transmembrane domain. In such embodiments, the fragment may be referred to as a soluble or secreted hemagglutinin protein or fragment.

[0176] In some embodiments, the ratio of (a):(b) is 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.

[0177] 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 between 2:1 and 4:1, suitably between 2:1 and 3:1, suitably 2:1 or 3:1.

[0178] In some embodiments, the ratio of (a):(b) is about 2:1, suitably 2.1.

[0179] In some embodiments, the ratio of (a):(b) is about 3:1, suitably 3.1.

[0180] In some embodiments, the immunogenic composition comprises:

[0181] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a strain of influenza B virus; and

[0182] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a strain of influenza A virus,

[0183] wherein (a) and (b) are different, and wherein the ratio of (a):(b) is between 2:1 and 4:1, suitably between 2:1 and 3:1, suitably 2:1 or 3:1.

[0184] In some embodiments, the immunogenic composition further comprises:

[0185] (c) at least one other antigen or at least one other nucleic acid (suitably mRNA) encoding the at least one other antigen, wherein the at least one other antigen is derived from a strain of influenza virus.

[0186] In some embodiments, the influenza virus strain of (c) is selected from the group consisting of influenza A virus and influenza B virus.

[0187] In some embodiments, the strain of influenza virus of (c) is a strain of influenza A virus.

[0188] As described above, in some embodiments, the strain of influenza A virus is selected from an influenza A virus characterized by hemagglutinin (HA), which is 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, suitably selected from the group consisting of H1, H3, H5, H7, H9 and H10, and more suitably selected from the group consisting of H1 and H3.

[0189] In some embodiments, the strain of influenza A virus is selected from influenza A virus characterized by neuraminidase (NA), which is selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11, suitably selected from the group consisting of N1, N2 and N8, and more suitably selected from the group consisting of N1 and N2.

[0190] 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, suitably H1N1 and H3N2.

[0191] In some embodiments, the strain of influenza A virus is selected from the group consisting of H1N1 and H3N2.

[0192] 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 / 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, influenza A(H3N2) viruses antigenically similar to the cell-propagated prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like virus, A / Darwin / 9 / 2021(H3N2)-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, A / Cambodia / e0826360 / 2020(H3N2)-like virus, A / Hong Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus 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.

[0193] In some embodiments, the strain of influenza A virus is H1N1.

[0194] 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 virus, A / Michigan / 45 / 2015(H1N1)pdm09-like virus, 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.

[0195] In some embodiments, the strain of influenza A virus is H3N2.

[0196] 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, influenza A(H3N2) viruses antigenically similar to the cell-propagated prototype virus A / Victoria / 361 / 2011, A / Texas / 50 / 2012(H3N2)-like virus, A / Darwin / 9 / 2021(H3N2)-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, A / Cambodia / e0826360 / 2020(H3N2)-like virus, A / Hong Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus, A / South Africa / 9715294 / 2013(H3N2)-like virus, A / South Africa / 9715295 / 2013(H3N2)-like virus, A / South Africa / 9715296 / 2013(H3N2)-like virus 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.

[0197] In some embodiments, the strain of influenza A virus is selected from the influenza A viruses listed in Table 1 and / or Table 2.

[0198] In some embodiments, the strain of influenza A virus is selected from the influenza A virus recommended by WHO for use in influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0199] In some embodiments, the strain of influenza virus of (c) is a strain of influenza B virus.

[0200] In some embodiments, the strain of influenza B virus is selected from the group consisting of B / Victoria lineage and B / Yamagata lineage.

[0201] 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 / 2004virus, B / Ohio / 1 / 2005, B / Florida / 4 / 2006-like virus, B / Brisbane / 3 / 2007, B / Brisbane / 60 / 20 08-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 virus, B / Phuket / 3073 / 2013-like virus, B / Austria / 1359417 / 2021-like virus, B / Washington / 02 / 2019-like virus and B / Colorado / 06 / 2017-like virus.

[0202] In some embodiments, the strain of influenza B virus is selected from the influenza B virus listed in Table 1 and / or Table 2.

[0203] In some embodiments, the strain of influenza B virus is selected from the influenza B virus recommended by WHO for use in influenza virus vaccine compositions (https: / / www.who.int / teams / global-influenza-programme / vaccines / who-recommendations).

[0204] In some embodiments, the at least one other antigen comprises or is composed 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), non-structural protein 1 (NS1), non-structural 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.

[0205] In some embodiments, the at least one other 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.

[0206] In some embodiments, the immunogenic composition comprises a combination of an HA antigen or a nucleic acid (suitably mRNA) encoding the HA antigen; the at least one other antigen comprises or consists of a peptide or protein selected from or derived from influenza virus HA or a fragment or variant thereof.

[0207] In some embodiments, the immunogenic composition comprises a combination of an HA antigen and a NA antigen or nucleic acids (suitably mRNA) encoding said HA antigen and NA antigen; said at least one other antigen comprises or consists of a peptide or protein selected from or derived from influenza virus NA or a fragment or variant thereof.

[0208] Similar to HA, neuraminidase (NA) is the major surface glycoprotein of influenza virus. Naturally acquired or vaccine-induced NA inhibitory (NAI) antibodies have been shown to contribute to influenza disease protection in naturally occurring influenza or experimental human challenge studies. Compared with hemagglutinin inhibitory antibodies, NAI antibodies appear to have an independent role in vaccine efficacy / effectiveness. Antigenic drift of HA and NA has been reported to be independent, suggesting that NA-specific immunity may provide a certain level of protection when HA drifts.

[0209] 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.

[0210] 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 headless hemagglutinin, meaning that the fragment does not include a head domain. In some embodiments, the fragment includes a portion of a head domain. In some embodiments, the fragment is a stem domain. In some embodiments, the fragment does not include a cytoplasmic domain. In some embodiments, the fragment does not include a transmembrane domain. In such embodiments, the fragment may be referred to as a soluble or secreted hemagglutinin protein or fragment.

[0211] In some embodiments, the NA antigen is a polypeptide comprising a full-length influenza NA protein. Suitably, the NA antigen is a polypeptide consisting of a full-length influenza NA protein.

[0212] In some embodiments, the NA antigen is a fragment of the neuraminidase protein, such as a truncated neuraminidase protein.

[0213] In some embodiments, the HA antigen and NA antigen or nucleic acids encoding said HA antigen and NA antigen (suitably mRNA) are present in an equimolar ratio.

[0214] In some embodiments, the HA antigen and NA antigen or nucleic acids (suitably mRNA) encoding said HA antigen and NA antigen are not present in an equimolar ratio.

[0215] In some embodiments, the dose (e.g. a weight dose or a molar dose, suitably a weight dose) of the at least one NA antigen or a nucleic acid encoding the same (suitably an mRNA) is different from the dose (e.g. a weight dose or a molar dose, suitably a weight dose) of the HA antigen or a nucleic acid encoding the HA antigen (suitably an mRNA).

[0216] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is between 4:1 and 1:4, suitably 3:1 and 1:3, suitably 2:1 and 2:1.

[0217] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is 4:1 or 1:4.

[0218] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is 3:1 or 1:3.

[0219] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is 2:1 or 1:2.

[0220] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is 3:2 or 2:3.

[0221] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is 4:3 or 3:4.

[0222] In some embodiments, the ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is about 1: 1. In some embodiments, the dosage ratio of HA:NA antigen or nucleic acid encoding the same (suitably mRNA) is 1:1.

[0223] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0224] In some embodiments, the HA of the ratio of HA:NA antigens or nucleic acid (suitably mRNA) encoding the same is HA derived from a strain of influenza A virus (suitably H1N1 and / or H3N2).

[0225] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 44, or a fragment thereof.

[0226] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 44 or a fragment thereof.

[0227] In some embodiments, the at least one other antigen comprises, or consists of, an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 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.

[0228] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 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.

[0229] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 5, 7, 17 or 35, or a fragment or variant thereof.

[0230] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 5, 7, 17 or 35, or a fragment or variant thereof.

[0231] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 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.

[0232] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 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.

[0233] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 1, 11, 19, 23, 27, 29, 39, 41 or 43, or a fragment or variant thereof.

[0234] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 1, 11, 19, 23, 27, 29, 39, 41 or 43, or a fragment or variant thereof.

[0235] In some embodiments, the at least one other antigen comprises, or consists of, an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 3, 9, 13, 15, 21, 25, 31, 33 or 37, or a fragment or variant thereof.

[0236] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 3, 9, 13, 15, 21, 25, 31, 33 or 37, or a fragment or variant thereof.

[0237] In some embodiments, the at least one other antigen comprises, or consists of, an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 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.

[0238] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 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.

[0239] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 6, 8, 18, 36, or a fragment or variant thereof.

[0240] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 8, 18, 36, or a fragment or variant thereof.

[0241] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 2, 12, 20, 24, 28, 30, 40, 42 or 44, or a fragment or variant thereof.

[0242] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NO: 2, 12, 20, 24, 28, 30, 40, 42 or 44, or a fragment or variant thereof.

[0243] In some embodiments, the at least one other antigen comprises, or consists of, an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO:4, 10, 14, 16, 22, 26, 32, 34, 38, or a fragment or variant thereof.

[0244] In some embodiments, the at least one other antigen comprises or consists of an amino acid sequence as described in any one of SEQ ID NOs: 4, 10, 14, 16, 22, 26, 32, 34, 38, or a fragment or variant thereof.

[0245] In some embodiments, the composition is a multivalent composition, and the strain of influenza virus of (a) and / or the strain of influenza virus of (b) and / or the strain of influenza virus of (c) are different.

[0246] In some embodiments, the strain of influenza virus of (c) is a strain of influenza A virus, and the ratio of (a):(b):(c) is 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.

[0247] In some embodiments, the strain of influenza virus of (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, 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.

[0248] In some embodiments, the strain of influenza virus of (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, 4:1:1, 4.2:1:1, 4.4:1:1, 4.6:1:1, 4.8:1:1, or 5:1:1.

[0249] In some embodiments, the strain of influenza virus of (c) is a strain of influenza A virus, and the ratio of (a):(b):(c) is between 2:1:1 and 4:1:1, suitably between 2:1:1 and 3:1:1, suitably 2:1:1 or 3:1:1.

[0250] In some embodiments, (c) is a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2.

[0251] In some embodiments, (c) is a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2, and the ratio of (a):(b):(c) is 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, suitably between 2:1:1 and 3:1:1, suitably 2:1:1 or 3:1:1.

[0252] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0253] In some embodiments, the immunogenic composition comprises:

[0254] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus;

[0255] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1, and

[0256] (c) at least one other antigen or at least one other nucleic acid (suitably mRNA) encoding the at least one other antigen, wherein the at least one other antigen is derived from a strain of influenza virus,

[0257] wherein (a), (b) and (c) are different and wherein the ratio of (a):(b):(c) is 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, suitably between 2:1:1 and 3:1:1, suitably 2:1:1 or 3:1:1.

[0258] In some embodiments, the immunogenic composition comprises:

[0259] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus, suitably from the B / Victoria lineage;

[0260] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1, and

[0261] (c) a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2,

[0262] wherein (a), (b) and (c) are different and wherein the ratio of (a):(b):(c) is 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, suitably between 2:1:1 and 3:1:1, suitably 2:1:1 or 3:1:1.

[0263] In some embodiments, the immunogenic composition comprises a plurality of (c), for example, (c) as defined herein. 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ).

[0264] In some embodiments, the composition comprises at least four, five, six, seven or eight antigens or nucleic acids encoding the same (suitably mRNA), optionally four to ten antigens or nucleic acids encoding the same (suitably mRNA), optionally four, seven or eight antigens or nucleic acids encoding the same (suitably mRNA).

[0265] In some embodiments, the antigens of (a), (b) and / or (c) are derived from at least two, three or four strains of influenza virus.

[0266] In some embodiments, the composition comprises four antigens or nucleic acids (suitably mRNA) encoding the same.

[0267] In some embodiments, the immunogenic composition comprises a combination of four HA antigens or four nucleic acids (suitably mRNAs) encoding said four HA antigens.

[0268] In some embodiments, the immunogenic composition comprises:

[0269] (a) and (b) as defined herein; suitably, (a) is a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus, and / or (b) is a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1;

[0270] (c 1 ) a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2; and

[0271] (c 2 ) a fourth HA antigen or a fourth nucleic acid (suitably mRNA) encoding the fourth HA antigen, wherein the fourth HA antigen is derived from a second strain of influenza B virus,

[0272] wherein the ratio of (a):(b) is 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, suitably between 2:1 and 3:1, suitably 2:1 or 3:1.

[0273] In some embodiments, the immunogenic composition comprises:

[0274] (a) and (b) as defined herein; suitably, (a) is a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus, and / or (b) is a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1;

[0275] (c 1 ) a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2; and

[0276] (c 2 ) a fourth HA antigen or a fourth nucleic acid (suitably mRNA) encoding the fourth HA antigen, wherein the fourth HA antigen is derived from a second strain of influenza B virus,

[0277] Where (a):(b):(c 1 ) is 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, suitably between 2:1:1 and 3:1:1, suitably 2:1:1 or 3:1:1.

[0278] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0279] In some embodiments, the immunogenic composition comprises:

[0280] (a) and (b) as defined herein; suitably, (a) is a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus, and / or (b) is a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1;

[0281] (c 1 ) a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2, and

[0282] (c 2 ) a fourth HA antigen or a fourth nucleic acid (suitably mRNA) encoding the fourth HA antigen, wherein the fourth HA antigen is derived from a second strain of influenza B virus,

[0283] Among them (a), (b), (c 1 ) and (c 2 ) are different, and where (a):(b):(c 1 ):(c 2 ) ratio is between 1.5:1:1:1.5 and 5:1:1:5.

[0284] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0285] In some embodiments, the immunogenic composition comprises:

[0286] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus;

[0287] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1;

[0288] (c 1 ) a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2, and

[0289] (c 2 ) a fourth HA antigen or a fourth nucleic acid (suitably mRNA) encoding the fourth HA antigen, wherein the fourth HA antigen is derived from a second strain of influenza B virus,

[0290] Among them (a), (b), (c 1 ) and (c 2 ) are different, and where (a):(b):(c 1 ):(c 2 ) ratio is between 1.5:1:1:1.5 and 5:1:1:5.

[0291] In some embodiments, (a): (b): (c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, optionally between 2:1:1:2 and 5:1:1:5, optionally between 3:1:1:3 and 5:1:1:5, optionally between 1.5:1:1:1.5 and 3:1:1:3, optionally between 2:1:1:2 and 4:1:1:4, optionally between 2:1:1:2 and 3:1:1:3.

[0292] In some embodiments, (a): (b): (c 1 ):(c 21:1:4, 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, (a):(b):(c): 1 ):(c 2 ) in a ratio of 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.

[0293] In some embodiments, (a): (b): (c 1 ):(c 2 ) in a ratio of between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0294] In some embodiments, the composition comprises seven antigens or nucleic acids (suitably mRNA) encoding the same.

[0295] In some embodiments, the immunogenic composition comprises a combination of four HA antigens or four nucleic acids (suitably mRNAs) encoding said four HA antigens and three NA antigens or three nucleic acids (suitably mRNAs) encoding said three NA antigens.

[0296] In some embodiments, the immunogenic composition further comprises:

[0297] (c 3 ) a first NA antigen or a first nucleic acid (suitably mRNA) encoding the first NA antigen, wherein the first NA antigen is derived from a first strain of the influenza A virus;

[0298] (c 4) a second NA antigen or a second nucleic acid (suitably mRNA) encoding the second NA antigen, wherein the second NA antigen is derived from a second strain of the influenza A virus; and

[0299] (c 5 ) a third NA antigen or a third nucleic acid (suitably mRNA) encoding the third NA antigen, wherein the third NA antigen is derived from the first strain of influenza B virus.

[0300] In some embodiments, the immunogenic composition further comprises:

[0301] (c 3 ) a first NA antigen or a first nucleic acid (suitably mRNA) encoding the first NA antigen, wherein the first NA antigen is derived from a first strain of the influenza A virus;

[0302] (c 4 ) a second NA antigen or a second nucleic acid (suitably mRNA) encoding the second NA antigen, wherein the second NA antigen is derived from a second strain of the influenza A virus; and

[0303] (c 5 ) a third NA antigen or a third nucleic acid (suitably mRNA) encoding the third NA antigen, wherein the third NA antigen is derived from the first strain of influenza B virus,

[0304] Among them (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 ) and (c 5 ) are different, and where (a):(b):(c 1 ):(c 2 ) ratio is between 1.5:1:1:1.5 and 5:1:1:5.

[0305] In some embodiments, (a): (b): (c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, optionally between 2:1:1:2 and 5:1:1:5, optionally between 3:1:1:3 and 5:1:1:5, optionally between 4:1:1:4 and 5:1:1:5, optionally between 1.5:1:1:1.5 and 4:1:1:4, optionally between 1.5:1:1:1.5 and 3:1:1:3, optionally between 2:1:1:2 and 4:1:1:4, optionally between 2:1:1:2 and 3:1:1:3.

[0306] In some embodiments, (a): (b): (c 1 ):(c 2 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, (a):(b):(c): 1 ):(c 2 ) in a ratio of 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.

[0307] In some embodiments, (a): (b): (c 1 ):(c 2 ) in a ratio of between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0308] In some embodiments, (a): (b): (c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ) in a ratio between 9:3:3:9:1:1:1 and 3:1:1:3:3:3:3, suitably between 6:2:2:6:1:1:1 and 3:1:1:3:2:2:2, suitably 6:2:2:6:1:1:1 or 3:1:1:3:2:2:2.

[0309] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0310] In some embodiments, the composition comprises eight antigens or nucleic acids (suitably mRNA) encoding the same.

[0311] In some embodiments, the immunogenic composition comprises a combination of four HA antigens or four nucleic acids encoding said four HA antigens (suitably mRNA) and four NA antigens or four nucleic acids encoding said four NA antigens (suitably mRNA).

[0312] In some embodiments, the composition further comprises:

[0313] (c 6 ) a fourth NA antigen or a fourth nucleic acid (suitably mRNA) encoding the fourth NA antigen, wherein the fourth NA antigen is derived from a second strain of the influenza B virus.

[0314] In some embodiments, the composition comprises:

[0315] (c 6 ) a fourth NA antigen or a fourth nucleic acid (suitably mRNA) encoding the fourth NA antigen, wherein the fourth NA antigen is derived from a second strain of the influenza B virus

[0316] Among them (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and (c 6 ) are different, and where (a):(b):(c 1 ):(c 2 ) ratio is between 1.5:1:1:1.5 and 5:1:1:5.

[0317] In some embodiments, (a): (b): (c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, optionally between 2:1:1:2 and 5:1:1:5, optionally between 3:1:1:3 and 5:1:1:5, optionally between 4:1:1:4 and 5:1:1:5, optionally between 1.5:1:1:1.5 and 4:1:1:4, optionally between 1.5:1:1:1.5 and 3:1:1:3, optionally between 2:1:1:2 and 4:1:1:4, optionally between 2:1:1:2 and 3:1:1:3.

[0318] In some embodiments, (a): (b): (c 1 ):(c 21:1:4, 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, (a):(b):(c): 1 ):(c 2 ) in a ratio of 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.

[0319] In some embodiments, (a): (b): (c 1 ):(c 2 ) in a ratio of between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0320] In some embodiments, (a): (b): (c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ):(c 6 ) in a ratio between 9:3:3:9:1:1:1:1 and 3:1:1:3:3:3:3:3, suitably between 6:2:2:6:1:1:1:1 and 3:1:1:3:2:2:2:2, suitably 6:2:2:6:1:1:1:1 or 3:1:1:3:2:2:2:2.

[0321] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0322] In some embodiments, the composition comprises six antigens or nucleic acids (suitably mRNA) encoding the same.

[0323] In some embodiments, the immunogenic composition comprises a combination of three HA antigens or three nucleic acids encoding said three HA antigens (suitably mRNA) and three NA antigens or three nucleic acids encoding said three NA antigens (suitably mRNA).

[0324] In some embodiments, the immunogenic composition comprises:

[0325] (a) a first hemagglutinin (HA) antigen or a first nucleic acid (suitably mRNA) encoding the first HA antigen, wherein the first HA antigen is derived from a first strain of influenza B virus, suitably from the B / Victoria lineage;

[0326] (b) a second HA antigen or a second nucleic acid (suitably mRNA) encoding the second HA antigen, wherein the second HA antigen is derived from a first strain of influenza A virus, suitably H1N1;

[0327] (c 1 ) a third HA antigen or a third nucleic acid (suitably mRNA) encoding the third HA antigen, wherein the third HA antigen is derived from a second strain of influenza A virus, suitably H3N2,

[0328] (c 3 ) a first NA antigen or a first nucleic acid (suitably mRNA) encoding the first NA antigen, wherein the first NA antigen is derived from a first strain of influenza A virus, suitably H1N1;

[0329] (c 4 ) a second NA antigen or a second nucleic acid (suitably mRNA) encoding the second NA antigen, wherein the second NA antigen is derived from a second strain of the influenza A virus, suitably H3N2; and

[0330] (c 5 ) a third NA antigen or a third nucleic acid (suitably mRNA) encoding the third NA antigen, wherein the third NA antigen is derived from the first strain of influenza B virus, suitably from the B / Victoria lineage,

[0331] Among them (a), (b), (c 1 )、(c 3 )、(c 4 ) and (c 5 ) are different, and where (a):(b):(c 1 ) ratio is between 1.5:1:1 and 5:1:1.

[0332] It must be noted that specific 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 set of parts of the invention) or other aspects (including, for example, medical uses (the first medical use and the second medical use) and, for example, methods of treatment).

[0333] Nucleic Acids

[0334] In some embodiments, at least one nucleic acid of the immunogenic composition (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is DNA or RNA, suitably mRNA. In some embodiments, at least one nucleic acid of the immunogenic composition (suitably (a), (b), (c), (d) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s nucleic acid is DNA.

[0335] In some embodiments, at least one nucleic acid of the immunogenic composition (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) The nucleic acid) is an artificial nucleic acid, such as an artificial DNA or an artificial RNA, suitably mRNA.

[0336] Nucleic acid-based vaccines (including DNA or RNA, suitably mRNA) represent a promising technology for novel vaccines against emerging viruses and for providing combination vaccines. Nucleic acids can be genetically engineered and administered to human subjects. Transfected cells directly produce encoded antigens (e.g., provided by DNA or RNA (particularly mRNA)), which results in a protective immune response.

[0337] A nucleic acid (eg DNA or RNA, suitably mRNA) according to the invention forms the basis of a nucleic acid-based immunogenic composition or a nucleic acid-based vaccine.

[0338] Such nucleic acid-based immunogenic compositions (first aspect) or nucleic acid-based vaccines (second aspect) as provided herein have advantages over classical vaccine approaches.

[0339] In general, protein-based vaccines or live attenuated vaccines are not ideal for developing countries because they are expensive to produce. In addition, protein-based vaccines or live attenuated vaccines require a long development time and are not suitable for rapid response to epidemic virus outbreaks (e.g., influenza virus outbreaks). In fact, because the traditional method of producing standard inactivated influenza vaccines takes a long time, GISRS recommends that it be made six to seven months before the start of the influenza season, during which the influenza virus may continue to evolve.

[0340] On the contrary, according to the immunogenic composition based on nucleic acid and vaccine of the present invention, allow very fast and cost-effective manufacturing.Therefore, compared with known vaccines, composition / vaccine based on nucleic acid can significantly cheaper and more quickly produce and manufacture, which is particularly advantageous for being used in developing countries or in the context of annual popular or global epidemic.Composition / vaccine based on nucleic acid provides GISRS with extra time to monitor the virus of transmission, and makes its recommendation when closer to flu season.This extension of GISRS monitoring timeline should allow GISRS to predict more accurately, thereby produce more effective vaccine, and its designated targeting is closer to the virus of transmission when flu season.In addition, different nucleic acids encoding different antigens (for example antigens of different influenza strains) can be combined in a kind of immunogenic composition / vaccine, to ensure or increase the effectiveness of the immune response for influenza virus.

[0341] The use of RNA (suitably mRNA) in or as a vaccine overcomes the shortcomings of conventional gene vaccination involving the incorporation of DNA into cells in terms of safety, feasibility, suitability and effectiveness of generating immune responses. RNA molecules (suitably mRNA) are considered significantly safer than DNA vaccines because RNA (suitably mRNA) is more easily degraded. They are quickly cleared from the body and cannot be incorporated into the genome and affect the gene expression of cells in an uncontrollable manner. RNA (suitably mRNA) vaccines are also less likely to cause serious side effects, such as the generation of autoimmune diseases or anti-DNA antibodies. Transfection with RNA (suitably mRNA) only requires insertion into the cytoplasm of the cell, which is easier to achieve than insertion into the nucleus.

[0342] In some embodiments, at least one nucleic acid of the immunogenic composition (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c6 )'s nucleic acid is RNA.

[0343] Thus, in some embodiments, (a) is a first RNA encoding the first HA antigen and / or (b) is a second RNA encoding the second HA antigen.

[0344] In some embodiments, (c) is at least one other RNA encoding the at least one other antigen.

[0345] In some embodiments, the immunogenic composition comprises a plurality of (c) which is RNA.

[0346] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is RNA.

[0347] Messenger RNA (mRNA) is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by the ribosome in the process of producing protein. 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 enables intracellular RNA to amplify.

[0348] mRNA-based influenza vaccine candidates are currently undergoing clinical trials. For example, mRNA-1010 is an mRNA vaccine candidate that encodes the HA glycoprotein of four influenza strains recommended by the WHO. In a Phase I study, mRNA-1010 was evaluated at equimolar total dose levels of 50, 100, and 200 μg in young and elderly adult cohorts.

[0349] In some embodiments, at least one nucleic acid of the immunogenic composition (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s nucleic acid is mRNA.

[0350] In some embodiments, (a) is a first mRNA encoding the first HA antigen and / or (b) is a second mRNA encoding the second HA antigen.

[0351] In some embodiments, the dose of each of the first mRNA and / or the second mRNA is 1 to 200 μg, suitably 1 to 60 μg, suitably 2 to 25 μg.

[0352] In some embodiments, the dose of each of the first mRNA and / or the second mRNA is 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, optionally 3 to 6 μg.

[0353] In some embodiments, the dose of each of the first mRNA and / or the 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.

[0354] In some embodiments, the dose of each of the first mRNA and / or the second mRNA is 3, 6, 9, 12 or 18 μg.

[0355] In some embodiments, (c) is at least one other mRNA encoding the at least one other antigen.

[0356] In some embodiments, the dose of each of the at least one other mRNA is 1 to 200 μg, suitably 1 to 60 μg, suitably 2 to 25 μg.

[0357] In some embodiments, the dose of each of the at least one additional mRNA is 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, optionally 3 to 6 μg.

[0358] 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.

[0359] In some embodiments, the dose of each of the at least one additional mRNA is 3, 6, 9, 12, or 18 μg.

[0360] In some embodiments, the immunogenic composition comprises a plurality of (c) which is mRNA.

[0361] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is mRNA.

[0362] In some embodiments, each of (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is 1 to 200 μg, suitably 1 to 60 μg, suitably 1 to 25 μg, suitably 2 to 25 μg.

[0363] In some embodiments, each of (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is 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, optionally 3 to 6 μg.

[0364] 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.

[0365] In some embodiments, each of (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) at a dose of 1, 2, 3, 6, 9, 12 or 18 μg.

[0366] The present invention also provides an immunogenic composition comprising:

[0367] (a) a first mRNA encoding HA of a first strain of influenza B virus;

[0368] (b) a second mRNA encoding HA of a first strain of influenza A virus (suitably H1N1);

[0369] (c 1 ) a third mRNA encoding HA of a second strain of influenza A virus (suitably H3N2); and

[0370] (c 2 ) a fourth mRNA encoding HA of a second strain of influenza B virus,

[0371] Where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0372] In some embodiments, the ratio is a weight / weight ratio or a molar ratio. Suitably, the ratio is a weight / weight ratio.

[0373] In some embodiments, (a) and (c) 2 ) is in a dose of 5 to 50 μg, optionally 10 to 40 μg, optionally 12 to 36 μg.

[0374] 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.

[0375] In some embodiments, (b) and (c) 1 ) is in a dose of 2 to 20 μg, optionally 5 to 15 μg, optionally 6 to 12 μg.

[0376] 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.

[0377] In some embodiments, (b) and (c)1 ) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μg.

[0378] In some embodiments, (a), (b), (c 1 ) and (c 2 ) is in a dose of 5 to 75 μg, optionally 10 to 60 μg, optionally 12 to 48 μg.

[0379] In some embodiments, (a), (b), (c 1 ) and (c 2 ) is in a dose range of 35 to 75 μg.

[0380] In some embodiments, (a), (b), (c 1 ) and (c 2 ) 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.

[0381] In some embodiments, (a), (b), (c 1 ) and (c 2 ) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 24, 25, 26, 27, 28, 29, 30, 45, 46, 47, 48, 49, 50, 55, 60 μg.

[0382] In some embodiments, (a) and (c) 2 ) is 5 to 50 μg, optionally 10 to 40 μg, optionally 12 to 36 μg, and (b) and (c) 1 ) is in a dose of 2 to 20 μg, optionally 5 to 15 μg, optionally 6 to 12 μg.

[0383] 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 ) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μg.

[0384] In some embodiments, the immunogenic composition further comprises:

[0385] (c3 ) a first mRNA encoding the NA of a first strain of influenza A virus (suitably H1N1);

[0386] (c 4 ) a second mRNA encoding NA of a second strain of said influenza A virus (suitably H3N2); and

[0387] (c 5 ) a third mRNA encoding the NA of the first strain of influenza B virus,

[0388] Where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0389] In some embodiments, (a): (b): (c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ) in a ratio between 9:3:3:9:1:1:1 and 3:1:1:3:3:3:3, suitably between 6:2:2:6:1:1:1 and 3:1:1:3:2:2:2, suitably 6:2:2:6:1:1:1 or 3:1:3:2:2:2.

[0390] In some embodiments, (a), (b), (c 1 ) and (c 2 ) is in a dose of 5 to 75 μg, optionally 10 to 60 μg, optionally 12 to 48 μg.

[0391] In some embodiments, (a), (b), (c 1 ) and (c 2 ) is in a dose range of 35 to 75 μg.

[0392] In some embodiments, (a), (b), (c 1 ) and (c 2 ) 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. In some embodiments, (a), (b), (c) 1 ) and (c 2) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 24, 25, 26, 27, 28, 29, 30, 45, 46, 47, 48, 49, 50, 55, 60 μg.

[0393] In some embodiments, (c 3 )、(c 4 ) and (c 5 ) is 2 to 50 μg, optionally 2 to 30 μg, optionally 5 to 20, optionally 9 to 18 μg. In some embodiments, (c 3 )、(c 4 ) and (c 5 )'s dose is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0394] In some embodiments, (c 3 )、(c 4 ) and (c 5 ) doses ranged from 9 to 36 μg.

[0395] In some embodiments, (c 3 )、(c 4 ) and (c 5 ) at doses of 9, 18, 27 or 36 μg.

[0396] In some embodiments, the immunogenic composition further comprises:

[0397] (c 6 ) a fourth mRNA encoding NA of a second strain of the influenza B virus,

[0398] Where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0399] In some embodiments, (a): (b): (c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ):(c 6) in a ratio between 9:3:3:9:1:1:1:1 and 3:1:1:3:3:3:3:3, suitably between 6:2:2:6:1:1:1:1 and 3:1:1:3:2:2:2:2, suitably 6:2:2:6:1:1:1:1 or 3:1:1:3:2:2:2:2.

[0400] In some embodiments, (c 3 )、(c 4 ) and (c 5 ) is in a dose of 5 to 50 μg, optionally 10 to 30 μg, optionally 12 to 24 μg.

[0401] In some embodiments, (c 3 )、(c 4 ) and (c 5 )'s dose is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 μg.

[0402] In some embodiments, (c 3 )、(c 4 )、(c 5 ) and (c 6 ) is in a dose of 5 to 50 μg, optionally 10 to 50 μg, optionally 12 to 48 μg.

[0403] In some embodiments, (c 3 )、(c 4 )、(c 5 ) and (c 6 )'s dose is 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, 48 μg.

[0404] In some embodiments, each of (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is 1 to 200 μg, suitably 1 to 60 μg, suitably 1 to 25 μg, suitably 2 to 25 μg.

[0405] In some embodiments, each of (a), (b), (c), (c 1 )、(c 2 )、(c3 )、(c 4 )、(c 5 ) and / or (c 6 ) is 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, optionally 3 to 6 μg.

[0406] 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.

[0407] In some embodiments, each of (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) at a dose of 1, 2, 3, 6, 9, 12 or 18 μg.

[0408] The mRNA used herein is suitably 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), and the like, generally at least about 50% pure (by weight), and usually at least 90% pure, such as at least 95% or at least 98% pure.

[0409] The mRNA used herein can be prepared in a variety of ways, such as by all or part of chemical synthesis, by digesting longer nucleic acids using nucleases (e.g., restriction endonucleases), by joining shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), preparing from genomic libraries or cDNA libraries, etc. In particular, mRNA can be prepared enzymatically using a DNA template.

[0410] The mRNA used herein can be an artificial nucleic acid. As used herein, the term "artificial nucleic acid" is intended to refer to non-natural nucleic acids. In other words, artificial nucleic acids can be understood as non-natural nucleic acid molecules. Such nucleic acid molecules can be non-natural due to their individual sequences (coding sequences, UTRs modified by G / C content for example) and / or due to other modifications (structural modifications of nucleotides for example). Generally, artificial nucleic acids can be designed and / or generated by genetic engineering, corresponding to required artificial nucleotide sequences. In this context, artificial nucleic acids are sequences that may not be naturally occurring, i.e., sequences that differ from wild-type or reference sequences / naturally occurring sequences by at least one nucleotide (by, for example, as further defined below, codon modifications). The term "artificial nucleic acid" is not limited to representing "a single molecule", but is understood to include a set of substantially identical nucleic acid molecules. Therefore, it can relate to a variety of substantially identical nucleic acid molecules.

[0411] Alternatively, or in addition, the sequence or chemical structure of the nucleic acid can be modified compared to the naturally occurring sequence of the encoding antigen. The sequence of the nucleic acid molecule can be modified, for example, modified to increase the efficiency of the expression or replication of the nucleic acid, or modified to provide additional stability or tolerance to degradation.

[0412] In some embodiments, the mRNA used herein may be a modified and / or stabilized nucleic acid, suitably a modified and / or stabilized artificial nucleic acid.

[0413] According to some embodiments, the mRNA used herein can therefore be provided as a "stabilized artificial nucleic acid" or a "stabilized encoding nucleic acid", that is, a nucleic acid that exhibits improved tolerance to in vivo degradation and / or a nucleic acid that exhibits improved in vivo stability, and / or a nucleic acid that exhibits improved in vivo translatability. Specific suitable modifications / adaptations in this context that are suitable for "stabilizing" nucleic acids are described below.

[0414] Suitable modifications that can "stabilize" mRNA are described below.

[0415] The mRNA used herein can 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. Suitably, 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.

[0416] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c4 )、(c 5 ) and / or (c 6 ) 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.

[0417] In some embodiments, the mRNA used herein can be codon optimized for expression in human cells." Codon optimization "is intended to indicate that the modification used about codons can increase the translation efficiency and / or half-life of nucleic acids. The term "codon-modified coding sequence" refers to a coding sequence that differs from the corresponding wild type or reference coding sequence by at least one codon (triplet nucleotides encoding an amino acid). Suitably, in the context of the present invention, the coding sequence modified by codons can show improved tolerance to in vivo degradation and / or improved in vivo stability, and / or improved in vivo translatability. The most general codon modification utilizes the degeneracy of the genetic code, wherein a variety of codons can encode the same amino acid, and can be used interchangeably (see Table 1 of WO2020002525), to optimize / modify the coding sequence as outlined herein, for in vivo application.

[0418] In some embodiments, the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a coding sequence adapted to human codon usage, a G / C content modified coding sequence, and a G / C optimized coding sequence, or any combination thereof.

[0419] In some embodiments, the codon-modified coding sequence has a G / C content of at least about 45%, 50%, 55% or 60%. In a specific embodiment, the G / C content of at least one coding sequence of the mRNA is 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%.

[0420] When transfected into mammalian host cells, the mRNA used herein comprising a codon-modified coding sequence has a stability of between 12-18 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 mammalian host cells (e.g., muscle cells).

[0421] When transfected into a mammalian host cell, the mRNA used herein comprising a codon modified coding sequence is translated into protein, wherein the amount of protein is at least comparable to the amount of protein obtained from a naturally occurring or wild-type or reference coding sequence transfected into a mammalian host cell, or suitably 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, or more.

[0422] In some embodiments, the mRNA used herein may be modified, wherein the C content of the at least one coding sequence may be increased, suitably 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 suitably 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 be suitably performed using the modification method described in WO2015 / 062738. In this context, the disclosure of WO2015 / 062738 is incorporated herein by reference.

[0423] In some embodiments, the mRNA used herein can be modified, wherein the G / C content of the at least one coding sequence can be optimized compared to the G / C content of the corresponding wild type or reference coding sequence (referred to herein as "G / C content optimized coding sequence"). In this context, "optimized" refers to a coding sequence in which the G / C content is suitably increased to the highest possible G / C content. The amino acid sequence encoded by the G / C content optimized coding sequence of the mRNA is suitably unmodified compared to the amino acid sequence encoded by the respective wild type or reference coding sequence. The generation of the mRNA sequence optimized by the method described in WO2002 / 098443 for G / C content can be performed. In this context, the disclosure of WO2002 / 098443 is included in the present invention with its full scope.

[0424] In some embodiments, the mRNA used herein can be modified, wherein the codons in the at least one coding sequence can be adapted to human codon usage (referred to herein as "coding sequences adapted to human codon usage"). Codons encoding the same amino acid appear at different frequencies in humans. Therefore, the coding sequence of the mRNA used herein is suitably modified so that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of this codon used according to human codons. For example, in the case of amino acid Ala, the wild type or reference coding sequence is suitably adapted so 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, and the codon "GCG" is used at a frequency of 0.10, etc. (see, for example, Table 1 of WO2020002525). Therefore, this procedure (as exemplified for Ala) is applied to each amino acid encoded by the coding sequence of the RNA to obtain a sequence adapted to human codon usage.

[0425] In some embodiments, the mRNA used herein may be modified, wherein the G / C content of the at least one coding sequence may be modified compared to the G / C content of the corresponding wild type or reference coding sequence (referred to herein as "G / C content modified coding sequence"). In this context, the term "G / C optimization" or "G / C content modification" relates to a nucleic acid comprising modified (suitably increased in number) guanosine and / or cytosine nucleotides compared to the corresponding wild type or reference coding sequence. This increase in number can be generated by replacing codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. Suitably, nucleic acid sequences with increased G / C content are more stable or show better expression than sequences with increased A / U. The amino acid sequence encoded by the G / C content modified coding sequence of the mRNA is suitably 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%, suitably at least 40% compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence.

[0426] In some embodiments, the mRNA used herein can be modified, wherein the codon adaptation index (CAI) can be increased or suitably maximized in the at least one coding sequence (referred to herein as "CAI maximized coding sequence"). In some embodiments, all codons of a wild-type or reference nucleic acid sequence that is relatively rare in, for example, humans are exchanged for respective codons that are frequent in, for example, humans, wherein the frequent codons encode the same amino acid as the relatively rare codon. Suitably, the most frequent codons are used for each amino acid of the encoded protein (see Table 1 of WO2020002525, the most frequent human codons are marked with asterisks). Suitably, the mRNA used herein comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the 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 adaptation index (CAI) of the at least one coding sequence is 1 (CAI=1). For example, in the case of amino acid Ala, the wild type or reference coding sequence can be adapted in a way that the most frequent human codon "GCC" is always used for this amino acid. Therefore, this procedure (as exemplified for Ala) can be applied to each amino acid encoded by the coding sequence of the mRNA to obtain a coding sequence that maximizes CAI.

[0427] In some embodiments, relative to the number of A and / or U nucleotides in the original nucleic acid sequence (e.g., wild type or reference sequence), the mRNA used herein can be modified by changing the number of A and / or U nucleotides in the nucleic acid sequence. In some embodiments, this AU change is performed to modify the retention time of individual nucleic acids in the composition to (i) allow co-purification using an HPLC method, and / or allow analysis of the obtained nucleic acid composition. This method is described in detail in the published PCT application WO2019092153A1. Claims 1 to 70 of WO2019092153A1 are incorporated herein by reference.

[0428] In some embodiments, the at least one coding sequence of the 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 coding sequence adapted to human codon usage, or a G / C modified coding sequence.

[0429] A poly A tail (eg, a poly A tail of about 30 or more adenosine residues) can be attached to the 3' end of the RNA to increase its half-life.

[0430] In some embodiments, the mRNA used herein comprises at least one poly (N) sequence, such as at least one poly (A) sequence, at least one poly (U) sequence, at least one poly (C) sequence, or a combination thereof.

[0431] In some embodiments, the mRNA used herein comprises at least one poly(A) sequence.

[0432] The terms "poly(A) sequence", "poly(A) tail" or "3'-poly(A) tail" as used herein will be recognized and understood by a person of ordinary skill in the art, and are intended, for example, to be a sequence of adenosine nucleotides, typically located at the 3'-end of a linear RNA (or in a circular RNA), of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is substantially homopolymeric, for example, a poly(A) sequence of, for example, 100 adenosine nucleotides has a length of substantially 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide other than 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 additionally comprising the at least one nucleotide or a stretch of nucleotides other than adenosine nucleotides).

[0433] The poly (A) sequence may comprise about 10 to about 500 adenylic acids, about 10 to about 200 adenylic acids, about 40 to about 200 adenylic acids, or about 40 to about 150 adenylic acids. In some embodiments, the poly (A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides in length.

[0434] In some embodiments, the mRNA 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.

[0435] In a further embodiment, the mRNA used herein comprises at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, suitably by 10 non-adenosine nucleotides (A30-N10-A70).

[0436] The poly(A) sequence as defined herein may be located directly at the 3' end of the mRNA. In some embodiments, the 3'-terminal nucleotide (i.e., the last 3'-terminal nucleotide in the polynucleotide chain) is the 3'-terminal A nucleotide of the at least one poly(A) sequence. The term "directly located at the 3' end" must be understood as being located precisely at the 3' end, in other words, the 3' end of the nucleic acid consists of a poly(A) sequence terminated with an A nucleotide.

[0437] In one embodiment, the mRNA used herein comprises a poly(A) sequence of at least 70 adenosine nucleotides, suitably at least 70 consecutive adenosine nucleotides, wherein the 3' terminal nucleotide is an adenosine nucleotide.

[0438] In some embodiments, the poly (A) sequence of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly (A) sequence is obtained in vitro by conventional methods of chemical synthesis without transcription from a DNA template. In other embodiments, a commercially available polyadenylation kit and corresponding protocols known in the art are used to generate a poly (A) sequence by enzymatic polyadenylation of the RNA (after RNA in vitro transcription), or alternatively, the poly (A) sequence is generated by using an immobilized poly (A) polymerase, for example using methods and means as described in WO2016174271.

[0439] The mRNA used herein may comprise a poly(A) sequence obtained by enzymatic polyadenylation, wherein most nucleic acid molecules comprise about 100 (+ / -20) to about 500 (+ / -50), suitably about 250 (+ / -20) adenosine nucleotides.

[0440] In some embodiments, the mRNA used herein comprises a poly(A) sequence derived from a template DNA, and optionally, additionally comprises at least one additional poly(A) sequence generated by enzymatic polyadenylation (eg, as described in WO2016091391).

[0441] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) comprises 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.

[0442] In some embodiments, the mRNA used herein comprises at least one polyadenylation signal.

[0443] In some embodiments, the mRNA used herein comprises at least one poly(C) sequence.

[0444] As used herein, the term "poly (C) sequence" is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly (C) sequence comprises 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 comprises about 30 cytosine nucleotides.

[0445] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) comprises a poly(A) tail sequence, preferably comprising 30 to 200 adenosine nucleotides and / or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides.

[0446] In some embodiments, the mRNA used herein comprises at least one histone stem-loop (hSL) or histone stem-loop structure.

[0447] The term "histone stem-loop" (abbreviated as "hSL" in, for example, the sequence listing) is intended to refer to a nucleic acid sequence that forms a stem-loop secondary structure that is predominantly found in histone mRNAs.

[0448] The histone stem-loop sequence / structure may be suitably selected from the histone stem-loop sequences disclosed in WO2012019780, the disclosures relating to histone stem-loop sequences / histone stem-loop structures being 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.

[0449] In some embodiments, the first mRNA and / or the second mRNA comprises at least one histone stem-loop.

[0450] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) contains at least one histone stem-loop.

[0451] In other embodiments, the mRNA used herein does not comprise hsL as defined herein.

[0452] In some embodiments, the mRNA used herein comprises a 3'-terminal sequence element. The 3'-terminal sequence element comprises a poly(A) sequence and optionally a histone stem-loop sequence.

[0453] The 5' end of the mRNA used herein may be capped. The mRNA used herein may be modified by adding a 5'-cap structure, which suitably 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).

[0454] For example, the 5' end of the RNA can be capped with a modified ribonucleotide or derivative thereof having the structure m7G(5')ppp(5')N (cap0 structure), which can be incorporated during RNA synthesis, or can be enzymatically engineered after RNA transcription (e.g., by using vaccinia virus capping enzyme (VCE), which is composed of mRNA triphosphatase, guanylyl transferase, and guanine-7-methyltransferase, which catalyzes the construction of N7-monomethylated cap 0 structure). Cap 0 structure plays an important role in maintaining the stability and translation efficiency of the RNA molecule. The 5' cap of the mRNA molecule can be further modified by 2'-O-methyltransferase, which results in the generation of cap 1 structure (m7Gppp[m2'-O]N), which can further improve translation efficiency.

[0455] In some embodiments, the mRNA used herein (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) comprises a 5'cap, preferably m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure, suitably a 5'-cap1 structure.

[0456] The term "5'-cap structure" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, 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 linked to the RNA via a 5'-5'-triphosphate bond.

[0457] Potentially suitable 5'-cap structures are cap0 (methylation of the first nucleobase, such as m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the second nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the third nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the fourth nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (such as phosphorothioate-modified ARCA), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine and 2-azidoguanosine.

[0458] The 5'-cap (cap0 or cap1) structure can be formed during chemical RNA synthesis or during RNA in vitro transcription (co-transcriptional capping) using cap analogs.

[0459] The term "cap analogue" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a non-polymerizable dinucleotide or trinucleotide that has a cap function because when it is incorporated at the 5'-end of a nucleic acid molecule (particularly an RNA molecule), it promotes translation or localization, and / or prevents degradation of the nucleic acid molecule. Non-polymerizable means that the cap analogue will only be incorporated 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, by 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 tetraphosphate derivatives thereof). Other cap analogs have been previously described (WO2008016473, WO2008157688, WO2009149253, WO2011015347, and WO2013059475). In this context, other suitable cap analogs are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017 / 053297, WO2017066782, WO2018075827 and WO2017066797, the disclosures of which referring to cap analogs are incorporated herein by reference.

[0460] In some embodiments, a modified cap1 structure is generated using trinucleotide cap analogs as disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, and WO2017066797. In particular, any cap structure that can be derived from the structure disclosed in claims 1-5 of WO2017053297 can be suitably used to co-transcriptionally generate a modified cap1 structure. In addition, any cap structure that can be derived from the structure defined in claim 1 or claim 21 of WO2018075827 can be suitably used to co-transcriptionally generate a modified cap1 structure.

[0461] In some embodiments, the mRNA used herein comprises a cap1 structure.

[0462] In some embodiments, a trinucleotide cap analogue as defined herein may be used, suitably to co-transcriptionally add a 5'-cap structure in an RNA in vitro transcription reaction as defined herein.

[0463] In some embodiments, the cap1 structure of the mRNA is formed by co-transcriptional capping using the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. In this context, a suitable cap1 analog is m7G(5')ppp(5')(2'OMeA)pG.

[0464] In other embodiments, co-transcriptional capping is used to form the cap1 structure of the mRNA using the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0465] In other embodiments, co-transcriptional capping is used using the cap analog 3'OMe-m7G(5')ppp(5')G to form the cap0 structure of the mRNA used herein.

[0466] In other embodiments, the 5'-cap structure is formed by enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase) to generate a cap0 or cap1 or cap2 structure. The 5'-cap structure (cap0 or cap1) can be added using the methods and methods disclosed in WO2016193226 using an immobilized capping enzyme and / or a cap-dependent 2'-O methyltransferase.

[0467] To determine the presence / absence of cap0 or cap1 structures, a capping assay as described in published PCT application WO2015101416 may be used, in particular a capping assay as described in 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 of RNA are described in PCT / EP2018 / 08667 or published PCT applications WO2014152673 and WO2014152659.

[0468] In some embodiments, the mRNA used herein comprises a m7G(5')ppp(5')(2'OMeA)cap structure. In such embodiments, these mRNAs comprise a 5'-terminal m7G cap, and additional methylation of the ribose of the adjacent nucleotide of the m7GpppN, in this case 2'O methylated adenosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprise this cap1 structure as determined using a capping assay.

[0469] In other embodiments, the mRNA used herein comprises a m7G(5')ppp(5')(2'OMeG)cap structure. In such embodiments, these mRNAs comprise a 5'-terminal m7G cap, and additional methylation of the ribose of the adjacent nucleotide, in this case 2'O-methylated guanosine. In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the coding RNA (species) comprise this cap1 structure as determined using a capping assay.

[0470] Therefore, the first nucleotide of the mRNA sequence, ie, the nucleotide downstream of the m7G(5')ppp structure, can be 2'O-methylated guanosine or 2'O-methylated adenosine.

[0471] In some embodiments, the A / U (A / T) content in the environment of the ribosome binding site of the mRNA used herein can 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. The efficient binding of these ribosomes to the ribosome binding site in turn has the effect of efficiently translating the mRNA.

[0472] Thus, in some embodiments, the mRNA used herein comprises a ribosome binding site, also known as a "Kozak sequence."

[0473] In some embodiments, the mRNA used herein may comprise at least one heterologous untranslated region (UTR), such as a 5'UTR and / or a 3'UTR.

[0474] The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to 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 can be part of a nucleic acid (e.g., DNA or RNA). A UTR can contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, promoter elements, etc.

[0475] In some embodiments, the mRNA used herein comprises a protein coding region ("coding sequence" or "cds") and a 5'-UTR and / or a 3'-UTR. Notably, the UTR may comprise regulatory sequence elements that determine the turnover, stability and positioning of nucleic acids (e.g., RNA). In addition, the UTR may comprise sequence elements that enhance translation. In the medical applications of nucleic acid sequences (including DNA and RNA), it is essential for therapeutic efficacy to translate the nucleic acid into at least one peptide or protein. Certain combinations of 3'-UTR and / or 5'-UTR can enhance the expression of operably connected coding sequences encoding the peptides or proteins of the present invention. Nucleic acid molecules comprising these UTR combinations advantageously enable rapid and transient expression of antigenic peptides or proteins after administration to a subject, suitably after intramuscular administration. Therefore, mRNA comprising certain combinations of 3'-UTR and / or 5'-UTR as provided herein is particularly suitable for administration as a vaccine, in particular, suitable for administration to the muscle, dermis or epidermis of a subject.

[0476] In some embodiments, the mRNA used herein comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. These heterologous 5'-UTR or 3'-UTR can be derived from naturally occurring genes or can be synthetically engineered. In some embodiments, the mRNA comprises at least one coding sequence as defined herein, which is operably linked to at least one (heterologous) 3'-UTR and / or at least one (heterologous) 5'-UTR.

[0477] In some embodiments, the mRNA used herein comprises at least one heterologous 3'-UTR.

[0478] In some embodiments, the first mRNA and / or the second mRNA comprises a 3'UTR.

[0479] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c6 )'s mRNA contains a 3'UTR.

[0480] The term "3'-untranslated region" or "3'-UTR" or "3'-UTR element" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a portion of a nucleic acid molecule that is located 3' (i.e., downstream) of a coding sequence and that is not translated into protein. A 3'-UTR can be a portion of a nucleic acid (e.g., DNA or RNA) that is located between a coding sequence and an (optional) terminal poly(A) sequence. A 3'-UTR can contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, etc.

[0481] In some embodiments, the mRNA used herein comprises a 3'-UTR, which may be derived from a gene associated with an RNA having an increased half-life (ie, which provides a stable RNA).

[0482] In some embodiments, the 3'-UTR comprises one or more polyadenylation signals, protein binding sites that affect the stability of the nucleic acid position in the cell, or one or more miRNAs or miRNA binding sites.

[0483] In some embodiments, the mRNA 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, α-globin (referred to as "muag"), CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homolog, fragment or variant of any of these genes.

[0484] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) comprises a 3'UTR containing a nucleic acid sequence or consisting of a 3'UTR derived from a gene selected from the group consisting of PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homolog, fragment or variant of any one of these genes.

[0485] The nucleic acid sequence in this context may 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.

[0486] In some embodiments, the mRNA used herein may include a 3'-UTR as described in WO2016107877, and the disclosure of WO2016107877 involving 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 includes a 3'-UTR as described in WO2017036580, and the disclosure of WO2017036580 involving 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 relating to 3'-UTR sequences is incorporated herein by reference. Particularly suitable 3'-UTRs are nucleic acid sequences according to SEQ ID NO: 20-36 of WO2016022914, or fragments or variants of these sequences.

[0487] In some embodiments, the mRNA used herein comprises at least one heterologous 5'-UTR.

[0488] In some embodiments, the mRNA used herein (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) contains a 5' untranslated region (UTR).

[0489] The term "5'-untranslated region" or "5'-UTR" or "5'-UTR element" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer 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 can be a portion of a nucleic acid that is located 5' of a coding sequence. Typically, a 5'-UTR starts at the transcription start site and ends before the start codon of the coding sequence. The 5'-UTR can contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, etc. The 5'-UTR can be modified post-transcriptionally, for example by enzymatic or post-transcriptional addition of a 5'-cap structure (e.g., for mRNA as defined herein).

[0490] In some embodiments, the mRNA used herein comprises a 5'-UTR, which may be derived from a gene associated with an RNA having an increased half-life (ie, it provides a stable RNA).

[0491] In some embodiments, the 5'-UTR comprises one or more protein binding sites that affect RNA stability or RNA location in the cell, or one or more miRNAs or miRNA binding sites.

[0492] In some embodiments, the mRNA used herein (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) 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 the group consisting of HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homolog, fragment or variant of any one of these genes.

[0493] The nucleic acid sequence in this context may be selected from the 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 (eg, SEQ ID NO: 1-20 of WO2019077001A1, or a fragment or variant thereof).

[0494] In some embodiments, the mRNA used herein may include a 5'-UTR as described in WO2013143700, and the disclosure of WO2013143700 involving 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences derived from SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421, and SEQ ID NO:1422 of WO2013143700, or fragments or variants of these sequences. In other embodiments, the mRNA used herein includes a 5'-UTR as described in WO2016107877, and the disclosure of WO2016107877 involving 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NO:25-30 and SEQ ID NO: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, and the disclosure of WO2017036580 relating to 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NO: 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, and the disclosure of WO2016022914 relating to 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NO: 3-19 of WO2016022914, or fragments or variants of these sequences.

[0495] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) comprises a heterologous 5'-UTR comprising or consisting of a nucleic acid sequence derived from a 5'-UTR of HSD17B4, and at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence derived from a 3'-UTR of PSMB3.

[0496] In some embodiments, the mRNA used herein (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c6 ) of mRNA) comprising from 5' to 3':

[0497] i) 5'-cap1 structure;

[0498] ii) a 5'-UTR derived from the 5'-UTR of the HSD17B4 gene;

[0499] iii) coding sequence;

[0500] iv) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene;

[0501] v) optionally, a histone stem-loop sequence; and

[0502] vi) a poly(A) sequence comprising about 100 A nucleotides, wherein the 3' terminal nucleotide of the RNA is adenosine.

[0503] In some embodiments, the RNA (suitably mRNA) can be prepared using any method known in the art, including chemical synthesis (eg, solid phase RNA synthesis) and in vitro methods (eg, RNA in vitro transcription reaction).

[0504] Thus, in some embodiments, the RNA (suitably mRNA) used herein is in vitro transcribed RNA.

[0505] 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 can be obtained by DNA-dependent in vitro transcription of a suitable DNA template (which can be a linearized plasmid DNA template or a PCR-amplified DNA template). The promoter used to control RNA in vitro transcription can be any promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6 or Syn5 RNA polymerases. In one embodiment of the invention, the DNA template is linearized with a suitable restriction endonuclease and then subjected to RNA in vitro transcription.

[0506] Reagents for RNA in vitro transcription generally include: a DNA template (linearized plasmid DNA or PCR product) having a promoter sequence that has a high binding affinity for its respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6 or Syn5); ribonucleotide triphosphates (NTPs) of 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 capable of binding to the promoter sequence within the DNA template. (e.g., T7, T3, SP6 or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor for inactivating any potential contaminating ribonucleases; optionally, a pyrophosphatase that degrades pyrophosphate, which can inhibit RNA in vitro transcription; MgCl2, which provides Mg2+ ions as a cofactor for the polymerase; a buffer (TRIS or HEPES) that maintains a suitable pH value, which may also contain an antioxidant (e.g., DTT) and / or a polyamine (e.g., spermidine) at an optimal concentration, such as a buffer system comprising TRIS-citrate disclosed in WO2017109161.

[0507] In some embodiments, co-transcriptional capping is used to form the cap1 structure of the mRNA used herein using the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A suitable cap1 analog that can be used to make the coding RNA (suitably mRNA) used herein is m7G(5')ppp(5')(2'OMeA)pG.

[0508] In other embodiments, co-transcriptional capping is used to form the cap1 structure of the RNA (suitably mRNA) used herein using the trinucleotide cap analogue 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0509] In other embodiments, co-transcriptional capping is used to form the capO structure of the RNA (suitably mRNA) used herein using the cap analog 3'OMe-m7G(5')ppp(5')G.

[0510] In some embodiments, the nucleotide mixture for RNA in vitro transcription may additionally contain modified nucleotides as defined herein. In this context, suitable modified nucleotides may be selected from pseudouridine (ψ), N1-methyl pseudouridine (m1ψ), 5-methylcytosine and 5-methoxyuridine. In some embodiments, the uracil nucleotides in the nucleotide mixture are (partially or completely) replaced with pseudouridine (ψ) and / or N1-methyl pseudouridine (m1ψ) to obtain modified RNA.

[0511] In some other embodiments, the nucleotide mixture for RNA in vitro transcription does not include modified nucleotides as defined herein. In some embodiments, the nucleotide mixture for RNA in vitro transcription includes only G, C, A and U nucleotides, and optionally includes cap analogs as defined herein.

[0512] In some embodiments, the nucleotide mixture used for the RNA in vitro transcription reaction (i.e., the ratio of each nucleotide in the mixture) can be optimized for a given RNA sequence, suitably as described in WO2015188933.

[0513] In this context, the in vitro transcription is performed in the presence of a sequence-optimized nucleotide mixture and optionally a cap analog.

[0514] In this context, a sequence-optimized nucleoside triphosphate (NTP) mixture is a mixture of nucleoside triphosphates (NTPs) for an in vitro transcription reaction of an RNA molecule of a given sequence, comprising four nucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP, wherein the ratio of each of these four nucleoside triphosphates (NTPs) in the sequence-optimized nucleoside triphosphate (NTP) mixture corresponds to the ratio of the respective nucleotides in the RNA molecule. If a ribonucleotide is not present in the RNA molecule, the corresponding nucleoside triphosphate is also not present in the sequence-optimized nucleoside triphosphate (NTP) mixture.

[0515] In embodiments where more than one different RNA (suitably mRNA) as defined herein has to be produced, for example where 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different RNAs have to be produced, the procedure as described in WO2017109134 may be suitably used.

[0516] In the context of producing nucleic acid-based vaccines, it may be necessary to provide GMP-grade nucleic acids, such as GMP-grade RNA or DNA. GMP-grade RNA or DNA can be produced using a production process approved by regulatory authorities. Therefore, in some embodiments, RNA is produced according to current good manufacturing practices (GMP), and various quality control steps are implemented at the DNA and RNA levels, suitably according to WO2016180430. In some embodiments, the mRNA of the present invention is GMP-grade mRNA. Therefore, the RNA used for vaccines is suitably GMP-grade RNA.

[0517] The RNA product obtained can be purified using PUREMESSENGER (CureVac, Tübingen, Germany; RP-HPLC according to WO2008077592) and / or tangential flow filtration (as described in WO2016193206) and / or oligo d(T) purification (see WO2016180430).

[0518] In some embodiments, RP-HPLC is used, suitably reverse phase high pressure liquid chromatography (RP-HPLC), using a macroporous styrene / divinylbenzene column (e.g., particle size 30 μm, pore size ) and additionally using a filtration cassette with a cellulose-based membrane with a molecular weight cut-off of approximately 100 kDa to purify the RNA (suitably mRNA) used herein.

[0519] In a further embodiment, the RNA used herein (suitably mRNA) is freeze-dried (e.g., as described in WO2016165831 or WO2011069586) to produce temperature-stable dried RNA (suitably mRNA) (powder). The RNA used herein (suitably mRNA) can also be dried using spray drying or spray freeze drying (e.g., according to WO2016184575 or WO2016184576) to produce temperature-stable RNA (suitably mRNA) (powder) as defined herein. Therefore, in the context of manufacturing and purifying RNA, the disclosures of WO2017109161, WO2015188933, WO2016180430, WO2008077592, WO2016193206, WO2016165831, WO2011069586, WO2016184575 and WO2016184576 are incorporated herein by reference.

[0520] Thus, in some embodiments, the RNA (suitably mRNA) used herein is dried RNA (suitably mRNA).

[0521] The term "dried RNA (or mRNA)" as used herein has to be understood as RNA (or mRNA) which has been freeze-dried, or spray-dried, or spray-freeze-dried as defined above to obtain a temperature-stable dried mRNA (powder).

[0522] In some embodiments, the RNA (suitably mRNA) used herein is purified RNA (suitably mRNA).

[0523] As used herein, the term "purified RNA (or mRNA)" must be understood as an RNA having a higher purity than the starting material (e.g., in vitro transcribed RNA) after certain purification steps (e.g., HPLC, TFF, Oligo d (T) purification, precipitation steps). Typical impurities that are substantially absent from purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, such as RNA polymerases, ribonucleases, pyrophosphatases, restriction endonucleases, deoxyribonucleases), spermidine, BSA, abortive RNA sequences, RNA fragments (short double-stranded RNA fragments, abortive sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other potential impurities that may originate from, for example, fermentation processes include bacterial impurities (bioburden, bacterial DNA) or impurities derived from the purification process (organic solvents, etc.). Therefore, in this regard, it is desirable that "RNA purity" be as close to 100% as possible. It is also desirable that the amount of full-length RNA transcripts be as close to 100% as possible for RNA purity. Thus, as used herein, "purified RNA" has a purity greater than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most advantageously 99% or greater. Purity can be determined, for example, by analytical HPLC, wherein the percentages provided above correspond to the ratio between the peak area of ​​the target RNA and the total area of ​​all peaks representing byproducts. Alternatively, purity can be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0524] It must 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 excellent stability characteristics (in vitro, in vivo) and improved efficacy (e.g. better translatability of the mRNA in vivo), and are therefore particularly suitable for pharmaceutical purposes, such as vaccines.

[0525] In some embodiments, the RNA (suitably mRNA) has been purified by RP-HPLC and / or TFF to remove double-stranded RNA, uncapped RNA and / or RNA fragments.

[0526] The formation of double-stranded RNA as a byproduct during, for example, RNA in vitro transcription can lead to the induction of innate immune responses, particularly IFNα, which is a major factor in inducing fever in vaccinated subjects, which is of course an unwanted side effect. Current techniques for immunoblotting of dsRNA (e.g., by dot blotting, serum-specific electron microscopy (SSEM) or ELISA) are used to detect dsRNA species from a mixture of nucleic acids and determine their size.

[0527] In some embodiments, the RNA (suitably mRNA) has been purified by RP-HPLC and / or TFF as described herein to reduce the amount of dsRNA.

[0528] In some embodiments, the RNA (suitably mRNA) comprises about 5%, 10% or 20% less double stranded RNA byproduct than RNA (suitably mRNA) that has not been purified using RP-HPLC and / or TFF.

[0529] In some embodiments, the RP-HPLC and / or TFF purified RNA (suitably mRNA) comprises about 5%, 10% or 20% less double-stranded RNA byproduct than RNA (suitably mRNA) that has been purified using Oligo dT, precipitation, filtration and / or AEX.

[0530] In some embodiments, the RNA (suitably mRNA) of the composition has an RNA integrity ranging from about 40% to about 100%.

[0531] The term "RNA integrity" generally describes whether there is a complete RNA sequence in a composition. Low RNA integrity may 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 temperature, ribonucleases, pH or other factors (e.g., nucleophilic attack, hydrolysis, etc.), which can reduce RNA integrity and, therefore, functionality of the RNA.

[0532] The technician can choose from a variety of different chromatographic or electrophoretic methods to determine RNA integrity. Chromatographic and electrophoretic methods are well known in the art. In the case of using chromatography (e.g., RP-HPLC), the analysis of the integrity of the RNA can be based on determining the peak area (or "area under the peak") of the 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 referred to as integration. The peak area representing the full-length RNA is usually set relative to the peak area of ​​the total RNA in the respective sample. RNA integrity can be expressed as RNA integrity %.

[0533] In the context of aspects of the present invention, RNA integrity can be determined using analytical (RP) HPLC. Typically, a test sample comprising a composition of a lipid-based carrier that encapsulates RNA can 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 can be captured using a suitable binding compound (e.g., Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA)) substantially according to the manufacturer's instructions. After the RNA sample is prepared, analytical (RP) HPLC can be performed to determine the integrity of the RNA. Typically, in order to determine RNA integrity, the RNA sample can be diluted to a concentration of 0.1 g / l using, for example, water for injection (WFI). The RNA sample diluted by about 10 μl can be injected into an HPLC column (e.g., a monolithic poly (styrene-divinylbenzene) matrix). Analytical (RP) HPLC can 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 from 30% buffer B, the gradient extends to 32% buffer B in 2 min, followed by 55% buffer B in 15 minutes at a flow rate of 1 ml / min. HPLC chromatograms are usually recorded at a wavelength of 260 nm. The chromatograms obtained can be evaluated using software, and the relative peak areas can be determined as percentages (%) as is well 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 usually known, analysis of the relative peak area provides information about the integrity of the RNA. Thus, if, for example, a total of 100 ng of RNA is injected, and 100 ng is determined as the relative peak area, the RNA integrity will be 100%. If, for example, the relative peak area will correspond to 80 ng, the RNA integrity will be 80%. Therefore, in the context of the present invention, RNA integrity is determined using analytical HPLC, suitably analytical RP-HPLC.

[0534] In some embodiments, the RNA integrity of the RNA (suitably mRNA) of the composition ranges from about 40% to about 100%. In some embodiments, the RNA integrity of the RNA (suitably mRNA) ranges from about 50% to about 100%. In some embodiments, the RNA integrity of the RNA (suitably mRNA) ranges from about 60% to about 100%. In some embodiments, the RNA integrity of the RNA (suitably mRNA) ranges from about 70% to about 100%. In some embodiments, the RNA integrity of the RNA (suitably mRNA) ranges from about 50%, about 60%, about 70%, about 80% or about 90%. RNA integrity is suitably determined using analytical HPLC (suitably analytical RP-HPLC).

[0535] In some embodiments, the RNA integrity of the RNA (suitably mRNA) of the composition is at least about 50%, suitably at least about 60%, more suitably at least about 70%, most suitably at least about 80% or about 90%. RNA integrity is suitably determined using analytical HPLC (more suitably analytical RP-HPLC).

[0536] After co-transcriptional capping as defined herein, and after purification as defined herein, the extent of capping of the RNA obtained can be determined using a capping assay as described in published PCT application WO2015101416 (in particular, as described in claims 27 to 46 of published PCT application WO2015101416). Alternatively, a capping assay described in PCT / EP2018 / 08667 can be used.

[0537] In some embodiments, an automated device for performing in vitro transcription of RNA can be used to produce and purify the mRNA of the present invention. Such a device can also be used to produce the composition or the vaccine (as further described below). In some embodiments, an apparatus as described in WO2020002598, in particular claims 1 to 59 and / or 68 to 76 (and Figure 1-Figure 1 8) The device described.

[0538] The methods described herein can be applied to methods of producing such immunogenic compositions or vaccines as described in further detail below.

[0539] In various embodiments, the mRNA used herein comprises (suitably in 5' to 3' direction) the following elements:

[0540] A) a 5'-cap structure, suitably as defined herein;

[0541] B) a 5'-terminal initiation element, suitably as defined herein;

[0542] C) optionally, a 5'-UTR, suitably as specified herein;

[0543] D) a ribosome binding site, suitably as defined herein;

[0544] E) at least one coding sequence, suitably as specified herein;

[0545] F) a 3'-UTR, suitably as specified herein;

[0546] G) optionally, a poly(A) sequence, suitably as defined herein;

[0547] H) optionally, a poly(C) sequence, suitably as defined herein;

[0548] I) optionally, a histone stem-loop structure, suitably as defined herein;

[0549] J) Optionally, a 3'-terminal sequence element, suitably as defined herein.

[0550] In some embodiments, the RNA (suitably mRNA) used herein does not comprise a replicase element (eg, a nucleic acid encoding a replicase).

[0551] In some embodiments, the RNA used herein (suitably the mRNA used herein, suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA, optionally each ) is not self-replicating.

[0552] In some embodiments, the RNA used herein (suitably the mRNA used herein, suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA, optionally each ) is self-replicating.

[0553] Chemical modification

[0554] In some embodiments, the RNA (suitably mRNA) used herein does not comprise chemically modified nucleotides.

[0555] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA does not contain chemically modified nucleotides.

[0556] In some embodiments, the RNA used herein (suitably mRNA) comprises a coding sequence consisting of only G, C, A and U nucleotides, and therefore comprises no modified nucleotides (except for the 5' terminal cap structure (cap0, cap1, cap2)).

[0557] In some embodiments, the RNA (suitably mRNA) used herein is a modified RNA (suitably mRNA), wherein the modification refers to a chemical modification, including backbone modification and sugar modification or base modification.

[0558] The modified RNA (suitably mRNA) may comprise one or more nucleotide analogs or modified nucleotides (nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications). As used herein, "nucleotide analogs" or "modified nucleotides" refer to nucleotides containing one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of a nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)) and / or containing one or more chemical modifications in or on the phosphate group of the backbone. Nucleotide analogs may contain further chemical modifications in or on the sugar portion of the nucleoside (e.g., ribose, modified ribose, hexameric sugar analogs or open-chain sugar analogs) or the phosphate group. The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art, see the following references: U.S. Patent Nos. 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.

[0559] A backbone modification as described herein is a modification in which the phosphate groups of the backbone of the nucleotides of the RNA (suitably mRNA) are chemically modified. A sugar modification as described herein is a chemical modification of the sugar of the nucleotides of the RNA (suitably mRNA). In addition, a base modification as described herein is a chemical modification of the base moiety of the nucleotides of the RNA (suitably mRNA). In this context, nucleotide analogs or modifications are suitably selected from nucleotide analogs applicable to transcription and / or translation.

[0560] In some embodiments, the RNA (suitably mRNA) used herein comprises at least one chemical modification.

[0561] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) contains at least one chemical modification.

[0562] Modified nucleobases (chemical modifications) that may be incorporated into modified nucleosides and nucleotides and present in the RNA (suitably mRNA) molecule include: 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-isopentenyladenosine); ms2i6A (2-methylthio-N6 isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio =Adenosine); g6A (N6-glycylaminoformyladenosine); t6A (N6-threonylaminoformyladenosine); ms2t6A (2-methylthio-N6-threonylaminoformyladenosine); m6t6A (N6-methyl-N6-threonylaminoformyladenosine); hn6A (N6-hydroxynorvalylaminoformyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2'-O-methylcytidine); s2 C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2-O-methylcytidine); k2C (lysine); 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 (whitingoside); o2yW (peroxywhitingoside); OHyW (hydroxy whitinoside); OHyW* (undermodified hydroxywhitinoside); imG (whitinoside); mimG (methylguanosine); Q (quercetin); oQ (epoxyquercetin); galQ (galactosylquercetin); manQ (mannosylquercetin); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaurine); 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-methoxycarbonylmethyl uridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyl uridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-aminomethyluridine) glycoside); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-seleno-uridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine); cmnm5U (5-carboxymethylaminomethyl uridine); cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyl uridine); 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,N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2'-O-dimethyluridine); m5D (5-methyldihydrouridine); f 5Cm (5-formyl-2'-O-methylcytidine); mlGm (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyladenosine) isomethyluridine); tm5s2U (S-taurinemethyl-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-(C; 1 -C 6 ) alkyl uracil, 5-methyl uracil, 5-(C 2 -C 6 ) alkenyluracil, 5-(C 2 -C 6 ) alkynyl uracil, 5-(hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C 1 -C 6 )alkylcytosine, 5-methylcytosine, 5-(C 2 -C6 ) alkenylcytosine, 5-(C 2 -C 6 ) alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C 2 -C 6 ) alkynylguanine, 7-deaza-8-substituted 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), m5C, m5U, m6A, s2U, W or 2'-O-methyl-U. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers.

[0563] In some embodiments, the nucleotide analogs / modifications that can be incorporated into the modified RNA (suitably mRNA) are selected from 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-methyl-inosine-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, xanthine riboside-5'-triphosphate. Particularly preferred are base-modified nucleotides selected from the group consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridine-4-ketoribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynyluridine, pseudouridine, 5-tauromethyluridine, 1-tauromethylpseudouridine, 5-tauromethyl-2-thiouridine, 1-tauromethyl-4-thiouridine, 5-methyluridine, 1-methylpseudouridine, 4-thiol-1-methylpseudouridine, 2-thiol-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thiol-1-methyl-1-deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thioldihydrouridine, 2-thioldihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine,and 4-methoxy-2-thiopseudouridine, 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopseudoisocytidine, 4-thio-1-methylpseudoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebulin, 5- azazebulin, 5-methylzebulin, 5-aza-2-thiozebulin, 2-thiozebulin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, and 4-methoxy-1-methylpseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza adenosine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylaminoformyladenosine, N6-threonylaminoformyladenosine, 2-methylthio-N6-threonylaminoformyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxyadenine, inosine, 1-methyl Inosine, wyosine, wyosine, 7-deazaguanosine, 7-deaza-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine,and N2,N2-dimethyl-6-thioguanosine, 5'-O-(1-thiophosphorothioate)-adenosine, 5'-O-(1-thiophosphorothioate)-cytidine, 5'-O-(1-thiophosphorothioate)-guanosine, 5'-O-(1-thiophosphorothioate)-uridine, 5'-O-(1-thiophosphorothioate)-pseudouridine, 6-azacytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyl uridine, 5-iodouridine, N1-methyl pseudouridine, 5,6-dimethoxyur ...uridine, 5'-O-( Hydrogen uridine, α-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, α-thioguanosine, 6-methylguanosine, 5-methylcytidine, 8-oxoguanosine, 7-deazaguanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudoisocytidine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azidoadenosine, 7-deazaadenosine. ,

[0564] In some embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methylpseudouridine, 2-thiol-5-azauridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxypseudouridine, 4-thiol-1-methylpseudouridine, 4-thiol-pseudouridine, 5-azauridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0565] Particularly suitable in this context are pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and 5-methoxyuridine, more preferably pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), still more preferably N1-methylpseudouridine (m1ψ).

[0566] In some embodiments, substantially all (eg substantially 100%) of the uracils in the coding sequence of the RNA (suitably mRNA) used herein have a chemical modification, suitably a chemical modification at the 5-position of the uracil.

[0567] In some embodiments, the chemical modification comprised by the RNA (suitably mRNA) used herein is a uridine modification, preferably wherein 100% of the uridine positions in the mRNA are modified.

[0568] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c5 ) and / or (c 6 )'s mRNA comprises a chemical modification that is a uridine modification, preferably wherein 100% of the uridine positions in the mRNA are modified.

[0569] It may be advantageous to incorporate modified nucleotides (e.g. pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and / or 5-methoxyuridine) into the coding sequence of the RNA (suitably mRNA) used herein, as unwanted innate immune responses may be modulated or reduced (if necessary) upon administration of the encoding mRNA or the vaccine.

[0570] In some embodiments, the coding sequence of the RNA (suitably mRNA) used in the present invention comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), suitably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides, optionally wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0571] In some embodiments, the RNA used herein (suitably mRNA) does not include a position substituted with N1-methylpseudouridine (m1ψ). In further embodiments, the RNA used herein (suitably mRNA) does not include a position substituted with pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and 5-methoxyuridine.

[0572] In some embodiments, the chemical modification is N1-methylpseudouridine and / or pseudouridine. In some embodiments, the chemical modification is N1-methylpseudouridine.

[0573] Carrier

[0574] A series of vector systems have been described that encapsulate or compound mRNA to facilitate mRNA delivery and subsequent expression of encoded antigens compared to mRNA that is not encapsulated or compounded. The present invention may utilize any suitable vector system. Specific vector systems worth noting are further described below.

[0575] In some embodiments, the RNA used herein (suitably mRNA) is complexed with, encapsulated with, partially encapsulated with, or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids) to form a lipid-based carrier, such as a liposome, a lipid nanoparticle (LNP), a cationic lipid complex (lipoplex) and / or a nanoliposome, suitably a lipid nanoparticle.

[0576] In some embodiments, (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) are formulated separately or together in lipid nanoparticles (LNPs).

[0577] In some embodiments, the RNA (suitably mRNA) used herein is formulated separately (in any formulation or complexing agent defined herein), suitably wherein the RNA (suitably mRNA) used herein is formulated in separate liposomes, lipid nanoparticles (LNPs), cationic lipid complexes and / or nanoliposomes.

[0578] In some embodiments, the RNA used herein (suitably mRNA, suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) were prepared separately.

[0579] In some embodiments, the RNA (suitably mRNA) used herein is co-formulated (in any formulation or complexing agent defined herein), wherein the RNA (suitably mRNA) used herein is formulated in separate liposomes, lipid nanoparticles (LNPs), cationic lipid complexes and / or nanoliposomes.

[0580] In some embodiments, the RNA used herein (suitably mRNA, suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) are co-formulated, i.e., prepared together.

[0581] LNP

[0582] The term "lipid nanoparticle", also referred to as "LNP", is not limited to any particular morphology, and includes any morphology generated when a cationic lipid and optionally one or more other lipids are combined (e.g., in an aqueous environment and / or in the presence of nucleic acids (e.g., RNA). For example, liposomes, lipid complexes, cationic lipid complexes, and the like are within the scope of lipid nanoparticles (LNPs).

[0583] Lipid nanoparticles (LNPs) are non-virion liposomal particles in which mRNA can be encapsulated. Incorporation of nucleic acids into LNPs is also referred to herein as "encapsulation," wherein the nucleic acid (e.g., RNA) is contained within the interior space of a liposome, lipid nanoparticle (LNP), cationic lipid complex, and / or nanoliposome.

[0584] LNP delivery systems and methods for their preparation are known in the art.

[0585] The particles may contain some external RNA (suitably mRNA) (eg on the surface of the particles), but ideally at least half of the RNA (suitably mRNA, and suitably at least 85%, especially at least 95%, such as all) is encapsulated.

[0586] LNP is suitably characterized as having the microvesicle of the internal water space isolated from the external medium by one or two double-layered films. The double-layer membrane of LNP is formed by amphipathic molecules usually, such as synthetic or natural lipids, which comprise spatially separated hydrophilic domains and hydrophobic domains. The double-layer membrane of liposome can also be formed (such as polymerosome, vesicle (niosome) etc.) by amphipathic polymers and surfactants. In the context of the present invention, LNP is generally used for transporting RNA (suitably mRNA) to target tissue.

[0587] Therefore, in some embodiments, the RNA used herein (suitably mRNA) is complexed with one or more lipids to form lipid nanoparticles (LNPs), liposomes, nanoliposomes, cationic lipid complexes, suitably LNPs. In some embodiments, LNPs are suitable for intramuscular and / or intradermal administration.

[0588] In some embodiments, at least about 80%, 85%, 90%, 95% of the lipid-based carrier, suitably LNP, has a spherical morphology, suitably comprising a solid core or a partially solid core.

[0589] LNP generally comprises cationic lipid and one or more excipients selected from neutral lipid, charged lipid, steroid and polymer conjugated lipid (for example PEGylated lipid).This RNA (suitably mRNA) can be encapsulated in the lipid part of this LNP or in the aqueous space encapsulated by some or all of the lipid parts of this LNP.This RNA (suitably mRNA) or its part also can be associated with this LNP and compound with it.LNP can comprise any lipid that can form the particle that these nucleic acids are attached to or encapsulate these one or more nucleic acids.In some embodiments, this LNP comprising nucleic acid (suitably RNA, more suitably mRNA) comprises one or more cationic lipid and one or more lipids with stabilization.The lipid with stabilization comprises neutral lipid and PEGylated lipid.

[0590] In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and a cationic lipid.

[0591] LNP can, for example, be formed by a mixture of: (i) a PEG-modified lipid, (ii) a non-cationic lipid, (iii) a sterol, (iv) an ionizable cationic lipid. Alternatively, LNP can, for example, be formed by a mixture of: (i) a PEG-modified lipid, (ii) a non-cationic lipid, (iii) a sterol, (iv) an ionizable cationic lipid.

[0592] In some embodiments, the non-cationic lipid is a neutral lipid.

[0593] In some embodiments, the cationic lipid is ionizable.

[0594] The in vivo characteristics and performance of LNPs can be modified by adding a hydrophilic polymer coating (e.g., polyethylene glycol (PEG)) to the LNP surface to impart steric stabilization. In addition, LNPs (or liposomes, nanoliposomes, cationic lipid complexes) can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to the LNP surface or to the ends of attached PEG chains (e.g., by PEGylated lipids or PEGylated cholesterol).

[0595] In one embodiment, RNA (suitably mRNA) is complexed with one or more lipids to form lipid nanoparticles, wherein the LNP (or liposome, nanoliposome, cationic lipid complex) comprises a polymer-conjugated lipid, suitably a PEGylated lipid / PEG lipid.

[0596] 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) and similar. The terms "PEGylated lipid" and "PEG-modified lipid" are used interchangeably herein.

[0597] Polymer-conjugated lipids (eg, PEG lipids) as defined herein may serve as aggregation-reducing lipids.

[0598] In certain embodiments, the LNP comprises a lipid with a stabilizing effect, which is a polyethylene glycol lipid (PEGylated lipid). Suitable polyethylene glycol lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol lipid is N-[(methoxy poly (ethylene glycol) 2000) aminoformyl]-1,2-dimyristoyloxypropane-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 PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); PEGylated phosphatidylethanolamine (PEG-PE); PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); PEGylated ceramide (PEG-cer); or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0599] In some embodiments, the PEG-modified lipid comprises PEG-DMG or PEG-cDMA.

[0600] In some embodiments, the PEGylated lipid is suitably derived from formula (IV) of published PCT patent application WO2018078053A1. Therefore, the PEGylated lipid of formula (IV) derived from published PCT patent application WO2018078053A1 and the respective disclosures related thereto are incorporated herein by reference.

[0601] In some embodiments, the PEG-modified lipid has Formula IV:

[0602]

[0603] Where R 8 and R 9 Each is independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;

[0604] And the average value of w is 30 to 60.

[0605] In some embodiments, the PEG-modified lipid R 8 and R 9 It is a saturated alkyl chain.

[0606] In some embodiments, the RNA (suitably mRNA) is complexed with one or more lipids to form LNPs, wherein the LNP comprises a polymer-conjugated lipid, suitably a PEGylated lipid, wherein the PEGylated lipid is suitably derived from formula (IVa) of published PCT patent application WO2018078053A1. Therefore, the PEGylated lipid of formula (IVa) derived from published PCT patent application WO2018078053A1 and the respective disclosures related thereto are incorporated herein by reference.

[0607] In some embodiments, the PEG lipid or PEGylated lipid has formula (IVa):

[0608]

[0609] wherein n has an average value ranging from 30 to 60, such as 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 is of formula (IVa), wherein n is an integer selected such that the average molecular weight of the PEG lipid is from about 2000 g / mol to about 3000 g / mol or from about 2300 g / mol to about 2700 g / mol, suitably about 2500 g / mol.

[0610] In some embodiments, the PEG-modified lipid has Formula IVa:

[0611]

[0612] wherein the average value of n ranges from 30 to 60, suitably wherein the average value of n is about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most suitably wherein the average value of n is 49 or 45; or

[0613] wherein n is an integer selected such that the average molecular weight of the PEG lipid is about 2500 g / mol.

[0614] The lipid of formula IVa as suitably used herein has the chemical designation 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also known as ALC-0159.

[0615] 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.

[0616] In some embodiments, the LNP comprises less than about 3, 2, or 1 mole percent PEG or PEG-modified lipid based on the total moles of lipid in the LNP.

[0617] In further embodiments, the LNP comprises about 0.1% to about 20% by mole of the PEG-modified lipid, such as 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% by mole (based on the total moles of 100% lipid in the LNP). In some embodiments, LNP comprises about 1.0% to about 2.0% of the PEG-modified lipid by mole, for example, 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%, most suitably 1.7% (based on the total moles of 100% lipid in the LNP). In various embodiments, the molar ratio of the cationic lipid to the PEGylated lipid ranges from about 100:1 to about 25:1.

[0618] In some embodiments, the LNP comprises about 0.5 to 10 mol%, optionally 0.5 to 5 mol%, or 0.5 to 3 mol% of PEG-modified lipid.

[0619] In some embodiments, the LNP comprises one or more additional lipids that stabilize the formation of the particle during formulation of the particle or during the manufacturing process (eg, a neutral lipid and / or one or more steroids or steroid analogs).

[0620] In some embodiments, the RNA (suitably mRNA) is complexed with one or more lipids to form lipid nanoparticles, wherein the LNP comprises one or more neutral lipids and / or one or more steroids or steroid analogs.

[0621] Suitable lipids with stabilizing effects include neutral lipids and anionic lipids. The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.

[0622] 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.

[0623] In some embodiments, the LNP (or liposome, nanoliposome, cationic lipid complex) comprises one or more neutral lipids, wherein the neutral lipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE). PE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (transDOPE), or a mixture thereof.

[0624] In some embodiments, the LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1.

[0625] In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Suitably, the molar ratio of the cationic lipid to DSPC may range from about 2:1 to about 8:1.

[0626] In some embodiments, the steroid is a sterol, suitably cholesterol.

[0627] In some embodiments, the steroid is cholesterol. Suitably, the molar ratio of the cationic lipid to cholesterol may range from about 2: 1 to about 1: 1. In some embodiments, the cholesterol may be PEGylated.

[0628] The sterol can account for about 10mol% to about 60mol% of the lipid particle, or about 25mol% to about 55mol%, or about 25mol% to about 40mol%. In one embodiment, the sterol accounts for about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or about 60mol% of the total lipid present in the lipid particle. In another embodiment, the LNP comprises about 5% to about 50% of the sterol by mole, for example, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% (based on the total moles of 100% lipid in the lipid nanoparticle) by mole.

[0629] The cationic lipid of LNP can be ionizable, i.e., it is protonated when the pH is reduced to a pK below the ionizable group in the lipid, but gradually becomes more neutral at higher pH values. At a pH value below the pK, the lipid can be associated with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid, which exhibits a positive charge when the pH is reduced.

[0630] Such cationic lipids (for use in liposomes, lipid nanoparticles (LNPs), cationic lipid complexes and / or nanoliposomes) include, but are not limited to, DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium chloride propane (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride), N-(1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium ... N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenoyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane 1,2-Dilinoleoyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleoyl-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride (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-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane) HGT4003, 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2 -bis((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxolane-5-amine, (6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (MC3), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxolane-5-amine), 1,1'-(2-(4-(2-( (2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)dodecan-2-ol (C12-200), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N,N-diundecyl-4,7,10,1 3-tetraazahexadecane-1,16-diamide), (6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether),. (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); (commercially available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE from GIBCO / BRL); and (commercially available cationic lipids, comprising an ethanol solution of distearylamido glycylcarboxyspermine (DOGS), from Promega Corp., Madison, Wis.) or any combination of any of the above. Other suitable cationic lipids for the compositions and methods of the present invention include those described in International Patent Publication WO2010053572 (and particularly CI 2-200 described in

[00225] ) and WO2012170930 (both of which are incorporated herein by reference), HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1).

[0631] In some embodiments, the cationic lipid of the liposomes, lipid nanoparticles (LNPs), cationic lipid complexes and / or nanoliposomes can be an amino lipid.

[0632] Representative amino lipids include, but are not limited to, 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-( N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); dilinoleoyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120).

[0633] In some embodiments, the cationic lipids of the liposomes, lipid nanoparticles (LNPs), cationic lipid complexes and / or nanoliposomes can be amino alcohol lipids (lipidoids).

[0634] Amino alcohol lipids can be prepared by the method described in U.S. Pat. No. 8,450,298 (incorporated herein by reference in its entirety). Suitable (ionizable) lipids may also be compounds as disclosed in Table 1, Table 2 and Table 3 of WO2017075531A1 and defined in Claims 1-24, which are incorporated herein by reference.

[0635] In another embodiment, suitable lipids may also be compounds disclosed in WO2015074085A1 (i.e., ATX-001 to ATX-032 or compounds as defined in claims 1-26), U.S. Patent Application Nos. 61 / 905,724 and 15 / 614,499, or U.S. Patent Nos. 9,593,077 and 9,567,296 (incorporated herein in their entirety by reference).

[0636] In other embodiments, suitable cationic lipids may also be compounds as disclosed in WO2017117530A1 (incorporated herein by reference in its entirety) (i.e. lipids 13, 14, 15, 16, 17, 18, 19, 20 or compounds as defined in the claims).

[0637] In some embodiments, ionizable lipids or cationic lipids may also be selected from lipids disclosed in WO2018078053A1 (i.e., lipids derived from formulas I, II, and III of WO2018078053A1, or lipids as specified in claims 1 to 12 of WO2018078053A1), and the disclosure of WO2018078053A1 is incorporated herein by reference as a whole. In this context, lipids disclosed in Table 7 of WO2018078053A1 (e.g., lipids derived from formulas I-1 to I-41) and lipids disclosed in Table 8 of WO2018078053A1 (e.g., lipids derived from formulas II-1 to II-36) can be used appropriately in the context of the present invention. Therefore, Formulas I-1 to I-41 and II-1 to II-36 of WO2018078053A1 and specific disclosures related thereto are incorporated herein by reference.

[0638] In some embodiments, the cationic lipid can be derived from Formula III of published PCT patent application WO2018078053A1. Therefore, Formula III of WO2018078053A1 and the specific disclosures related thereto are incorporated herein by reference.

[0639] In some embodiments, the RNA (suitably mRNA) is complexed with one or more lipids to form LNPs (or liposomes, nanoliposomes, cationic lipid complexes), wherein the cationic lipid of the LNP is selected from structures III-1 to III-36 of Table 9 of published PCT patent application WO2018078053A1. Therefore, Formula III-1 to Formula III-36 of WO2018078053A1 and the specific disclosures related thereto are incorporated herein by reference.

[0640] In some embodiments, the ionizable cationic lipid has Formula III:

[0641]

[0642] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0643] L 1 or L 2 are each independently -O(C=O)- or -(C=O)O-;

[0644] G 1 and G 2 Each independently is unsubstituted C 1 -C 12 Alkylene or C 1 -C 12 alkenylene;

[0645] G 3 C 1 -C 24 Alkylene, C 1 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene or C 3 -C 8 Cycloalkenylene;

[0646] R 1 and R 2 Each independently is a branched or straight chain C 6 -C 24 Alkyl or C 6 -C 24 alkenyl;

[0647] R 3 H, OR 5 、CN、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0648] R 4 C 1 -C 12 alkyl;

[0649] R 5 H or C 1 -C 6 alkyl.

[0650] In some embodiments, the ionizable cationic lipid has Formula III:

[0651]

[0652] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0653] L 1 or L 2 are each independently -O(C=O)- or -(C=O)O-;

[0654] G 1 and G 2 Each independently is unsubstituted C 1 -C 12 Alkylene;

[0655] G 3 C 1 -C 24 Alkylene;

[0656] R 1 and R 2 Each independently is a branched or straight chain C 6 -C 24 alkyl;

[0657] R 3 OR 5 ;and

[0658] R 5 For H.

[0659] In some embodiments, the ionizable cationic lipid has Formula III, and wherein R 1 , R 2 or R 1 and R 2 Both have one of the following structures:

[0660]

[0661] In some embodiments, R 2 Has the following structure:

[0662]

[0663] In some embodiments, the cationic lipid has the formula:

[0664]

[0665] In some embodiments, the ionizable cationic lipid has the formula:

[0666]

[0667]

[0668] In some embodiments, the ionizable cationic lipid has Formula III-3:

[0669]

[0670] The lipid of formula III-3 as suitably used herein has the chemical term ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also known as ALC-0315, CAS number 2036272-55-4.

[0671] In certain embodiments, the cationic lipid as defined herein, more suitably the cationic lipid compound III-3 ((4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate)), is present in the LNP in an amount of about 30 mol% to about 80 mol%, suitably about 30 mol% to about 60 mol%, more suitably about 40 mol% to about 55 mol%, more suitably about 47.4 mol% relative to the total lipid content of the LNP. If more than one cationic lipid is incorporated into the LNP, such percentages apply to the combined cationic lipids.

[0672] In some embodiments, the cationic lipid as defined herein is present in the LNP in an amount of about 20 mol % to about 60 mol %.

[0673] In some embodiments, the LNP comprises a cationic lipid having the following structure:

[0674]

[0675] In some embodiments, the cationic lipid is present in the LNP with an amount of about 30mol% to about 70mol%. In one embodiment, the cationic lipid is present in the LNP with an amount of about 40mol% to about 60mol%, such as being respectively about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60mol%. In some embodiments, the cationic lipid is present in the LNP with an amount of about 47mol% to about 48mol%, such as being respectively about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0mol%, wherein 47.4mol% is particularly suitable.

[0676] In some embodiments, the cationic lipid is present in a ratio of about 20mol% to about 70mol% or 75mol% or about 45mol% to about 65mol% or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or about 70mol% of the total lipid present in the LNP. In a further embodiment, the LNP comprises a cationic lipid based on mole about 25% to about 75%, for example, based on mole (based on the total mole number of 100% lipid in the lipid nanoparticle) 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%. In some embodiments, the ratio of the cationic lipid to nucleic acid (suitably RNA, more suitably mRNA) is about 3 to about 15, such as about 5 to about 13 or about 7 to about 11.

[0677] Other suitable (cationic or ionizable) lipids are disclosed in WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO2013063468, US20110256175, US20120128760, U S20120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017070620, W O2017099823, WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8 ,466,122 and 8,569,256 and U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, US20140039032 and WO2017112865.In this context, in particular, the invention relates to (cationic) lipids suitable for LNPs (or liposomes, nanoliposomes, cationic lipid complexes) in WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017070620, WO2017099823 , WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO201104391 3. WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO20100807 The disclosures of 24, 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 U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, and US20140039032, and WO2017112865 are incorporated herein by reference.

[0678] In other embodiments, the cationic lipid or ionizable lipid is

[0679]

[0680]

[0681] In some embodiments, amino lipids or cationic lipids as defined herein have at least one protonatable group or deprotonatable group, so that the lipid is positively charged at a pH equal to or lower than physiological pH (e.g., pH 7.4), and neutral at a second pH (suitably equal to or higher than physiological pH). Of course, it will be understood that the addition or removal of protons is an equilibrium process as the pH changes, and the mention of charged lipids or neutral lipids refers to the properties of the main species, and does not require that all lipids must exist in a charged form or neutral form. Lipids or zwitterionic lipids with more than one protonatable group or deprotonatable group are not excluded, which can be suitable in the context of the present invention. In some embodiments, the pKa range of the protonatable group of these protonatable lipids is from about 4 to about 11, for example, and pKa is from about 5 to about 7.

[0682] LNP (or liposome, nanoliposome, cationic lipid complex) can comprise two or more (different) cationic lipids as defined herein. Cationic lipids can be selected to contribute to different favorable properties. For example, cationic lipids with different properties (such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue or toxicity) can be used in the LNP (or liposome, nanoliposome, cationic lipid complex). In particular, these cationic lipids can be selected so that the properties of the mixed LNP are more ideal than the properties of the single LNP of the individual lipids.

[0683] The amount of permanent cationic lipid or lipidoid can be selected taking into account the amount of nucleic acid cargo. In one embodiment, these amounts are selected so that the N / P ratio of the (these) nanoparticles or the composition ranges from about 0.1 to about 20, or

[0684] (i) an amount enabling an N / P ratio in the range of about 1 to about 20, suitably about 2 to about 15, more suitably about 3 to about 10, even more suitably about 4 to about 9, most suitably about 6;

[0685] (ii) an amount enabling an N / P ratio in the range of about 5 to about 20, more suitably about 10 to about 18, even more suitably about 12 to about 16, and most suitably about 14;

[0686] (iii) the amount is such as to achieve a lipid:mRNA weight ratio in the range of 20 to 60, suitably about 3 to about 15, 5 to about 13, about 4 to about 8 or about 7 to about 11; or

[0687] (iv) For the lipid nanoparticles according to the present invention, in particular the lipid nanoparticles comprising the cationic lipid III-3, this amount enables to achieve an N / P ratio in the range of about 6.

[0688] 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 based on, for example, that 1 μg RNA typically contains about 3 nmol of phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The "N" value can be calculated based on the molecular weight of the cationic lipid or lipidoid and the relative content of permanent cationic groups and (if present) cationizable groups. If more than one cationic lipid is present, the N value should be calculated based on all cationic lipids contained in the lipid nanoparticles.

[0689] 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.

[0690] 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).

[0691] In some embodiments, the LNP comprises: (a) RNA used herein, suitably mRNA, (b) a cationic lipid, (c) an aggregation reducing agent (such as a polyethylene glycol (PEG) lipid or a PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally a sterol.

[0692] In some embodiments, these cationic lipids (as defined above), non-cationic lipids (as defined above), cholesterol (as defined above) and / or PEG-modified lipids (as defined above) can be combined in different relative molar ratios. For example, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid can be between about 30-60:20-35:20-30:1-15, respectively, or a ratio of 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 a ratio of 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.

[0693] In some embodiments, these LNPs (or liposomes, nanoliposomes, cationic lipid complexes) comprise ALC-0315, RNA used herein (suitably mRNA), a neutral lipid which is DSPC, a steroid which is cholesterol, and a PEGylated lipid which is ALC-0159.

[0694] In some embodiments, the LNP comprises about 0.5 to 15 mol % of a PEG-modified lipid, about 5 to 25 mol % of a non-cationic lipid, about 25 to 55 mol % of a sterol, and about 20 to 60 mol % of an ionizable cationic lipid.

[0695] 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: 0.5-15% PEG lipid.

[0696] In some embodiments, the RNA (suitably mRNA) is complexed with one or more lipids to form lipid nanoparticles, wherein the LNP comprises

[0697] I. at least one cationic lipid as defined herein, suitably a lipid of formula III-3 (ALC-0315);

[0698] II. at least one neutral lipid as defined herein, suitably 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC);

[0699] III. at least one steroid or steroid analogue as defined herein, suitably cholesterol; and

[0700] IV. At least one polymer conjugated lipid, suitably a PEG lipid as defined herein, such as PEG-DMG or PEG-cDMA, suitably a PEGylated lipid of or derived from formula (IVa-ALC-0159).

[0701] In some embodiments, the mRNA is complexed with one or more lipids to form a lipid nanoparticle (LNP), wherein the LNP comprises (i) to (iv) in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol: 0.5-15% polymer-conjugated lipid (suitably PEG lipid).

[0702] In some embodiments, the lipid nanoparticle (or liposome, nanoliposome, cationic lipid complex) comprises: a cationic lipid having formula (III-3) and / or a PEG lipid having formula (IVa), optionally a neutral lipid, suitably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and optionally a steroid, suitably cholesterol, wherein the molar ratio of the cationic lipid to DSPC is optionally in the range of about 2:1 to 8:1, wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range of about 2:1 to 1:1.

[0703] In one embodiment, the composition comprises the RNA (suitably mRNA), lipid nanoparticles (LNP) in a molar ratio of about 50:10:38.5:1.5, suitably 47.5:10:40.8:1.7 or more suitably 47.4:10:40.9:1.7 (i.e. the ratio (mol %) of a cationic lipid (suitably a lipid of formula III-3 (ALC-0315)), DSPC, cholesterol and a polymer-conjugated lipid (suitably a PEG lipid, suitably a PEG lipid of formula (IVa) wherein n=49, even more suitably a PEG lipid of formula (IVa) wherein n=45; ALC-0159); dissolved in ethanol).

[0704] WO2017 / 070620 provides general information about 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.

[0705] In various embodiments, the average diameter of the LNPs that suitably encapsulate the mRNA of the invention is 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. As used herein, the average diameter can be about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1500 nm, about 2000 nm, about 3000 nm, about 3500 nm, about 4000 nm, about 5000 nm, about 6000 nm, about 7000 nm, about 8000 nm, about 9000 nm, about 10000 nm, about 15000 nm, about 10000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm, about 15000 nm

[0706] In some embodiments, the LNP has a diameter of 50 to 200 nm.

[0707] Suitably, the polydispersity of the LNP is 0.4 or less, such as 0.3 or less. Typically, the PDI is determined by dynamic light scattering.

[0708] In some embodiments, the composition has a polydispersity index (PDI) value of less than about 0.4, suitably less than about 0.3, more suitably less than about 0.2, and most suitably less than about 0.1.

[0709] Vaccines and combination vaccines

[0710] The immunogenic compositions described herein are suitable for use as vaccines.

[0711] In a second aspect, the invention relates to a vaccine comprising the immunogenic composition as described herein.

[0712] The vaccine may be a live attenuated vaccine, an inactivated vaccine, a recombinant vaccine or a nucleic acid-based vaccine.

[0713] The vaccine is suitable for active immunization against disease caused by the influenza viruses contained in the vaccine, suitably influenza A and influenza B viruses.

[0714] In some embodiments, the vaccine is a multivalent vaccine.

[0715] In some embodiments, the vaccine is a trivalent (ie, comprises immunogenic components derived from 3 strains of influenza virus) or a quadrivalent influenza virus vaccine (ie, comprises immunogenic components derived from 4 strains of influenza virus).

[0716] In some embodiments, the vaccine is a trivalent influenza virus vaccine.

[0717] In some embodiments, the trivalent influenza virus vaccine comprises three HA antigens or RNA (suitably mRNA) encoding the same.

[0718] In some embodiments, the trivalent influenza virus vaccine comprises two HA antigens derived from strains of influenza A virus or nucleic acids encoding the same (suitably mRNA), and one HA antigen derived from a strain of influenza B virus or nucleic acid encoding the same (suitably mRNA).

[0719] In some embodiments, the trivalent influenza virus vaccine comprises three mRNAs encoding three HA antigens.

[0720] In some embodiments, the trivalent influenza virus vaccine comprises two mRNAs encoding two HA antigens derived from strains of influenza A virus and one mRNA encoding one HA antigen derived from a strain of influenza B virus.

[0721] In some embodiments, the trivalent influenza virus vaccine comprises 2 HA and 2 NA antigens derived from a strain of influenza A virus or nucleic acids encoding the same (suitably mRNA), and 1 HA and 1 NA antigen derived from a strain of influenza B virus or nucleic acids encoding the same (suitably mRNA).

[0722] In some embodiments, the trivalent influenza virus vaccine comprises 6 mRNAs encoding 3 HA and 3 NA antigens.

[0723] In some embodiments, the trivalent influenza virus vaccine comprises four mRNAs encoding two HA and two NA antigens derived from strains of influenza A virus, and two mRNAs encoding one HA and one NA antigen derived from a strain of influenza B virus.

[0724] In some embodiments, the trivalent influenza virus vaccine comprises (a), (b) and (c) as defined herein, wherein the ratio of (a):(b):(c) is between 1.5:1:1 and 5:1:1, suitably between 2:1:1 and 4:1:1, suitably between 2:1:1 and 3:1:1, suitably 2:1:1 or 3:1:1. In some embodiments, the vaccine is a quadrivalent influenza virus vaccine.

[0725] In some embodiments, the quadrivalent influenza virus vaccine comprises four HA antigens or nucleic acids (suitably mRNA) encoding the same.

[0726] In some embodiments, the quadrivalent influenza virus vaccine comprises two HA antigens derived from a strain of influenza A virus or nucleic acids encoding the same (suitably mRNA), and two HA antigens derived from a strain of influenza B virus or nucleic acids encoding the same (suitably mRNA).

[0727] In some embodiments, the quadrivalent influenza virus vaccine comprises four mRNAs encoding four HA antigens.

[0728] In some embodiments, the quadrivalent influenza virus vaccine comprises two mRNAs encoding two HA antigens derived from a strain of influenza A virus and two mRNAs encoding two HA antigens derived from a strain of influenza B virus.

[0729] In some embodiments, the quadrivalent influenza virus vaccine comprises (a), (b), (c) as defined herein. 1 ) and (c 2 ), where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0730] In some embodiments, the quadrivalent influenza virus vaccine comprises four HA antigens or nucleic acids encoding the same (suitably mRNA), and three NA antigens or nucleic acids encoding the same (suitably mRNA), such as (i.e. a seven-component quadrivalent influenza virus vaccine).

[0731] In some embodiments, the quadrivalent influenza virus vaccine comprises four mRNAs encoding four HA antigens and three mRNAs encoding three NA antigens.

[0732] In some embodiments, the quadrivalent influenza virus vaccine comprises (a), (b), (c) as defined herein. 1 )、(c 2 )、(c 3 )、(c 4 ) and (c 5 ), where (a):(b):(c 1 ):(c 2) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0733] In some embodiments, the quadrivalent influenza virus vaccine comprises four HA antigens or nucleic acids encoding the same (suitably mRNA), and four NA antigens or nucleic acids encoding the same (suitably mRNA) (ie, an eight-component quadrivalent influenza virus vaccine).

[0734] In some embodiments, the quadrivalent influenza virus vaccine comprises four mRNAs encoding four HA antigens and four mRNAs encoding four NA antigens.

[0735] In some embodiments, the quadrivalent influenza virus vaccine comprises (a), (b), (c) as defined herein. 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and (c 6 ), where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:3.

[0736] In some embodiments, the vaccine further comprises 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 another pathogen, suitably a virus, suitably a respiratory virus.

[0737] In some embodiments, the antigen is from other viruses 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 suitably, the antigen from other viruses is a spike protein or antigenic fragment thereof from a SARS-CoV-2 virus, or mRNA encoding a spike protein or antigenic fragment thereof from a SARS-CoV-2 virus. For example, the antigen can be a SARS-CoV-2 virus spike protein or antigenic fragment thereof selected from those provided in Table 1 of published PCT application WO2021156267A1 or Table 1 of published PCT application WO2022137133A1 (each of which is incorporated herein by reference).

[0738] Test kit or kit

[0739] In a third aspect, the invention relates to a kit or a set of parts comprising an antigen or a nucleic acid and / or an mRNA as defined herein (suitably (a), (b), (c), (d) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )), optionally containing a liquid carrier for dissolution, and optionally technical instructions providing information about the administration and dosage of the components.

[0740] The technical instructions for the kit may contain information about administration and dosage and patient populations. Such kits (suitably kits of parts) may be applied, for example, to any of the applications or uses mentioned herein, suitably to immunogenic compositions or vaccines for treating or preventing infection or disease caused by influenza viruses (suitably influenza A and / or influenza B viruses).

[0741] In some embodiments, the immunogenic composition or the vaccine is provided in separate parts of the kit, wherein the immunogenic composition or the vaccine is suitably freeze-dried or spray-dried or spray-freeze-dried.

[0742] The kit may further contain as a part a carrier (eg, a buffer solution) for dissolving the dried or freeze-dried nucleic acid composition or the vaccine.

[0743] In some embodiments, an antigen or nucleic acid and / or mRNA as defined herein (suitably (a), (b), (c), (d) 1 )、(c 2 )、(c3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) were prepared separately.

[0744] In some embodiments, an antigen or nucleic acid and / or mRNA as defined herein (suitably (a), (b), (c), (d) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) is provided as part of the kit.

[0745] In some embodiments, an antigen or nucleic acid and / or mRNA as defined herein (suitably (a), (b), (c), (d) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) are each provided as a separate part of the kit. Suitably, the kit or set 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 containing a nucleic acid and / or mRNA as defined herein (suitably (a), (b), (c), (d), (e), or (f). 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA).

[0746] In some embodiments, a kit or set of parts as defined herein comprises a multi-dose container and / or an administration device (eg, a syringe for intramuscular and / or intradermal injection) for administering the composition / the vaccine.

[0747] Preparation and administration

[0748] In some embodiments, an antigen or nucleic acid (suitably mRNA) as defined herein is co-formulated. Suitably, (a), (b), (c), (d) as defined herein are co-formulated. 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6)'s mRNA are co-formulated, that is, prepared together.

[0749] In some embodiments, the antigens or nucleic acids (suitably mRNA) as defined herein of the kit or set of parts are formulated separately. In some embodiments, (a), (b), (c), (d) as defined herein are 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA were prepared separately.

[0750] In some embodiments, an antigen or nucleic acid (suitably mRNA) as defined herein is co-filled. Suitably, (a), (b), (c), (d) as defined herein are co-filled. 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNAs are co-filled, i.e. filled together, optionally after separate formulation.

[0751] In some embodiments, the antigen or nucleic acid as defined herein (suitably mRNA) is formulated as a bedside mixed preparation. Suitably, the mRNA as defined herein (suitably (a), (b), (c), (d) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNA) are formulated as a bedside mixed preparation.

[0752] As used herein, a "point-of-care mixed formulation" must be understood as a formulation in which some (e.g. one or more) immunogenic components (e.g. mRNA) are (suitably each) formulated separately (e.g. in LNP) and then mixed to form the point-of-care mixed formulation.

[0753] In some embodiments, the bedside mixed formulation is obtained by a process comprising the following steps: (1) independently formulating (e.g., in LNP) each antigen or nucleic acid (suitably mRNA), and (2) mixing each (LNP) formulated antigen or nucleic acid (suitably mRNA).

[0754] In some embodiments, the bedside mixed formulation is obtained by a process comprising the following steps: (1) co-formulating (e.g., in LNP) the antigen derived from a strain of influenza A virus or a nucleic acid encoding it (suitably mRNA), (2) co-formulating the antigen derived from a strain of influenza B virus or a nucleic acid encoding it (suitably mRNA), and (3) mixing the co-formulated antigen (LNP) derived from a strain of influenza A virus or a nucleic acid encoding it (suitably mRNA) with the co-formulated antigen (LNP) derived from a strain of influenza B virus or a nucleic acid encoding it (suitably mRNA).

[0755] In some embodiments, the bedside mixed formulation is obtained by a method comprising the following steps: (1) co-formulating (e.g., in LNP) the antigen derived from a strain of influenza A virus or a nucleic acid encoding the same (suitably mRNA), (2) independently formulating each antigen derived from a strain of influenza B virus or a nucleic acid encoding the same (suitably mRNA), and (3) mixing the (LNP) co-formulated antigen derived from a strain of influenza A virus or a nucleic acid encoding the same (suitably mRNA) with each (LNP) formulated antigen derived from a strain of influenza B virus or a nucleic acid encoding the same (suitably mRNA).

[0756] The immunogenic composition may be administered by a variety of suitable routes, including parenteral (such as intramuscular, intradermal, intranasal or subcutaneous) administration. Suitably, the immunogenic composition, vaccine or kit or set of parts as described herein is administered intramuscularly and / or intradermally.

[0757] In some embodiments, intramuscular administration of an immunogenic composition as described herein results in expression of the encoded antigenic construct in a subject. Administration of an immunogenic composition as described herein results in translation of the mRNA and production of the encoded antigen in a subject.

[0758] The immunogenic compositions described herein may be provided in liquid form or in dried (eg, freeze-dried) form.

[0759] In some embodiments, the immunogenic composition is provided in liquid form.

[0760] In some embodiments, the immunogenic composition can be freeze-dried to improve the storage stability of the preparation and / or the RNA (suitably mRNA). In some embodiments, the immunogenic composition as described herein can be spray-dried to improve the storage stability of the preparation and / or the RNA (suitably mRNA). The lyoprotectant for freeze drying and / or spray drying can be selected from trehalose, sucrose, mannose, dextran and inulin.

[0761] Suitably, the immunogenic composition as described herein is freeze-dried (e.g., according to WO2016165831 or WO2011069586) to produce a temperature-stable dry RNA (suitably mRNA) (powder) composition as defined herein. The immunogenic composition can also be dried using spray drying or spray freeze drying (e.g., according to WO2016184575 or WO2016184576) to produce a temperature-stable composition (powder) as defined herein.

[0762] Thus, in some embodiments, the immunogenic composition is a dry composition.

[0763] The term "dried composition" as used herein has to be understood as a composition which has been freeze-dried, or spray-dried, or spray-freeze-dried as defined above to obtain a temperature-stable dry composition (powder), e.g. comprising RNA (suitably mRNA) complexed with LNPs (as defined above).

[0764] In some embodiments, the freeze-dried or spray-dried composition has a water content of less than about 10%.

[0765] In some embodiments, the freeze-dried or spray-dried composition has a water content of between about 0.5% and 5%.

[0766] In some embodiments, the freeze-dried or spray-dried composition is stable for at least 2 months, suitably at least 3 months, 4 months, 5 months, 6 months after storage at about 5°C.

[0767] The liquid used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like. The requirements for buffer and / or tonicity adjusting agent will depend on the contents of the container 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 / Na 2 PO 4 、Na / K 2 PO 4 or K / K 2 PO 4 .

[0768] Suitably, the dosage volume of the preparation used in the present invention is between 0.05ml and 1ml, such as between 0.1 and 0.6ml, in particular a dosage volume of 0.45 to 0.55ml, such as 0.5ml. The volume of the composition used may depend on the subject, the delivery route and the location, with smaller doses given by the intradermal route. A typical human dose for administration by a route such as intramuscular is about 200μl to 750μl, such as 400μl to 600μl, in particular about 500μl, such as 500μl.

[0769] The immunogenic compositions described herein can be provided in a variety of physical containers, such as vials or pre-filled syringes.

[0770] In some embodiments, the immunogenic composition is provided in a single dose. In other embodiments, the immunogenic composition, the vaccine, or the kit or set of parts is provided in a multiple dose form containing 2, 5 or 10 doses.

[0771] Typically, liquids will be transferred between containers, such as from vials to syringes, to provide an "overage" that ensures that the full volume required can be conveniently transferred. The level of overage required will depend on the circumstances, but excessive overage should be avoided to reduce waste, while insufficient overage may lead to practical difficulties. Overage may be in the order of 20 to 100 μl per dose, such as 30 μl or 50 μl.

[0772] Stabilizers may be present. Stabilizers may be of particular relevance when multi-dose containers are provided, since doses of the final formulation(s) may be administered to a subject over a period of time.

[0773] The formulation is suitably sterile.

[0774] Methods for establishing strong and lasting immunity generally include repeated immunizations, i.e., boosting the immune response by administering one or more further doses. Such further administrations may be performed with the same immunogenic composition (homologous boost) or with different immunogenic compositions (heterologous boost). The present invention may be applied as part of a homologous or heterologous prime / boost regimen, either as a priming immunization or a boosting immunization.

[0775] Thus, the administration of an immunogenic composition as described herein can be part of a multiple dose administration regimen. For example, an immunogenic composition as described herein can be provided as a priming dose in a multiple dose regimen (especially a two-dose or three-dose regimen, especially a two-dose regimen). An immunogenic composition as described herein can be provided as a booster dose in a multiple dose regimen (especially a two-dose or three-dose regimen, such as a two-dose regimen).

[0776] The priming dose and booster dose can be homologous or heterologous. Therefore, the immunogenic composition as described herein can be provided as a priming dose and (one or more) booster dose in a homologous multiple dose regimen (especially a two-dose regimen or a three-dose regimen, especially a two-dose regimen). Alternatively, the immunogenic composition as described herein can be provided as a priming dose or a booster dose in a heterologous multiple dose regimen (especially a two-dose regimen or a three-dose regimen, especially a two-dose regimen), and the (these) booster doses can be different (for example, immunogenic compositions as described herein; or alternative antigen presentation (with or without adjuvant), such as squalene emulsion adjuvant).

[0777] The time between doses may be from two weeks to six months, such as from three weeks to three months. Periodic longer term booster doses may also be provided, such as once every 2 to 10 years.

[0778] In some embodiments, the immunogenic composition further comprises at least one pharmaceutically acceptable carrier.

[0779] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein suitably includes a liquid or non-liquid base for the composition to be administered. If the composition is provided in liquid form, the carrier may be water, such as pyrogen-free water; isotonic saline or a buffered (aqueous) solution, such as a phosphate, citrate, or other buffered solution. Water may be used, or suitably a buffer, more suitably an aqueous buffer, containing a sodium salt, suitably at least 50 mM of sodium salt; a calcium salt, suitably at least 0.01 mM of calcium salt; and an optional potassium salt, suitably at least 3 mM of potassium salt. According to some embodiments, the sodium salt, calcium salt, and optional potassium salt may be present in the form of its halide (e.g., chloride, iodide, or bromide), in the form of its hydroxide, carbonate, bicarbonate, or sulfate, etc. Examples of sodium salts include NaCl, NaI, NaBr, NaBr, and NaBr. 2 CO 3 、NaHCO 3 、Na 2 SO 4 Examples of the optional potassium salt include KCl, KI, KBr, K 2 CO 3 , KHCO 3 , K 2 SO 4 , while examples of calcium salts include CaCl 2 ,CaI 2 , CaBr 2 、CaCO 3 、CaSO 4 , Ca(OH) 2 .

[0780] In addition, the organic anion of the above-mentioned cation can be in the buffer.Therefore, in some embodiments, the immunogenic composition can include a pharmaceutically acceptable carrier or excipient, use one or more pharmaceutically acceptable carriers or excipients to, for example, increase stability, increase cell transfection, allow lasting or delayed, increase the translation of the encoded antigenic peptide or protein in vivo, and / or change the release spectrum of the encoded antigenic peptide or protein in vivo.Except traditional excipients (such as any and all solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives), excipients can include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, cationic lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics and combinations thereof.In some embodiments, one or more compatible solids or liquid fillers or diluents or encapsulated compounds suitable for application to experimenter can also be used. The term "compatible" as used herein means that the components of the composition are capable of being mixed with the at least one nucleic acid of component A and / or component B and optionally the plurality of nucleic acids of the composition in a manner such that no interaction occurs which would significantly reduce the biological activity or pharmaceutical effectiveness of the composition under normal conditions of use (e.g., intramuscular or intradermal administration). A pharmaceutically acceptable carrier or excipient must be of sufficiently high purity and sufficiently low toxicity to make it suitable for administration to the subject to be treated. Compounds that can be used as pharmaceutically acceptable carriers or excipients can be 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, ethylcellulose, cellulose acetate; tragacanth powder; malt; gelatin; animal fats and oils; solid glidants, such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from the cocoa genus; polyols, such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

[0781] The 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 suitably a composition suitable for intramuscular administration to a subject.

[0782] Subjects contemplated for administration of these immunogenic compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese and / or turkeys.

[0783] In various embodiments, the immunogenic composition contains no more than a certain proportion of free RNA (suitably mRNA).

[0784] In this context, the term "free RNA (suitably mRNA)" or "non-complexed RNA (suitably mRNA)" or "non-encapsulated RNA (suitably mRNA)" includes RNA (suitably mRNA) that is not encapsulated in a lipid-based carrier as defined herein. Free RNA (suitably mRNA) may represent contamination or impurities during formulation of the composition (e.g. during encapsulation of the RNA (suitably mRNA) into the lipid-based carriers).

[0785] In some embodiments, the immunogenic composition comprises free RNA (suitably mRNA) in the range of about 30% to about 0%. In some embodiments, the composition comprises about 20% free RNA (suitably mRNA) (and about 80% encapsulated RNA (suitably mRNA)), about 15% free RNA (suitably mRNA) (and about 85% encapsulated RNA (suitably mRNA)), about 10% free RNA (suitably mRNA) (and about 90% encapsulated RNA (suitably mRNA)), or about 5% free RNA (suitably mRNA) (and about 95% encapsulated RNA (suitably mRNA)). In some embodiments, the composition comprises less than about 20% free RNA (suitably mRNA), suitably less than about 15% free RNA (suitably mRNA), more suitably less than about 10% free RNA (suitably mRNA), and most suitably less than about 5% free RNA (suitably mRNA).

[0786] The term "encapsulated RNA (suitably mRNA)" comprises RNA (suitably mRNA) molecules encapsulated in a lipid-based carrier as defined herein. In the context of the present invention, the ratio of encapsulated RNA (suitably mRNA) is typically determined using a RiboGreen assay.

[0787] Medical uses (first medical uses and second / further medical uses) and methods of treatment

[0788] In a fourth aspect, the invention relates to an immunogenic composition, vaccine or kit or set of parts as described herein for use as a medicament.

[0789] Also described herein is the use of an immunogenic composition, vaccine or kit or set of parts as described herein as a medicament.

[0790] In a fifth aspect, the invention relates to an immunogenic composition, vaccine or kit or set of parts as described herein for use in the treatment or prevention of influenza virus infection, suitably influenza A and / or influenza B.

[0791] The present invention also describes the use of the immunogenic composition, vaccine or kit or set of parts of the present invention in the treatment or prevention of influenza virus infection (suitably influenza A and / or influenza B).

[0792] In some embodiments, a single dose of the immunogenic composition is 0.1 to 1000 μg, particularly 1 to 500 μg, especially 2 to 500 μg, particularly 10 to 250 μg, suitably 25 to 150 μg of total mRNA.

[0793] 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 and each mRNA is 1 to 200 μg, suitably 1 to 60 μg, suitably 1 to 25 μg, suitably 2 to 25 μg, suitably 3 to 18 μg.

[0794] In some embodiments, a single dose of the composition is 2 to 500 μg, in particular 10 to 250 μg, such as 10 to 75 μg of total mRNA.

[0795] In some embodiments, a single dose of the immunogenic composition is 10 to 100 μg.

[0796] 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.

[0797] In some embodiments, for young adults (eg, 18 to 64 years old), a single dose of the composition is 1 to 10 μg of each mRNA.

[0798] In some embodiments, for young adults (eg, 18 to 64 years old), a single dose of the composition is 1, 2, 3, 6, or 9 μg of each mRNA.

[0799] In some embodiments, for young adults (eg, 18 to 64 years old), a single dose of the composition is 15 to 50 μg of total mRNA.

[0800] In some embodiments, for young adults (eg, 18 to 64 years old), a single dose of the composition is 16, 32, or 48 μg of total mRNA.

[0801] In some embodiments, for elderly adults (eg, 65 years and older), a single dose of the composition is 2 to 20 μg of each mRNA.

[0802] In some embodiments, for elderly adults (eg, 65 years and older), a single dose of the composition is 2, 3, 6, 9, or 18 μg of each mRNA.

[0803] In some embodiments, for elderly adults (eg, 65 years and older), a single dose of the composition is 30 to 100 μg of total mRNA.

[0804] In some embodiments, for elderly adults (eg, 65 years and older), a single dose of the composition is 32, 48, or 96 μg of total mRNA.

[0805] In some embodiments, the use is for intramuscular administration and / or intradermal administration, suitably intramuscular administration.

[0806] In some embodiments, an antigen or nucleic acid and / or mRNA as described herein (suitably (a), (b), (c), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) mRNA) was administered at different injection sites.

[0807] 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 of the antigen or nucleic acid and / or mRNA derived from a strain of influenza B virus.

[0808] In some embodiments, the antigens or nucleic acids and / or mRNA derived from a strain of influenza B virus are administered separately, suitably at different injection sites.

[0809] In some embodiments, an immune response is elicited, suitably an adaptive immune response, more suitably a protective adaptive immune response against influenza virus (suitably influenza A and / or influenza B).

[0810] In some embodiments, an immune response is elicited.

[0811] In some embodiments, an adaptive immune response is elicited.

[0812] In some embodiments, a protective adaptive immune response against influenza virus is elicited.

[0813] In some embodiments, a protective adaptive immune response against influenza A virus and / or influenza B virus is elicited.

[0814] In some embodiments, a protective adaptive immune response is elicited against one or more influenza A virus subtypes and / or influenza B virus lineages, suitably against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage, and influenza B Victoria lineage.

[0815] In some embodiments, the immune response elicited comprises neutralizing antibodies against influenza virus (suitably influenza A virus and / or influenza B virus, more suitably one or more influenza A virus subtypes and / or influenza B virus lineages, more suitably against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage, and influenza B Victoria lineage).

[0816] In some embodiments, the immune response elicited includes functional antibodies that are able to effectively neutralize the respective virus.

[0817] In some embodiments, the immune response elicited is a cross-reactive immune response, wherein these functional antibodies that are capable of effectively neutralizing the respective viruses further neutralize viruses belonging to the same and / or other influenza A subtypes and / or influenza B lineages.

[0818] In some embodiments, the cross-reactive immune response is homologous, heterologous, and / or heterosubtypic.

[0819] In the context of an immune response elicited, the term "homologous" will be recognized and understood by a person of ordinary skill in the art, and is, for example, an immune response elicited against the same strain (such as the same influenza A strain or the same influenza B strain). For example, the immunogenic composition may comprise an HA antigen (or a nucleic acid encoding the same, suitably RNA, suitably mRNA) derived from A / Michigan / 45 / 2015 (H1N1pdm9), which may elicit an immune response against the A / Michigan / 45 / 2015 (H1N1pdm9) strain.

[0820] In the context of an immune response elicited, the term "heterologous" will be recognized and understood by those of ordinary skill in the art, and is, for example, an immune response elicited against different strains within a subtype (for influenza A virus) or lineage (for influenza B virus), such as different influenza A strains within a subtype (such as an H1 subtype or an H3 subtype). For example, the immunogenic composition may comprise an HA antigen (or a nucleic acid encoding it, suitably RNA, suitably mRNA) derived from A / Michigan / 45 / 2015 (H1N1pdm9), which may elicit an immune response against an A / New Caledonia / 20 / 1999 (H1N1) strain.

[0821] In the context of an immune response elicited, the term "heterosubtypic" will be recognized and understood by those of ordinary skill in the art, and is, for example, an immune response elicited against one or more different subtypes (for influenza A viruses) or different strains within a lineage (for influenza B viruses). For example, the immunogenic composition may comprise an HA antigen (or a nucleic acid encoding the same, suitably RNA, suitably mRNA) derived from A / Michigan / 45 / 2015 (H1N1pdm9), which may elicit an immune response against Hong Kong / 4801 / 2014 (H3N2).

[0822] In a further embodiment, the immune response elicited comprises a broad functional cellular T cell response against the respective virus. In particular, the immune response elicited comprises a CD4+ T cell immune response and / or a CD8+ T cell immune response.

[0823] In a further embodiment, the immune response elicited comprises a well-balanced B cell and T cell response against the respective virus.

[0824] In some embodiments, the immune response elicited comprises an antigen-specific immune response.

[0825] In some embodiments, the immune response elicited partially or completely reduces the severity of one or more symptoms and / or the duration that the subject experiences one or more symptoms of influenza virus infection.

[0826] In some embodiments, the immune response elicited reduces the likelihood of developing a confirmed influenza virus infection following challenge.

[0827] In some specific embodiments, the immune response elicited slows the progression of influenza (suitably influenza A and / or influenza B).

[0828] In a sixth aspect, the present invention relates to a method of treating or preventing a disorder caused by influenza virus (suitably influenza A and / or influenza B), wherein the method comprises applying or administering to a subject in need thereof an immunogenic composition, vaccine or kit or set of parts as described herein.

[0829] Prevention (inhibition) or treatment of disease (particularly viral infection) involves inhibiting the complete occurrence of the disease or condition, for example, in a subject with a risk of disease (such as viral infection). "Treatment" refers to a therapeutic intervention to alleviate the signs or symptoms of a disease or pathological condition after it has begun to occur. The term "alleviation", with respect to a disease or pathological condition, refers to any observable beneficial effect of treatment. Inhibiting a disease may include preventing the disease or reducing the risk of the disease, such as preventing viral infection or reducing the risk of viral infection. The beneficial effect may be, for example, delaying the onset of clinical symptoms of the disease in susceptible subjects, reducing the severity of some or all clinical symptoms of the disease, slowing the progression of the disease, reducing viral load, improving the overall health or well-being of the subject, or demonstrating other parameters specific to the particular disease. "Preventive" treatment is a treatment applied to subjects who do not show signs of the disease or only show early signs for the purpose of reducing the risk of pathology.

[0830] In some embodiments, the composition, vaccine, or kit or set of parts is administered in a therapeutically effective amount.

[0831] In some embodiments, the disorder is an influenza virus (suitably influenza A virus and / or influenza B virus) infection.

[0832] In some embodiments, the subject in need thereof is a mammalian subject, suitably a human subject.

[0833] In a seventh aspect, the present invention relates to a method of eliciting an immune response, wherein the method comprises applying or administering to a subject in need thereof an immunogenic composition, vaccine or kit or set of parts as described herein.

[0834] In some embodiments, the immune response is an adaptive immune response, suitably a protective adaptive immune response against influenza virus (suitably against influenza A virus and / or influenza B virus).

[0835] In some embodiments, an immune response is elicited.

[0836] In some embodiments, an adaptive immune response is elicited.

[0837] In some embodiments, a protective adaptive immune response against influenza virus is elicited.

[0838] In some embodiments, a protective adaptive immune response against influenza A virus and / or influenza B virus is elicited.

[0839] In some embodiments, a protective adaptive immune response is elicited against one or more influenza A virus subtypes and / or influenza B virus lineages (suitably against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage, and influenza B Victoria lineage).

[0840] In some embodiments, the immune response elicited comprises neutralizing antibodies against influenza virus (suitably influenza A virus and / or influenza B virus, more suitably one or more influenza A virus subtypes and / or influenza B virus lineages, more suitably against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage and influenza B Victoria lineage).

[0841] In some embodiments, the immune response elicited includes functional antibodies that are able to effectively neutralize the respective virus.

[0842] In some embodiments, the immune response elicited is a cross-reactive immune response, wherein these functional antibodies that are capable of effectively neutralizing the respective viruses further neutralize viruses belonging to the same and / or other influenza A subtypes and / or influenza B lineages.

[0843] In some embodiments, the cross-reactive immune response is homologous, heterologous, and / or heterosubtypic.

[0844] In a further embodiment, the immune response elicited comprises a broad functional cellular T cell response against the respective virus. In particular, the immune response elicited comprises a CD4+ T cell immune response and / or a CD8+ T cell immune response.

[0845] In a further embodiment, the immune response elicited comprises a well-balanced B cell and T cell response against the respective virus.

[0846] In some embodiments, the immune response elicited comprises an antigen-specific immune response.

[0847] In some embodiments, the immune response elicited partially or completely reduces the severity of one or more symptoms and / or the length of time that the subject experiences one or more symptoms of influenza virus infection.

[0848] In some embodiments, the immune response elicited reduces the likelihood of developing a confirmed influenza virus infection following challenge.

[0849] In some specific embodiments, the immune response elicited slows the progression of influenza (suitably influenza A and / or influenza B).

[0850] In some embodiments, the subject in need thereof is a mammalian subject, suitably a human subject.

[0851] In some embodiments, a composition, vaccine, or kit or set of parts as described herein is administered in an amount effective to induce a T cell response against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage, and influenza B Victoria lineage.

[0852] In some embodiments, the composition, vaccine, or kit or set of parts as described is administered in an amount effective to induce a neutralizing antibody response against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage, and influenza B Victoria lineage.

[0853] In some embodiments, administration of the immunogenic composition, the vaccine, or the kit or set of parts to a subject elicits neutralizing antibodies without eliciting antibodies that enhance disease. In particular, administration of the immunogenic composition, the vaccine, or the kit or set of parts to a subject does not induce immunopathological effects, such as enhanced disease and / or antibody-dependent enhancement (ADE).

[0854] Further definition

[0855] For the sake of clarity and readability, the following definitions are provided. Any technical features mentioned for these definitions can be read in each and all embodiments of the present invention. Additional definitions and explanations can be specifically provided in the context of these embodiments.

[0856] Throughout the specification (including claims), where the context permits, the term "comprising" and variations thereof (such as "comprises") will be interpreted as including the stated element (e.g., integer) or elements (e.g., multiple integers) without necessarily excluding any other elements (e.g., integers). Thus, a composition "comprising" X may consist of X alone, or may include additional ingredients, such as X+Y.

[0857] The word " basically " does not exclude "completely", for example, a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0858] As used herein, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0859] Unless otherwise specified, include The step of mixing two or more components method There is no requirement for any particular order of mixing. Thus, the components can be mixed in any order. Where there are three components, then two components can be combined with each other, and then that combination can be combined with the third component, and so on.

[0860] the term" Immunogenic fragments "or" Immunogenic variants ” has to be understood as any fragment / variant of the corresponding influenza antigen that is able to elicit an immune response in a subject.

[0861] In the context of numbers, percentage This is to be understood as referring to the total amount of the respective item. In other cases, and unless the context dictates otherwise, percentages are to be understood as percentages by weight (wt.-%).

[0862] About: When the Determinants or values ​​do not need to be identical (i.e., 100% identical), the term "about" is used. Thus, "about" means that the Determinants or values ​​can differ by 1% to 20%, for example, 1% to 10%; in particular, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. It is known to those skilled in the art that, for example, certain parameters or Determinants can vary slightly based on how the parameters are determined. For example, if a Determinant or value is defined herein as, for example, "about 100 nucleotides" in length, the length can differ by 1% to 20%. Thus, the skilled artisan knows that in this specific example, the length can differ by 1 to 20 nucleotides. Thus, a length of "about 100 nucleotides" can cover sequences ranging from 80 to 120 nucleotides.

[0863] Adaptive immune response : The term "adaptive immune response" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to the antigen-specific response of the immune system (adaptive immune system). Antigen specificity allows the generation of responses specifically directed against specific pathogens or pathogen-infected cells. The ability to organize these specialized responses is usually maintained in the body by "memory cells" (B cells).

[0864] antigen: The term "antigen" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a substance that can be recognized by the immune system (e.g., by the adaptive immune system) and can trigger an antigen-specific immune response (e.g., by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response). Typically, an antigen can be or can include a peptide or protein presented by MHC to a T cell. Fragments, variants, and derivatives of a peptide or protein comprising at least one epitope are also understood to be antigens.

[0865] Antigenic peptide, polypeptide or protein : The term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by those of ordinary skill in the art, and is intended to refer, for example, to a peptide or protein derived from a (antigenic or immunogenic) protein that stimulates the body's adaptive immune system to provide an adaptive immune response. Therefore, an antigen / 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.

[0866] Cationic : Unless there is a clear different meaning in the specific context, the term "cationic" means that the respective structure carries a positive charge that is permanent or non-permanent but responsive to certain conditions (such as pH). Therefore, the term "cationic" covers both "permanent cationic" and "cationizable". The term "permanent cationic" 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.

[0867] Cationizable: The term "cationizable" as used herein means that a compound, or a group, or an atom is positively charged at a lower pH of its environment, and uncharged at a higher pH. Similarly, in a non-aqueous environment 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, in particular the pKa of the respective cationizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In a dilute aqueous environment, the so-called Henderson-Hasselbalch equation, well known to those skilled in the art, can be used to estimate the proportion of cationizable compounds, groups, or atoms that are positively charged. For example, in some embodiments, if the compound or moiety is cationizable, it is suitable that it is positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, such as a pH of equal to or below 9, equal to or below 8, equal to or below 7, such as at a physiological pH, such as about 7.3 to 7.4, i.e., under physiological conditions, particularly physiological salt conditions of cells in vivo. In other embodiments, it is suitable that the cationizable compound or moiety is predominantly neutral at physiological pH (e.g., about 7.0-7.4), but becomes positively charged at lower pH values. In some embodiments, the pKa of the cationizable compound or moiety ranges from about 5 to about 7.

[0868] Coding sequence / coding region : The term "coding sequence" or "coding region" and the corresponding abbreviation "cds" as used herein will be recognized and understood by those of ordinary skill in the art, and are intended to refer, for example, to a sequence of several nucleotide triplets that can be translated into a peptide or protein. In the context of the present invention, a coding sequence can be an RNA sequence consisting of a number of nucleotides that can be divided by three, which begins with a start codon, and, for example, ends with a stop codon.

[0869] From: The term "derived from" as used in the context of nucleic acids throughout this specification, i.e. for a nucleic acid "derived from" (another) nucleic acid, means that the 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 derived from it. It is known to those skilled in the art that the sequence identity is usually calculated for nucleic acids of the same type (i.e. for DNA sequences or for RNA sequences). Therefore, it is understood that if DNA is "derived from" RNA, or if RNA is "derived from" DNA, then in a first step, the RNA sequence is converted into a corresponding DNA sequence (particularly by replacing uracil (U) with thymine (T) in the entire sequence), or vice versa, the DNA sequence is converted into a corresponding RNA sequence (particularly by replacing T with U in the entire sequence). " derived from " refers to a nucleic acid that is derived from a nucleic acid and is modified compared to the nucleic acid derived from it, for example, so as to even further increase RNA stability and / or extend and / or increase protein production. In the context of amino acid sequences (such as antigenic peptides or proteins), the term "derived from" refers to an amino acid sequence derived from (another) amino acid sequence and has a common amino acid sequence derived from it of, 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.

[0870] Epitope: The term "epitope" (also referred to as "antigenic determinant" in the art) as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to T cell epitopes and B cell epitopes. T cell epitopes are parts of these antigenic peptides or proteins, and may comprise a fragment preferably having a length of about 6 to about 20 or even more amino acids, such as a fragment processed and presented by an MHC class I molecule, preferably a length of about 8 to about 10 amino acids, such as 8, 9 or 10 (or even 11 or 12 amino acids), or a fragment processed and presented by an MHC class II molecule, preferably a length of about 13 to about 20 or even more amino acids. These fragments are usually recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule, i.e., these fragments are usually not recognized in their native form. B cell epitopes are usually fragments located on the outer surface of (native) proteins or peptide antigens, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which can be recognized by antibodies, i.e., recognized in their native form. Furthermore, such epitopes of proteins or peptides may be selected from any variants of such proteins or peptides mentioned herein. In this context, epitopes may be conformational epitopes or discontinuous epitopes consisting of segments of a protein or peptide as defined herein, which are discontinuous in the amino acid sequence of the protein or peptide as defined herein, but are brought together in the three-dimensional structure; or continuous epitopes or linear epitopes consisting of a single polypeptide chain.

[0871] Snippet: The term "fragment" as used throughout this specification in the context of a nucleic acid sequence (e.g., RNA or DNA) or an amino acid sequence can generally be a shorter portion of the full-length sequence of, for example, a nucleic acid sequence or an amino acid sequence. Thus, a fragment generally consists of a sequence identical to a corresponding extended sequence within the full-length sequence. In the context of the present invention, a particular fragment of a sequence consists of a continuous extended sequence of an entity, such as nucleotides or amino acids corresponding to a continuous extended sequence of an entity in a molecule from which the fragment is derived, which represents at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e., full-length) molecule (e.g., a viral protein) from which the fragment is derived. The term "fragment" as used throughout this specification in the context of a protein or peptide can, generally, include a sequence of a protein or peptide as defined herein, which, 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 this specification in the context of RNA sequences may, in general, include RNA sequences that are truncated at the 5'-end and / or 3'-end compared to a reference RNA sequence. Thus, this truncation may occur at the amino acid level, or correspondingly at the nucleic acid level. Thus, sequence identity relative to such a fragment as defined herein may, for example, refer to the entire protein or peptide as defined herein, or to the entire (coding) nucleic acid molecule of such a protein or peptide. A fragment of a protein or peptide may contain at least one epitope of these proteins or peptides.

[0872] Heterogeneous : The term "heterologous" or "heterologous sequence" as used throughout this specification in the context of nucleic acid sequences or amino acid sequences refers to a sequence (e.g., RNA, DNA, amino acid) that must be understood as being derived from another gene, another allele, or, for example, another species or virus. If two sequences are not derived from the same gene or from the same allele, they are generally understood to be "heterologous". That is, although heterologous sequences may be derivable from the same organism or virus, in nature, they do not occur in the same nucleic acid or protein.

[0873] Humoral immune response : The term "humoral immunity" or "humoral immune response" will be recognized and understood by those of ordinary skill in the art, and is intended to refer, for example, to B cell-mediated antibody production, and optionally to the auxiliary processes that accompany antibody production. Humoral immune responses can generally be characterized by: 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, which include pathogen and toxin neutralization, phagocytosis and pathogen elimination promoted by classical complement activation and opsonins.

[0874] (Sequence) identity: The term "identity" as used in the context of nucleic acid sequences or amino acid sequences throughout this specification will be recognized and understood by those of ordinary skill in the art, and is intended to refer to, for example, the percentage of identity of two sequences. In order to determine the percentage of identity of two sequences (for example, nucleic acid sequences or amino acid (aa) sequences as defined herein, for example, aa sequences encoded by nucleic acid sequences as defined herein or these aa sequences themselves), these sequences can be compared (aligned) so as to be subsequently compared with each other. Therefore, the position of, for example, the first sequence can be compared with the corresponding position of the second sequence. If the position in the first sequence is occupied by the same residue as the position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences are different at this position. If, compared with the first sequence, insertion occurs in the second sequence, then a gap can be inserted into the first sequence to allow further comparison. If, compared with the first sequence, deletion occurs in the second sequence, then a gap can be inserted into the second sequence to allow further comparison. Thus, the percentage of identity of two sequences is a function of the number of identical positions divided by the total number of positions (including only the position occupied in one sequence). The percentage ratio of two sequence identities can be determined using an algorithm (such as the algorithm integrated in the BLAST program).Sequence identity can be determined by using the EMBOSS Water sequence alignment tool on the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_water / (parameter is gap opening (gap open)=12, gap extension (gap extend)=1, and for protein sequences, it is matrix (matrix)=BLOSUM62, or for DNA / RNA sequences, it is matrix=fullDNA), or by using the EMBOSS Needle sequence alignment tool on the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / (default parameters are such as gap opening=10, gap extension=0.5, end gap penalty (end gap penalty)=false, end gap opening=10, and end gap extension=0.5, and for protein sequences, it is matrix=BLOSUM62, or for DNA / RNA sequences, it is matrix=fullDNA) to determine. Unless otherwise specified, when this application refers to sequence identity to a particular reference sequence, it is intended that the identity be calculated over the entire length of this reference sequence.

[0875] Immunogen, Immunogen : The term "immunogen" or "immunogenicity" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a compound capable of stimulating / inducing an (adaptive) immune response. An immunogen may be a peptide, polypeptide or protein.

[0876] Immune response : The term "immune response" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to the specific response of the adaptive immune system to a particular antigen (the so-called specific or adaptive immune response) or the non-specific response of the innate immune system (the so-called non-specific or innate immune response), or a combination thereof.

[0877] 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 of ordinary skill in the art, and is intended, for example, to refer to a system that generally comprises cells and mechanisms that protect a host from infection by other organisms in a non-specific manner. This means that the cells of the innate system can recognize and respond to pathogens in a universal manner, but unlike the adaptive immune system, it does not confer durable or protective immunity to the host. The innate immune system can be activated by ligands of pattern recognition receptors (e.g., Toll-like receptors, NOD-like receptors, or RIG-I-like receptors, etc.).

[0878] Lipid compounds : Lipidoid compounds (also lipidoids for short) are lipid-like compounds, ie amphiphilic compounds with lipid-like physical properties. In the context of the present invention, the term "lipid" is considered to encompass lipidoid compounds.

[0879] Nucleic acid, nucleic acid molecule : The term "nucleic acid" or "nucleic acid molecule" as used herein will be recognized and understood by those of ordinary skill in the art. The term "nucleic acid" or "nucleic acid molecule" refers in particular to a DNA (molecule) or an RNA (molecule). The term is used synonymously 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 phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid" or "nucleic acid molecule" also encompasses modified nucleic acids (molecules), for example, base-modified, sugar-modified or backbone-modified DNA or RNA (molecules) as defined herein.

[0880] Nucleic acid sequence, DNA sequence, RNA sequence : The terms "nucleic acid sequence", "DNA sequence", "RNA sequence" will be recognized and understood by those of ordinary skill in the art, and for example refer to a specific and individual order of a succession of nucleotides thereof.

[0881] Permanent cationic: The term "permanent cationic" as used herein will be recognized and understood by those of ordinary skill in the art, and means, for example, that the respective compound, or group, or atom is positively charged under any pH value or hydrogen ion activity of its environment. Typically, this positive charge is caused by the presence of a quaternary nitrogen atom. When a compound carries a plurality of such positive charges, it can be referred to as a permanent polycationic.

[0882] Stabilized RNA : The term "stabilized RNA" refers to a modified RNA such that it is more stable to breakdown or degradation (e.g., by environmental factors or enzymatic digestion, such as exonuclease or endonuclease degradation) than an RNA without such modification. Preferably, in the context of the present invention, the stabilized RNA is stabilized in a cell (e.g., a prokaryotic or eukaryotic cell), preferably in a mammalian cell (e.g., a human cell). The stabilizing effect can also be exerted outside the cell, e.g., in a buffer solution or the like, e.g., for storing a composition comprising the stabilized RNA.

[0883] T cell response : The term "cellular immunity" or "cellular immune response" or "cellular T cell response" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended to refer, for example, to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. More generally, cellular immunity is not based on antibodies, but on the activation of cells of the immune system. Typically, cellular immune responses can be characterized by, for example, activating antigen-specific cytotoxic T lymphocytes that can induce apoptosis in cells (e.g., specific immune cells, such as dendritic cells or other cells), displaying epitopes of foreign antigens on their surfaces.

[0884] RNA : The term "RNA" is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer composed of nucleotide monomers. These nucleotides are typically adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or their analogs, which are interconnected by 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 portion of the second adjacent monomer. The specific order of these monomers, i.e., the order in which the bases are connected to the sugar / phosphate backbone, is called an RNA sequence. In general, 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, the RNA can be obtained by RNA in vitro transcription. Alternatively, RNA can be obtained by chemical synthesis.

[0885] RNA in vitro transcription: The term "RNA in vitro transcription" or "in vitro transcription" refers to the process of synthesizing RNA in an in vitro cell-free system. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template (which is typically a linear DNA template, such as a linearized plasmid DNA or PCR product). The promoter used to control RNA in vitro transcription can be any promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6 or Syn5 RNA polymerases. In one embodiment of the invention, the DNA template is linearized with a suitable restriction endonuclease and then subjected to RNA in vitro transcription. Reagents commonly used for RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) having a promoter sequence with high binding affinity for its respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6 or Syn5); ribonucleotide triphosphates (NTPs) of the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analog as defined herein; optionally, a modified nucleotide 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 for inactivating any potential contaminating ribonucleases; optionally, pyrophosphatase; MgCl 2 ; A buffer (TRIS or HEPES) maintaining an appropriate pH value, which may also contain an antioxidant (eg DTT) and / or a polyamine, such as spermidine.

[0886] Variant (of a sequence) : The term "variant" as used in the context of a nucleotide sequence throughout this specification will be recognized and understood by those of ordinary skill in the art, and is intended to refer, for example, to a variant of a nucleotide sequence derived from another nucleotide sequence. For example, a variant of a nucleotide sequence can show one or more nucleotide deletions, insertions, additions and / or substitutions compared to the nucleotide sequence from which the variant is derived. A variant of a nucleotide sequence can be at least 50%, 60%, 70%, 80%, 90% or 95% identical to the nucleotide sequence from which the variant is derived. In the sense that the variant retains at least 50%, 60%, 70%, 80%, 90% or 95% or more of the function of the sequence from which it is derived, the variant is a functional variant. A "variant" of a nucleotide sequence can have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity to an extended sequence of at least 10, 20, 30, 50, 75 or 100 nucleotides of such a nucleotide sequence.

[0887] As used throughout this specification, the term "variant" in the context of a protein or peptide is intended to refer to a protein or peptide variant having an amino acid sequence that differs from the original sequence by one or more mutations / substitutions (such as one or more substitutions, insertions and / or deletions). Suitably, these fragments and / or variants have the same or comparable specific antigenic properties (immunogenic variants, antigenic variants). Insertions and substitutions are possible, in particular, at sequence positions that do not result in modifications of the three-dimensional structure or that do not affect the binding region. Modifications to the three-dimensional structure by one or more 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 with an extended sequence of at least 10, 20, 30, 50, 75 or 100 amino acids of such a protein or peptide. Alternatively, a "variant" of a protein or polypeptide may have 1 to 20, e.g. 1 to 10, single amino acid mutations, e.g. 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 a protein or peptide. By mutation we mean or include substitutions, insertions or deletions. In one embodiment, a variant of a protein comprises a functional variant of the protein, meaning that, in the context of the present invention, the variant exerts substantially the same or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity of the protein from which it is derived.

[0888] Multivalent vaccines / combinations : The multivalent vaccines or combinations of the invention provide more than one valency (eg, antigen) derived from more than one virus (eg, at least one influenza virus as defined herein and at least one other influenza virus as defined herein).

[0889] Example

[0890] In the following, specific examples illustrating various embodiments and aspects of the present invention are presented. However, the scope of the present invention should not be limited to the specific embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and practice the present invention. However, the scope of the present invention is not limited to the exemplary embodiments, which are only intended to illustrate a single aspect of the present invention, and functionally equivalent methods are also within the scope of the present invention. In fact, various modifications of the present invention other than those described herein will become apparent to those skilled in the art based on the foregoing description, the accompanying drawings and the following examples. All such modifications fall within the scope of the appended claims.

[0891] Example 1: Phase 1 Quadrivalent Influenza Vaccination Trial - Unmodified mRNA

[0892] The Phase 1 trial was designed to evaluate the safety, reactogenicity, and immunogenicity of CVSQIV when administered as a single dose at different dose levels using an adaptive dose-finding design.

[0893] 1. Trial objectives and endpoints

[0894] Main Objectives

[0895] To evaluate the safety and reactogenicity profile of CVSQIV at different dose levels.

[0896] Secondary Objectives

[0897] The humoral immune response to different dose levels of CVSQIV was evaluated in terms of hemagglutination inhibition (HAI) antibody titers.

[0898] Exploratory goals

[0899] The humoral immune response to different dose levels of CVSQIV was evaluated in terms of microneutralizing (MN) and NA inhibitory (NI) antibody titers.

[0900] To evaluate the innate immune response of all sentinel subjects to different dose levels of CVSQIV.

[0901] • To evaluate cross-reactivity to influenza antigens not included in the vaccine.

[0902] end

[0903] main

[0904] The frequency of Grade 3 adverse reactions (ARs) and any serious adverse reactions (SARs) by dose level within at least 20 hours after administration of the investigational vaccine, which is used to decide on subsequent vaccination of additional sentinel subjects at the same dose level.

[0905] The frequency of grade 3 ARs and any SARs by dose level within at least 60 hours after investigational vaccine administration was used to decide on dose escalation and continued enrollment at the same dose level.

[0906] The frequency, intensity, and duration of actively solicited local AR by dose level on the day of vaccination and for the following 7 days to characterize the safety and reactogenicity profile.

[0907] The frequency, intensity, duration, and relationship to the investigational vaccination of actively solicited 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.

[0908] The incidence, intensity, and relationship to the investigational vaccination of unsolicited AEs by dose level on the day of vaccination and up to 28 days thereafter to characterize the safety and reactogenicity profile.

[0909] The incidence of serious adverse events (SAEs) and adverse events of special interest (AESIs) throughout the trial and their relationship to the investigational vaccination were used to characterize the safety and reactogenicity profile.

[0910] secondary

[0911] Anti-HA antibody titers measured by HAI assay on days 22 and 183

[0912] · Geometric mean titer (GMT) of antigen-specific anti-HA antibody titers.

[0913] · Proportion of subjects with antigen-specific seroconversion*.

[0914] *Seroconversion of HA antigen as measured by the HAI assay was defined as a post-vaccination titer ≥1:40 for subjects with a baseline titer ≤1:10 and a post-vaccination titer increase of at least 4-fold from baseline for subjects with a baseline titer ≥1:10.

[0915] Proportion of subjects with a 2-fold increase in anti-HA antibody titer relative to baseline following antigen-specific vaccination.

[0916] Proportion of subjects with a 4-fold increase in anti-HA antibody titer relative to baseline following antigen-specific vaccination.

[0917] Proportion of subjects with anti-HA antibody titers ≥1:40 and ≥1:80 after antigen-specific vaccination.

[0918] Exploratory

[0919] Anti-HA antibody titers measured by microneutralization assay on days 22 and 183

[0920] GMT of antigen-specific anti-HA antibody titer.

[0921] Proportion of subjects with a 2-fold increase in anti-HA antibody titer relative to baseline following antigen-specific vaccination.

[0922] Proportion of subjects with a 4-fold increase in anti-HA antibody titer relative to baseline following antigen-specific vaccination.

[0923] Anti-neuraminidase (NA) antibody titers measured by enzyme-linked lectin assay (ELLA) on days 22 and 183

[0924] GMT of antigen-specific anti-NA titers.

[0925] Proportion of subjects with a 2-fold increase in anti-NA titer relative to baseline following antigen-specific vaccination.

[0926] Proportion of subjects with a 4-fold increase in anti-NA titer relative to baseline following antigen-specific vaccination.

[0927] Proportion of subjects with anti-NA titers ≥1:40 and ≥1:80 after vaccination.

[0928] Cross-reactivity with antigens not included in the vaccine

[0929] · Anti-NA antibody titers against B-Phuket.

[0930] Innate immune response (only in sentinel subjects)

[0931] • Serum cytokine concentrations on days 2 and 22, including but not limited to IFN-α, IFN-γ, IL-6, chemokine ligand (CCL) 2, and IFN-γ-induced protein 10 (IP-10).

[0932] 2. Experimental Design

[0933] For the Phase 1 trial, subjects were enrolled in a staggered manner at 5 dose levels (3, 6, 12, 20, and 28 μg dose levels). All subjects received a single dose of CVSQIV on Day 1. Subjects were enrolled in 2 age groups, including a young adult group aged 18-55 years and an elderly adult group aged ≥65 years. Each dose level was tested in 48 subjects (24 in each age group), as follows:

[0934] 12 sentinel subjects (6 in each age group), and

[0935] · 36 additional subjects (18 in each age group) after no safety issues were identified in the sentinel subjects.

[0936] Three protocol-scheduled visits were conducted on Day 1 (the day of vaccination), Day 22 (21 days after vaccination), and Day 183 (6 months after vaccination). An additional visit was conducted on Day 2 (1 day after vaccination) for sentinel subjects to collect safety, reactogenicity, and immunogenicity data. At each protocol-scheduled visit, blood samples were taken for safety and / or immunogenicity testing. In addition, 4 protocol-scheduled telephone contacts were made for all subjects (on Days 3, 8, 29, and 92) to collect safety data, and an additional telephone contact was made on Day 2 for non-sentinel subjects.

[0937] To ensure the safety of the subjects participating in the trial, the enrollment of each dose level began with sentinel subjects (i.e., safety data after vaccination was collected and evaluated from a limited number of subjects, and then a large number of subjects were exposed to the same dose level). Within each dose level, safety data was collected and evaluated up to a minimum of 20 hours after vaccination from the first 4 sentinel subjects (2 in each age group), and then vaccination continued for an additional 8 sentinel subjects (4 in each age group). Subsequently, data was collected and evaluated for a minimum of 60 hours from all 12 sentinel subjects at each dose level, and then a large number of subjects were exposed to the same dose level.

[0938] 3. Experimental population

[0939] Inclusion criteria

[0940] Subjects were included in this trial only if they met all of the following criteria:

[0941] 1. Healthy male or female subjects aged between 18 and 55 years (inclusive) at the time of inclusion (young adult group) or aged ≥ 65 years (elderly adult group) at the time of inclusion.

[0942] Healthy subjects were defined as individuals who were in good general health as assessed by the investigator. Chronic health conditions were acceptable if they were considered stable and well controlled with treatment in the investigator's judgment.

[0943] 2. Obtain signed informed consent prior to any trial procedures.

[0944] 3. Expected to comply with protocol procedures and available for clinical follow-up through last scheduled contact.

[0945] 4. According to the investigator's assessment, no clinically significant findings were found during the physical examination.

[0946] 5. Body mass index (BMI) ≥ 18.0 and ≤ 32.0 kg / m 2 .

[0947] 6. Females: At inclusion, for women presumed to be of childbearing potential on the day of inclusion, a negative human chorionic gonadotropin (hCG) pregnancy test (serum). On Day 1 (before vaccination): a negative urine pregnancy test (hCG) (required only if a serum pregnancy test was performed more than 3 days ago).

[0948] Note: Women who are postmenopausal (defined as amenorrhea for ≥ 12 consecutive months without an alternative medical reason prior to enrollment) or permanently sterilized will be considered to be of no reproductive potential.

[0949] 7. Females of childbearing potential must use a highly effective birth control method from 1 month before to 3 months after administration of the investigational vaccine.

[0950] Exclusion criteria

[0951] If a subject met any of the exclusion criteria, they were not included in the trial.

[0952] 1. Any investigational or unregistered product (vaccine or drug) other than the investigational vaccine has been used within 28 days prior to administration of the investigational vaccine, or is planned to be used during the trial.

[0953] 2. Received any influenza vaccine within 90 days of inclusion.

[0954] 3. Received any mRNA vaccine within 2 months of inclusion.

[0955] 4. Receipt of any other vaccine within 28 days prior to enrollment, or planned to receive any vaccine within 28 days of trial vaccine administration.

[0956] 5. Any treatment with immunosuppressants or other immunomodulatory drugs (including, but not limited to, corticosteroids, biologics, and methotrexate) for a total of >14 days within 6 months prior to investigational vaccine administration, or planned use during the trial, except for inhaled or topically applied steroids. For use of corticosteroids, this means prednisone or equivalent, 0.5 mg / kg / day, for 14 days or longer.

[0957] 6. Any medically diagnosed or suspected immunosuppression or immunodeficiency condition based on history and physical examination, including known human immunodeficiency virus infection.

[0958] 7. Chronic hepatitis B virus infection and chronic hepatitis C virus infection.

[0959] 8. History of AEs of suspected immune-mediated etiology (pIMD).

[0960] 9. History of angioedema.

[0961] 10. Any history of neurological disorder or epilepsy (including Guillain-Barré syndrome), except childhood febrile epilepsy.

[0962] 11. History of hypersensitivity to any component of CVSQIV or to aminoglycoside or β-lactam antibiotics.

[0963] 12. Any history of severe allergic reaction or anaphylaxis.

[0964] 13. History or current status of alcohol and / or drug abuse.

[0965] 14. Administration of immune globulin and / or any blood products within 3 months prior to administration of the investigational vaccine.

[0966] 15. The presence or evidence of significant acute or chronic medical or psychiatric illness.

[0967] 16. Current or past malignancy, unless completely eliminated without sequelae for >5 years.

[0968] 17. For women: pregnancy or breastfeeding.

[0969] 18. Subjects with impaired coagulation function or any bleeding disorders are prohibited from intramuscular injection or blood drawing.

[0970] 19. Subjects employed by the sponsor, investigator or trial site, or relatives of researchers working for this trial.

[0971] 4. Experimental vaccines

[0972] CVSQIV is an investigational LNP-formulated RNACTIVE quadrivalent seasonal influenza vaccine containing 4 HA antigens and 3 NA antigens according to the WHO recommendations for the composition of cell-based or recombinant influenza virus vaccines for the 2020-2021 Northern Hemisphere influenza season.

[0973] This IMP consists of the following active pharmaceutical ingredients:

[0974] Four mRNAs encoding HA of influenza virus strains A / Hawaii / 70 / 2019 (H1N1), A / Hong Kong / 45 / 2019 (H3N2), B / Washington / 02 / 2019, and B / Phuket / 3073 / 2013;

[0975] · Three mRNAs encoding NA of influenza virus strains A / Hawaii / 70 / 2019 (H1N1), A / Hong Kong / 45 / 2019 (H3N2), and B / Washington / 02 / 2019;

[0976] · Four lipid components: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), PEGylated lipids and cationic lipids.

[0977] All subjects received a single dose of CVSQIV on Day 1. The injection was given intramuscularly (IM) by needle in the deltoid region.

[0978] 5. Trial Evaluation and Procedures

[0979] The following trial visits / contacts were used:

[0980] For sentinel subjects: 4 protocol-scheduled visits on Days 1, 2, 22, and 183; and 4 protocol-scheduled telephone calls on Days 3, 8, 29, and 92.

[0981] • For all additional subjects: 3 protocol-scheduled visits on Days 1, 22, and 183; and 5 protocol-scheduled telephone calls on Days 2, 3, 8, 29, and 92.

[0982] At each protocol-scheduled visit, blood samples were obtained for safety and / or immunogenicity testing.

[0983] The purpose of the phone call was to inquire about the subject's general health and assess safety. Electronic logs were used to efficiently collect both solicited and unsolicited adverse events (AEs) following vaccination in real time.

[0984] 5.1 Safety Assessment

[0985] Active collection of adverse events

[0986] Reactogenicity was assessed on the day of vaccination and daily for 7 days thereafter by collecting unsolicited local adverse reactions (ARs) (injection site pain, redness, swelling, and itching) and unsolicited systemic AEs (fever, headache, fatigue, chills, myalgia, arthralgia, nausea / vomiting, and diarrhea) using an electronic diary.

[0987] Body temperature was measured orally and using a thermometer provided to the subjects at Visit 1.

[0988] Solicited AEs were assessed according to the intensity criteria of absent, mild, moderate, and severe ( Tables 3 and 4 ).

[0989] By definition, all unsolicited local ARs occurring from the time of vaccination were considered related to the investigational vaccination. For unsolicited systemic AEs, the investigators assessed the relationship between the investigational vaccine and each occurrence of each AE.

[0990] Table 3 - Intensity grading of active solicitation of local AR

[0991]

[0992]

[0993] Based on the US Food and Drug Administration toxicity classification criteria Coates et al. 2020.

[0994] Table 4 - Intensity Grading of Actively Solicited Systemic Adverse Events

[0995]

[0996]

[0997] iv = intravenous

[0998] Based on the US Food and Drug Administration toxicity classification criteria Coates et al. 2020.

[0999] Unsolicited adverse events and serious adverse events

[1000] Electronic diaries were used to collect unsolicited AEs on the day of vaccination and for the following 28 days.

[1001] The occurrence of AEs (serious and non-serious) was assessed by non-indicative questioning of subjects at each visit / contact. AEs voluntarily raised by subjects in the diary or detected by observation, physical examination, laboratory testing, or other assessments throughout the trial during or between visits / contacts were recorded in the electronic case report form (eCRF) if they fell into the reporting period. Subjects were instructed to report any AE with serious symptoms, chief complaint, or objective change in their health status immediately to the investigator or site personnel, regardless of the relationship between the event and the investigational vaccine, to assess the occurrence of SAEs, AESIs, and non-serious concurrent medical conditions (including influenza-like illness) that may affect the immune response. Non-serious AEs occurring after Day 29 will not be collected unless they are classified as pIMDs, non-serious concurrent medical conditions, or if they lead to trial discontinuation.

[1002] For all AEs, the investigator assessed the relationship between the investigational vaccine and each occurrence of the AE / SAE. In addition, for unsolicited AEs reported on the day of vaccination and within 28 days thereafter, the investigator or site personnel also recorded whether the subject received medical care for the AE.

[1003] SAEs, non-serious intercurrent medical conditions that may affect the immune response (including influenza-like illness), and AEs leading to trial discontinuation will be collected throughout the trial.

[1004] The results of the reactogenicity assessment of subjects in the CVSQIV trial are shown in Figure 2A-2C Actively solicited adverse events were assessed for subjects at the mRNA dose levels studied and are shown in Figure 2A Overall, a dose-dependent increase in reactogenicity was observed, with a low incidence of grade 3 reactogenicity. Results were also analyzed separately between younger and older adults. Figure 2BAs shown, higher rates of reactogenicity were observed in younger adults when compared to older adults. Grade 3 reactogenicity was observed only in younger adults (note that one case of grade 3 diarrhea / vomiting in an older adult at 3 μg was found to be caused by amebiasis and was considered unrelated to the investigational vaccine). These results were also differentiated between local and systemic events, as Figure 2C These results show that the severity of the commonly observed local reactogenicity (almost exclusively pain at the injection site) was low. The overall reactogenicity profile was driven primarily by systemic reactogenicity.

[1005] Physical examination, vital signs, and electrocardiogram

[1006] Physical examinations and vital signs were performed / measured by qualified healthcare professionals.

[1007] At each visit, vital signs (temperature, systolic / diastolic blood pressure, and heart rate) were recorded in a standardized manner after the subject rested in a seated position for 5 minutes. At the vaccination visit on Day 1, vital signs were measured before vaccination and before discharge after vaccination. Subjects were observed for 4 hours after vaccination. Vital signs must be within normal or clinically irrelevant abnormal ranges or have returned to pre-vaccination values ​​before subjects can be discharged.

[1008] A complete physical examination was performed on Day 1 unless the results of a complete physical examination performed within 21 days before Day 1 were available and sufficient based on protocol requirements, in which case a symptom-directed physical examination was performed on Day 1 before vaccination. The complete physical examination include...

Claims

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

1.

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

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

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

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

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

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

1.

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

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

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

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

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

14. The immunogenic composition according to any one of claims 10 to 13, wherein the at least one other 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), non-structural protein 1 (NS1), non-structural 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. The immunogenic composition according to any one of claims 10 to 14, wherein the at least one other 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. The immunogenic composition of claim 16, wherein the composition comprises at least four, five, six, seven or eight antigens or nucleic acids encoding the same, optionally four to ten antigens or nucleic acids encoding the same, optionally four, seven or eight antigens or nucleic acids encoding the same.

18. The immunogenic composition of claim 16 or 17, wherein the composition comprises at least four, five, six, seven or eight mRNAs, optionally four to ten mRNAs, optionally four, seven or eight mRNAs.

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

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

22. The immunogenic composition according to any one of claims 4 to 21, wherein the strain of influenza A virus is selected from influenza A virus characterized by neuraminidase (NA), wherein the neuraminidase (NA) is selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11, suitably selected from the group consisting of N1, N2 and N8, more suitably selected from the group consisting of N1 and N2.

23. An immunogenic composition according to any one of claims 4 to 22, wherein 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, suitably H1N1 and H3N2.

24. The immunogenic composition of any one of claims 4 to 23, wherein 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)pd m09-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, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09-like virus, A / Hong 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 / 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 9-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 / 2019(H1N1)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 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 of any one of claims 4 to 24, wherein the strain of influenza B virus is selected from the group consisting of the B / Victoria lineage and the B / Yamagata lineage.

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 virus, B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus, B / Washington / 02 / 2019 (B / Victoria lineage)-like virus, B / Colorado / 06 / 2017-like virus (B / Victoria / 2 / 87 lineage), B / Brisbane / 60 / 2008-like virus, B / Colorado / 06 / 2019 (B / Victoria lineage)-like virus.

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

3.

28. The immunogenic composition of claim 27, further comprising: (c 3 ) a first mRNA encoding the NA of the first strain of influenza A virus; (c 4 ) a second mRNA encoding 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, Where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:

3.

29. The immunogenic composition of claim 28, further comprising: (c 6 ) an eighth mRNA encoding the NA of the second strain of influenza B virus, Where (a):(b):(c 1 ):(c 2 ) in a ratio of between 1.5:1:1:1.5 and 5:1:1:5, suitably between 2:1:1:2 and 4:1:1:4, suitably between 2:1:1:2 and 3:1:1:3, suitably 2:1:1:2 or 3:1:1:

3.

30. The immunogenic composition of claim 28, wherein (a): (b): (c 1 ):(c 2 ):(c 3 ):(c 4 ):(c 5 ):(c 6 ) in a ratio between 9:3:3:9:1:1:1:1 and 3:1:1:3:3:3:3:3, suitably between 6:2:2:6:1:1:1:1 and 3:1:1:3:2:2:2:2, suitably 6:2:2:6:1:1:1:1 or 3:1:1:3:2:2:2:

2.

31. The immunogenic composition according to any one of claims 27 to 29, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) are formulated separately or unseparated in lipid nanoparticles (LNPs).

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

34. The immunogenic composition of claim 33, wherein the ionizable cationic lipid has Formula III: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L 1 or L 2 are each independently -O(C=O)- or -(C=O)O-; G 1 and G 2 Each is independently unsubstituted C1-C 12 Alkylene or C1-C 12 alkenylene; G 3 C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R 1 and R 2 Each independently is a branched or straight chain C6-C 24 Alkyl or C6-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 ; R 4 C1-C 12 alkyl; R 5 It is H or C1-C6 alkyl.

35. The immunogenic composition of claim 34, wherein the ionizable cationic lipid has Formula III: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L 1 or L 2 are each independently -O(C=O)- or -(C=O)O-; G 1 and G 2 Each is independently unsubstituted C1-C 12 Alkylene; G 3 C1-C 24 Alkylene; R 1 and R 2 Each independently is a branched or straight chain C6-C 24 alkyl; R 3 OR 5 ;and R 5 For H.

36. The immunogenic composition of claim 32, wherein the ionizable cationic lipid has Formula III, and wherein R 1 , R 2 or R 1 and R 2 Both have one of the following structures:

37. The immunogenic composition of claim 36, wherein R 2 Has the following structure:

38. The immunogenic composition of claim 32, wherein the cationic lipid has the formula:

39. The immunogenic composition of claim 32, wherein the cationic lipid has the formula:

40. The immunogenic composition of claim 33, wherein the ionizable cationic lipid has the formula:

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 immunogenic composition of any one of claims 32 to 40, wherein the PEG-modified lipid has Formula IV: Where R 8 and R 9 Each is independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; And the average value of w ranges from 30 to 60.

43. The immunogenic composition of claim 42, wherein R 8 and R 9 A saturated alkyl chain.

44. The immunogenic composition of claim 32, wherein the PEG-modified lipid has Formula IVa: wherein the average value of n ranges from 30 to 60, suitably wherein the average value of n is about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most suitably wherein the average value of n is 49 or 45; or wherein n is an integer selected such that the average molecular weight of the PEG lipid is about 2500 g / mol.

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

48. 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. 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 about 2 to about 12, optionally an N / P ratio of 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. The immunogenic composition according to any one of claims 27 to 50, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) of the mRNA, optionally each, is not self-replicating.

52. The immunogenic composition according to any one of claims 27 to 51, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) comprises a coding sequence, which 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. The immunogenic composition of claim 53, wherein the G / C content of the codon-modified coding sequence is at least about 45%, 50%, 55% or 60%.

55. The immunogenic composition according to any one of claims 27 to 54, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) contains a 5'cap, preferably m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure, preferably a 5'-cap1 structure.

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

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

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

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

60. The immunogenic composition of claim 59, wherein the 5'UTR comprises or consists of a nucleic acid sequence derived from a 5'-UTR of a gene selected from the group consisting of: HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homolog, fragment or variant of any of these genes.

61. The immunogenic composition according to any one of claims 27 to 60, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) contains a 3'UTR.

62. The immunogenic composition of claim 61, wherein the 3'UTR comprises or consists of a nucleic acid sequence derived from a 3'-UTR of a gene selected from the group consisting of: Selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homologue, fragment or variant of any of these genes.

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

64. The immunogenic composition according to any one of claims 27 to 63, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) wherein the mRNA comprises from 5' to 3': i) 5'-cap1 structure; ii) a 5'-UTR derived from the 5'-UTR of the HSD17B4 gene; iii) the coding sequence; 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 comprising about 100 A nucleotides, wherein the 3' terminal nucleotide of the RNA is adenosine.

65. The immunogenic composition according to any one of claims 27 to 64, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) does not contain chemically modified nucleotides.

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

67. The immunogenic composition of claim 66, wherein the chemical modification is selected from pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methylpseudouridine, 2-thiol-5-azauridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxypseudouridine, 4-thiol-1-methylpseudouridine, 4-thiol-pseudouridine, 5-azauridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

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

69. The immunogenic composition according to any one of claims 66 to 68, wherein (a), (b), (c 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) wherein said chemical modification comprised by said mRNA is a uridine modification, preferably wherein 100% of the uridine positions in said mRNA are modified.

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

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

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

73. The vaccine according to 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 another pathogen, suitably said pathogen is a virus.

74. The vaccine according to claim 73, wherein the antigen other virus is selected from the group consisting of coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), Pneumoviridae virus (e.g., respiratory syncytial virus, metapneumovirus) and Paramyxoviridae virus (e.g., parainfluenza virus, Henipavirus), and suitably the antigen from other virus is a spike protein or an antigenic fragment thereof from SARS-CoV-2 virus, or an mRNA encoding a spike protein or an antigenic fragment thereof from SARS-CoV-2 virus.

75. A kit or set of parts comprising an antigen or nucleic acid as defined in any one of claims 1 to 26 or an mRNA as defined in any one of claims 3 to 71, suitably (a), (b), (c) as defined in any one of claims 27 to 71 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ), optionally containing a liquid carrier for dissolution, and optionally a technical instruction sheet providing information about the administration and dosage of the components.

76. The kit or kit of parts according to claim 75, wherein the antigen or the nucleic acid or the mRNA is suitably (a), (b), (c) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 )'s mRNAs were prepared separately.

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 set of parts of claims 75 or 76, wherein the antigen or the nucleic acid or the mRNA is suitably (a), (b), (c) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) is formulated as a bedside mixed preparation.

78. 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 set of parts of claims 75 or 76, wherein the antigen or the nucleic acid and / or (a), (b), (c) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) were co-formulated.

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 a set of parts according to claim 75 or 76 for use as a medicament.

80. 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 a set of parts according to claim 75 or 76 for use in the treatment or prevention of influenza virus infection, suitably influenza A and / or influenza B.

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

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

83. A kit or a kit of parts for use according to any one of claims 80 to 82, wherein the antigen or the nucleic acid or the mRNA is suitably (a), (b), (c) 1 )、(c 2 )、(c 3 )、(c 4 )、(c 5 ) and / or (c 6 ) of the mRNA and administered to different injection sites.

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

85. A method of treating or preventing a disorder caused by influenza virus, suitably influenza A and / or influenza B, wherein the method comprises 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 set of parts according to claims 75 or 76.

86. A method of eliciting an immune response, wherein the method comprises 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 set of parts according to claims 75 or 76.

87. A method according to claim 86, wherein the immune response is an adaptive immune response, suitably a protective adaptive immune response against influenza virus, suitably a protective adaptive immune response against influenza A virus and / or influenza B virus.

88. The method of claim 85 or the method of claim 86 or 87, wherein the subject in need thereof is a mammalian subject, suitably a human subject.

89. 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 set 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 lineage, and influenza B Victoria lineage.

90. 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 set of parts of claim 75 or 76 is administered in an amount effective to induce a neutralizing antibody response against influenza A H1N1, influenza A H3N2, influenza B Yamagata lineage, and influenza B Victoria lineage.

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