Potency assay

Abstract

The present disclosure relates to methods for determining potency of multivalent RNA vaccines.

Description

[0001] POTENCY ASSAY

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from U.S. Provisional Application No. 63 / 658,115 entitled “Potency assay” filed 10 June 2024, the entire contents of which are hereby incorporated by reference.

[0004] FIELD

[0005] The present disclosure relates to methods for determining potency of multivalent RNA vaccines.

[0006] BACKGROUND

[0007] Influenza, commonly known as the flu, is a highly contagious respiratory illness caused by influenza viruses. Influenza viruses are members of the Orthomyxoviridae family and are categorized into three main types: A, B, and C. Influenza A viruses are further classified into subtypes based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Conventional influenza vaccines are typically trivalent or quadrivalent, targeting the virus strains that are predicted to be prevalent in a given influenza season.

[0008] Prior to batch release, it is a requirement that influenza vaccines meet certain potency (also known as “release”) criteria. The standard assay for HA content in inactivated influenza vaccines is based on single radial immunodiffusion ("SRID"). However, the SRID is slow to perform, has poor dynamic range, is susceptible to considerable variability, and can create bottle necks in development because it requires specific anti-HA serum to be obtained and prepared. These assays are also limited to quantification of only one antigen, not each antigen provided in the vaccine.

[0009] RNA-based vaccines have gained recent popularity following their approval for use during the COVID-19 pandemic. The rise in popularity of RNA-based vaccines is in part due to the ability of these vaccines to encode for multiple viral antigens. However, standardised methods to determine the potency of these vaccines and their ability to express multiple antigens in a cell are limited.

[0010] Accordingly, there remains a need in the art for improved potency assays with the ability to determine the amount of each antigen present in multivalent vaccines, particularly RNA-based vaccines. SUMMARY

[0011] The present disclosure is based on present inventors’ production of a potency assay for multivalent immunogenic compositions. Advantageously, the methods of the present disclosure can be used to determine the amount of each antigen expressed by cells incubated with multivalent RNA vaccines.

[0012] Accordingly, the present disclosure provides a method for determining the potency of a multivalent immunogenic composition, wherein the immunogenic composition comprises: (i) a RNA encoding one or more viral antigens from a first viral strain; (ii) a RNA encoding one or more viral antigens from a second viral strain; and wherein the method comprises: (i) incubating the immunogenic composition in a culture medium with a population of cells under culture conditions; (ii)(a) contacting a first sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the first viral strain; (ii)(b) contacting a second sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the second viral strain; and (iii) determining the level of each viral antigen in each sample, comprising detecting the labelled detection antibody in each sample and determining the number or percentage of cells that are positive for each viral antigen in each sample.

[0013] In an example, the immunogenic composition further comprises (iii) a RNA encoding one or more viral antigens from a third viral strain; and (ii)(c) contacting a third sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the third viral strain.

[0014] In an example, the immunogenic composition further comprises (iii) a RNA encoding one or more viral antigens from a third viral strain; (iv) a RNA encoding one or more viral antigens from a fourth viral strain; and wherein the method further comprises (ii)(c) contacting a third sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the third viral strain; (ii)(d) contacting a fourth sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the fourth viral strain.

[0015] In an example, step (iii) comprises determining the level of each viral antigen in each sample, comprising detecting the labelled detection antibody in each sample and determining the number of cells that are positive for each viral antigen in each sample. In an example, step (iii) comprises determining the level of each viral antigen in each sample, comprising detecting the labelled detection antibody in each sample and determining the percentage of cells that are positive for each viral antigen in each sample. In an example, potency is calculated by correlating the percentage of cells that are positive for each viral antigen with the amount of RNA encoding the antigen.

[0016] In an example, potency is calculated according to the formula: specific potency (ng-1) = slope x (linear regression slope x 105) ± standard error, wherein the slope is calculated from a two-fold serial dilution of RNA.

[0017] In an example, step (ii) comprises: (ii) treating the sample of cells with a fixation and permeabilization solution so as to fix and permeabilize the cells; (iii) washing the cells to remove the fixation and permeabilization solution; (iv) contacting the cells with the labelled detection antibodies under suitable conditions; (v) washing the cells to remove any unbound labelled detection antibody; and (vi) transferring the cells to a suitable medium for determining the level of each viral antigen in each sample.

[0018] In an example, each label is a fluorescent label. In an example, each fluorescent label is selected from a group consisting of: a fluorescent label with an excitation wavelength of 300-379 nm; a fluorescent label with an excitation wavelength of 380-439 nm; a fluorescent label with an excitation wavelength of 440-509 nm; a fluorescent label with an excitation wavelength of 510-599 nm; a fluorescent label with an excitation wavelength of 600-729 nm; and a fluorescent label with an excitation wavelength of over 730 nm.

[0019] In an example, each detection antibody comprises a distinct fluorescent label. In an example, the fluorescent labels do not have substantial spectral overlap. In an example, each labelled detection antibody comprises a distinct fluorescent label, wherein the distinct fluorescent labels do not have substantial spectral overlap. In an example, each fluorescent label has a difference in excitation wavelength of between about 100 nm and about 400 nm. In an example, each fluorescent label has a difference in excitation wavelength of between about 100 nm and about 300 nm. In an example, each fluorescent label has a difference in excitation wavelength of between about 100 nm and about 200 nm.

[0020] In an example, each sample is contacted with a first labelled detection antibody and a second labelled detection antibody, wherein the first and second labelled detection antibodies comprise a distinct fluorescent label. In an example, the first labelled detection antibody comprises a fluorescent label with an excitation wavelength of 440-509 nm and the second labelled detection antibody comprises a fluorescent label with an excitation wavelength of 600-729 nm.

[0021] In an example, the labelled detection antibody comprises a primary antibody that is directly linked to the fluorescent label, or a primary antibody that is contacted with a secondary antibody that is directly linked to the fluorescent label. In an example, the labelled detection antibody comprises a primary antibody that is directly linked to the fluorescent label. In an example, the labelled detection antibody comprises a primary antibody that is contacted with a secondary antibody that is directly linked to the fluorescent label.

[0022] In an example, each fluorescent label is detected by flow cytometry. In an example, the flow cytometry is fluorescence activated cell sorting (FACS). In an example, the flow cytometry is multicolour flow cytometry.

[0023] In an example, the viral strain is an influenza virus strain, a coronavirus strain, a respiratory syncytial virus strain, a parainfluenza virus stain, a human metapneumovirus strain, or an Epstein-Barr virus strain. In an example, the first and second viral strains are selected from the group consisting of an influenza virus strain, a coronavirus strain, a respiratory syncytial virus strain, a parainfluenza virus stain, a human metapneumovirus strain, or an Epstein-Barr virus strain. In an example, the first, second, and third viral strains are selected from the group consisting of an influenza virus strain, a coronavirus strain, a respiratory syncytial virus strain, a parainfluenza virus stain, a human metapneumovirus strain, or an Epstein-Barr virus strain. In an example the first, second, third and fourth viral strain is an influenza virus strain, a coronavirus strain, a respiratory syncytial virus strain, a parainfluenza virus stain, a human metapneumovirus strain, or an Epstein-Barr virus strain.

[0024] In an example, the first and second viral strains are an influenza viral strain. In an example, the first, second, and third viral strains are an influenza viral strain. In an example, the first, second, third, and fourth viral strains are an influenza strain.

[0025] In an example, the first and second viral strains are an influenza viral strain and a coronavirus strain. In an example, the first, second, and third viral strains are an influenza viral strain and a coronavirus strain. In an example, the first, second, third, and fourth viral strains are an influenza strain and a coronavirus strain.

[0026] In an example, the first and second viral strains are an influenza viral strain and a respiratory syncytial virus strain. In an example, the first, second, and third viral strains are an influenza viral strain and a respiratory syncytial virus strain. In an example, the first, second, third, and fourth viral strains are an influenza strain and a respiratory syncytial virus strain.

[0027] In an example, the first, second, and third viral strains are an influenza viral strain, a coronavirus strain, and a respiratory syncytial virus strain. In an example, the first, second, third, and fourth viral strains are an influenza strain, a respiratory syncytial virus strain, and a coronavirus strain. In an example, the influenza strains are selected from the group consisting of influenza A and influenza B. In an example, the influenza strains are selected from the group consisting of influenza A H1N1, influenza A H3N1, influenza B / Victoria, and influenza B / Yamagata. In an example, the viral antigen is a hemagglutinin (HA) protein from an influenza strain. In an example, the viral antigen is a neuraminidase (NA) protein from an influenza strain.

[0028] In an example, the RNA encoding the one or more viral antigens from each viral strain are provided in a multi ci str onic RNA construct.

[0029] In an example, the RNA encoding each antigen is provided in a monocistronic RNA construct.

[0030] In an example, the immunogenic composition comprises: (i) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a first influenza strain; (ii) a RNA encoding a HA protein and a RNA encoding an NA protein from a second influenza strain; and the method comprises: (i) incubating the immunogenic composition in a culture medium with a population of cells under culture conditions; (ii)(a) contacting a first sample of the population of cells with a labelled detection antibody that binds the HA protein from the first influenza strain, and a labelled detection antibody that binds the NA protein from the first influenza strain; (ii)(b) contacting a second sample of the population of cells with a labelled detection antibody that binds the HA protein from the second influenza strain, and a labelled detection antibody that binds the NA protein from the second influenza strain; (iii) determining the level of the HA protein and the NA protein in each sample, comprising detecting the labelled detection antibody in each sample and determining the number or percentage of cells that are positive for both the HA label and the NA label in each sample.

[0031] In an example, the immunogenic composition further comprises (iii) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a third influenza strain; (iv) a RNA encoding a HA protein and a RNA encoding an NA protein from a fourth influenza strain; and wherein the method further comprises (ii)(c) contacting a third sample of the population of cells with a labelled detection antibody that binds the HA protein from the first influenza strain, and a labelled detection antibody that binds the NA protein from the third influenza strain; (ii)(d) contacting a fourth sample of the population of cells with a labelled detection antibody that binds the HA protein from the second influenza strain, and a labelled detection antibody that binds the NA protein from the fourth influenza strain.

[0032] In an example, step (iii) comprises detecting the labelled detection antibody in each sample and determining the number of cells that are positive for both the HA label and the NA label in each sample. In an example, step (iii) comprises detecting the labelled detection antibody in each sample and determining the percentage of cells that are positive for both the HA label and the NA label in each sample.

[0033] In an example, the RNA encoding the HA protein and the RNA encoding the NA protein are provided in a multi ci stronic RNA construct. In an example, the immunogenic composition comprises: (i) a multi ci stronic RNA encoding the HA protein and the NA protein from a first influenza strain; (ii) a multicistronic RNA encoding the HA protein and the NA protein from a second influenza strain; (iii) a multicistronic RNA encoding the HA protein and the NA protein from a third influenza strain; and (iv) a multicistronic RNA encoding the HA protein and the NA protein from a fourth, influenza strain.

[0034] In an example, the RNA encoding the HA protein and the RNA encoding the NA protein are provided in a monocistronic RNA construct. In an example, the immunogenic composition comprises: (i) a monocistronic RNA encoding the HA protein and a monocistronic RNA encoding the NA protein from a first influenza strain; (ii) a monocistronic RNA encoding the HA protein and a monocistronic encoding the NA protein from a second influenza strain; (iii) a monocistronic RNA encoding the HA protein and a monocistronic RNA encoding the NA protein from a third influenza strain; and (iv) a monocistronic RNA encoding the HA protein and a monocistronic RNA encoding the NA protein from a fourth, influenza strain.

[0035] In an example, the RNA is a mRNA. In an example, the RNA is a self-amplifying RNA. In an example, the RNA is a conventional RNA.

[0036] In an example, the RNA is formulated in a delivery vehicle. In an example, the RNA is formulated in a lipid nanoparticle (LNP). In an example, the RNA is naked RNA.

[0037] In an example, the labelled detection antibody that binds to the HA protein is: an influenza A H1N1 anti -HA antibody; influenza A H3N1 anti -HA antibody; influenza B / Victoria anti-HA antibody; or influenza B / Yamagata anti-HA antibody. In an example, the labelled detection antibody that binds to the HA protein is an influenza A H1N1 anti- HA antibody. In an example, the labelled detection antibody that binds to the HA protein is an influenza A H3N1 anti-HA antibody. In an example, the labelled detection antibody that binds to the HA protein is an influenza B / Victoria anti-HA antibody.

[0038] In an example, the labelled detection antibody that binds to the HA protein is an influenza B / Yamagata anti-HA antibody.

[0039] In an example, the labelled detection antibody that binds to the NA protein is: an influenza A H1N1 anti -NA antibody; an influenza A H3N1 anti -NA antibody; an influenza B / Victoria anti-NA antibody; or an influenza B / Yamagata anti-NA antibody. In an example, the labelled detection antibody that binds to the NA protein is an influenza A H1N1 anti -NA antibody. In an example, the labelled detection antibody that binds to the NA protein is an influenza A H3N1 anti -NA antibody. In an example, the labelled detection antibody that binds to the NA protein is an influenza B / Victoria anti-NA antibody. In an example, the labelled detection antibody that binds to the NA protein is an influenza B / Yamagata anti-NA antibody.

[0040] In an example, the cells are mammalian cells. In an example, the mammalian cells are HEK293 cells or BHK-21 cells. In an example, the mammalian cells are HEK293 cells. In an example, the mammalian cells are BHK-21 cells.

[0041] The present disclosure also provides a method for selecting a multivalent immunogenic composition suitable for use in in method of inducing an immune response in a subject, comprising: (i) determining the potency of the immunogenic composition according to the method of the disclosure, and (ii) selecting an immunogenic composition having a potency value of between 5 and 2500.

[0042] In an example, the potency value is between 5 and 200. In an example, the potency value is between 10 and 150. In an example, the potency value is between 50 and 150. In an example, the potency value is between 10 and 50. In an example, the potency value is between 5 and 15.

[0043] In another example, the potency value is between 200 and 1000. In an example, the potency value is between 200 and 800. In an example, the potency value is between 100 and 400.

[0044] In another example, the potency value is between 500 and 2500. In an example, the potency value is between 600 and 1200. In an example, the potency value is between 500 and 1000.

[0045] The present disclosure provides an immunogenic composition selected for use in method of inducing an immune response against influenza in a subject in need thereof, wherein the immunogenic composition comprises (i) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a first influenza strain; (ii) a RNA encoding a HA protein and a RNA encoding an NA protein from a second influenza strain; (iii) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a third influenza strain; and (iv) a RNA encoding a HA protein and a RNA encoding an NA protein from a fourth influenza strain; wherein the first, second, third, and fourth influenza strains comprising influenza A H1N1, influenza A H3N1, influenza B / Victoria, and influenza B / Yamagata; and wherein selection is performed according to the method of the disclosure.

[0046] In an example, the disclosure provides a method of inducing an immune response in a subject, the method comprising selecting an immunogenic composition suitable for use according to the methods disclosed herein, and administering the immunogenic composition to a subject.

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1: Schematic of potency assay.

[0049] Figure 2: Titration of HA antibodies (H1N1).

[0050] Figure 3: Titration of HA antibodies (H3N2).

[0051] Figure 4: Titration of HA antibodies (B / Vic).

[0052] Figure 5: Titration of HA antibodies (B / Yam)

[0053] Figure 6: HA antibodies titration (quantification).

[0054] Figure 7: HA antibodies - mock stains.

[0055] Figure 8: Titration of NA antibodies (H1N1).

[0056] Figure 9: Titration of NA antibodies (H3N2).

[0057] Figure 10: Titration of NA antibodies (B / Vic).

[0058] Figure 11: Titration of NA antibodies (B / Yam).

[0059] Figure 12: NA antibodies titration (quantification).

[0060] Figure 13: NA antibodies - mock stains.

[0061] Figure 14: Cross reactivity - DS-B (H1N1).

[0062] Figure 15: Cross reactivity - DS-B (H3N2).

[0063] Figure 16: Cross reactivity - DS-B (B / Yam).

[0064] Figure 17: Cross reactivity - DS-B (B / Vic).

[0065] Figure 18: Cross reactivity -B / Yam antibody.

[0066] Figure 19: HA and NA double positive populations for all 4 strains- DP-1.

[0067] Figure 20: HA and NA double positive populations for all 4 strains- DP -2.

[0068] Figure 21: HEK293 Assay for Quadrivalent DP-1.

[0069] Figure 22: HEK293 Assay for Quadrivalent DP-2.

[0070] Figure 23: HEK293 Assay for Quadrivalent DP-3.

[0071] Figure 24: DP-1 potency.

[0072] Figure 25: DP-2 potency.

[0073] Figure 26: DP-3 potency.

[0074] Figure 27: DP-4 potency.

[0075] Figure 28: Summary of DP comparison by FACS potency in BHK21 and HEK293.

[0076] Figure 29: Observation of superior NA performance by DP-3 except B / Vic, generally lower HA performance by DP-3 in BHK21.

[0077] Figure 30: Observation of superior NA performance by DP-3 except B / Vic, generally lower HA performance by DP-3 in HEK293. Figure 31: Similar HA expression for DP-3 and DP-l / DP-2. Higher NA expression for DP-3.

[0078] Figure 32: Similar HA expression for DP-3 and DP-l / DP-2. Higher NA expression for DP-3.

[0079] Figure 33: DS-B constructs perform marginally higher in the BHK21 and DS-A perform higher in HEK293.

[0080] Figure 34: IDMS data confirmed the differential expression by DS-B and DS-A in BHK21 and HEK293.

[0081] Figure 35: IDMS data confirmed the differential expression by DS-B and DS-A in BHK21 and HEK293.

[0082] Figure 36: SAM / LNP-transfected cells: dose titration.

[0083] Figure 37: Data analysis and calculation of specific potency.

[0084] Figure 38: Data analysis and calculation of specific potency - natural log.

[0085] DETAILED DESCRIPTION

[0086] General

[0087] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0088] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0089] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0090] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive). Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0091] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0092] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0093] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0094] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0095] The term “substantially” does not exclude “completely” (e.g., a composition which is “substantially free” from Y may be completely free from Y).

[0096] The term “about” in relation to a numerical value x is optional and means, for example, any number within 1, 5 or 10% of the referenced number. In certain examples, the term “about” encompasses the exact number recited.

[0097] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0098] As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source. Similarly, the term “based on” shall be taken to indicate that a specified integer may be developed or used from a particular source albeit not necessarily directly from that source.

[0099] The terms “level” and “amount” are used to define the amount of a particular substance in a sample from a subject or in a cell culture media (or sample therefrom). For example, a particular concentration, weight, percentage (e.g. v / v%) or ratio can be used to define the level of a particular substance / cell in a sample. In an example, the level is expressed in terms of how much of a particular marker, such as an antigen, is expressed by a population of cells under culture conditions.

[0100] Selected Definitions

[0101] As used herein, the term “immunogenic” will be understood to refer to a compound or composition that induces or generates an immune response. A compound or composition that induces or generates an immune response is typically one that induces a humoral or cell-mediated response in a subject to which the compound or composition is administered. The humoral or cell-mediated response may be specific to the compound or composition. Such a response may be detected and / or quantified by determining the induction of antibodies in the subject and / or cellular responses. A quantitative antibody measurement may be determined. Alternatively, or in addition, a qualitative antibody measurement may be determined. For example, but without limitation, a measure of one or more functional features of antibodies elicited in a subject to which the compound or composition is administered, may be determined. Suitable functional features which can be measured are known in the art and include, without limitation, hemagglutinin agglutination-inhibition. A quantitative method for the determination of expression or secretion of cytokines or alteration in the phenotype of immune cells may also be used.

[0102] The term “vaccine” is used herein to refer to a composition that is administered to a subject to produce or increase immunity to a particular disease, for example, a virus. In some examples, vaccines include a pharmaceutically acceptable adjuvant and / or a pharmaceutically acceptable carrier. For the avoidance of doubt, the terms “immunogenic composition” and “vaccine” in the context of the present disclosure can be used interchangeably unless otherwise specified. The immune response may be a protective immune response. By “protective immune response” it is meant an immune response that is sufficient to prevent or at least reduce the severity or extent of one or more symptoms of an influenza-associated disease, disorder or condition in a subject. A protective immune response may be determined directly through experimentation (such as by performing challenge studies to a subject vaccinated with a particular compound or composition). Alternatively or in addition, a protective immune response may be determined via a measurement of one or more suitable correlates of protection. Suitable correlates of protection will be appreciated by one of skill in the art, and may include, for example but without limitation, antibody binding titre, HAI titre, microneutralisation (MN) titre, cytokine assays, cellular responses, antibody dependent cellular cytotoxicity assays. A protective immune response may be demonstrated by an HAI titre of about 40, or about 50, or about 60 according to known, standard HAI assay protocols, including any of those described herein. Thus, in one example, a protective immune response may be demonstrated by an HAI titre of about 40 according to known, standard HAI assay protocols, or according to any HAI assay protocols described herein.

[0103] The term “multivalent” refers to an immunogenic composition that immunizes (i.e. elicits an immune response) against more than one antigen. The person skilled in the art will appreciate a multivalent immunogenic composition immunises against any number of antigens. The terms “bivalent”, “trivalent”, “quadrivalent” etc, refer to immunogenic compositions that elicit an immune response against two, three, or four antigens, respectively. In an example, the immunogenic composition is a multivalent immunogenic composition. In an example, the immunogenic composition is a bivalent immunogenic composition. In an example, the immunogenic composition is a trivalent immunogenic composition. In an example, the immunogenic composition is a quadrivalent immunogenic composition.

[0104] The term “potency” as used herein refers to an indicator of the therapeutic potential of an immunogenic composition or vaccine. This value can be used as a parameter for determining whether a manufactured batch of a vaccine is suitable for release to the public, or whether it has experienced a production failure and so should not be used. Assays of the disclosure are particularly useful for analysing vaccines which contain multiple different antigens. In an example, potency is determined by measuring the level of each antigen encoded by each RNA in the immunogenic composition in a population of cells that have been incubated with the immunogenic composition under culture conditions. In an example, potency can be expressed as “specific potency”. Specific potency can be calculated by performing a serial dilution of RNA and measuring the number or percentage of cells positive for each antigen. A linear regression analysis is then performed to determine a slope which is then multiplied by a constant. Specific potency can be calculated according to the following formula: specific potency (ng-1) = slope x (linear regression slope x 105) ± standard error. The term “potency value” as used herein refers to the number determined according to the above formula. In some examples, potency value of a composition is the “accumulative potency”. In this example, the “accumulative potency” is the sum of the potency value determined for each strain according to the above formula.

[0105] As used herein, the term “antigen” refers to a molecule or structure containing one or more epitopes that induce, elicit, augment or boost a cellular and / or humoral immune response. Antigens can include, for example, proteins and peptides from a pathogen such as a virus, bacteria, fungus, protozoan, plant or from a tumour. In an example, the antigen is a viral antigen, for example, an influenza antigen, a coronavirus antigen, a respiratory syncytial virus antigen, a parainfluenza virus antigen, a human metapneumovirus antigen, or an Epstein-Barr virus antigen.

[0106] To facilitate a clear description, antigens, viral strains, and / or particular sequence components are referred to as e.g., a “first”, “second”, “third”, fourth”, etc. It is to be understood that the first, second, third, and / or fourth antigen, viral strain, and / or nucleotide sequence can appear in any desired order or orientation, unless otherwise specified, and that no particular order or orientation is intended by the words “first”, “second” etc.

[0107] As used herein, the term “strain”, “type” and “subtype” refer to viruses having different characteristics and / or are genetically distinct, and are used interchangeably unless otherwise specified. For example, influenza A virus is a different strain and different type of virus to influenza B virus. Likewise, influenza A H1N1 is a different strain and different type of virus than influenza A H2N1, H2N2 and H3N2.

[0108] The skilled artisan will be aware that an “antibody” is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a light chain variable region (VL) and a polypeptide comprising a heavy chain variable region (VH). An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A VH and a VL interact to form a Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a K light chain or a light chain and a heavy chain from mammals is a, 5, a, y, or p. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG2, IgGs, IgG4, IgAi and IgA2) or subclass. The term “antibody” also encompasses humanized antibodies, primatized antibodies, human antibodies, synhumanized antibodies and chimeric antibodies.

[0109] As used herein, the term “detect” or “detecting” refers to the identification of the presence or existence of antigen, e.g. a viral antigen, in a sample.

[0110] As used herein, a “detectable label” is a molecular or atomic tag or marker that generates or can be induced to generate an optical or other signal or product that can be detected visually or by using a suitable detector. Detectable labels are well known in the art and include, for example, a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a prosthetic group, a contrast agent and an ultrasound agent. In an example, the detectable label is a fluorescent label. In an example, the fluorescent label is a fluorophore or fluorochrome. For the avoidance of doubt and unless otherwise specified, the terms “fluorophore” and “fluorochrome” are used interchangeably.

[0111] As used herein, the term “binds” in reference to the interaction of an antibody with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.

[0112] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that an antibody of the disclosure reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen than it does with alternative antigens. For example, an antibody binds to a particular viral antigen with materially greater affinity (e.g., 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold greater affinity) avidity, more readily, and / or with greater duration than it binds to other antigens, e.g., to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

[0113] The term “culture conditions” is used in the context of the present disclosure to refer to the controlled conditions required for the growth and survival of a population of cells in culture. In certain examples, an immunogenic composition of the disclosure is incubated with a population of cells under culture conditions in order to determine potency of said immunogenic composition.

[0114] As used herein, the term “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone.

[0115] As used herein, the term “RNA” or “ribonucleic acid” refers to a single stranded molecular chain of nucleotides chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar in an adjacent nucleotide. Suitable forms of RNA will be apparent to the skilled person, for example, monocistronic mRNA or multi ci stronic mRNA. The monocistronic mRNA or multi ci str onic mRNA may be conventional mRNA (cRNA) or a self-replicating RNA. Any suitable RNA constructs or vectors known in the art may be used in the context of the present disclosure to deliver viral antigens to a subject.

[0116] As used herein, the term “monocistronic” refers to a RNA that encodes one polypeptide.

[0117] As used herein, the term “multicistronic” (also known as “polycistronic”) refers to a RNA that encodes two or more polypeptides. The term encompasses “bicistronic” (or “di ci stronic”; i.e., encoding two polypeptides) and “tri ci stronic” (i.e., encoding three polypeptides) molecules. By “bicistronic” is meant a single nucleic acid that is capable of encoding two distinct polypeptides from different regions of the nucleic acid.

[0118] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0119] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0120] As used herein, the term “lipid nanoparticle” or “LNP” shall be understood to refer to lipid-based particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and which comprises a compound of any formulae described herein. In embodiments, LNPs are formulated in a composition for delivery of a polynucleotide to a desired target such as a cell, tissue, organ, tumor, and the like. For example, the lipid nanoparticle or LNP any lipid composition, including, may be selected from, but not limited to, liposomes or vesicles, where an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single; unilamellar or multiple; multilam ellar), micelle- like lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles , wherein solid lipid nanoparticles lack lipid bilayers

[0121] As used herein, the terms “treating”, “treat” or “treatment” include administering an immunogenic composition described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition.

[0122] As used herein, the term “preventing”, “prevent” or “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a specified disease or condition in an individual. An individual may be predisposed to or at risk of developing the disease but has not yet been diagnosed with the disease.

[0123] As used herein, the phrase “delaying progression of’ includes reducing or slowing down the progression of the disease or condition in an individual and / or at least one symptom of a disease or condition.

[0124] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic or prophylactic result. In an example, the desired result is induce an immune response in a subject. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect treatment of a disease or condition as hereinbefore described. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect a change associated with a disease or condition as hereinbefore described. The effective amount may vary according to the disease or condition to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of RNA. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

[0125] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0126] Potency assay

[0127] The present disclosure relates to methods for determining the potency of multivalent immunogenic compositions. Potency assays are used to measure the ability of a drug (e.g. an immunogenic composition) to elicit a particular response at a certain dose in a relevant biological system. Potency assays are usually required by regulatory agencies for release of drug product under GMP. They are also useful in drug discovery to rank potential therapeutic candidates.

[0128] The potency assay of the present disclosure can be described as having three stages: (i) incubating a population of cells with an immunogenic composition under culture conditions; (ii) contacting cell samples with labelled detection antibodies that bind each viral antigen present in the immunogenic composition; and (iii) determining the level of antigen in each sample. For ease of reference, these stages can be described as the “cell culture” stage, the “sample preparation” stage, and the “analysis” stage.

[0129] Cell culture

[0130] Populations of cells according to the disclosure can be cultured in a suitable culture medium according to standard cell culture techniques known in the art.

[0131] In an example, the population of cells are mammalian cells. The skilled person will appreciate that any suitable mammalian cells can be used. In an example, the mammalian cells are immortalized. Non-limiting examples of immortalized mammalian cells include human embryonic kidney (HEK293) cells, baby hamster kidney cells (BHK21), Chinese hamster ovary (CHO) cells, and murine myeloma (e.g. NSO, Sp2 / 0) cells. In an example, the population of cells are HEK293 cells or BHK21 cells. In an example, the population of cells are HEK293 cells. In an example, the population of cells are BHK21 cells.

[0132] Populations of cells can be cultured under conditions suitable for the particular cell. Relevant conditions include a regulated physico-chemical environment (optimal pH, osmotic pressure, temperature), gases (02 and C02 amounts) and a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors and hormones. The term “medium” or “media” as used in the context of the present disclosure, includes the components of the environment surrounding the cells. The media contributes to and / or provides the conditions suitable to allow cells to grow. Media can include liquid growth media and gelatinous media such as agar, agarose, gelatin and collagen matrices. In an example, the cell culture media is a basal media. Examples of basal media include Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI), and / or any modified versions thereof.

[0133] Cell populations can be cultured in a cell culture medium and seeded on a cell culture substrate at a desired cell density prior to incubation with an immunogenic composition. Suitable culture substrates include tissue culture flasks, tissue culture plates, or other culture materials known in the art including glass, plastic, gels, scaffolds, or membranes. Cells can be seeded at suitable cell densities or confluency for given substrate. In an example, cells are seeded at a density of 0.5 x 106cells / well of a tissue culture plate on the day of incubation with the immunogenic composition. In an example, cells are passaged in tissue culture flasks to be at 80-90% confluency on the day of incubation with the immunogenic composition.

[0134] Immunogenic compositions can be added to the population of cells and incubated under suitable culture conditions. An example suitable culture conditions is a temperature of about 37 degrees Celsius and about 5% CO2. In an example, the immunogenic composition is incubated with the population of cells for between about 8 and about 30 hours. In an example, the immunogenic composition is incubated with the population of cells for between about 10 and about 24 hours. In an example, the immunogenic composition is incubated with the population of cells for between about 12 and about 24 hours. In an example, the immunogenic composition is incubated with the population of cells for between about 12 and about 20 hours. In an example, the immunogenic composition is incubated with the population of cells for between about 16 and about 19 hours.

[0135] The amount of immunogenic composition added to the population is not particularly limited, provided that the RNA is in an amount sufficient for effective transfection. In some examples, the immunogenic composition is prepared in predilutions of the RNA / LNP complexes in media which is then added to the population of cells. In an example, the immunogenic composition is added in an amount between 800 ng RNA / 100 pL media to 0.39 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 800 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 400 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 200 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 100 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 50 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 25 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 12.5 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 6.25 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 3.125 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 1.56 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 0.78 ng RNA / 100 pL media. In an example, the immunogenic composition is added an amount of 0.39 ng RNA / 100 pL media.

[0136] In some examples, the immunogenic composition may be added as part of a serial dilution. For, the immunogenic composition is added at 12, 10, 8, 6, 5, 4, or 3 different dilutions. In an example, the dilutions are a two-fold dilution. In an example, the immunogenic composition is added in at 12 two-fold serial dilutions. In an example, the immunogenic composition is added in at 12 two-fold serial dilutions across a concentration of 800 ng RNA / 100 pL media to 0.39 ng RNA / 100 pL media. In an example, the immunogenic composition is added in an amount of 800 ng RNA / 100 pL media, 400 ng RNA / 100 pL media, 200 ng RNA / 100 pL media, 100 ng RNA / 100 pL media, 50 ng RNA / 100 pL media, 25 ng RNA / 100 pL media, 12.5 ng RNA / 100 pL media, 6.25 ng RNA / 100 pL media, 3.125 ng RNA / 100 pL media, 1.56 ng RNA / 100 pL media, 0.78 ng RNA / 100 pL media, and 0.39 ng RNA / 100 pL media.

[0137] Following incubation, cells can be detached from the culture substrate with a suitable detaching agent. Non-limiting examples of detaching agents include EDTA or EDTA based reagents (e.g. Versene), trypsin or trypsin-based reagents (e.g. TrypLE), or mechanical agents such as cell scrapers.

[0138] Sample preparation

[0139] Samples of the cell population are then prepared and labelled with suitable detection antibodies. Samples can be prepared and labelled using routine methods known in art.

[0140] In an example, the cell samples are fixed using a suitable fixation reagent, such as paraformaldehyde (PF A). In another example, the cell samples are fixed using a suitable fixation reagent, e.g. paraformaldehyde (PF A), and permeabilised using a suitable permeabilization reagent, such as a detergent (e.g. saponin, Triton X-100, or Tween-20). In an example, the cell samples are fixed prior to the labelling step. In an example, the cell samples are fixed and permeabilised prior to the labelling step.

[0141] Samples are then contacted with a labelled detection antibody for each viral antigen present in the immunogenic composition. Suitable detection antibodies will be apparent to the person skilled in the art and can be selected according to each viral antigen present in the immunogenic composition.

[0142] In an example, the sample is contacted with a labelled detection antibody that binds the HA protein of an influenza A H1N1 strain, and a labelled detection antibody that binds the NA protein of an influenza A H1N1 strain. In an example, the sample is contacted with a labelled detection antibody that binds the HA protein of an influenza A H3N2 strain, and a labelled detection antibody that binds the NA protein of an influenza A H3N2 strain.

[0143] In an example, the sample is contacted with a labelled detection antibody that binds the HA protein of an influenza B / Victoria strain, and a labelled detection antibody that binds the NA protein of an influenza B / Victoria strain.

[0144] In an example, the sample is contacted with a labelled detection antibody that binds the HA protein of an influenza B / Yamagata strain, and a labelled detection antibody that binds the NA protein of an influenza B / Yamagata strain.

[0145] For the avoidance of doubt, the term “labelled detection antibody” in the context of the present disclosure encompasses both a primary antibody that is directly conjugated to the label, and a primary antibody that is contacted with a secondary antibody which is conjugated to the label. Accordingly, in an example, the labelled detection antibody is a primary antibody directly conjugated to the label. In another example, the labelled detection antibody comprises a primary antibody that binds a viral antigen, and a secondary antibody that binds the primary antibody, wherein the secondary antibody is directly conjugated to the label. In an example, the labelled detection antibody comprises a primary antibody complexed (or conjugated) to a fluorescently labelled secondary antibody. Methods for performing such complexions are known in the art and can be performed using commercially available kits, such as a Zenon™ kit (Thermo Fisher).

[0146] Detectable labels are well known in the art and include, for example, a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a prosthetic group, a contrast agent and an ultrasound agent. In an example, the detectable label is a fluorescent label.

[0147] Fluorescent labels useful in the practice of the present disclosure can include, without limitation, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5 -Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5- HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6C; 6- CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7- Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange+DNA; Acridine Orange+RNA; Acridine Orange, both DNA & RNA; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350; Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 532; Alexa Fluor 546; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 633; Alexa Fluor 647; Alexa Fluor 660; Alexa Fluor 680; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio Dye, Zn2+; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG CBQCA; ATTO-TAG FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisamninophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET Bimane; Bisbenzamnide; Bisbenzimide (Hoechst); bis-BTC=Ratio Dye, Zn2+; Blancophor FFG; Blancophor SV; BOBO-1; BOBO-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO-1; BO-PRO-3; Brilliant Sulphoflavin FF; BTC-Ratio Dye Ca2+; BTC-5N-atio Dye, Zn2+; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue; Cascade Yellow 399; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP— Cyan Fluorescent Protein; CFP / YFP; FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine; Coelenterazine cp (Ca2+Dye); Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2; Cy3.1 8; Cy3.5; Cy3; Cy5.1 8; Cy5.5; Cy5; Cy7; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4- ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DilCl 8(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC 18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilCl 8(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; Red fluorescent protein; DTAF; DY- 630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; FITC Antibody; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1-43; FM 4-46; Fura Red (high pH); Fura Red / Fluo-3; Fura-2, high calcium; Fura-2, low calcium; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted (rsGFP), GFP wild type, non- UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LIVE / DEAD Kit Animal Cells, Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue, LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag- Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Greene 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP; sgBFP (super glow BFP); sgGFP; sgGFP (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYT; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOXBlue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red; Texas Red- X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE- Cy5); TRITC (TetramethylRodamine-IsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3.

[0148] Fluorescent labels can be classified according to their excitation and emission wavelength. The classifications include UV (excitation 300-379), violet (380-439 nm), blue (440-509 nm), green / yellow (510-599 nm), red (600-729), and near infrared / infrared (730+ nm). Non-limiting examples of fluorescent labels in each of these categories is provided in Table 1.

[0149] Table 1 : Classification of fluorescent labels according to excitation spectra

[0150] In an example, each fluorescent label is selected from a group consisting of: a fluorescent label with an excitation wavelength of 300-379 nm; a fluorescent label with an excitation wavelength of 380-439 nm; a fluorescent label with an excitation wavelength of 440-509 nm; a fluorescent label with an excitation wavelength of 510-599 nm; a fluorescent label with an excitation wavelength of 600-729 nm; and a fluorescent label with an excitation wavelength of over 730 nm.

[0151] In the context of the present disclosure, it is preferable to select fluorescent labels with different excitation and emission wavelengths so that the respective labels are able to be visually distinguished from each other. Accordingly, each labelled detection antibody in a sample should comprise a distinct fluorescent label, wherein the distinct fluorescent labels do not have substantial spectral overlap. “Spectral overlap” as used herein refers to the phenomenon when a fluorescent label exhibits fluorescence that “spills over” into a detector channel. This occurs because most fluorochromes have broad emission spectra. Spectral overlap of emission spectra between fluorophores can be removed by compensating for the expected amount of spectral overlap. Compensation is the process of correcting the spillover from our primary signal in each secondary channel it is measured in. However, it is generally considered good practice to select fluorophores which have minimal overlap in their emission spectra to minimize the amount of compensation required.

[0152] There are various online tools available that assist with designing and selecting suitable fluorescent labels that do not have substantial spectral overlap. Examples include Thermofisher Fluorescence SpectraViewer (https: / / www.thermofisher.com / order / fluorescence-spectraviewer) and BD® Spectrum Viewer (https: / / www.bdbiosciences.com / en-au / resources / bd-spectrum-viewer).

[0153] In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 440-509 nm and a second antibody comprising a fluorescent label that has an excitation wavelength of 600- 729 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 440-509 nm and a second antibody comprising a fluorescent label with an excitation wavelength of over

[0154] 730 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 440-509 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 510- 599 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 440-509 nm and a second antibody comprising a fluorescent label with an excitation wavelength of SOO- 379 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 440-509 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 3 SO- 439 nm.

[0155] In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 300-379 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 510- 599 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 380-439 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 510- 599 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 600-729 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 510- 599 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of over 730 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 510- 599 nm.

[0156] In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 600-729 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 300- 379 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 600-729 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 3 SO- 439 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 600-729 nm and a second antibody comprising a fluorescent label with an excitation wavelength of over 730 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 380-439 nm and a second antibody comprising a fluorescent label with an excitation wavelength of 300- 379 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 380-439 nm and a second antibody comprising a fluorescent label with an excitation wavelength of over 730 nm. In an example, a sample of the disclosure is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 300-379 nm and a second antibody comprising a fluorescent label with an excitation wavelength of over 730 nm.

[0157] In an example, the sample is contacted with a first antibody comprising a fluorescent label that has an excitation wavelength of 440-509 nm and a second antibody comprising a fluorescent label that has an excitation wavelength of 600-729 nm. In this example, the fluorescent label that has an excitation wavelength of 440-509 nm is selected from Green Fluorescent Protein (GFP), BODIPY FL, NovaFluor Blue 510, Fluorescein (FITC), Alexa Fluor 488, Oregon Green 488, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, PerCP-Cyanine5.5, or PerCP-eFluor 710. In an example, the fluorescent label that has an excitation wavelength of 440-509 nm is Fluorescein (FITC), Alexa Fluor 488, or Oregon Green 488. In an example, the fluorescent label that has an excitation wavelength of 440-509 nm is Fluorescein (FITC). In an example, the fluorescent label that has an excitation wavelength of 440-509 nm is Alexa Fluor 48. The person skilled in the art will appreciate that any other fluorescent label may be used provided that it has an excitation wavelength of 440-509 nm.

[0158] In an example, the fluorescent label that has an excitation wavelength of 600-729 nm is selected from NovaFluor Red 660, Allophycocyanin (APC), Cy5, eFluor 660, Alexa Fluor 647, NovaFluor Red 685, NovaFluor Blue 690, Alexa Fluor 660, NovaFluor Red 700, Alexa Fluor 680, NovaFluor Red 710, Alexa Fluor 700, NovaFluor Red 725, or NovaFluor Red 755. In an example, the fluorescent label that has an excitation wavelength of 600-729 nm is Allophycocyanin (APC). The person skilled in the art will appreciate that any other fluorescent label may be used provided that it has an excitation wavelength of 440-509 nm.

[0159] In one example, one sample of the cell population is taken for each viral strain of the multivalent immunogenic composition. For example, where there are two viral strains (i.e., a first and second viral strain), two samples are taken and contacted with antibodies that bind each of the viral antigens encoded by the RNA. Where there are three viral strains (i.e., a first, second, and third viral strain), three samples are taken and contacted with antibodies that bind each of the viral antigens encoded by the RNA. Where there are four viral strains (i.e., a first, second, third, and fourth viral strain), four samples are taken and contacted with antibodies that bind each of the viral antigens encoded by the RNA, and so on.

[0160] However, as the person skilled in the art would appreciate, the number of samples taken is not particularly limited, provided that the sample is able to be contacted by a separate labelled detection antibody for each antigen. For example, where there are 8 antigens (e.g. two antigens for four separate viral strains), one sample may be taken and contacted with 8 different labelled detection antibodies that bind to each antigen, wherein each labelled detection antibody comprises a distinct fluorescent label.

[0161] Cells can be labelled with labelled detection antibodies using routine techniques in the art. Generally, the recommended quantity of each detection antibody is added to a sample comprising cells resuspended in an appropriate volume of a suitable buffer and incubated under recommended conditions. After the labelling reaction is complete, cells can be washed by centrifugation in a suitable buffer to remove unbound antibody. The cell pellet can be re-suspended in an appropriate volume of suitable buffer and passed through a cell strainer to ensure a suspension of single cells in suitable buffer for analysis.

[0162] Analysis

[0163] In an example, the potency assay of the present disclosure is a flow cytometrybased assay. In an example, the flow cytometry is fluorescence-activated cell sorting (FACS). In general, flow cytometry involves the passage of individual cells through the path of one or more laser beams. A scattering of a beam and excitation of any fluorescent molecule attached to, or found within, a cell is detected by photomultiplier tubes to create a readable output. Often optical filters and beam splitters direct various scattered light to detectors, which generate electronic signals proportional to intensity of light signals received. Data can be collected, stored in computer memory, and cell characteristics analyzed based on fluorescent and light scattering properties.

[0164] In some examples, the number of cells that are measured by flow cytometry is about 1,000 cells, about 5,000 cells, about 10,000 cells, about 40,000 cells, about 100,000 cells, about 500,000 cells, about 1,000,000 cells, or more than 1,000,000 cells. In some instances, the number of cells that are measured by flow cytometry is up to about 1,000 cells, up to about 5,000 cells, up to about 10,000 cells, up to about 40,000 cells, up to about 100,000 cells, up to about 500,000 cells, up to about 1,000,000 cells, or more than 1,000,000 cells.

[0165] Cell samples of the disclosure may be labelled with two or more fluorescent labels and then excited by one or more lasers to emit light at the fluorophore emission frequency or frequencies. In some examples, fluorescence is measured as cells pass through multiple laser beams simultaneously. Several detection elements, e.g. fluorophore- conjugated antibodies or fluorescence markers, can be used simultaneously, so measurements made as one cell passes through a laser beam may consist of scattered light intensities as well as light intensities from each fluorophore. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more different fluorescent labels are used. In an example, a combination of 2 fluorescent labels is used for each sample analysed. In an example, a combination of 3 fluorescent labels is used for each sample analysed. In an example, a combination of 4 fluorescent labels is used for each sample analysed. In an example, a combination of 5 fluorescent labels is used for each sample analysed. In an example, a combination of 6 fluorescent labels is used for each sample analysed. In an example, a combination of 7 fluorescent labels is used for each sample analysed. In an example, a combination of 8 fluorescent labels is used for each sample analysed.

[0166] In some examples, flow cytometry data is presented as 2-dimensional (2D) plots of parameters called cytograms. Often in cytograms, two measurement parameters are depicted such as one on an x-axis and one on a y-axis. In some instances, parameters depicted comprise at least one of side scatter signals (SSCs), forward scatter signals (FSCs), and fluorescence. In other examples, data in a cytogram is displayed as at least one of a dot plot, a pseudo-colour dot plot, a contour plot, or a density plot. Alternatively, or additionally, flow cytometry data can be presented as a single parameter histogram.

[0167] Flow cytometry data is conventionally analyzed by gating. Often sub-populations of cells are gated or demarcated within a plot. Gating can be performed manually or automatically. Manual gates, by way of non-limiting example, can take the form of polygons, squares, or dividing a cytogram into quadrants or other sectional measurements. In some instances, an operator can create or manually adjust the demarcations to generate new sub-populations of cells. Alternately or in combination, gating is performed automatically. Gating can be performed, in some part, manually or in some part automatically.

[0168] In some examples, gating involves using scatter signals, for example forward scatter (FSC) and side scatter (SSC), to differentiate subcellular debris from cells of interest. In some examples, single cells are gated from multiple or clumps of cells. In some examples, gating is applied to at least one fluorescent label to identify cells that are positive for a viral antigen of interest. In some examples, gating is applied to different fluorescent label combinations to identify cells that are positive for combinations of viral antigens of interest. In some examples, a subset of cells is gated for further analysis, for example, cells that express are positive for two fluorescent labels (e.g., “double-positive” cells).

[0169] The potency of immunogenic compositions disclosed herein can be determined by determining the level of each antigen by detecting each labelled detection antibody in each sample and determining the number or percentage of cells that are positive for each antigen in each sample.

[0170] In some examples, data analysis can be based on a single hit kinetics model (SHKM), also known as Poisson frequency distribution, or single hit Poisson model (SHPM). The SHPM model predicts how the percentage of cells positive for each antigen increases as the amount (or dose) of RNA increases in the transfection reaction. In an example, the SHPM model can be represented by the following formula (Equation 1):

[0171] % antigen positive = 100% x (l-e'cxDose) where:

[0172] Dose = RNA mass (nanograms (ng)) c is a proportionality constant (c has units of ng'1) e is Euler’s number, 2.71828.

[0173] Accordingly, the fraction of transfected cells that are antigen negative ( / o) can be expressed as:

[0174] , % antigen postive

[0175] Jto — I- - 100%

[0176] Equation 1 can be expressed as: fo= e'cxDose(Equation 2)

[0177] Equation 2 can be expressed as:

[0178] Info = - c x Dose Using this model, linear regression can be applied to values plotted on an x, y, graph that represent a straight line to calculate the slope. The value of the slope represents the specific activity, which is the increase in the probably of successful transfection per RNA unit mass.

[0179] In some examples, the potency value is calculated from a standard curve of serial RNA dilutions. In this example, a series of two-fold dilutions of the immunogenic composition (e.g. comprising RNA encoding each antigen formulated in LNPs at varying concentrations of RNA) is performed. The serial dilution is based on the concentration (e.g. ng) of RNA in the LNP / RNA sample. An exemplary 2-fold dilution series includes 800 ng RNA, 400 ng RNA, 200 ng RNA, 100 ng RNA, 50 ng RNA, 25 ng RNA, 12.5 ng RNA, 6.25 ng RNA, 3.125 ng RNA, 1.56 ng RNA, 0.78 ng RNA, and 0.39 ng RNA. Each dilution is incubated with a population of cells under culture conditions, stained with a labelled detection antibody for each antigen encoded by the RNA, and analysed by flow cytometry according to the methods disclosed herein. The percentage of cells positive for each antigen for each serial dilution is determined (e.g. in the context of influenza, cells that are positive for both HA and NA). The number of cells positive for each antigen in each serial dilution can be plotted as a standard curve. A linear regression analysis can be performed on the curve to calculate its slope. The specific potency can then be calculated by multiplying the value of the slope by a constant (linear regression slope x 105). In an example, specific potency can be calculated according to the following formula: specific potency (ng-1) = slope x (linear regression slope x 105) ± standard error.

[0180] Antigens

[0181] Methods of the present disclosure are particularly useful for determining the potency of immunogenic compositions comprising multiple RNA-encoded antigens. Antigens suitable for use in the immunogenic compositions described herein will be apparent to the skilled person and, for example, include proteins and peptides derived from any pathogen. For example, the antigen is a virus, bacteria, a fungus or a protozoan.

[0182] Viral antigens

[0183] Viral antigens that can be encoded by the RNA will be apparent to the skilled person and include, for example, proteins and peptides from a Orthomyxoviruses (e.g., Influenza A, B and C), Paramyxoviridae viruses (Pneumoviruses (e.g., Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkey rhinotracheitis virus), Paramyxovirus types 1-4 (PIV), Mumps, Sendai viruses, Simian virus 5)), Bovine parainfluenza virus, Nipahvirus, Henipavirus and Newcastle disease virus), Poxviridae (e.g., Variola vera, including but not limited to, Variola major and Variola minor, Metapneumoviruses, such as human metapneumovirus (hMPV) and avian metapneumoviruses (aMPV)), Morbilliviruses (e.g., Measles), Picomaviruses (e.g., Enteroviruses, Rhinoviruses, Heparnavirus, Parechovirus, Cardioviruses and Aphthoviruses), Enteroviruseses (e.g., Poliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus types 1 to 9, 11 to 27 and 29 to 34 and Enterovirus 68 to 71), Bunyaviruses (e.g., California encephalitis virus), Phlebovirus (e.g., Rift Valley Fever virus), Nairovirus (e.g., Crimean-Congo hemorrhagic fever virus), Hepamaviruses (e.g., Hepatitis A virus (HAV)), Togaviruses (e.g., Rubivirus, an Alphavirus, or an Arterivirus), Flaviviruses (e.g., Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus), Pestiviruses (e.g., Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV)), Hepadnaviruses (e.g., Hepatitis B virus, Hepatitis C virus), Rhabdoviruses (e.g., Lyssavirus (Rabies virus) and Vesiculovirus (VSV)), Caliciviridae (e.g., Norwalk virus, and Norwalk-like Viruses (e.g., Hawaii Virus and Snow Mountain Virus); Coronaviruses (e.g., severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), SARS coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV)), Retroviruses (e.g., Oncovirus, a Lentivirus or a Spumavirus), Reoviruses (e.g., Orthoreo virus, a Rotavirus, an Orbivirus, or a Coltivirus), Parvoviruses (e.g., Parvovirus B 19), Delta hepatitis virus (HDV), Hepatitis E virus (HEV), Human Herpesviruses (e.g., Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8)), Papovaviruses (e.g., Papillomaviruses and Polyomaviruses), Adenoviruess and Arenaviruses.

[0184] In an example, the antigen / s of the present disclosure is a viral antigen from a respiratory virus. Respiratory viral antigens that can be encoded by the RNA will be apparent to the skilled person and include, for example, proteins and peptides from a Orthomyxoviruses (e.g., Influenza A, B and C), Paramyxoviridae viruses (Pneumoviruses (e.g., Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkey rhinotracheitis virus), Paramyxoviruses (PIV), and Metapneumovirus such as human metapneumovirus (hMPV) and avian metapneumoviruses (aMPV)), Picomaviruses (e.g., Rhinoviruses) and Coronaviruses (e.g., severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), SARS coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV)).

[0185] Influenza

[0186] In one example, the antigen / s of the present disclosure is a viral antigen from an influenza virus. Influenza viruses are enveloped RNA viruses, belonging to the family Orthomyxoviridae. Three genera of this family, influenza virus A, B and C, cause influenza in humans. Influenza virus A and B are further classified, based on the viral surface proteins hemagglutinin (HA) and neuraminidase (NA).

[0187] Suitable influenza viral antigens include haemagglutinin (HA), neuraminidase (NA), matrix proteins (Ml, M2, NB and BM2), a heterotrimeric RNA-dependent RNA polymerase (made up of one polymerase acidic subunit (PA), and two polymerase basic subunits (PB1 and PB2)), nucleoprotein (NP), and two non- structural proteins (NS1 and NS2; NS2 is also known as nuclear export protein (NEP)), and pro-apoptotic peptide (PB1-F2).

[0188] At the time of filing there are 18 described HA (H1-H18) and 11 described NA (Nl-Nl l) subtypes (e.g. “strains”) of influenza A viruses. There are two antigenically and genetically distinct lineages of influenza B viruses: the Victoria lineage (“B / Victoria”) and the Yamagata lineage (“B / Yamagata).

[0189] Non limiting examples of influenza A subtypes include: H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1NS, H1N9, H1N10, H1N11, H2N1, H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8, H2N9, H2N10, H2N11, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8, H3N9, H3N10, H3N11, H4N1, H4N2, H4N3, H4N4, H4N5, H4N6, H4N7, H4N8, H4N9, H4N10, H4N11, H5N1H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H5N10, H5N11, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N9, H6N10, H6N11, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, N7N8, H7N9,

[0190] H7N10, H7N11, HSN!, H8N2, H8N3, H8N4, H8N5, H8N6, H8N7, H8N8, H8N9,

[0191] H8N10, H8N11, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9,

[0192] H9N10, H9N11, H10DN1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, HIONS,

[0193] H10N9, H10N10, H10N11, H11N1, H11N2, H11N3, H11N4, H11N5, H11N6, H11N7, HUNS, H11N9, H11N10, H11N11, H12N1, H12N2, H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H12N10, H12N11, H13N1, H13N2, H13N3, H13N4, H13N5, H13N6, H13N7, H13N8, H13N9, H13N10, H13N11, H14N1, H14N2, H14N3, H14N4, H14N5, H14N6, H14N7, H14N8, H14N9, H14N10, H14N11, H15N1, H15N2, H15N3, H15N4, H15N5, H15N6, H15N7, H15N8, H15N9, H15N10, H15N11, H16N1, H16N2, H16N3, H16N4, H16N5, H16N6, H16N7, H16N8, H16N9, H16N10, H16N11, H17N1, H17N2, H17N3, H17N4, H17N5, H17N6, H17N7, H17N8, H17N9, H17N10, H17N11, H18N1, H18N2, H18N3, H18N4, H18N5, H18N6, H18N7, H18N8, H18N9, H18N10, and H18Nll.

[0194] Influenza virus strains for use in seasonal vaccines change from season to season. Authorities such as the World Health Organisation (WHO) and Centre for Disease Control (CDC) publish a list of the predominant circulating influenza strains each year and further publish recommended influenza strains for inclusion in influenza vaccines, or reference strains to guide selection of the strains for inclusion in influenza vaccines. Accordingly, the first, second, and / or fourth influenza strain may be recommended for inclusion in an immunogenic composition by a public health authority (e.g., the WHO).

[0195] As used herein, the term “seasonal influenza virus strain” refers to a strain of influenza virus to which a subject population is exposed to on a seasonal basis. The immunogenic composition described herein may be suitable for protecting against seasonal virus strains that are presently being spread or are endemic within a human population. In addition, the immunogenic composition described herein may be suitable for protecting against emerging seasonal virus strains. Nucleotide sequences of all known influenza strains and antigens can be readily determined via online databases, such as the Influenza Virus Resource (https: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / ).

[0196] Non-limiting examples of influenza A strains include: influenza A / Califomia / 07 / 2009 (H1N1); influenza A / Michigan / 45 / 2015 / (HlNl); influenza A / Netherlands / 602 / 2009 (H1N1); influenza A / Vietnam / 1194 / 2004 (H5N1); influenza A / Vietnam / 1203 / 2004 (H5N1); influenza A / Hong Kong / 4801 / 2014 (H3N2); A / Hong Kong / 2671 / 2019 / (H3N2); influenza A / Panama / 2007 / 1999 (H3N2); influenza

[0197] A / Kansas / 14 / 2017 / (H3N2); influenza A / South Australia / 34 / 2019 / (H3N2); influenza A / Tasmania / 503 / 2020 / (H3N2); influenza A / Cambodia / e08263601 / 2020 / (H3N2); influenza A / Darwin / 6 / 2021 / (H3N2);. influenza A / Perth / 20 / 2020 / (H3N2).

[0198] Non-limiting examples of influenza B strains include B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus; B / Brisbane / 60 / 2008 (Victoria Lineage); B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0199] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two seasonal influenza virus strains. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from three seasonal influenza virus strains. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from four seasonal influenza virus strains.

[0200] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two influenza viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from three influenza viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from four influenza viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two influenza A viruses and one influenza B virus. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from two influenza A viruses and two influenza B viruses. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H1N1 virus, an influenza A H3N2 virus, and an influenza B virus. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H1N1 virus, an influenza A H3N2 virus, an influenza B / Victoria virus, and an influenza B / Yamagata virus.

[0201] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H1N1 strain selected from: A / Califomia / 07 / 2009 (H1N1); influenza A / Michigan / 45 / 2015 / (HlNl); influenza A / Netherlands / 602 / 2009 (H1N1); and A / Sydney / 5 / 2021 (H1N1).

[0202] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an influenza A H3N2 strain selected from: influenza A / Hong Kong / 4801 / 2014 (H3N2); A / Hong Kong / 2671 / 2019 / (H3N2); influenza A / Panama / 2007 / 1999 (H3N2); influenza A / Kansas / 14 / 2017 / (H3N2); influenza A / South Australia / 34 / 2019 / (H3N2); influenza A / Tasmania / 503 / 2020 / (H3N2); influenza A / Cambodia / e08263601 / 2020 / (H3N2); influenza A / Darwin / 6 / 2021 / (H3N2);. influenza A / Perth / 20 / 2020 / (H3N2).

[0203] In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B strain selected from: B / Austria / 1359417 / 2021 (B / Victoria lineage); B / Brisbane / 60 / 2008 (Victoria Lineage); B / Phuket / 3073 / 2013 (B / Yamagata lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from two influenza B strain selected from: B / Austria / 1359417 / 2021 (B / Victoria lineage); B / Brisbane / 60 / 2008 (Victoria Lineage); B / Phuket / 3073 / 2013 (B / Yamagata lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B / Victoria strain and an influenza B / Yamagata strain. In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B / Victoria strain selected from: B / Austria / 1359417 / 2021 (B / Victoria lineage); B / Brisbane / 60 / 2008 (Victoria Lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA and NA protein from an influenza B / Yamagata strain selected from: B / Phuket / 3073 / 2013 (B / Yamagata lineage). In an example, immunogenic compositions of the disclosure comprise one or more RNAs encoding a HA protein and NA protein from an A / Sydney / 5 / 2021 (H1N1); an A / Darwin / 6 / 2021 (H3N2); a B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus; and a B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0204] Coronavirus

[0205] Immunogenic compositions of the present disclosure can comprise an RNA encoding a viral antigen from a coronavirus, for example an alphacoronavirus, a betacoronavirus, a gammacoronavirus and / or a deltacoronavirus.

[0206] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from an alphacoronavirus. Examples of alphacoronaviruses include Alphacoronavirus 1, Human coronavirus 229E (HCoV 229E), Human coronavirus NL63 (HCoV NL63), Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2 and Scotophilus bat coronavirus 512.

[0207] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from a gammacoronavirus. Examples of gammacoronaviruses include Avian coronavirus and Beluga whale coronavirus SW 1.

[0208] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from a deltacoronavirus. Examples of deltacoronaviruses include Bulbul coronavirus HKU11 and Porcine coronavirus HKU15

[0209] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from a betacoronavirus. Examples of betacoronaviruses include Betacoronavirus 1 (Bovine Coronavirus, Human coronavirus OC43), Hedgehog coronavirus 1, Human coronavirus HKU1 (HCoV HKU1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS-CoV-2) and Tylonycteris bat coronavirus HKU4.

[0210] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from a betacoronavirus selected from the group consisting of Middle East respiratory syndrome-related coronavirus (MERS-CoV) and Severe acute respiratory syndrome-related coronavirus (SARS-CoV or SARS-CoV-2). For example, the viral antigen is from MERS-CoV. In another example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from SARS-CoV. In an example, immunogenic compositions of the disclosure comprise an RNA encoding a viral antigen from SARS-CoV-2.

[0211] In an example, immunogenic compositions of the disclosure comprise an RNA encoding a spike (S) protein and / or an RNA encoding a nucleocapsid (N) protein of a coronavirus. In an example, immunogenic compositions of the disclosure comprise an RNA encoding a spike (S) protein and / or an RNA encoding a nucleocapsid (N) protein of SARS-CoV-2. In an example, immunogenic compositions of the disclosure comprise an RNA encoding a spike (S) protein and / or an RNA encoding a nucleocapsid (N) protein from SARS-CoV-2 strain 2019-nCoV / USA-WAl / 2020.

[0212] The present disclosure contemplates that the viral antigen can be a modified S protein. In an example, a modified S protein comprises a mutation in the receptor binding domain. For example, the mutation is selected from the group consisting of S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, 1468 V, E4710, 1472 V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, P479L, P479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S and Y505H, Y508H. In an example, a modified S protein comprises a mutation in the receptor binding domain selected from the group consisting of N439K, N439L, L452R, S477N, T478I, V483 A and E484D.

[0213] In an example, a modified S protein comprises a mutation selected from the group consisting of P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V395I, F400C, R403K, R403S, D405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417R, K417N, 1418V, Y421S, Y423C, Y423F, Y423S, D427Y, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S and D614G. Respiratory syncytial virus

[0214] In an example, the immunogenic composition of the disclosure comprises an RNA encoding an antigen from a respiratory syncytial virus strain. In an example, respiratory syncytial virus strain is a RSV-A. In an example, respiratory syncytial virus strain is a RSV-B.

[0215] In an example, the antigen is an attachment glycoprotein (G), the fusion protein (F), or the small hydrophobic protein (SH).

[0216] In an example, immunogenic composition of the disclosure comprises an RNA encoding the G protein from an RSV-A strain. In an example, an immunogenic composition of the disclosure comprises an RNA encoding the G protein from an RSV- B strain.

[0217] Parainfluenza virus

[0218] In an example, the immunogenic composition of the disclosure comprises an RNA encoding an antigen from a parainfluenza virus (HPIV) strain. In an example, the parainfluenza virus strain is HPIV-1, HPIV-2, HPIV-3, or HPIV-4. In an example, HPIV- 4 is HPIV-4A or HPIV-4B. In an example, the antigen is the nucleocapsid protein (NP), the phosphoprotein (P), the fusion glycoprotein (F), the matrix protein (M), the hemagglutinin-neuraminidase (HN) glycoprotein, or the RNA polymerase (L). For example, the immunogenic composition of the disclosure comprises an RNA encoding the NP, P, F, M, HN, or L protein from HPIV-1, HPIV-2, HPIV-3, or HPIV-4.

[0219] Human metapneumovirus

[0220] In an example, the immunogenic composition of the disclosure comprises an RNA encoding an antigen from a human metapneumovirus (HMPV) strain. In an example, the human metapneumovirus virus strain is HMPV Al, HMPV A2a, HMPV A2b, HMPV Bl, or HMPV B2. In an example, the antigen is the nucleocapsid protein (N), the phosphoprotein (P), the fusion (F), the matrix protein (M), the M2 protein, the SH protein, the attachment glycoprotein (G), or the RNA polymerase (L). For example, the immunogenic composition of the disclosure comprises an RNA encoding the N, P, M, F, M2, SH, G, or L protein from HMPV Al, HMPV A2a, HMPV A2b, HMPV Bl, or HMPV B2.

[0221] Epstein-Barr virus

[0222] In an example, the immunogenic composition of the disclosure comprises an RNA encoding an antigen from a Epstein-Barr virus (EBV) strain. In an example, the EBV strain is EBV-1 or EBV-2. EBV-1 and EBV-2 can further be categorised according to the EBV nuclear antigen (EBNA-1). In an example, the EBV strain has nuclear antigen EBNA-2, EBNA-3A / 3, EBNA-3B / 4, or EBNA-3C / 6.

[0223] In an example, the antigen is EBNA-1. For example, the immunogenic composition of the disclosure comprises an RNA encoding the EBNA-1 protein from EBV-1 or EBV-2.

[0224] Polynucleotides

[0225] Immunogenic compositions comprise RNA, for example an mRNA, comprising a nucleotide sequence encoding at least two antigens as disclosed herein.

[0226] As used herein, the term “RNA (ribonucleic acid)” refers to a single stranded molecular chain of nucleotides chemically bonded by a series of ester linakges between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar in an adjacent nucleotide. Suitable forms of RNA will be apparent to the skilled person. In one example, the RNA is messenger RNA (mRNA). In one example, the mRNA encoding the antigen is a monocistronic mRNA. For example, the monocistronic mRNA is a conventional mRNA (cRNA) or a self-replicating RNA. In another example, the mRNA encoding the antigen is a multi ci str onic mRNA. For example, the multi ci str onic mRNA is a conventional mRNA (cRNA) or a self-replicating RNA.

[0227] Conventional (non-replicating) RNA

[0228] RNAs of the present disclosure encompass a non-replicating mRNA (also referred to as conventional mRNA (cRNA) or non-amplifying). The skilled person will understand that the cRNA of the present disclosure comprise in order from 5’ to 3’ : a 5 ’cap structure, a 5’-UTR, a fragment and / or a variant thereof, a first nucleotide sequence encoding a first antigen of interest, a second nucleotide sequence encoding a second antigen of interest, a 3’-UTR and a 3 ’tailing sequence (e.g. a polyadenylation signal or one or more poly-A tails). The cRNA of the present disclosure may further comprise a translation internal ribosome entry site (e.g. Kozak consensus sequence or IRES) operably linked to the chemottractant or antigen of interest. In an example, the present disclosure provides a monocistronic cRNA. In an example, the present disclosure provides a multi ci str onic cRNA.

[0229] Self-replicating RNA

[0230] RNAs of the present disclosure encompasses a self-replicating RNA (also known as self-amplifying RNA or sa-mRNA). In an example, the RNA is a self-replicating RNA (also known as a replicon). In an example, the present disclosure provides a monocistronic self-replicating RNA. In an example, the present disclosure provides a multi ci str onic self-replicating RNA.

[0231] The skilled person will understand that the self-replicating RNA of the present disclosure is based on the genomic RNA of RNA viruses. The RNA should be positive (+)-stranded so that it can be directly translated after delivery to a cell without the need for intervening replication steps (e.g., reverse transcription). Translation of the RNA results in the production of non- structural proteins (NSPs) which combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The complex then amplifies the original RNA, producing both antisense and sense transcripts, resulting in production of multiple daughter RNAs which may subsequently be translated and transcribed, enhancing overall protein expression.

[0232] In one example, the self-replicating RNA of the present disclosure comprises the non- structural proteins of the RNA virus, the 5’ and 3’ untranslated regions (UTRs) and the native subgenomic promoter.

[0233] In one example, the self-replicating RNA comprises one or more non- structural proteins of the RNA virus. For example, the RNA comprises at least one or more genes selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase and other non-structural viral proteins. For example, the selfreplicating RNA comprises a viral replicase (or viral polymerase).

[0234] It will be apparent to the skilled person that RNA suitable for use in the present disclosure may also include a 5' untranslated region (5’-UTR), a 3' untranslated region (3’UTR), and / or a coding or translating sequence. In addition, the RNA may comprise a 5' cap structure, a chain terminating nucleotide, a stem loop (e.g., a histone stem loop), a 3 ’ tailing sequence (e.g., a polyadenylation signal or one or more polyA tails. In another example, the self-replicating RNA comprises a 5'- and a 3 '-end UTR of the RNA virus. It will be apparent to the skilled person that the terms 5’ and a 3’UTR also encompasses the terms 5’ and 3’ conserved sequence elements (CSE). In one example, the selfreplicating RNA comprises a 5’- and a 3’-end CSE.

[0235] The self-replicating RNA of the present disclosure cannot induce production of infectious viral particles. For example, the self-replicating RNA of the present disclosure does not comprise viral genes encoding structural proteins necessary for production of viral particles.

[0236] In one example, the self-replicating RNA is derived from or based on an alphavirus. Suitable alphaviruses will be apparent to the skilled person and / or described herein. In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus, for example, a positive-stranded RNA virus. Suitable positive- stranded RNA viruses suitable for use in the present disclosure will be apparent to the skilled person and include, for example, a picornavirus, a flavivirus, a rubivirus, a pestivirus, a hepacivirus, a calicivirus, or a coronavirus.

[0237] Alphavirus

[0238] In one example, the self-replicating RNA of the present disclosure is derived from (or based on) an alphavirus.

[0239] Alphaviruses are the sole genus in the Togaviridae family and are an enveloped virus with a positive-sense, single-stranded RNA genome. The skilled person will understand that the alphavirus genome comprises two open reading frames (ORFs), non- structural and structural. The first ORF encodes four non-structural proteins (NSP1, NSP2, NSP3 and NSP4) necessary for transcription and replication of viral RNA. The second encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and El, which associate as a heterodimer. The viral membrane- anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion.

[0240] In one example, the self-replicating RNA of the present disclosure comprises a viral replicase (or viral polymerase). For example, the viral replicase is an alphavirus replicase, such as an alphavirus protein NSP4.

[0241] In one example, the self-replicating RNA of the present disclosure does not encode one or more alphavirus structural proteins (e.g., capsid and / or envelope glycoproteins). For example, the self-replicating RNA is unable to produce RNA- containing alphavirus virions (i.e., infectious viral particles).

[0242] In one example, the self-replicating RNA comprises a native alphavirus SG promoter. For example, the native alphavirus SG promoter is a minimal SG promoter (i.e., the minimal sequence required for initiation of transcription).

[0243] The skilled person will be aware of alphaviruses suitable for use in the present disclosure. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEE; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, S.A. AR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus may also include chimeric alphaviruses (e.g., as described by Perri et al, (2003) J. Virol. 77(19): 10394-403) that contain genome sequences from more than one alphavirus.

[0244] Regulatory elements

[0245] In an example, immunogenic compositions of the disclosure comprise a monocistronic mRNA (e.g., a cRNA or self-replicating RNA) comprising a nucleotide sequence encoding an antigen. In an example, immunogenic compositions of the disclosure comprise a monocistronic self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an internal ribosome entry site (IRES), or a subgenomic (SG) promoter.

[0246] In one example, immunogenic compositions of the disclosure comprise a monocistronic self-replicating RNA comprising a nucleotide sequence encoding an antigen, operably linked to a SG promoter.

[0247] In an example, immunogenic compositions of the disclosure comprise a multi ci str onic mRNA (e.g., a cRNA or self-replicating RNA) comprising a first nucleotide sequence encoding a first antigen and a second nucleotide sequence encoding a second antigen of interest. In one example, the nucleotide sequences encoding the antigens of interest are operably linked to a regulatory element selected from the group consisting of a Kozak consensus sequence, an IRES, a SG promoter and combinations thereof.

[0248] In one example, the first nucleotide sequence encoding a first and the second nucleotide sequence encoding a second antigen of interest are operably linked to a regulatory element selected from the group consisting of a SG promoter and an IRES.

[0249] In one example, immunogenic compositions of the disclosure comprise a multi ci str onic self-replicating RNA comprising a first nucleotide sequence encoding a first antigen operably linked to a SG promoter; and a second nucleotide sequence encoding a second antigen operably linked to a regulatory element a promoter selected from the group consisting of a SG promoter and an IRES.

[0250] Subgenomic Promoter

[0251] SG promoters (also known as ‘junction region’ promoters) suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein.

[0252] In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. In one example, the native SG promoter is a minimal SG promoter. For example, the minimal SG promoter is the minimal sequence required for initiation of transcription. . In one example, the native SG promoter is an extended SG promoter. For example, the extended SG promoter is a minimal SG promoter extended at the 5’ end with nucleotides occurring in a sequence encoding a non-structural protein (e.g., NSP4) of the RNA virus (e.g., an alphavirus). In one example, the extended SG promoter is a minimal SG promoter extended at the 5’ end with nucleotides occurring in a sequence encoding an alphavirus NSP4.

[0253] In one example, the polynucleotide of the disclosure comprises a SG promoter from any alphavirus. For example, the RNA of the disclosure (e.g., cRNA or selfreplicating RNA) comprises a SG promoter from any alphavirus.

[0254] In one example, the self-replicating RNA comprises a SG promoter from any alphavirus.

[0255] In one example, the polynucleotide of the present disclosure is a multi ci stronic mRNA that comprises two or more nucleotide sequences encoding a chemoattractant and an antigen of interest. In one example, the two or more nucleotide sequences are each operaby linked to SG promoters. When two or more SG promoters are present in the RNA of the present disclosure, the promoters can be the same or different. For example, the two or more SG promoters are derived from the same alphavirus. In another example, the two or more SG promoters are derived from different alphaviruses.

[0256] When two or more SG promoters are present in the self-replicating RNA of the present disclosure, the promoters can be the same or different. For example, the two or more SG promoters are derived from the same alphavirus. In another example, the two or more SG promoters are derived from different alphaviruses.

[0257] Internal Ribosomal Entry Site (IRES)

[0258] IRES suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein.

[0259] In one example, the IRES is derived from encephalomyocarditis virus (EMCV). For example, the IRES is a wild-type IRES from EMCV.

[0260] In one example, the IRES is derived from a fibroblast growth factor 1 A (FGF1 A) IRES.

[0261] In addition, synthetic IRES elements have been described, which can be designed, according to methods know in the art to mimic the function of naturally occurring IRES elements (see Chappell, SA et al. Proc. Natl Acad. Sci. USA (2000) 97(4): 1536-41). Kozak consensus sequence

[0262] As used herein, the term “Kozak consensus sequence” refers to a nucleotide sequence identified in eukaryotic genes that facilitates the translation of the gene by containing a start codon (also referred to as a translation initiation codon) which is recognised by a ribosome.

[0263] Exemplary Kozak consensus sequence are known in the art and / or described herein. In one example, the Kozak consensus sequence is GCCACC. In one example, the Kozak consensus sequence is ACCATGG. In another example, the Kozak consensus sequence is ACCATG.

[0264] 5’ untranslated region (5’UTR)

[0265] In one example, the self-replicating RNA comprises a 5'- UTR of the RNA virus.

[0266] As used herein, the term “5 ’-untranslated region” or “5 ’-UTR” refers to a noncoding region of an mRNA located at the 5 ’end of the translation initiation sequence (AUG).

[0267] Exemplary 5’-UTRs include, for example, 5 ’-UTR of haptoglobin (HP), fibrinogen beta chain (FGB), haptoglobin-related protein (HPR), albumin (ALB), complement component 3 (C3), fibrinogen alpha chain (FGA), alpha 6 collagen (C0I6A), alpha- 1 -antitrypsin (SERPINA1), alpha- 1 -anti chymotrypsin (SERPINA3) a fragment and / or a variant thereof.

[0268] In one example, the 5’UTR is a 5’UTR of a Venezuelan equine encephalitis virus (VEEV) or modified forms thereof.

[0269] In one example, the 5’UTR comprises at least one microRNA binding site, an AU rich element (ARE), a GC-rich element, a stem loop, and combinations thereof. microRNA binding site

[0270] As used herein, the term “microRNA binding site” refers to a sequence within a polyncleotide (e.g. within a DNA or RNA transcript) that has sufficient complementarity to all or one region of a miRNA to interact, associate or bind to the microRNA (miRNA).

[0271] As used herein, the term “microRNA” or “miRNA” refers to 19-25 nucleotide long non-coding RNAs that bind to the 5 ’-UTR of polynucleotides and down-regulate gene expression (e.g. by inhibiting translation). The presence of microRNA binding site(s) in the 5’UTR of the present disclosure can function to inhibit translation of the 5’- UTR.

[0272] Suitable miRNA binding sites for use in the present disclosure will be apparent to the skilled person and / or described herein. In one example, the miRNA binding site comprises a binding site for tissue specific microRNA or those regulating biological processes. For example, miRNA of the liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17- 92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). For example, microRNA that regulate biological processes such as angiogenesis (miR-132). Further exemplifying miRNA and miRNA binding sites are disclosed in US patent application US14 / 043,927.

[0273] 3 ’untranslated region (3’-UTR)

[0274] In one example, an RNA of the present disclosure comprises a 3 ’-untranslated region (3’-UTR).

[0275] As used herein, the term “3’-UTR” refers to a region of an mRNA located at the 3’end of the the translation termination codon (i.e. stop codon).

[0276] Exemplary 3’-UTRs include, for example, a 3’-UTR of arachidonate 5- lipoxygenase (AL0X5), alpha I collagen (C0L1A1), tyrosine hydroxylase (TH) gene, amino-terminal enhancer of split (AES), human mitochondrial 12S rRNA (mtRNRl), a fragment and / or a variant thereof.

[0277] In one example, the 3’UTR is a 3’UTR of a Sindbis virus (SINV) or modified forms thereof.

[0278] In one example, the 3’-UTR comprises or consists of a nucleotide sequence derived from a 3’-UTR of an albumin gene. In one example, the 3’-UTR comprises or consists of a nucleotide sequence derived from a 3’-UTR of a vertebrate a-globin gene. For example, the 3’-UTR comprises or consists of a nucleotide sequence derived from a 3’-UTR of a mammalian a-globin gene. For example, the 3’-UTR comprises or consists of a nucleotide sequence derived from a 3’-UTR of a human a-globin gene.

[0279] In one example, the 3’-UTR of the present disclosure further comprises at least one microRNA binding site, an AU rich element (ARE), a GC-rich element, a triple helix, a stem loop, one or more stop codons or a combination thereof.

[0280] Stop codon

[0281] As used herein, the term “stop codon” refers to a trinucleotide sequence within a mRNA that signals the stop of protein synthesis by a ribosome. In one example, the polynucleotide of the present disclosure comprises at least one stop codon at the 5’end of a 3’-UTR. For example, the stop codon is selected from UAG, UAA, and UGA.

[0282] In one example, the polynucleotide comprises two consecutive stop codons comprising a sequence UGAUGA.

[0283] In one example, the polynucleotide comprises two consecutive stop codons comprising a sequence UAAUAG.

[0284] 3 ’ tailing sequence

[0285] The RNA of the present disclosure comprises one or more 3’ tailing sequences located at the 3 ’end of the 3’UTR.

[0286] As described herein, the term “3’ tailing sequence” or “3’ tailing sequences” refers to a nucleotide sequence (e.g. polyadenylation signal) which induces the addition of non-encoded nucleotides to the 3 ’end of a mRNA or a nucleotide sequence (e.g. poly- A sequence) located at the 3’ end of a mRNA. A skilled person will appreciate that the 3 ’tailing sequence and / or products of the 3 ’tailing sequence in a mRNA functions to stabilise the mRNA and / or prevent the mRNA from degradation.

[0287] In one example, the one or more 3’ tailing sequences are selected from the group consisting of a poly-A sequence, polyadenylation signal, a G-quadruplex, a poly-C sequence, a stem loop and combinations thereof.

[0288] Poly-A sequence

[0289] As used herein, the term “polyA sequence” refers to a nucleotide sequence of Adenine (A) located at the 3 ’end of a mRNA. In the context of the present disclosure, the polyA sequence may be located within the mRNA or DNA (e.g. a DNA plasmid serving as a template for generating the mRNA by transcription of the vector).

[0290] Suitable poly-A sequence for use in the present disclosure will be apparent to the skilled person and / or are described herein. In one example, the poly-A sequence comprises consecutive (i.e. one after the other) adenosine nucleotides of any length (e.g. to 10 to 300). For example, the poly-A sequence comprises 36 consecutive adenosine nucleotides.

[0291] In one example, the poly-A sequence comprises consecutive adenosine nucleotides separated by one or more interrupting linkers. In one example, the poly-A sequence comprises consecutive adenosine nucleotides without an interrupting linker. Polyadenylation signal

[0292] As used herein, the term “polyadenylation signal” refers to a nucleotide sequence which induces polyadenylation. Polyadenylation is typically understood to be the addition of a polyA sequence to a RNA (e.g. to a premature mRNA to generate a mature mRNA). The polyadenylation signal may be located within a nucleotide sequence at the 3 ’-end of the polynucleotide (e.g. mRNA) to be polyadenylated.

[0293] Suitable polyadenylation signal for use in the present disclosure will be apparent to the skilled person and / or described herein.

[0294] In one example, the polyadenylation signal comprises a hexamer consisting of Adenine and Uracil / Thymidine nucleotides. In one example, the hexamer sequence comprises or consists of AAUAAA.

[0295] In one example, the 3 ’tailing sequence comprises a polyadenylation signal but does not comprise a polyA sequence.

[0296] 5 ’Cap

[0297] In one example, an mRNA according to the present disclosure comprises a 5 ’terminal cap structure.

[0298] As used herein, the term “5 ’cap structure” refers to a structure at the 5’ terminal end of a mRNA involved in nuclear export and binds a mRNA Cap Binding Protein (CBP). The 5 ’cap structure is known to stabilise mRNA through association of CBP with poly(A) binding protein to form a mature mRNA. Accordingly, the presence of a 5 ’cap structure in the mRNA of the present disclosure can further increase the stability of the mRNA compared to a mRNA without the 5 ’cap.

[0299] Exemplary 5 ’cap structure includes, for example, anti-reverse cap analogue (ARC A), N7,2'-0-dimethyl-guanosine (mCAP), inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), Capl, and Cap2.

[0300] Typically, an endogenous mRNA is 5 ’capped with a guanosine through a (5)’- ppp-(5)’ -triphosphate linkage attached to the 5 ’terminal nucleotide of the mRNA. The guanosine cap can then be methylated to a 7-methylguanosine (m7G) generating a 7mG(5’)ppp(5’)N,pN2p (CapO structure), where N represents the first and second 5 ’terminal nucleotide of the mRNA. The capO structure can be further 2’-O-methylated to produce 7mG(5’)ppp(5’)NlmpNp (Capl), and / or 7mG(5’)-ppp(5')NlmpN2mp (Cap2).

[0301] In one example, the polynucleotide of the present disclosure comprises an endogenous cap. As used herein, the term “endogenous cap” refers to a 5 ’cap synthesised in a cell. For example, endogenous cap is a natural 5’cap or a wild-type 5’cap. For example, the endogenous cap is a CapO, Capl, or Cap2 structure.

[0302] In one example, the polynucleotide of the present disclosure comprises an analog of an endogenous cap (also referred to as cap analog).

[0303] As used herein, the term “analogue thereof’ in the context of an endogenous cap or “cap analog” refers to a synthetic 5’cap. The cap analog can be used to produce 5’capped mRNA in in vitro transcription reactions. Cap analogs may be chemically (i.e. non-ezymatically) or enzymatically synthesized and / or linked to a nucleotide (e.g. 5 ’terminal nucleotide of an mRNA). Exemplary cap analogs are commercially available and include, for example, 3"-O-Me-m7G(5')ppp(5')G, G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs). In one example, the cap analog is N7,3'-O-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine (i.e. anti-reverse cap analogue (ARC A)).

[0304] Immunogenic compositions

[0305] It will be apparent to the skilled person and / or described herein, that the immunogenic composition comprising a polynucleotide, RNA, cRNA and / or selfreplicating RNA as disclosed herein may be present as naked RNA or in combination with lipids, polymers or other delivery system that facilitates entry into the cells.

[0306] In one example, the immunogenic composition of the present disclosure further comprises a LNP, a polymeric microparticle and an oil-in-water emulsion. For example, the polynucleotide, the cRNA and / or the self-replicating RNA is encapsulated in, bound to or adsorbed on a LNP, a polymeric microparticle, or an oil-in-water emulsion.

[0307] Lipid Nanoparticles

[0308] In one example, the immunogenic composition of the present disclosure further comprises a LNP.

[0309] It will be apparent that the term “lipid nanoparticle” or “LNP” refers to any lipid composition, including, but not limited to, liposomes or vesicles, where an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single; unilamellar or multiple; multilamellar) micelle-like lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles, wherein solid lipid nanoparticles lack lipid bilayers.

[0310] Lipid nanoparticles suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein. The lipids can have an anionic, cationic or zwitterionic hydrophilic head group. In one example, the lipid nanoparticle comprises a PEG-lipid, a sterol structural lipid and / or a neutral lipid. In one example, the lipid nanoparticle further comprises a cationic lipid. In one example, the lipid nanoparticle does not comprise a cationic lipid.

[0311] In one example, the LNP comprises a PEG-lipid. For example, the PEG-lipid is selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG- DMPE, PEG-DPPC, a PEG-DSPE lipid and combinations thereof.

[0312] In one example, the LNP comprises a structural lipid. For example, the structural lipid is selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and alpha-tocopherol and combinations thereof.

[0313] In one example, the LNP comprises a neutral lipid. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. For example, the neutral lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3 -phosphocholine

[0314] (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1 ,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3 -phosphoethanolamine (DSPE), l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero- 3 -phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3 -phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin and combinations thereof.

[0315] In one example, the LNP comprises a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy- N,Ndimethyl- 3 -aminopropane (DODMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5- bis((9z,12z)-octadeca-9,12,dien-l-yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-l- yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC) and dodecylphosphocholine. The lipids can be saturated or unsaturated.

[0316] Polymeric microparticles

[0317] In one example, the immunogenic composition of the present disclosure further comprises a polymeric microparticle.

[0318] The skilled person will be aware that various polymers can form microparticles to encapsulate or adsorb the polynucleotide, the cRNA and / or the self-replicating RNA of the present disclosure. It will be apparent that use of a substantially non-toxic polymer means that particles are safe, and the use of a biodegradable polymer means that the particles can be metabolised after delivery to avoid long-term persistence. Useful polymers are also sterilisable, to assist in the preparation of pharmaceutical grade formulations.

[0319] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(a- hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl- pyrrolidinones or polyester-amides, and combinations thereof.

[0320] Oil-in-water cationic emulsions

[0321] In one example, the immunogenic composition of the present disclosure further comprises an oil-in-water cationic emulsion.

[0322] Suitable oils for use in an oil-in-water emulsion will be apparent to the skilled person and / or are described herein. For example, the emulsion comprises one or more oils derived, for example, from an animal (e.g., fish) or a vegetable source (e.g., nuts, seeds, grains). The skilled person will recognise that biocompatible and biodegradable oils are preferentially used. Exemplary animal oils (i.e., fish oils) include cod liver oil, shark liver oils, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, corn oil.

[0323] In addition to the oil, the oil-in-water emulsion also comprises a cationic lipid to facilitate formation and stabilisation of the emulsion. Suitable cationic lipids will be apparent to the skilled person and / or are described herein. Exemplary cationic lipids include, but are not limited to, limited to: 1, 2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-Dimethylaminoethane)-carbamoyl] Cholesterol (DC Cholesterol), dimethyldioctadecyl-ammonium (DDA), l,2-Dimyristoyl-3-Trimethyl- AmmoniumPropane (DMTAP), dipalmitoyl[C16:0]trimethyl ammonium propane (DPTAP) and distearoyltrimethylammonium propane (DSTAP).

[0324] In some examples, the oil-in-water emulsion also comprises a non-ionic surfactant and / or a zwitterionic surfactant. The skilled person will be aware of surfactants suitable for use in the present disclosure. Exemplary surfactants include, but are not limited to: the polyoxyethylene sorbitan esters surfactants (e.g., polysorbate 20 and polysorbate 80) and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0325] Pharmaceutically acceptable carrier

[0326] Suitably, in immunogenic compositions of the disclosure or methods for administration of the same to a subject, the polynucleotide, RNA, cRNA and / or the selfreplicating RNA is combined with a pharmaceutically acceptable carrier as is understood in the art. Accordingly, one example of the present disclosure provides an immunogenic composition (e.g., a pharmaceutical composition) comprising the polynucleotide, RNA, cRNA and / or the self-replicating RNA of the disclosure (and any delivery system) combined with a pharmaceutically acceptable carrier.

[0327] In general terms, by “carrier” is meant a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating or lubricant that may be safely administered to any subject, e.g., a human. Depending upon the particular route of administration, a variety of acceptable carriers, known in the art may be used, as for example described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).

[0328] The immunogenic composition of the present disclosure are useful for parenteral, topical, oral, or local administration, intramuscular administration, aerosol administration, or transdermal administration, for prophylactic or for therapeutic treatment. In one example, the immunogenic composition is administered parenterally, such as intramuscularly, subcutaneously or intravenously. For example, the immunogenic composition may be administered intramuscularly. In another example, the immunogenic composition is administered parenterally, such as intramuscularly, subcutaneously or intravenously. For example, immunogenic composition is administered intramuscularly. Formulation of immunogenic composition to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising a immunogenic composition to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The immunogenic composition can be stored in the liquid stage or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

[0329] The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

[0330] Upon formulation, compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically / prophylactically effective. The dosage ranges for the administration of the immunogenic composition of the disclosure are those large enough to produce the desired effect. For example, the composition comprises an effective amount of the immunogenic composition. In one example, the immunogenic composition comprises a therapeutically effective amount of the self-replicating RNA. In another example, the immunogenic composition comprises a prophylactically effective amount of the selfreplicating RNA. In one example, the immunogenic composition comprises an effective amount of the RNA. In one example, the immunogenic composition comprises a therapeutically effective amount of the RNA. In another example, the immunogenic composition comprises a prophylactically effective amount of the RNA.

[0331] The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

[0332] Dosage can vary from about 0.1 mg / kg to about 300 mg / kg, e.g., from about 0.2 mg / kg to about 200 mg / kg, such as, from about 0.5 mg / kg to about 20 mg / kg, in one or more dose administrations daily, for one or several days.

[0333] In some examples, the immunogenic composition is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses). For example, the immunogenic composition is administered at an initial dose of between about lOmg / kg to about 30mg / kg. The immunogenic composition is then administered at a maintenance dose of between about O.OOOlmg / kg to about lOmg / kg. The maintenance doses may be administered every 7-35 days, such as, every 7 or 14 or 28 days.

[0334] In some examples, a dose escalation regime is used, in which the immunogenic composition is initially administered at a lower dose than used in subsequent doses. This dosage regime is useful in the case of subject’s initially suffering adverse events

[0335] In the case of a subject that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, increasing doses may be administered.

[0336] Methods of Treatment or Prevention

[0337] The present disclosure provides methods of using the immunogenic composition or the pharmaceutical composition of the present disclosure as a vaccine.

[0338] The present disclosure also provides methods of treating or preventing a disease or condition in a subject comprising administering the immunogenic composition or the pharmaceutical composition of the present disclosure. For example, the disease or condition is a respiratory virus infection, such as influenza or COVID-19. In one example, the disease or condition is ARDS.

[0339] Influenza

[0340] Influenza, also known as "the flu", is an infectious disease caused by an influenza virus. Symptoms can be mild to severe and the most common symptoms include high fever, runny nose, sore throat, muscle and joint pain, headache, coughing, and feeling tired. Symptoms typically begin two days after exposure to the virus and most last less than a week. Complications of influenza may include viral pneumonia, secondary bacterial pneumonia, sinus infections, and worsening of previous health problems such as asthma or heart failure. Viral pneumonia may also lead to acute respiratory distress syndrome (ARDS). It will be apparent to the skilled person that there are currently four influenza viruses - A, B, C and D. Influenza A virus is the most common flu virus infecting humans, animals, and birds, whilst influenza B virus infection mostly occurs in humans. Infection of influenza C virus does not cause any severe symptom in human or mammals and influenza D, to date, has only infected pigs and cattle.

[0341] Thus, in some examples of the present disclosure, the subject has an influenza virus infection. In one example, the subject has influenza. In particular, the influenza is associated with ARDS. In one example, the methods of the present disclosure can be used to treat or prevent ARDS in a subject suffering from an influenza virus infection. In one example, the methods of the present disclosure can be used to treat or prevent ARDS in a subject suffering from influenza.

[0342] Coronavirus Disease 2019 (COVID-19)

[0343] The present disclosure provides, for example, methods of treating or preventing COVID-19.

[0344] The present disclosure also provides, for example, methods of treating or preventing SARS-CoV-2 infection. In some examples of the present disclosure the subject has a SARS-CoV-2 infection but does not have clinically diagnosed COVID-19.

[0345] COVID-19 is an infectious disease caused by SARS-CoV-2. It was first identified in December 2019 in Wuhan, Hubei, China, and has resulted in an ongoing pandemic. Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. While the majority of cases result in mild symptoms, some progress to ARDS. The time from exposure to onset of symptoms is typically around five days, but may range from two to fourteen days. There are currently no vaccines nor specific antiviral treatments for COVID-19 and management involves the treatment of symptoms, supportive care, isolation, and experimental measures.

[0346] Thus, in some examples, the subject has a SARS-CoV-2) infection. In one example, the subject has COVID-19, for example, severe COVID-19. In particular, severe COVID-19 often results in ARDS. The methods of the present disclosure can be used to treat or prevent ARDS in a subject suffering from severe COVID-19.

[0347] Acute Respiratory Distress Syndrome (ARDS)

[0348] The present disclosure provides, for example, methods of treating or preventing ARDS in a subject.

[0349] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltrates, severe hypoxemia, and disruption of the alveolar-capillary membrane barrier (i.e., pulmonary vascular leak), leading to non-cardiogenic pulmonary edema. There is currently no effective pharmacological therapy.

[0350] Infectious etiologies, including influenza and coronavirus infection, are leading causes of ARDS. Accordingly, in one example of the present disclosure, the ARDS is associated with an influenza or a coronavirus infection. For example, the ARDS is associated with influenza. In another example, the ARDS is associated with a coronavirus infection, such as a SARS-COV infection. In one example, the ARDS is associated with a SARS-CoV-2 infection.

[0351] ARDS is classified according to the Berlin Definition, which includes:

[0352] (1) presentation within 1 week of clinical insult or onset of respiratory symptoms;

[0353] (2) acute hypoxemic respiratory failure, as determined by a PaO2 / FiO2 ratio of 300 mmHg or less on at least 5 cm of continuous positive airway pressure (CPAP) or positive end expiratory pressure (PEEP), where PaO2 is the partial pressure of oxygen in arterial blood and the FiO2 is the fraction of inspired oxygen;

[0354] (3) bilateral opacities on lung radiographs not fully explained by effusions, consolidation, or atelectasis; and

[0355] (4) edema / respiratory failure not fully explained by cardiac failure or fluid overload.

[0356] In one example, the subject has or suffers from ARDS (i.e., the subject satisfies the Berlin definition of ARDS). For example, the subject is in need of treatment (i.e., in need thereof).

[0357] In one example, the subject has or suffers from a symptom associated with ARDS. Symptoms associated with ARDS and methods of identifying subjects at risk of developing ARDS will be apparent to the skilled person and / or are described herein. For example, the subject has one or more or all of the following symptoms: a) a respiratory frequency of greater than 30 breaths per minute; b) an oxygen saturation (SpCh) of 93% or less on room air; c) a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaCh / FiCh) of less than 300 mmHg; d) a SpCh / FiCh ratio of less than 218; and e) radiographic lung infiltrates in an amount of greater than 50%.

[0358] Currently, ARDS is classified as mild, moderate or severe with an associated increased mortality. The severity of ARDS can be categorized according to the Berlin definition as follows:

[0359] (i) Mild ARDS: PaCh / FiCh of 200-300 mmHg on at least 5 cm CPAP or PEEP;

[0360] (ii) Moderate ARDS: PaCh / FiCh of 100-200 mmHg on at least 5 cm PEEP; and (iii)Severe ARDS: PaCh / FiCh of less than or equal to 100 mmHg on at least 5 cm PEEP.

[0361] In one example, the ARDS is mild ARDS. In another example, the ARDS is moderate ARDS. In a further example, the ARDS is severe ARDS.

[0362] The methods of the present disclosure can, in addition to treatment of existing ARDS, be used to prevent the onset of ARDS. Thus, in one example, the subject does not have ARDS.

[0363] Kits

[0364] Another example of the disclosure provides kits containing a self-replicating RNA of the present disclosure useful for the treatment or prevention of a disease or disorder as described above.

[0365] In one example, the kit comprises (a) a container comprising a self-replicating RNA optionally in a delivery system and / or a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating or preventing a disease or disorder (e.g., influenza, COVID-19 or ARDS) in a subject.

[0366] In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for a disease or disorder of the disclosure and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the self-replicating RNA. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to developing influenza, an influenza virus infection, a SARS-CoV-2 infection, COVID-19 and / or ARDS, with specific guidance regarding dosing amounts and intervals of treatment and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0367] The present disclosure includes the following non-limiting Examples.

[0368] Another example of the disclosure provides kits containing proteins or antibodies of the disclosure for use in any method described herein (e.g., determining the potency of an immunogenic composition). In one example, the panel or kit as described herein is for ex vivo analysis. In one example, the kit is suitable for use with whole blood, plasma, serum and / or sputum samples.

[0369] In one example, the panel or kit as described herein is suitable for high-throughput screening. The term “high-throughput screening” refers to screening methods that can be used to test or assess more than one sample at a time and that can reduce the time for testing multiple samples. In one example, the methods are suitable for testing or assessing at least 5 samples, at least 10, at least 20, at least 30, at least 50, at least 70, at least 90, at least 150, at least 200, at least 300 samples at a time. Such high-throughput screening methods can analyse more than one sample rapidly e.g. in at least 30 minutes, in at least 1 hour, in at least 2 hours, in at least 3 hours, in at least 4 hours, in at least 5 hours, in at least 6 hours, in at least 7 hours, in at least 8 hours, in at least 9 hours or in at least 10 hours. High-throughput screening may also involve the use of liquid handling devices. In one example, high-throughput analysis may be automated.

[0370] The present disclosure includes the following non-limiting examples.

[0371] EXAMPLES

[0372] Example 1: Development of a quadrivalent influenza potency assay

[0373] Quadrivalent influenza vaccines were prepared by encapsulating 4 seasonal influenza sa-mRNA bi-cistronic constructs in LNPs. The 4 seasonal influenza strains were influenza A H1N1, influenza A H3N2, influenza B / Yamagata (B / Yam), and influenza B / Victoria (B / Vic). Four vaccine drug products were prepared, denoted DP-1 to DP-4

[0374] Table 1 : Quadrivalent influenza vaccine preparations

[0375] DP = drug product; DS = drug substance. Antibodies specific for the HA and NA antigen for each influenza strain were obtained. HA antibodies contained the APC fluorescent label. NA antibodies contained the FITC fluorescent label. A complexation of primary antibodies to fluorescently tagged secondary antibodies was done using a Zenon™ kit (ThermoFisher). The details of the antibodies used are shown in Table 2.

[0376] Table 2: Antibodies used in quadrivalent influenza potency assay The experimental protocol follows. A schematic of the protocol is provided in Figure 1.

[0377] Day 0

[0378] The quadrivalent potency assay was developed in two cell lines: HEK293 and BHK-21 cells. HEK293 cells were pre-seeded at a density of 0.5 x 106cells / well in DMEM growth media supplemented with 10% FBS. BHK-21 cells were passaged in DMEM growth media supplemented with 10% FBS in a tissue culture flasks to 80-100% confluency for Day 1.

[0379] Day 1

[0380] Twelve 2-fold dilutions of the LNP stock were prepared in opti-MEM media. The dilutions are performed in 1.1 ml tubes. The dilution range was 800 ng / 100 pL - 0.39 ng / 100 pL of LNP-RNA in opti-MEM. It is envisioned that alternative linear ranges, for example 500 ng to 0.78 ng can also be used.

[0381] The HEK293 cells were washed with IX PBS. Media was replaced with DMEM (1 % FBS) growth media. 100 pL of the above dilutions were added to each well (one LNP-RNA dilution per well).

[0382] The BHK-21 cells were trypsinized and re-suspended to a concentration of 1 x 106cells / 250 pL of opti-MEM. 250 pL of the cell suspension in opti-MEM were added to 100 pL of the LNP-RNA dilution in 1.1 ml tubes (equivalent to 1 x 106cells per LNP- RNA dilution). The LNP-RNA-BHK-21 cells from each 1.1 ml tube was mixed and transferred to a well of a 6-well plate containing DMEM (1 % FBS) growth media.

[0383] The cells and LNP-RNA suspensions were then incubated for 16-19 hours at 37°C, 5% CO2.

[0384] Day 2

[0385] Following the 16-19 hour incubation, the cells were detached, fixed and permeabilized, and washed.

[0386] Post-wash, each sample cell sample was split into 4 sub-samples (corresponding to one sample for each influenza strain). Each sample was then immunostained with the corresponding pair of anti -HA and anti -NA antibody for each influenza construct. For example, one sub-set of the fixed sample was stained for Hl and N1 for the H1N1 sa- mRNA construct.

[0387] Samples were stained under appropriate conditions according to recommended procedures for each antibody. Following incubation, samples were washed and resuspended in a flow cytometry buffer. All subsamples were then analysed by flow cytometry.

[0388] Example 2: Optimization of the quadrivalent potency assay

[0389] Antibody titration

[0390] Antibody titration experiments were performed to determine the optimum concentration of antibody for the assay. Titration of the HA antibodies for each respective influenza strain are shown in Figures 2-7. Titration of the NA antibodies for each respective strain is shown in Figures 8-13.

[0391] Cross reactivity

[0392] Cross reactivity of each antibody against HA / NA antigens from other influenza strains was tested. The results are shown in Figures 14-17. The H1N1, H3N2, and B / Yam antibodies did not cross-react with other strains (Figures 14-16, respectively).

[0393] Notably, the B / Yam NA antibody was found to be cross-reactive with the B / Vic NA antigen (Figure 17). However, as shown in Figure 18, the cross-reactive population (APC+ / FITC-) is clearly distinguishable from the true double-positive population (APC+ / FITC+). Accordingly, the cross-reactive population can be easily excluded (i.e. can be gated out) and does not interfere with the assay.

[0394] Example 3: Characterisation of sa-mRNA quadrivalent influenza vaccines using the quadrivalent potency assay

[0395] Both HA and NA antigens were detected for all four influenza strains (shown as the FITC+ / APC+ double-positive population) in DP-1 (Figure 19) and DP -2 (Figure 20). Potency values and RNA activity for the DS (i.e. four RNA constructs) are shown in Figure 21, 22, and 23, for DP-1, DP -2, and DP-3, respectively.

[0396] Figures 24-27 show potency values for DP-1, DP-2, DP-3 and DP-4 (respectively) in BHK21 cells and HEK293 cells. Potency values for each strain in the vaccine were measured. Potency values were measured in vaccines prepared using fresh LNPs, LNPs stored at 4 degrees for 1 day, and LNPs which had undergone one freeze / thaw cycle.

[0397] Linear range

[0398] The linear range determination for the quadrivalent potency assay in BHK21 and HEK293 cells is shown in Table 3. ‘Linear Range from’ indicates the starting concentration of the LNP / RNA (in ng) for respective quadrivalent LNP formulation where the linear range for FACS potency begins for indicated cell line. Table 3: Linear range determination

[0399] Example 4: Comparison DPs in different cell lines

[0400] A summary of the data provided in Figures 24 to 26 is shown in Figure 28. The comparison indicates that (i) DP-1 (comprising DS-A and Lipid-A) have potency in HEK293 cells than BHK21 cells; (ii) DP-2 (comprising DS-A and Lipid B) has low potency in both HEK293 and BHK21 cells; and (iii) DP-3 (comprising DS-B and Lipid C) has good potency in both HEK293 and BHK21 cells.

[0401] It was also observed that DS-B generally had higher expression of the NA antigen across all strains and generally lower or similar expression of the HA antigen compared to DS-A, with the exception of the B / Vic strain where the opposite trend was observed (Figure 29 and 30). This observation was also reflected in protein concentration for each antigen (Figure 31 and Figure 32).

[0402] Finally, performance of DS-A and DS-B in different cell lines were also examined. It was observed that DS-B constructs had a marginally higher RNA activity (Figure 33) and protein concentration (Figure 34 and Figure 35) in BHK21 cells, whilst DS-A had a higher RNA activity (Figure 33) and protein concentration (Figure 34 and Figure 35) in HEK293 cells.

[0403] Together, these data show that the quadrivalent potency assay is particularly useful for evaluating and distinguishing between relative effectiveness of various vaccine preparations. Example 5: Data analysis

[0404] The potency values calculated in Examples 1-4 are based on a single hit kinetics model (Poisson frequency distribution). The SHPM model predicts how the percentage of cells positive for each antigen increases as the amount (or dose) of RNA increases in the transfection reaction. The SHPM model can be represented by the following formula (Equation 1):

[0405] % antigen positive = 100% x (l-e'cxDose) where:

[0406] Dose = RNA mass (nanograms (ng)) c is a proportionality constant (c has units of ng'1) e is Euler’s number, 2.71828.

[0407] Accordingly, the fraction of transfected cells that are antigen negative ( / o) can be expressed as: j. , % antigen postive

[0408] JJO — i - - 100%

[0409] Equation 1 can be expressed as:

[0410] / o= e'cxDose(Equation 2)

[0411] Equation 2 can be expressed as:

[0412] Info = - c x Dose

[0413] Using this model, linear regression is applied to values plotted on an x, y, graph that represent a straight line to calculate the slope. The value of the slope represents the specific RNA activity, which is the increase in the probably of successful transfection per RNA unit mass.

[0414] The potency value is calculated from a standard curve of the twelve 2-fold serial RNA dilutions (Example 1). The percentage of cells positive for each antigen (HA+ NA+ for each serial dilution is determined (Figure 36) and plotted against RNA concentration as a standard curve (Figure 37). A linear regression analysis can be performed on the curve to calculate its slope. The specific potency can then be calculated by multiplying the value of the slope by a constant (linear regression slope x 105according to the following formula: specific potency (ng-1) = slope x (linear regression slope x 105) ± standard error (Figure 38).

Claims

CLAIMS1. A method for determining the potency of a multivalent immunogenic composition, wherein the immunogenic composition comprises:(i) a RNA encoding one or more viral antigens from a first viral strain;(ii) a RNA encoding one or more viral antigens from a second viral strain; and wherein the method comprises:(i) incubating the immunogenic composition in a culture medium with a population of cells under culture conditions;(ii)(a) contacting a first sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the first viral strain;(ii)(b) contacting a second sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the second viral strain; and(iii) determining the level of each viral antigen in each sample, comprising detecting the labelled detection antibody in each sample and determining the number or percentage of cells that are positive for each viral antigen in each sample.

2. The method of claim 1, wherein the immunogenic composition further comprises(iii) a RNA encoding one or more viral antigens from a third viral strain;(iv) a RNA encoding one or more viral antigens from a fourth viral strain; and wherein the method further comprises(ii)(c) contacting a third sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the third viral strain;(ii)(d) contacting a fourth sample of the population of cells with one or more labelled detection antibodies that each bind to the one or more viral antigens from the fourth viral strain.

3. The method according to claim 1, wherein potency is calculated by correlating the percentage of cells that are positive for each viral antigen with the amount of RNA encoding the antigen.

4. The method according to claim 3, wherein the potency is calculated according to the formula: specific potency (ng-1) = slope x (linear regression slope x 105) ± standard error, wherein the slope is calculated from a two-fold serial dilution of RNA.

5. The method according to claim 1, wherein step (ii) comprises:(ii) treating the sample of cells with a fixation and permeabilization solution so as to fix and permeabilize the cells;(iii) washing the cells to remove the fixation and permeabilization solution;(iv) contacting the cells with the labelled detection antibodies under suitable conditions;(v) washing the cells to remove any unbound labelled detection antibody; and(vi) transferring the cells to a suitable medium for determining the level of each viral antigen in each sample.

6. The method according to claim 1, wherein each label is a fluorescent label.

7. The method according to claim 6, wherein each fluorescent label is selected from a group consisting of:- a fluorescent label with an excitation wavelength of 300-379 nm;- a fluorescent label with an excitation wavelength of 380-439 nm;- a fluorescent label with an excitation wavelength of 440-509 nm;- a fluorescent label with an excitation wavelength of 510-599 nm;- a fluorescent label with an excitation wavelength of 600-729 nm; and- a fluorescent label with an excitation wavelength of over 730 nm.

8. The method according to claim 6, wherein each labelled detection antibody comprises a distinct fluorescent label, wherein the distinct fluorescent labels do not have substantial spectral overlap.

9. The method according to claim 1, wherein each sample is contacted with a first labelled detection antibody and a second labelled detection antibody, wherein the first and second labelled detection antibodies comprise a distinct fluorescent label, preferably wherein first labelled detection antibody comprises a fluorescent label with an excitation wavelength of 440-509 nm and the second labelled detection antibody comprises a fluorescent label with an excitation wavelength of 600-729 nm.

10. The method according to claim 6, wherein each fluorescent label is detected by flow cytometry.

11. The method according to claim 10, wherein the flow cytometry is multicolour flow cytometry.

12. The method according to claim 2, wherein first, second, third or fourth viral strain is an influenza virus strain, a coronavirus strain, a respiratory syncytial virus strain, a parainfluenza virus stain, a human metapneumovirus strain, or an Epstein-Barr virus strain.

13. The method according to claim 1, wherein the RNA encoding the one or more viral antigens from each viral strain are provided in a multi ci str onic RNA construct.

14. The method according to claim 1, wherein the immunogenic composition comprises:(i) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a first influenza strain;(ii) a RNA encoding a HA protein and a RNA encoding an NA protein from a second influenza strain; and wherein the method comprises:(i) incubating the immunogenic composition in a culture medium with a population of cells under culture conditions;(ii)(a) contacting a first sample of the population of cells with a labelled detection antibody that binds the HA protein from the first influenza strain, and a labelled detection antibody that binds the NA protein from the first influenza strain;(ii)(b) contacting a second sample of the population of cells with a labelled detection antibody that binds the HA protein from the second influenza strain, and a labelled detection antibody that binds the NA protein from the second influenza strain;(iii) determining the level of the HA protein and the NA protein in each sample, comprising detecting the labelled detection antibody in each sample and determining the number or percentage of cells that are positive for both the HA label and the NA label in each sample.

15. The method of claim 14, wherein the immunogenic composition further comprises(iii) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a third influenza strain;(iv) a RNA encoding a HA protein and a RNA encoding an NA protein from a fourth influenza strain; and wherein the method further comprises(ii)(c) contacting a third sample of the population of cells with a labelled detection antibody that binds the HA protein from the first influenza strain, and a labelled detection antibody that binds the NA protein from the third influenza strain;(ii)(d) contacting a fourth sample of the population of cells with a labelled detection antibody that binds the HA protein from the second influenza strain, and a labelled detection antibody that binds the NA protein from the fourth influenza strain.

16. The method according to claim 15, wherein the RNA encoding the HA protein and the RNA encoding the NA protein are provided in a multi ci str onic RNA construct.

17. The method according to claim 16, wherein the immunogenic composition comprises:(i) a multi ci str onic RNA encoding the HA protein and the NA protein from a first influenza strain;(ii) a multi ci str onic RNA encoding the HA protein and the NA protein from a second influenza strain;(iii) a multi ci str onic RNA encoding the HA protein and the NA protein from a third influenza strain; and(iv) a multi ci str onic RNA encoding the HA protein and the NA protein from a fourth, influenza strain.

18. The method according to claim 1, wherein the RNA is a mRNA.

19. The method according to claim 1, wherein the RNA is a self-amplifying RNA.

20. The method according to claim 1, wherein the RNA is formulated in a delivery vehicle, preferably wherein the delivery vehicle is a lipid nanoparticle (LNP).

21. The method according to claim 14, wherein the influenza strains are selected from the group consisting of influenza A and influenza B.

22. The method according to claim 14, wherein the influenza strains are selected from the group consisting of influenza A H1N1, influenza A H3N1, influenza B / Victoria, and influenza B / Yamagata.

23. The method according to claim 14, wherein the labelled detection antibody that binds to the HA protein is:- an influenza A H1N1 anti-HA antibody;- influenza A H3N1 anti-HA antibody;- influenza B / Victoria anti-HA antibody; or- influenza B / Yamagata anti-HA antibody.

24. The method according to claim 14, wherein the labelled detection antibody that binds to the NA protein is:- an influenza A H1N1 anti-NA antibody;- an influenza A H3N1 anti-NA antibody;- an influenza B / Victoria anti-NA antibody; or- an influenza B / Yamagata anti-NA antibody.

25. The method according to claim 1, wherein the cells are mammalian cells.

26. The method according to claim 25, wherein the mammalian cells are HEK293 cells of BHK-21 cells.

27. A method for selecting a multivalent immunogenic composition suitable for use in in method of inducing an immune response in a subject, comprising:(i) determining the potency of the immunogenic composition according to the method of claim 1, and(ii) selecting an immunogenic composition having a potency value of between 5 and 2500.

28. An immunogenic composition selected for use in method of inducing an immune response against influenza in a subject in need thereof, wherein the immunogenic composition comprises(i) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a first influenza strain;(ii) a RNA encoding a HA protein and a RNA encoding an NA protein from a second influenza strain;(iii) a RNA encoding a hemagglutinin (HA) protein and a RNA encoding an neuraminidase (NA) protein from a third influenza strain; and(iv) a RNA encoding a HA protein and a RNA encoding an NA protein from a fourth influenza strain; wherein the first, second, third, and fourth influenza strains comprising influenza A H1N1, influenza A H3N1, influenza B / Victoria, and influenza B / Yamagata; and wherein selection is performed according to the method of claim 27.

29. A method of inducing an immune response in a subject, the method comprising selecting an immunogenic composition suitable for use according to claim 28, and administering the immunogenic composition to a subject.

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