Chimeric influenza b virus hemagglutinin mRNA vaccines and uses thereof

Abstract

Provided herein are nucleoside-modified ribonucleic acid molecules encoding a chimeric influenza virus hemagglutinin (HA) polypeptide that comprises a HA globular head domain and a HA stalk domain, wherein the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B, the HA globular head domain comprises an amino acid sequence of a HA globular head domain of an influenza A, or the HA globular head domain comprises an amino acid sequence of a HA globular head domain of the influenza B, wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A, and the nucleoside-modified RNA molecule comprises at least one pseudouridine. Also provided herein are methods of immunizing a subject against influenza virus disease and methods of preventing influenza virus symptoms in a subject using a composition described herein.

Description

Attorney Docket No. MSZ0003PCTCHIMERIC INFLUENZA B VIRUS HEMAGGLUTININ mRNA VACCINES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This applications claims priority to, and the benefit of, U. S. Provisional Patent Application No. 63 / 725,732, filed 27 November 2024, which is incorporated by reference herein in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under awards AI097092, AI145870, AI146101, and AI153064 awarded by the National Institutes of Health. The government has certain rights in this invention.INCORPORATION BY REFERENCE

[0003] In compliance with 37 C. F. R. 1.52(e), the sequence information contained in electronic file named “MSZ0003PCT_Sequence_Listing.xml”, which was created on 21 October 2025 and 17,217 bytes in size, is incorporated by reference herein in its entirety.BACKGROUND

[0004] 1. Field. Provided herein are nucleoside-modified messenger ribonucleic acid (mRNA) molecules encoding a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza vims HA polypeptide comprises a HA globular head domain and a HA stalk domain, wherein the HA stalk domain comprises an amino acid sequence of a HA stalk domain of a HA of an influenza B vims, and the HA globular head domain comprises an amino acid sequence of a HA globular head domain of a HA of an influenza A vims, or the HA globular head domain comprises an amino acid sequence of a HA globular head domain of the HA of the influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A vims.

[0005] 2. Background Information. Influenza is a contagious respiratory disease caused by influenza A and B vimses in humans. Seasonal influenza causes significant morbidity and mortality world-wide, despite the availability of a seasonal vaccine. Vaccination is one of the most effective countermeasures to reduce the clinical and socio-economic impacts of influenza infections. The efficacy of the seasonal influenza vaccine is highly dependent on itsAttorney Docket No. MSZ0003PCTsimilarity to circulating vims strains with an efficacy ranging from 40-60% (Trombetta, C. M., et al., Influenza Viruses and Vaccines: The Role of Vaccine Effectiveness Studies for Evaluation of the Benefits of Influenza Vaccines. Vaccines (Basel), 2022. 10(5)). The vaccine effectiveness (VE) of commercially available influenza vaccines is in the range of 10-60%, being generally lower for H3N2 in comparison to H1N1 and influenza B viruses (Belongia and McLean, Clin Infect Dis 69, 1817-1823 (2019); Okoli eta al., Vaccine 39, 1225-1240 (2021)). In those instances where there is a mismatch between the vaccine and the circulating stains, the vaccine is less effective (Lo, Y. C., et al., Surveillance and vaccine effectiveness of an influenza epidemic predominated by vaccine-mismatched influenza B / Yamagata-lineage viruses in Taiwan, 2011-12 season. PLoS One, 2013. 8(3): p. e58222; Krammer, F., et al., Influenza. Nat Rev Dis Primers, 2018. 4(1): p. 3; Paules, C. I., et al., Chasing Seasonal Influenza - The Need for a Universal Influenza Vaccine. N Engl J Med, 2018. 378(1): p. 7-9; Zost, S. J., et al., Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. Proc Natl Acad Sci U S A, 2017. 114(47): p. 12578-12583). Additionally, influenza recombination events give rise to pandemic strains as seen in 1918, 1957, 1968, and 2009 for which there is little pre-existing influenza immunity in the population (Khiabanian, H., V. Trifonov, and R. Rabadan, Reassortment patterns in Swine influenza viruses. PLoS Curr, 2009. 1: p. RRN1008;Shapshak, P., et al., The influenza pandemic of 2009: lessons and implications. Mol Diagn Ther. 2011. 15(2): p. 63-81).

[0006] Current influenza vims vaccines are composed of the matched influenza A group 1, influenza A group 2, and influenza B vims circulating strains. The immune response elicited by these vaccines mainly targets the immunodominant head domain of the most abundant influenza vims glycoprotein, the HA. The head domain of the HA is subject to strong antigenic drift and can accommodate mutations that facilitate escape from pre-existing immunity, hence annual revaccinations are required. Moreover, seasonal influenza vims vaccines would offer little to no protection against pandemic influenza vimses. The development of broadly protective vaccines is therefore of high importance (Kanekiyo and Graham, Next-Generation Influenza Vaccines. Cold Spring Harbor Perspectives in Medicine, a038448 (2020); F. Krammer, A. Garcia-Sastre, P. Palese, Is It Possible to Develop a " Universal" Influenza Vims Vaccine? Potential Target Antigens and Critical Aspects for a Universal Influenza Vaccine. Cold Spring Harb Perspect Biol 10, (2018)).

[0007] Historically, influenza B vimses have attracted less attention since they lack an animal reservoir and are less antigenically diverse than influenza A vimses. Despite their lowAttorney Docket No. MSZ0003PCTpandemic risk, influenza B viruses account for 20 to 30% of all influenza cases around the globe. The first influenza B virus was isolated in 1940 (B / Lee / 1940) and in the late 1980s two new antigenically distinct lineages emerged and co-circulated for several decades: the B / Yamagata / 16 / 1988-like (Yam) strains and the B / Victoria / 2 / 1 87-like (Vic) strains. Since the beginning of the Coronavirus disease-19 (COVID-19) pandemic, the B / Yamagata-lineage has not been identified, which has motivated the recommendation against its inclusion in the 2024-2025 influenza vaccination. Pre-existing immunity against that lineage will promptly disappear, which will bring a new risk of re-emergence of similar viruses against which we will not be protected. In this unprecedented epidemiological context, the development of a universal influenza B vaccine that protects against all (known) influenza B viruses is of great importance.

[0008] Influenza B viruses undergo frequent changes to evade host pre-existing immunity. These mutations are mainly accumulated in their surface glycoproteins, the hemagglutinin (HA) and neuraminidase (NA) (Figure 1 A), through a process known as antigenic drift. The HA glycoprotein is divided into two subunits: the HA1 subunit and the HA2 subunit. The HA1 subunit contains the globular head that encloses the receptor binding site, which allows influenza virus to bind to sialic acid and engage its entry into the host cell. The head domain also contains the four major antigenic regions, defined as the 120 loop, the 150 loop, the 160 loop, and the 190 helix (Figure IB). These immunodominant domains are the main target for neutralizing antibodies with hemagglutination inhibition (HI) activity and represent the main targets of the antigenic drift. Current seasonal influenza vaccines aim to match the vaccine strain with the circulating virus. The selection of these vaccines is based on Hi-active antibodies which makes their protection only strained-matched and poorly cross-reactive against other strains.

[0009] Therefore, a need exists for a more cross-reactive influenza virus vaccine, especially for influenza B virus, as well as methods for inducing an immune response against influenza virus hemagglutinin or preventing influenza virus disease.SUMMARY

[0010] Provided herein are ribonucleic acid (RNA) molecules (e.g., messenger RNA (mRNA) molecules, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) that are translated into a chimeric influenza virus hemagglutinin (HA) polypeptide described herein, associated methods of making use and formulation into therapeutic compositions. In any aspect or embodimentAttorney Docket No. MSZ0003PCTdescribed herein, provided herein is an mRNA molecule that is translated into a chimeric influenza virus HA polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide) described herein, wherein the mRNA molecule is a non-replicating. In any aspect or embodiment described herein, the non-replicating mRNA molecule comprises a single-stranded linear RNA with a 5’ cap, 3’ poly A tail, and 5’ and 3’ untranslated regions (UTR). In any aspect or embodiment described herein, the nonreplicating mRNA molecule comprises at least one modified nucleoside. In any aspect or embodiment described herein, the non-replicating mRNA molecule is a nucleoside-modified mRNA molecule described herein. In any aspect or embodiment described herein, provided herein is an mRNA molecule that is translated into a chimeric influenza virus HA polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide) described herein, wherein the mRNA molecule is a self-amplifying mRNA (samRNA). Generally, such a samRNA comprises a nucleotide sequence that encodes a chimeric influenza HA polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide) described herein and additional viral proteins, such as alphavirus replication machinery, required for functional replication activity (see, e.g., Papukashvili, D., et al., Self-Amplifying RNA Approach for Protein Replacement Therapy. Int J Mol Sci, 2022. 23(21); Blakney, A. K., S. Ip, and A. J. Geall, An Update on Self-Amplifying mRNA Vaccine Development. Vaccines (Basel), 2021. 9(2)). In any aspect or embodiment described herein, the samRNA persists once injected in a subject resulting in more antigen expression, requires a lower dose of vaccine as compared to a non-replicating mRNA vaccine. In any aspect or embodiment described herein, the samRNA induces broad influenza neutralizing antibodies and / or cellular immune response, and protects from lethal viral challenge. A further aspect provided herein is an RNA molecule (e.g., a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) molecule) that is translated into a chimeric influenza virus HA polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide) described herein, wherein the RNA molecule is a circular RNA (circRNA). In any aspect or embodiment described herein, the circRNA comprises an internal ribosome entry site (IRES) and an RNA sequence that is transcribed into a chimeric influenza virus HA polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide). In any aspect or embodiment described herein, the circRNA comprises N6-methyladenosine, an internal ribosome entry site (IRES), and an RNA sequence that is transcribed into a chimeric influenza virus HA polypeptide (e.g., a firstAttorney Docket No. MSZ0003PCTchimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide). In any aspect or embodiment described herein, provided herein is the nucleic acid (e.g., DNA) template that is in vitro transcribed to produce circRNA, which may be translated into a chimeric influenza virus hemagglutinin polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide) described herein. In any aspect or embodiment described herein, an RNA molecule (e.g., an mRNA molecule, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) described herein is encapsulated in a nanoparticle, such as, e.g., described herein (e.g., Example 1, 2 and / or 3). In any aspect or embodiment described herein, an RNA molecule (e.g., an mRNA molecule, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) described herein is complexed with peptides containing strings of positively charged amino acids, such as lysine and arginine, to form a protamine-RNA complex. In any aspect or embodiment described herein, an RNA molecule (e.g., an mRNA molecule, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) described herein is associated or encapsulated with a polymer, such as a cationic polymers (e.g., polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer and polysaccharides (e.g., chitosan). In any aspect or embodiment described herein, an RNA molecule (e.g., an mRNA molecule, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) described herein is associated or encapsulated in an emulsion, such as, e.g., a cationic nanoemulsion (CNE), which utilizes nanoemulsion with cationic lipids. In any aspect or embodiment described herein, an RNA molecule (e.g., an mRNA molecule, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, or a second nucleoside-modified RNA molecule) described herein is associated or encapsulated in a liposome.

[0011] An aspect of the present disclosure provides a nucleoside-modified ribonucleic acid (RNA) molecule encoding a chimeric influenza virus hemagglutinin (HA) polypeptide comprising a HA globular head domain and a HA stalk domain, wherein: (a) the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B virus, (b) the HA globular head domain comprises an amino acid sequence of (i) a HA globular head domain of a HA of an influenza A virus or (ii) a HA globular head domain of an Influenza B virus (e.g., the same influenza B virus as the HA stalk domain), wherein one or moreAttorney Docket No. MSZ0003PCTantigenic sites have been replaced with antigenic sites of an influenza A vims, and (c) the nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0012] In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, 120 loop, 150 loop, 160 loop, and 190 helix.

[0013] In any aspect or embodiment described herein, the influenza A vims is an influenza A vims that originates from a different species than the influenza B vims.

[0014] In any aspect or embodiment described herein, the influenza A vims is an avian Influenza A vims.

[0015] In any aspect or embodiment described herein, at least one of (a) the influenza B vims is B / Phuket / 3073 / 2013 Influenza vims or B / Brisbane / 20 / 2008 Influenza vims; (b) the influenza A vims is a hemagglutinin 5 (H5) Influenza A vims, a hemagglutinin 13 (H13) Influenza A vims, or subtype thereof; or (c) a combination thereof.

[0016] In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hll, H12, H13, H16, H17, or H18 (preferably H13 or H5). In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of the HA of the influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17, or H18 (preferably H13 or H5).

[0017] In any aspect or embodiment described herein, (a) the influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the influenza A vims is a hemagglutinin 5 (H5) Influenza A vims; or (b) the influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the Influenza A vims is a hemagglutinin 13 (H13).

[0018] In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of the influenza B vims and a HA cytoplasmic domain of the influenza B vims.

[0019] In any aspect or embodiment described herein, the HA globular head domain comprises the amino acid sequence of the HA globular head domain of the influenza A vims.Attorney Docket No. MSZ0003PCT

[0020] In any aspect or embodiment described herein, the HA globular head domain comprises the amino acid sequence of the HA globular head domain of the influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of the influenza A vims.

[0021] In any aspect or embodiment described herein, the nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine.

[0022] Another aspect of the present disclosure provides a nucleoside-modified RNA molecule encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially of, or consisting of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0023] A further aspect of the present disclosure provides a composition comprising, consisting essentially of, or consisting of, the nucleoside-modified RNA molecule (e.g., a first nucleoside-modified RNA molecule and / or a second nucleoside-modified RNA molecule) of the present disclosure, and a pharmaceutically acceptable carrier. Thus, for example, the composition comprises, consists essentially of, or consists of, the first nucleoside-modified RNA molecule of the present disclosure and / or a second nucleoside-modified RNA molecule) of the present disclosure, and a pharmaceutically acceptable carrier.

[0024] In any aspect or embodiment described herein, the composition further comprises a lipid component. For example, in any aspect or embodiment described herein, the lipid component is part of a lipid nanoparticle, such as described herein.

[0025] Another aspect of the present disclosure provides a method for inducing an immune against influenza vims hemagglutinin (HA) or preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising administering to the subject at least one dose of the nucleoside-modified RNA molecule of the present disclosure, or the composition of the present disclosure, wherein the method is effective for inducing an immune response against HA or preventing influenza vims symptoms in the subject.

[0026] In any aspect or embodiment described herein, the dose is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the nucleoside-modified RNA molecule.

[0027] In another aspect, provided herein are methods for inducing an immune response to influenza vims in a subject, the method comprising administering to the subject two or more compositions described herein. In another aspect, provided herein are methods for preventing influenza vims symptoms (e.g, disease) in a subject, the method comprising administering to the subject two or more compositions described herein. In another aspect,Attorney Docket No. MSZ0003PCTprovided herein are methods of immunizing a subject against influenza virus disease, the method comprising administering to the subject two or more compositions described herein. In any aspect or embodiment described herein, each composition comprises a different nucleoside-modified mRNA molecule described herein. In any aspect or embodiment described herein, each composition comprises a different nucleoside-modified mRNA molecule encoding a chimeric influenza virus HA polypeptide. In any aspect or embodiment described herein, the two or more compositions are administered intramuscularly to the subject. In any aspect or embodiment described herein, the subject is human.

[0028] An additional aspect of the present disclosure provides a method for inducing an immune response against influenza vims hemagglutinin (HA) or preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising: (a) administering to the subject a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (b) administering to the subject a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least oneAttorney Docket No. MSZ0003PCTpseudouridine, wherein the method is effective for inducing an immune response against HA or preventing influenza virus symptoms in the subject.

[0029] Yet another aspect of the present disclosure provides a method for inducing an immune response against influenza virus hemagglutinin (HA) or preventing influenza virus symptoms (e.g, disease) in a subject, the method comprising: (a) administering to the subject a does of a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza virus HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine: and (b) administering to the subject a dose of a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine, wherein the method is effective for inducing an immune response against HA or preventing influenza vims symptoms in the subject.

[0030] In any aspect or embodiment described herein, at least one of (1) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims; (2) the second HAAttorney Docket No. MSZ0003PCTglobular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; or (3) a combination thereof.

[0031] In any aspect or embodiment described herein, at least one of (1) the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus; (2) the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; or (3) a combination thereof.

[0032] In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, lat least one of 20 loop, 150 loop, 160 loop, 190 helix, or a combination thereof. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, 120 loop, 150 loop, 160 loop, and 190 helix.

[0033] In any aspect or embodiment described herein, at least one of (1 ) the first influenza A virus is an influenza A vims that originates from a different species than the first influenza B vims; (2) the second influenza A vims is an influenza A vims that originates from a different species than the second influenza B vims; (3) the first influenza A vims is an avian influenza A vims; (4) the second influenza A vims is an avian influenza A vims; or (5) a combination thereof.

[0034] In any aspect or embodiment described herein, at least one of (1) the first influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims: (2) the second influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims; (3) the first influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims, a hemagglutinin 5 (H5) Influenza A vims, or a subtype thereof; (4) the second influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims, a hemagglutinin 5 (H5) Influenza A vims, or a subtype thereof; or (5) a combination thereof.

[0035] In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide (e.g., a first chimeric influenza vims HA polypeptide and / or a chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, the amino acid sequence of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17, or H18 (preferably H13 or H5). In any aspect or embodiment described herein, the HA globularAttorney Docket No. MSZ0003PCThead domain of the chimeric influenza vims HA polypeptide (e.g., a first chimeric influenza vims HA polypeptide and / or a chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of the HA of the influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17, or H18 (preferably H13 or H5).

[0036] In any aspect or embodiment described herein, (1) at least one of the first influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the first influenza A vims is a hemagglutinin 13 (H13) Influenza A vims; the second influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the second influenza A vims is a hemagglutinin 5 (H5) Influenza A vims; or a combination thereof; or (2) at least one of the first influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the first influenza A vims is a hemagglutinin 5 (H5) Influenza A vims or a subtype thereof; the second influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the second influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims or a subtype thereof; or a combination thereof.

[0037] In any aspect or embodiment described herein, at least one of (1 ) the first chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B vims (e.g., the first Influenza B vims) and a HA cytoplasmic domain of an influenza B vims (e.g., the first Influenza B vims); (2) the second chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B vims (e.g., the second influenza B vims) and a HA cytoplasmic domain of an influenza B vims (e.g., the second influenza B vims); or (3) a combination thereof.

[0038] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the second nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof (e.g., wherein the first chimeric influenza vims HA polypeptide and the second chimeric influenza vims HA polypeptide have different amino acid sequence or SEQ ID NOs).

[0039] In any aspect or embodiment described herein, at least one of: (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, whereinAttorney Docket No. MSZ0003PCTthe transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide are transcribed from different nucleotide sequence or SEQ ID NOs).

[0040] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0041] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0042] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0043] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.Attorney Docket No. MSZ0003PCT

[0044] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0045] In any aspect or embodiment described herein, at least one of (1 ) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0046] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0047] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0048] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5,Attorney Docket No. MSZ0003PCTand wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0049] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0050] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6 or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2 or 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0051] In any aspect or embodiment described herein, at least one of (1 ) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2 or 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6 or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0052] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0053] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.Attorney Docket No. MSZ0003PCT

[0054] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0055] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0056] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0057] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0058] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0059] In any aspect or embodiment described herein, at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein theAttorney Docket No. MSZ0003PCTtranscription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

[0060] In any aspect or embodiment described herein, administering the second composition is performed after a period of time (e.g., about 25 days to about 90 days, about 40 days to about 75 days, about 45 days to about 65 days, or about 55 days to about 60 days) from administering the first composition.

[0061] In any aspect or embodiment described herein, the first composition and the second composition are co-administered (e.g., at the same time, in the same composition or within about from 1 to 24 hours).

[0062] In any aspect or embodiment described herein, at least one of (1) the dose of the first composition is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the first nucleoside-modified RNA molecule, (2) the dose of the second composition is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the second nucleoside-modified RNA molecule, or (3) a combination thereof.

[0063] In any aspect or embodiment described herein, the composition (e.g., the first composition, the second composition, or both the first composition and the second composition) of the present disclosure is administered intramuscularly to the subject.

[0064] In any aspect or embodiment described herein, the subject is human.

[0065] In any aspect or embodiment described herein, the method further comprises (a) prior to administering the nucleoside-modified RNA molecule of the present disclosure, administering an influenza vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) HA antigen (e.g., a seasonal quadrivalent influenza vaccine); or (b) prior to administering the first composition of the present disclosure, administering an influenza vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) HA antigen (e.g., a seasonal quadrivalent influenza vaccine).

[0066] Another aspect of the present disclosure provides a kit comprising a container including the nucleoside-modified RNA molecule of the present disclosure, the composition of the present disclosure, the first composition of the present disclosure, or the second composition of the present disclosure.

[0067] A further aspect of the present disclosure provides a kit comprising a first container including the first composition of the present disclosure and a second container including the second composition of the present disclosure.

[0068] Another aspect of the present disclosure provides a kit comprising two or more containers, wherein each container comprises a different nucleoside-modified mRNA molecule described herein and / or composition described herein.Attorney Docket No. MSZ0003PCT

[0069] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule comprises at least 1 modified nucleoside (e.g., pseudouridine). In any aspect or embodiment described herein, the nucleoside-modified RNA molecule comprises at least 2, at least 3, least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 modified nucleosides), such as, e.g., pseudouridine. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule comprises 1, 2, or 3 modified nucleosides (e.g., pseudouridine). In any aspect or embodiment described herein, the nucleoside-modified RNA molecule comprises 4, 5, 6, 7, 8, 9, 10 or more modified nucleosides (e.g., pseudouridine).

[0070] In any aspect or embodiment described herein, the nucleoside-modified mRNA molecule (e.g., the first nucleoside-modified RNA molecule and / or the second nucleoside-modified RNA molecule) of the present disclosure comprises an in vitro transcribed nucleoside-modified mRNA molecule comprising at least one modified nucleoside of uridine.BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. The drawings are only for the purpose of illustrating embodiments of the disclosure and are not to be construed as limiting the disclosure. Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure.

[0072] Figures 1A and IB. Universal influenza vaccine approach. (1A) Influenza B virus contains two main glycoproteins on its surface: the hemagglutinin (HA) and the neuraminidase (NA). Described herein is a universal vaccination targeting the subdominant epitopes of the HA protein employing the nucleoside-modified messenger-RNA-lipid nanoparticle (mRNA-LNP) platform. (IB) Sequential vaccination with chimeric hemagglutinin (HA) or mosaic HA vaccines constructs, where the major immunodominant epitopes are replaced in each vaccination with sequences from avian exotic HAs will refocus the immune response to subdominant head and stalk epitopes of the HA. The HA sequence can be divided into two main domains the HA1 and HA2 domain. The HA1 domain is completely replaced by an avian sequence in the cHA approach, whereas the four major antigenic domains in the HA1 (120 loop, 150 loop, 160 loop and 190 helix) are replaced in the mHA approach (TM: transmembrane domain, CT: cytoplasmatic tail).Attorney Docket No. MSZ0003PCT

[0073] Figures 2A, 2B, 2C, 2D, and 2E. In vitro expression of cHA and mHA immunogens. The surface expression of the four vaccine candidates was measured by immune staining with anti-HA polyclonal sera and analyzed by flow cytometry.

[0074] Figures 3A, 3B, 3C, 3D, 3E, 3F, and 3G. Universal influenza B mRNA-LNP vaccination in mice. (3A) Vaccination regimen and experimental workflow. (3B) Vaccination groups. BALB / c mice were vaccinated in a two-dose vaccination scheme after a priming vaccination with 1 pg of QIV (Flulaval) followed by two doses mRNA-LNP of the different vaccines in a 3-4-week interval. Mice vaccinated with an mRNA-LNP expressing luciferase and an unvaccinated group (PBS) were included as controls. (3C-3E) Binding of serum antibody titers against subdominant and cross-reactive epitopes (n = 5). Binding of serum antibodies towards the immuno-subdominant epitopes. (3C) The cH7 / BYam protein with a group 2 avian H7 head and the B / Yamagata / 16 / 1988 HA stalk was used to measure stalkspecific antibodies. (3D) A mHl 1 / BYam protein displaying the Hl 1 sequences at the major antigenic sites within the B / Yamagata / 16 / 1988 HA was used to measure antibody binding to conserved epitopes in the head and stalk domains. (3E) Antibodies against B / Lee / 40 HA protein were measured as a surrogate of cross-reactive antibodies. (3F) IgG2a / lgGl ratio serum antibody titers against B / Lee / 40 HA recombinant protein. Kruskal-Wallis test corrected using Dunn’s test for multiple comparisons is depicted in these graphs (*P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001). (3G) HI active antibodies against mH5 / Phuket, mH13 / Brisbane and B / Lee / 40.

[0075] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H. Antibody-mediated protection of universal influenza B mRNA-LNP vaccines in mice. BALB / c mice (n = 5) received 100 pL of vaccinated pooled sera intraperitoneally and 2 hours later were challenged IN as described in Fig.3. Weight loss and survival of mice were monitored for 2 weeks with a humane endpoint of >25% loss of the initial weight. Weight loss and survival for low dose groups (4A and 4E), intermediate dose (4B and 4F), high dose groups (4C and 4G) and control groups (4D and 4H) are depicted.

[0076] Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, and 5H. T -cell immunity of universal influenza B mRNA-LNP vaccines in mice measured by intracellular cytokine staining. BALB / c mice were vaccinated in a two-dose vaccination as described in Figures 2A-2E. One week after the last boost, mice were euthanized and splenocytes were harvested. Splenocytes were ex -vivo stimulated with HA peptide pool and intracellular cytokine staining was performed and analyzed by FACS. Intracellular cytokine staining was used to measure antigen-specific production of CD4+-specific IFN-y (5A), IL-2 (5B), TNF-a (5C) and IL-4Attorney Docket No. MSZ0003PCT(5D), and CD8+-specific IFN-y (5E), IL-2 (5F), TNF-a (5G) and CD107 (5H). Kruskal-Wallis test corrected using Dunn’s test for multiple comparisons is depicted in these graphs (*P < 0.05; **P <0.01; ***P <0.001; ****P<0.0001).

[0077] Figures 6A and 6B. Polyfunctional T-cell immunity of universal influenza B mRNA-LNP vaccines in mice measured by intracellular cytokine staining. Intracellular cytokine staining was performed as previously described in Figures 5A-5H. Polyfunctional antigen-specific production of CD4+ (6A) and CD8+ (6B) were measured for each vaccination group.

[0078] Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H. In vivo protection of universal influenza B mRNA-LNP vaccines in mice. BALB / c mice (n=5) were vaccinated as described in Figures 2A-2D and were challenged intranasally (IN) in a total volume of 30 pL 4 weeks after second boost. Weight loss and survival of mice were monitored for 2 weeks with a humane endpoint of >25% loss of the initial weight. Weight loss and survival for low dose groups (7A and 7E), intermediate dose (7B and 7F), high dose groups (7C and 7G) and control groups (7D and 7H) are depicted.

[0079] Figures 8A, 8B, 8C, 8D, 8E, and 8F. In vitro expression of cHA and mHA immunogens. The surface expression of the four vaccine candidates was measured by immuno-staining with anti-HA polyclonal sera and analyzed by flow cytometry for cH13 / BBris (8 A), mH13 / BBris(8B), cH5 / BPhu (8C) mH5 / BPhu (8D) and negative control firefly luciferase (Luc, 8E). The percentage of HA positive cells was measured in triplicate for each condition (8F). The figure was created with Biorender.com. Kruskal -Wallis test corrected using Dunn’s test for multiple comparisons is depicted in these graphs (*P < 0.05; **P < 0.01).

[0080] Figures 9A, 9B, 9C, 9D, 9E, and 9F. Universal influenza B virus mRNA-LNP vaccination in mice. (9A) Vaccination regime and experimental workflow. Vaccination groups are shown in Table 3: 8- 10- week-old naive BALB / c mice were vaccinated in a two-dose vaccination scheme after a priming with 1 pg of QIV (Flulaval) followed by two doses mRNA-LNP of the different vaccines in a 3-4-week interval. Mice vaccinated with an mRNA-LNP expressing Luc (prime only), with mHA split vaccines (mHA split) and an unvaccinated group (naive) were included as controls. (9B-9D) Binding of serum antibody titers against subdominant and cross-reactive epitopes (n = 5); (9B) A cH7 / BYam protein with a group 2 avian H7 head and the B / Yamagata / 16 / 1988 HA stalk was used to measure HA stalk-specific antibodies, (9C) AmHll / BYam protein displaying the Hll sequences at the major antigenic sites within the B / Yamagata / 16 / 1988 HA was used to measure antibodyAttorney Docket No. MSZ0003PCTbinding to conserved epitopes in the head and stalk domains and (9D) Antibodies against the B / Lee / 40 HA protein were measured as a surrogate of cross-protection. (9E) IgG2a / IgG1 ratio serum antibody titers against B / Lee / 40 the HA protein. (9F) Measurement of antibodies with HI activity against mH5 / Phuket, mH13 / Brisbane and B / Lee / 40 viruses. Kruskal-Wallis test corrected using Dunn’s test for multiple comparisons is depicted in these graphs (*P < 0.05; **P < 0.0k ***P < 0.001; ****P<0.0001).

[0081] Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H. T-cell immunity of universal influenza B mRNA-LNP vaccines in mice measured by intracellular cytokine staining. 8-10-week-old naive BALB / c mice were vaccinated in a two-dose vaccination scheme as described in Figures 8A-8F. One week after the last boost, mice were euthanized and splenocytes were harvested. Splenocytes were ex-vivo stimulated with an HA peptide pool and intracellular cytokine staining was performed and analyzed by flow cytometry. Intracellular cytokine staining was used to measure IFN-y(lOA), IL-2 (10B), TNF-a (10C), and IL-4 (10D) production by HA-specific CD4+ T cells and IFN-y (10E), IL-2 (10F), TNF-a (10G), and CD107 (10H) production by HA-specific CD8+ T cells. Kruskal-Wallis test corrected using Dunn’s test for multiple comparisons is depicted in these graphs (*P < 0.05; **P < 0.01; ***P < 0.001; ****P.0001).

[0082] Figures 11A and 11B. Polyfunctional T-cell immune responses of universal influenza B mRNA-LNP vaccines in mice measured by intracellular cytokine staining. Intracellular cytokine staining was performed as previously described in Fig. 10A-10H. One week after the last boost, mice were euthanized and splenocytes were harvested. Splenocytes were ex-vivo stimulated with an HA peptide pool and intracellular cytokine staining was performed and analyzed by flow cytometry. Polyfunctional production of cytokines by antigen-specific CD4+- (11A) and CD8+-T cells (1 IB) was measured for each vaccination group.

[0083] Figures 12A, 12B, 12C, and 12D. Antibody-mediated protection of universal influenza B mRNA-LNP vaccines in a serum passive transfer experiment mice. 8-10-week -old naive BALB / c mice (n = 5) received 100 pL of vaccinated pooled sera intraperitoneally and 2 hours later were challenged IN as described in Figures 9A-9F. Body weight loss and survival of mice were monitored for 2 weeks with a humane endpoint of >25% loss of the initial body weight. Body weight loss and survival for low dose groups (12A), intermediate dose (12B), high dose groups (12C) and control groups: mice vaccinated with an mRNA-LNP expressing Luc (prime only), with mHA split vaccines (mHA split) and an unvaccinatedAttorney Docket No. MSZ0003PCTgroup (naive) (12D) are depicted. Survival curves were compared with Mantel-Cox test (*P V 0.05; **P V 0.01).

[0084] Figures 13A, 13B, 13C, and 13D. In vivo protection of universal influenza B mRNA-LNP vaccines in mice. 8-10-week-old naive BALB / c mice (n=5) were vaccinated as described in Figures 9A-9F and were challenged intranasally (IN) in a total volume of 30 pL 4 weeks after second boost. Weight loss and survival of mice were monitored for 2 weeks with a humane endpoint of >25% loss of the initial weight. Weight loss and survival for low dose (13A), intermediate dose (13B), high dose(13C) and control groups: mice vaccinated with an mRNA-LNP expressing Luc (prime only), with mHA split vaccines (mHA split) and an unvaccinated group (naive) (13D) are shown. Survival curves were compared with Mantel-Cox test (*P 0.05; **P < 0.01).

[0085] Figures 14A and 14B. In vitro CR8033, CR8059 and CR9114 binding of cHA and mHA immunogens. The surface expression of the four vaccine candidates was measured by immuno-staining with human monoclonals a double staining of influenza B HA head monoclonal antibodies (CR8033 and CR8059) (14A) or a pan-stem monoclonal antibody (CR9114) (14B) and analyzed by flow cytometry for cH13 / BBris, mH13 / BBris, cH5 / BPhu and mH5 / BPhu. Percentage of HA positive cells was measured in triplicate for each condition.

[0086] Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, 151, and 15J. Humoral immune responses to QIV, cHA, and mHA mRNA-LNP vaccines. (15 A) Schematic representation of the vaccination and sample collection schedule. Seven days before the first immunization, serum samples were collected (pre-study samples). On day 0, all 20 nonhuman primates (NHPs) received two full human doses of the seasonal QIV, administered four weeks apart. On day 56, six NHPs received a third dose of the QIV, seven the cH5 / Bpilu, and seven the mH5 / BPhu mRNA-LNP vaccine. On day 84, the same groups received their respective vaccines, either a fourth dose of QIV, cH13 / BBris, or mH13 / BBris. Serum samples were collected at multiple time points. (15B-15F) HA-specific total binding IgG levels against various influenza B strains were measured by ELISA using serum collected from NHPs. Area under the curve (AUC) with a cutoff value of the average background plus three standard deviations (SDs) are shown. (15G and 15H) Antibody-dependent cellular cytotoxicity (ADCC) activity against various influenza B virus strains, shown as AUC. (151 and 15J) Hemagglutination inhibition (HAI) titers against various influenza B vims strains. (15B-15J) n=6 / 7 / 7 (QIV / cHA / mHA) NHP per group in a single experiment. Each symbolAttorney Docket No. MSZ0003PCTrepresents one animal on the left panels, and each symbol represents a group of animals on the right panels, and the data represent mean ± standard error of the mean (SEM). Kruskal-Wallis with Dunn’s multiple comparisons test was performed. Detailed statistical analyses and significance values are provided in Tables 4 and 5.

[0087] Figures 16A, 16B, 16C, and 16D. Antibody-mediated protection in mice following serum transfer from vaccinated NHPs. BALB / c mice received 150 pL of pooled serum from vaccinated NHPs collected before the study (pre-study), four weeks post-prime (day 84) or four weeks post-boost (day 112) via intraperitoneal injection. Four hours later the mice were challenged intranasally with influenza B viruses. Weight loss and survival were monitored for 14 days. A humane endpoint was defined as >20% loss of initial body weight. (16A and 16B) Weight loss and survival curve of the mice after challenge with B / Florida / 4 / 2006 virus. (16C and 16D) Weight loss and survival curve of the mice after challenge with B / Malaysia / 2506 / 2004 virus. Each symbol represents a group of animals. Data are presented as mean ± SEM. n=5 / group in a single experiment. Weight loss was compared between groups on individual days. One-way ANOVA followed by Tukey’s multiple comparisons test or Kruskal-Wallis with Dunn’s multiple comparisons test was applied. Survival curves were compared using the log-rank (Mantel-Cox) test. Detailed statistical analyses and significance values are provided in Tables 6 and 7.DETAILED DESCRIPTION

[0088] While various embodiments of the present disclosure are described herein, it will be understood by those skilled in the art that such embodiments are provided by way of example only. It will be understood by those skilled in the art that numerous modifications and changes to, and variations and equivalent substitutions of, the embodiments described herein can be made without departing from the scope of the disclosure. It is understood that various alternatives to the embodiments described herein may be employed in practicing the disclosure, and modifications may be made to adapt a particular structure or material to the teachings of the disclosure. It is also understood that every embodiment of the disclosure may optionally be combined with any one or more of the other embodiments described herein which are consistent with that embodiment.

[0089] Where a combination is disclosed, it is understood that each possible subcombination of the elements of that combination is also disclosed. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed.Attorney Docket No. MSZ0003PCT

[0090] Where elements are presented in list format (e.g., in a Markush group), it is understood that each possible subgroup of the elements is also disclosed, and any one or more elements can be removed from the list or group.

[0091] It is also understood that, unless clearly indicated to the contrary, in any method described or claimed herein that includes more than one act or step, the order of the acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are recited, but the disclosure encompasses embodiments in which the order is so limited.

[0092] It is further understood that, in general, where an embodiment in the description or the claims is referred to as comprising one or more features, the disclosure also encompasses embodiments that consist of, or consist essentially of, such feature(s).

[0093] It is also understood that any embodiment of the disclosure, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether or not the specific exclusion is recited in the specification.

[0094] Headings are included herein for reference and to aid in locating certain sections. Headings are not intended to limit the scope of the embodiments and concepts described in the sections under those headings, and those embodiments and concepts may have applicability in other sections throughout the entire disclosure.

[0095] Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0096] The articles "a" and "an" as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0097] The term “exemplary” as used herein means “serving as an example, instance or illustration”. Any embodiment or feature characterized herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or features.

[0098] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that areAttorney Docket No. MSZ0003PCTconjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to " A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0099] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0100] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. It is expressly contemplated that all embodiments and claims reciting an open-ended transitional phrase can be written with any other transitional phrase, which may be more limiting, unless clearly precluded by the context or art. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi -closed transitional phrases, respectively.

[0101] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related orAttorney Docket No. MSZ0003PCTunrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0102] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0103] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within one standard deviation. In some embodiments, when no particular margin of error (e.g., a standard deviation to a mean value given in a chart or table of data) is recited, the term “about” or “approximately” means that range which would encompass the recited value and the range which would be included by rounding up or down to the recited value as well, taking into account significant figures. In certain embodiments, the term “about” or “approximately” means within 10% or 5% of the specified value.Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values or in a series of two or more ranges of numerical values, the term “about” or “approximately” applies to each one of the numerical values in that series of numerical values or in that series of ranges of numerical values.

[0104] Whenever the term “at least” or “greater than” precedes the first numerical value in a series of two or more numerical values, the term “at least” or “greater than” applies to each one of the numerical values in that series of numerical values.

[0105] Whenever the term “no more than” or “less than” precedes the first numerical value in a series of two or more numerical values, the term “no more than” or “less than” applies to each one of the numerical values in that series of numerical values.

[0106] All patent literature and all non-patent literature cited herein are incorporated herein by reference in their entirety to the same extent as if each patent literature or non-Attorney Docket No. MSZ0003PCTpatent literature were specifically and individually indicated to be incorporated herein by reference in its entirety.

[0107] Provided herein are nucleoside-modified messenger ribonucleic acid (mRNA) molecules encoding a chimeric influenza vims hemagglutinin (HA) polypeptide, wherein the chimeric influenza vims HA polypeptide comprises a HA globular head domain and a HA stalk domain, wherein the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B vims, and the HA globular head domain comprises an amino acid sequence of (i) the HA globular head domain of an influenza A vims or (ii) the HA globular head domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain), wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A vims. Also provided herein are compositions comprising a nucleoside-modified mRNA molecule of the present disclosure. Further provided herein are methods of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by influenza B vims) and methods of preventing influenza vims disease (e.g., influenza vims disease caused by influenza B vims) in a subject using a nucleoside-modified RNA molecules of the present disclosure or composition of the present disclosure.Terminology

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] As used herein, the term “about” or “approximately” when used in conjunction with a number refers to any number within 1%, 5%, or 10% of the referenced number and includes the referenced number.

[0110] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0111] The terms "chimeric influenza vims hemagglutinin," "chimeric influenza vims HA," "chimeric hemagglutinin," "chimeric HA," "chimeric influenza hemagglutinin", and “cHA” are used herein interchangeably.

[0112] The terms "mosaic influenza virus hemagglutinin," " mosaic influenza virus HA," " mosaic hemagglutinin," " mosaic HA," "chimeric influenza hemagglutinin", and “mHA” areAttorney Docket No. MSZ0003PCTused herein interchangeably, and in reference to a type of chimeric influenza virus hemagglutinin as described herein.

[0113] As used herein, the terms “comprising” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude the presence or addition of one or more features, or components, or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting essentially of’ and / or “consisting of’. Consequently, the term “consisting essentially of” or “consisting of’ can be used in place of the terms “comprising” and “including” to provide for more specific embodiments.

[0114] As used herein, the term “elderly human” refers to a human that is 65 years old or older.

[0115] As used herein, the terms " HA" and "hemagglutinin" refer to any influenza virus hemagglutinin known to those of skill in the art or a derivative thereof. Typically, a precursor influenza virus hemagglutinin comprises a signal peptide, a stalk domain (or stem domain), a globular head domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, a hemagglutinin consists of a single polypeptide chain, such as HAO. In some embodiments, a hemagglutinin consists of more than one polypeptide chain in quaternary association, e.g., HA1 and HA2. Those of skill in the art will recognize that an immature HAO may be cleaved to release a signal peptide (generally approximately 15 to approximately 20 amino acids) to yield a mature hemagglutinin HAO (z.e., HAO without a signal peptide). In the context of an influenza A virus hemagglutinin, a mature hemagglutinin HAO is generally cleaved by proteolytic enzymes to yield an HA1 subunit (generally approximately 320 to approximately 328 amino acids, including the globular head domain and a portion of the stem domain) and an HA2 subunit (generally approximately 220 to approximately 222 amino acids, including the remainder of the stem domain, a transmembrane domain and a cytoplasmic domain). The terms "hemagglutinin" and " HA", as used herein, encompass an influenza virus HA monomer as well as trimers of influenza virus HA. In addition, the terms "hemagglutinin" and " HA", as used herein, encompass influenza virus hemagglutinin that are modified by, e.g., glycosylation (e.g., N-linked glycosylation) and lipid modification (e.g., S-palmitoylation).

[0116] The terms “influenza virus globular head domain”, “influenza virus HA globular head domain”, “influenza HA globular head domain”, “globular head domain”, “headAttorney Docket No. MSZ0003PCTdomain”, “HA head domain”, “HA globular head”, and “HA globular head domain” are used herein interchangeably.

[0117] The terms “influenza virus stem domain”, “influenza stem domain”, “influenza HA stem domain”, “stem domain”, “stalk domain”, ’’influenza vims HA stem domain”, “HA stalk”, “HA stalk domain”, and “HA stem domain” are used herein interchangeably.

[0118] As used herein, the term “HA1” refers to the HA1 subunit / domain of an influenza vims hemagglutinin. Typically, an HA1 subunit / domain includes the globular head domain and a portion of the stem domain of influenza vims hemagglutinin (see, e.g., Sriwilaijaroen and Suzuki, Proc Jpn Acad Ser B Phys Biol Sci. 2012 Jun 11; 88(6): 226-249, the contents of which is incorporated by reference in its entirety).

[0119] As used herein, the term “HA2" refers to the HA2 subunit / domain of an influenza vims hemagglutinin. Typically, an HA2 subunit / domain includes a portion of the stem domain, a transmembrane domain, and a cytoplasmic domain of influenza vims hemagglutinin (see, e.g., Sriwilaijaroen and Suzuki, Proc Jpn Acad Ser B Phys Biol Sci. 2012 Jun 11; 88(6): 226-249, the contents of which is incorporated by reference in its entirety).

[0120] As used herein, the term “HA2 stem domain” refers to the stem domain of the HA2 domain of an influenza vims hemagglutinin. An exemplary HA2 stem domain may be found in Table 1 (see, e.g., a HA2 domain from SEQ ID NO: 1, 3, 5, or 7).

[0121] As used herein, the tern " HA1 C-terminal stem segment" refers to a polypeptide segment that corresponds to the carboxy -terminal portion of the stem domain of HA1. In some embodiments, an HA1 C-terminal stem segment consists of amino acid residues corresponding approximately to amino acids Aq through Acterm of an HA1 of an influenza A virus hemagglutinin. Aq is the cysteine residue in the HA1 C-terminal stem segment that forms or is capable of forming a disulfide bond with a cysteine residue in an influenza A virus HA1 N-terminal stem segment. Acterm or otherwise referred to herein as HAlc-term is the C-terminal amino acid of the HA1 domain as recognized by those of skill in the art. In some embodiments, Aq is the Cys at amino acid position 277 of an HA1 according to H3 numbering. In some embodiments, an HA1 C-terminal stem segment consists of amino acid residues corresponding approximately to amino acids 277-329 of HA1 according to H3 numbering. Note that, in this numbering system, 1 refers to the N-terminal amino acid of the mature HAO protein, from which the signal peptide has been removed. Those of skill in the art will readily be able to recognize the amino acid residues that correspond to the HA1 C-terminal stem segment of other influenza HA polypeptides, e.g., the amino acid residues that correspond to the HA1 C-terminal stem segment of HA1 from an Hl hemagglutinin. ForAttorney Docket No. MSZ0003PCTexample, an alignment of HAs may be done to identify the residue that corresponds to the Cys at amino acid position 277 according to H3 numbering. See, e.g., SEQ ID NO: 1 and / or 5 in Table 1 for the cysteine that corresponds to Cys at amino acid position 277 according to H3 numbering. In some embodiments, the HA1 C-terminal stem segment comprises the amino acid sequence of a HA1 C-terminal stem segment from SEQ ID NO: 1 or 5.

[0122] As used herein, the term " HA1 N-terminal stem segment" refers to a polypeptide segment that corresponds to the amino-terminal portion of the stem domain of an influenza virus hemagglutinin HA1. In some embodiments, an HA1 N-terminal stem segment consists of amino acid residues corresponding approximately to amino acids AN-term through Apof an HA1 of an influenza A vims hemagglutinin. AN-term otherwise referred to herein as HAIN-term is the N-tenuinal amino acid of HA1 as recognized by those of skill in the art. Apis the cysteine residue in the HA1 N-terminal stem segment that forms or is capable of forming a disulfide bond with a cysteine residue in an influenza A vims HA1 C-terminal stem segment. In some embodiments, residue Apis the Cys at amino acid position 52 of an HA1 according to H3 numbering. In some embodiments, an HA1 N-terminal stem segment consists of amino acid residues corresponding approximately to amino acids 1-52 of HA1 according to H3 numbering. Note that, in this numbering system, 1 refers to the N-terminal amino acid of the mature HAO protein, from which the signal peptide has been removed. Those of skill in the art will readily be able to recognize the amino acid residues that correspond to the HA1 N-terminal stem segment of other influenza HA polypeptides, e.g., the amino acid residues that correspond to the HA1 N-terminal stem segment of HA1 from an Hl hemagglutinin. For example, an alignment of HAs may be done to identify the residue that corresponds to the Cys at amino acid position 52 according to H3 numbering. See, e.g., SEQ ID NO: 1 or 5 in Table 1 for the cysteine that corresponds to Cys at amino acid position 52 according to H3 numbering. In some embodiments, the HA1 N-terminal stem segment comprises the amino acid sequence of a HA1 N-terminal stem segment from SEQ ID NO: 1 or 5.

[0123] As used herein, the tern "heterologous" in the context of a polypeptide, nucleic acid, or vims refers to a polypeptide, nucleic acid, or vims, respectively, that is not normally found in nature or not normally associated in nature with a polypeptide, nucleic acid, or vims of interest. For example, an HA globular head domain that is heterologous to an HA stalk domain refers to an HA globular head domain that would not be found in nature associated with the HA stalk domain or would not normally be associated in nature with the HA stalk domain.

[0124] As used herein, the term “human adult” refers to a human 18 years old and older.Attorney Docket No. MSZ0003PCT

[0125] As used herein, the term “human child” refers to a human 1 years old to 18 years old.

[0126] As used herein, the term “human infant” refers to a newborn human to 1 years old.

[0127] Immunologically distinct: In any aspect or embodiment described herein, the HA globular head domain of a chimeric HA is immunologically distinct from the HA globular head domain normally associated with an HA stem domain if there is no cross-reactivity in an immunoassay described herein or known to one of skill in the art. In any aspect or embodiment described herein, the HA globular head domain of a chimeric HA is immunologically distinct from the HA globular head domain normally associated with an HA stem domain if there is no cross-reactivity in a hemagglutinin inhibition assay known to one of skill in the art. In any aspect or embodiment described herein, the HA globular head domain of a chimeric HA is immunologically distinct from the HA globular head domain normally associated with an HA stem domain if there is no cross-reactivity in an immunoassay and a hemagglutinin inhibition assay known to one of skill in the art. In any aspect or embodiment described herein, the HA globular head domain of a chimeric HA is immunologically distinct from the HA globular head domain normally associated with an HA stem domain if the HA globular head domain of the chimeric HA is considered of a different influenza A vims subtype than the HA globular head domain normally associated with the HA stem domain using Center for Disease Control and Prevention (CDC), Food and Drug Administration (FDA), or World Health Organization (WHO) criteria.

[0128] As used herein, the term “nucleic acid” and “nucleotide” is intended to include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) (e.g., messenger RNA (mRNA)) and analogs of the DNA or RNA generated using modified nucleosides or nucleotide analogs. In any aspect or embodiment described herein, the nucleic acid is a RNA. In any aspect or embodiment described herein, the nucleic acid is complementary DNA (cDNA).

[0129] As used herein, the term “or” is to be interpreted as an inclusive “or” meaning any one or any combination. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0130] Percent identity: Techniques known to one of skill in the art can be used to determine the percent identity between two amino acid sequences or between two nucleotide sequences. Generally, to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g.,Attorney Docket No. MSZ0003PCTgaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = numberof identical overlapping positions / total number of positions X 100%). In some embodiments, the two sequences are the same length. In some embodiments, the percent identity is determined over the entire length of an amino acid sequence or nucleotide sequence. The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U. S. A.87:22642268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U. S. A.90:58735877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol.215:403.

[0131] BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison puiposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:33893402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). In another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1 88, CABIOS 4: 1117. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniquesAttorney Docket No. MSZ0003PCTsimilar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0132] As used herein, the terms "replication," "viral replication," and "vims replication" in the context of a vims refer to one or more, or all, of the stages of a viral life cycle which result in the propagation of vims. The steps of a viral life cycle include, but are not limited to, virus attachment to the host cell surface, penetration or entry of the host cell (e.g., through receptor mediated endocytosis or membrane fusion), uncoating (the process whereby the viral capsid is removed and degraded by viral enzymes or host enzymes thus releasing the viral genomic nucleic acid), genome replication, synthesis of viral messenger RNA (mRNA), viral protein synthesis, and assembly of viral ribonucleoprotein complexes for genome replication, assembly of vims particles, post-translational modification of the viral proteins, and release from the host cell by lysis or budding and acquisition of a phospholipid envelope which contains embedded viral glycoproteins. In some embodiments, the terms "replication," "viral replication" and "vims replication" refer to the replication of the viral genome. In some embodiments, the terms "replication," "viral replication" and "vims replication" refer to the synthesis of viral proteins.

[0133] As used herein, terms “subject” or “patient” are used interchangeably to refer to an animal (e.g., birds, reptiles, and / or mammals). In some embodiments, a subject is a bird. In some embodiments, a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, a subject is a non-human animal. In some embodiments, a subject is a non-human primate (e.g., a monkey, such as a rhesus monkey). In some embodiments, a subject is a farm animal or pet. In specific embodiments, a subject is a human. In some embodiments, a subject is a human adult. In some embodiments, a subject is an elderly human. In some embodiments, a subject is a human child. In some embodiments, a subject is a human infant.

[0134] As used herein, the term “seasonal influenza vims strain” refers to a strain of influenza vims to which a subject population is exposed to on a seasonal basis. In specific embodiments, the term seasonal influenza vims strain refers to a strain of influenza A vims. In some embodiments, the term seasonal influenza vims strain refers to a strain of influenza A vims that belongs to the Hl or H3 subtype, i.e., the two subtypes that presently persist in the human subject population.

[0135] The terms “tertiary structure” and “quaternary structure” have the meanings understood by those of skill in the art. Generally, tertiary stmcture refers to the three-Attorney Docket No. MSZ0003PCTdimensional structure of a single polypeptide chain. Generally, quaternary structure refers to the three-dimensional structure of a polypeptide having multiple polypeptide chains.

[0136] As used herein, in some embodiments, the phrase “wild-type” in the context of a viral polypeptide refers to a viral polypeptide that is found in nature and is associated with a naturally occurring vims.

[0137] In some embodiments, the phrase “wild-type” in the context of a vims refers to a vims that is prevalent, circulating naturally and producing typical outbreaks of disease. In some embodiments, the term “wild-type” in the context of a vims refers to a parental vims Nucleoside-Modified RNA Molecules

[0138] In one aspect, provided herein are nucleoside-modified RNA molecules encoding a chimeric influenza vims hemagglutinin (HA) polypeptide described herein. An aspect of the present disclosure provides a nucleoside-modified ribonucleic acid (RNA) molecule encoding a chimeric influenza vims hemagglutinin (HA) polypeptide comprising a HA globular head domain and a HA stalk domain, wherein: (a) the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B vims, (b) the HA globular head domain comprises an amino acid sequence of (i) the HA globular head domain of an influenza A vims or (ii) the HA globular head domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain), wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A vims, and (c) the nucleoside-modified RNA molecule comprises at least one pseudouridine. A further aspect of the present disclosure provides a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine. Another aspect of the present disclosure provides a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the secondAttorney Docket No. MSZ0003PCTHA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A virus of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0139] In any aspect or embodiment described herein, provided herein are nucleoside-modified RNA molecules comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide described herein. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially or, or consisting of, the amino acid sequence of SEQ ID NO: 1. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially or, or consisting of, the amino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially or, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially or, or consisting of, the amino acid sequence of SEQ ID NO: 5. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially or, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially or, or consisting of, the amino acid sequence of SEQ ID NO: 7.

[0140] In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 80%, at least 85%, or least 90% identical to the amino acid sequence of SEQ ID NO: 1. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 80%, at least 85%, or least 90% identical to theAttorney Docket No. MSZ0003PCTamino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 95%, at least 98%, or least 99% identical to the amino acid sequence of SEQ ID NO: 1. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 95%, at least 98%, or least 99% identical to the amino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 80%, at least 85%, or least 90% identical to the amino acid sequence of SEQ ID NO: 5. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 80%, at least 85%, or least 90% identical to the amino acid sequence of SEQ ID NO: 7. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 95%, at least 98%, or least 99% identical to the amino acid sequence of SEQ ID NO: 5. In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule comprising, consisting essentially of, or consisting of, a codon optimized sequence encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 95%, at least 98%, or least 99% identical to the amino acid sequence of SEQ ID NO: 7.

[0141] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) comprises at least 1 modified nucleoside. In any aspect or embodiment described herein, the nucleoside-modified RNAAttorney Docket No. MSZ0003PCTmolecule comprises at least 2, at least 3, least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 modified nucleosides. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule comprises 1, 2, or 3 modified nucleosides. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule comprises 4, 5, 6, 7, 8, 9, 10 or more modified nucleosides. In any aspect or embodiment described herein, the modified nucleoside(s) is pseudouridine. In any aspect or embodiment described herein, the modified nucleosides includes pseudouridine and one or more other modified nucleosides (e.g., one or more other modified nucleosides described herein).

[0142] In any aspect or embodiment described herein, a modified nucleoside is a modified nucleoside of uridine. In any aspect or embodiment described herein, a modified nucleoside is pseudouridine. In any aspect or embodiment described herein, a modified nucleoside is a monophosphate, diphosphate, or triphosphate of a pseudouridine e.g., a monophosphate, diphosphate, or triphosphate of mlacp3 (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine, m I (1 -methylpseudouridine), Tm (2’-O-methylpseudouridine), m5D (5-methyldihydrouridine), or m3T (3-methylpseudouridine)). In any aspect or embodiment described herein, a modified nucleoside is a modified nucleoside of cytidine. In any aspect or embodiment described herein, a modified nucleoside is m5C (5-methylcytidine). In any aspect or embodiment described herein, a modified nucleoside is a modified nucleoside of adenosine. In any aspect or embodiment described herein, a modified nucleoside is m6A (N6-methyladenosine). In some embodiments, a modified nucleoside is a modified nucleoside of guanosine.

[0143] In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises a modified nucleoside of uridine (e.g., s2U (2-thiouridine), T (pseudouridine), m5U (5-methyluridine), and / or Um (2’-O-methyluridine)). In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza vims HA polypeptide described herein comprises a modified nucleoside of cytidine (e.g., m5C (5-methylcytidine)). In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modifiedAttorney Docket No. MSZ0003PCTRNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises a modified nucleoside of adenosine (e.g., m‘A (N6-methyladenosine)). In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises a modified nucleoside of guanosine.

[0144] In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) comprises pseudouridine. In any aspect or embodiment described herein, the pseudouridine is naturally occurring. In any aspect or embodiment described herein, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (see, e.g., Kariko et al., 2008, Mol Ther 16: 1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0145] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (see, e.g., Kariko et al., 2005, Immunity 23:165-175). Further, protein-encoding, in vitro transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Also, it is shown that the presence of pseudouridine improves the stability of RNA (see, e.g., Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (see, e.g., Anderson et al., 2010, Nucleic Acids Res 38:5884-5892).

[0146] Similar effects as described for pseudouridine have also been observed for RNA containing 1 -methyl -pseudouridine.

[0147] In any aspect or embodiment described herein, a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises pseudouridine known to one of skill in the art or described herein. In any aspect or embodiment described herein, the pseudouridine isAttorney Docket No. MSZ0003PCTmlacp3lP (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine, ml'P (1-methylpseudouridine), Tm (2’-O-methylpseudouridine), m5D (5-methyldihydrouridine), or 1113d1(3-methylpseudouridine). In any aspect or embodiment described herein, a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises a monophosphate, diphosphate, or triphosphate of a pseudouridine (e.g., a monophosphate, diphosphate, or triphosphate of mlacp3 (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine, ml'P (1-methylpseudouridine), Tm (2’-O-methylpseudouridine), m5D (5-methyldihydrouridine), or m3T (3-methylpseudouridine)). In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises m IT-5-triphosphate. In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises a pseudouridine-like nucleoside known in the art.

[0148] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein further comprises, consists essentially of, or consists of, a cap on the 5’ end and / or a 3’ poly(A) tail. In any aspect or embodiment described herein, the poly(A) tail is about 100 nucleotides to about 300, or about 100 nucleotides to about 400 nucleotides in length. In any aspect or embodiment described herein, the poly(A) tail is about 100 nucleotides to about 110 nucleotides in length. In any aspect or embodiment described herein, the poly(A) tail is 101 nucleotides in length. In any aspect or embodiment described herein, such a poly(A) tail results in about a two-fold increase in the translation efficiency of the RNA. In any aspect or embodiment described herein, different chemical groups are attached to the 3’ end to increase mRNA stability. For example, in any aspect or embodiment described herein, ATP analogs are incorporated into the poly(A) tail using poly(A)Attorney Docket No. MSZ0003PCTpolymerase. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein further comprises, consists essentially of, or consists of, a 5’ capl structure.

[0149] In any aspect or embodiment described herein, a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein comprises pseudouridine, a cap on the 5’ end, and a 3’ poly (A) tail. In any aspect or embodiment described herein, the pseudouridine is one described herein. In any aspect or embodiment described herein, the pseudouridine is ml'P-5-triphosphate. In any aspect or embodiment described herein, the poly (A) tail is about 100 nucleotides to about 300, or about 100 nucleotides to about 400 nucleotides in length. In any aspect or embodiment described herein, the poly(A) tail is about 100 nucleotides to about 110 nucleotides in length. In any aspect or embodiment described herein, the poly(A) tail is 101 nucleotides in length.

[0150] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza virus HA polypeptide described herein further comprises, consists essentially of, or consists of, a 5’ untranslated region (UTR) and a 3’ UTR. In any aspect or embodiment described herein, the 5’ UTR is between zero and 3000 nucleotides in length. In any aspect or embodiment described herein, the UTR sequences are associated, or found in nature, with an influenza virus gene segment encoding a wild-type influenza virus HA. In any aspect or embodiment described herein, the UTR sequences are not found in nature associated with an influenza virus gene segment encoding a wild-type influenza virus HA. In any aspect or embodiment described herein, the 3’ UTR is selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art (e.g., AU-rich elements in 3’ UTR sequences can decreased). In any aspect or embodiment described herein, the 5’ UTR contains a Kozak sequence. For example, in any aspect or embodiment described herein, the Kozak sequence may be designed or naturally occurring.Attorney Docket No. MSZ0003PCT

[0151] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) encoding a chimeric influenza vims HA polypeptide described herein is a nucleoside-modified RNA described in the Examples.

[0152] In any aspect or embodiment described herein, provided herein is a nucleoside-modified RNA molecule (e.g., a first nucleoside-modified RNA molecule or a second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is a transcription product generated from transcription of the DNA sequence that encodes an amino acid sequence of SEQ ID NO: 1 in the presence of a modified nucleoside of uridine. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is a transcription product generated from transcription of the DNA sequence that encodes an amino acid sequence of SEQ ID NO: 3 in the presence of a modified nucleoside of uridine. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is a transcription product generated from transcription of the DNA sequence that encodes an amino acid sequence of SEQ ID NO: 5 in the presence of a modified nucleoside of uridine. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is a transcription product generated from transcription of the DNA sequence that encodes an amino acid sequence of SEQ ID NO: 7 in the presence of a modified nucleoside of uridine.

[0153] An aspect of the present disclosure provides a nucleoside-modified ribonucleic acid (RNA) molecule encoding a chimeric influenza virus hemagglutinin (HA) polypeptide comprising a HA globular head domain and a HA stalk domain, wherein: (a) the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B virus, (b)Attorney Docket No. MSZ0003PCTthe HA globular head domain comprises an amino acid sequence of (i) a HA globular head domain of an influenza A vims, or (ii) the HA globular head domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain), wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A vims, and (c) the nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0154] In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain) and a HA cytoplasmic domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain).

[0155] Another aspect of the present disclosure relates to a nucleoside-modified RNA molecule encoding a chimeric influenza vims HA polypeptide comprising, consisting essentially or, or consisting of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0156] A further aspect of the present disclosure relates to a nucleoside-modified RNA molecule transcribed from a DNA sequence that encodes an amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, wherein the transcription occurred in the presence of a modified nucleoside of uridine.Methods for Producing Nucleoside-Modified RNA Molecules

[0157] Techniques known to one of skill in the art may be used to produce the nucleoside-modified RNA molecule described herein. In any aspect or embodiment described herein, a codon optimized nucleic acid molecule encoding the chimeric influenza vims HA polypeptide described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is synthesized using techniques known in the art and the codon optimized nucleic acid molecule is litigated into an mRNA production vector. In any aspect or embodiment described herein, the codon optimized nucleic acid molecule is operably linked to a promoter for an RNA polymerase. “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. In any aspect or embodiment described herein, the promoter is a T7 RNA polymerase promoter. In any aspect or embodiment described herein, the promoter is a T3 RNA polymerase promoter or SP6 RNA polymerase promoter. In any aspect or embodiment described herein, theAttorney Docket No. MSZ0003PCTpromoter comprises a consensus nucleotide sequence of a T7 RNA polymerase promoter, T3 RNA polymerase promoter, or SP6 RNA polymerase promoter known to one of skill in the art.

[0158] In any aspect or embodiment described herein, the codon optimized nucleic acid molecule encoding the chimeric influenza virus HA polypeptide of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is operably linked to a 5’ untranslated region (UTR) and a 3’ UTR. In any aspect or embodiment described herein, the 5’ UTR is between zero and 3000 nucleotides in length. The length of 5’ and 3’ UTR sequences can be altered by different methods, including, but not limited to, designing primers for polymerase chain reaction (PCR) that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA. In any aspect or embodiment described herein, the UTR sequences are associated, or found in nature, with an influenza virus gene segment encoding a wild-type influenza virus HA. In any aspect or embodiment described herein, the UTR sequences are not found in nature associated with an influenza virus gene segment encoding a wild-type influenza virus HA. In any aspect or embodiment described herein, the 3’ UTR is selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art e.g., AU-rich elements in 3’ UTR sequences can decreased). In any aspect or embodiment described herein, the 5’ UTR contains a Kozak sequence. The Kozak sequence may be designed or naturally occurring.

[0159] In any aspect or embodiment described herein, the mRNA production vector is linearized and is in vitro transcribed (IVT) into RNA using techniques known to one of skill in the art. In any aspect or embodiment described herein, the linearized mRNA production vector is transcribed using an RNA polymerase (e.g., T7 RNA polymerase). In any aspect or embodiment described herein, one or more modified nucleosides are included in the in vitro transcription reaction. In any aspect or embodiment described herein, a modified nucleoside of uridine (e.g., s2U (2-thiouridine), ‘P (pseudouridine), m5U (5-methyluridine), and / or Um (2’-O-methyluridine)) is included in the in vitro transcription reaction. In any aspect or embodiment described herein, a modified nucleoside of cytidine (e.g., m5C (5-methylcytidine)) is included in the in vitro transcription reaction. In any aspect or embodiment described herein, a modified nucleoside of adenosine (e.g., m6A (N6-Attorney Docket No. MSZ0003PCTmethyladenosine)) is included in the in vitro transcription reaction. In any aspect or embodiment described herein, a modified nucleoside of guanosine is included in the in vitro transcription reaction.

[0160] In any aspect or embodiment described herein, pseudouridine is used in place of uridine in the in vitro transcription reaction. In any aspect or embodiment described herein, the pseudouridine is mlacp3T (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine, m I (1 -methylpseudouridine), Tin (2’-O-methylpseudouridine), m5D (5-methyldihydrouridine), or m3T (3-methylpseudouridine). In any aspect or embodiment described herein, a monophosphate, diphosphate, or triphosphate of a pseudouridine (e.g., a monophosphate, diphosphate, or triphosphate of mlacp3T (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine, mlT (1-methylpseudouridine), Tm (2’-O-methylpseudouridine), m5D (5-methyldihydrouridine), or m3T (3-methylpseudouridine)) is used in place of uridine in the in vitro transcription reaction. In any aspect or embodiment described herein, mlT-5-triphosphate is used in the in vitro transcription reaction. In any aspect or embodiment described herein, pseudouridine-like nucleoside known in the art is included in the in vitro transcription reaction.

[0161] In any aspect or embodiment described herein, in vitro transcribed RNA encoding a chimeric influenza virus HA polypeptide comprises a cap on the 5’ end and / or a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability of mRNA in the cell. In any aspect or embodiment described herein, the poly (A) tail is about 100 nucleotides to about 300, or about 100 nucleotides to about 400 nucleotides. In any aspect or embodiment described herein, such a poly(A) tail results in about a two-fold increase in the translation efficiency of the RNA. In any aspect or embodiment described herein, different chemical groups are attached to the 3’ end to increase mRNA stability. For example, in any aspect or embodiment described herein, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA. Techniques known to one of skill in the art may be used to produce in vitro transcribed RNA encoding a chimeric influenza virus HA polypeptide including a 5’ cap (see, e.g., Cougot, el al.. Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001): Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)). In any aspect or embodiment described herein, in vitro transcribed RNA encoding a chimeric influenza virus HA polypeptide include a 5’ capl structure. Techniques known to one of skill in the art may be used to produce a 5’ capl structure. For example, in any aspect or embodiment describedAttorney Docket No. MSZ0003PCTherein, a 5' capl structure can be generated using Vaccinia capping enzyme and 2’-0-methyltransferase enzymes (CellScript, Madison, WI).

[0162] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule and / or the second nucleoside-modified RNA molecule) is an mRNA molecule.

[0163] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is purified using techniques known to one of skill in the art. In any aspect or embodiment described herein, chromatography methods, including but not limited to HPLC, cellulose, and fast protein liquid chromatography (FPLC) may be used to purify a nucleoside-modified RNA molecule described herein. An exemplary FPLC -based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. Exemplary purification procedures are also described in U. S. Patent Application Publication No. 2016 / 0032316, which is hereby incorporated by reference in its entirety. In any aspect or embodiment described herein, a non-HPLC method is used to purify the nucleoside-modified RNA molecule described herein. In any aspect or embodiment described herein, the methodology described in the Examples is used to purify the nucleoside-modified RNA molecule described herein. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is purified to remove double-stranded contaminants.

[0164] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is produced as described in the Examples.

[0165] In any aspect or embodiment described herein, a nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modifiedAttorney Docket No. MSZ0003PCTRNA molecule and the second nucleoside-modified RNA molecule) exhibits enhanced ability to be translated by a target cell. In any aspect or embodiment described herein, translation is enhanced by a factor of 2-fold or more relative to its unmodified counterpart. In any aspect or embodiment described herein, translation is enhanced by a factor of 3 -fold or more relative to its unmodified counterpart. In any aspect or embodiment described herein, translation is enhanced by a factor of 4-fold or more relative to its unmodified counterpart. In any aspect or embodiment described herein, translation is enhanced by a factor of 5-fold or more relative to its unmodified counterpart. In any aspect or embodiment described herein, translation is enhanced by a factor of 10-fold or more relative to its unmodified counterpart. In any aspect or embodiment described herein, translation is enhanced by a factor of 20-fold or more relative to its unmodified counterpart.

[0166] In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) induces a significantly more robust humoral immune response and / or adaptive immune response as compared with an unmodified in vitro synthesized RNA molecule of the same sequence. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) induces a humoral immune response and / or an adaptive immune response that is 2-fold or greater than its unmodified counterpart. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) induces a humoral immune response and / or an adaptive immune response that is 3-fold or greater than its unmodified counterpart. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) induces a humoral immune response and / or an adaptive immune response that is 4-fold or greater than its unmodified counterpart. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and theAttorney Docket No. MSZ0003PCTsecond nucleoside-modified RNA molecule) induces a humoral immune response and / or an adaptive immune response that is 5-fold or greater than its unmodified counterpart. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) induces a humoral immune response and / or an adaptive immune response that is 10-fold or greater than its unmodified counterpart. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule) induces a humoral immune response and / or an adaptive immune response that is 20-fold or greater than its unmodified counterpart.Chimeric Influenza Virus HA Polypeptides

[0167] In any aspect or embodiment described herein, the nucleoside-modified ribonucleic acid (RNA) molecule encoding a chimeric influenza vims hemagglutinin (HA) polypeptide that comprises a HA globular head domain and a HA stalk domain, wherein: (a) the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B vims, (b) the HA globular head domain comprises an amino acid sequence of (i) a HA globular head domain of an influenza A vims or (i) a HA globular head domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain), wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A vims, and (c) the nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the HA globular head domain comprises the amino acid sequence of the HA globular head domain of an influenza A vims. In any aspect or embodiment described herein, the HA globular head domain comprises the amino acid sequence of the HA globular head domain of an influenza B vims (e.g., the same influenza B vims as the HA stalk domain), wherein one or more antigenic sites have been replaced with antigenic sites of the influenza A vims.

[0168] In any aspect or embodiment described herein, the HA globular head domain is one described in the Example. In any aspect or embodiment described herein, the HA stalk domain is one described in the Examples.

[0169] In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof. In any aspect or embodiment described herein, the antigenic sitesAttorney Docket No. MSZ0003PCTcomprise, consist essentially of, or consist of, 120 loop, 150 loop, 160 loop, and 190 helix. In any aspect or embodiment described herein, the influenza A virus is an influenza A virus that originates from a different species than the influenza B virus. In any aspect or embodiment described herein, the influenza A vims is an avian influenza A vims. In any aspect or embodiment described herein, at least one of (a) the influenza B vims is B / Phuket / 3073 / 2013 Influenza virus or B / Brisbane / 20 / 2008 Influenza vims; (b) the influenza A vims is a hemagglutinin 5 (H5) Influenza A vims, a hemagglutinin 13 (H13) Influenza A vims, or subtype thereof; or (c) a combination thereof.

[0170] In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hll, H12, H13, H16, H17, or H18 (preferably H13 (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6) or H5 (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP)). In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of the HA of the influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hll, H12, H13, H16, H17, or H18 (preferably H13 (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6) or H5 (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP)).

[0171] In any aspect or embodiment described herein, the influenza vims HA stalk domain comprises the HA stalk domain of an HA of an influenza B vims. In any aspect or embodiment described herein, the HA stalk domain comprises the HA stalk domain of Influenza vims B / Phuket / 3073 / 2013 HA or B / Phuket / 3073 / 2013-like HA. In any aspect or embodiment described herein, the HA stalk domain comprises the HA stalk domain of Influenza vims B / Brisbane / 20 / 2008 HA or B / Brisbane / 20 / 2008 -like HA.

[0172] In any aspect or embodiment described herein, (a) the influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the influenza A vims is a hemagglutinin 5 (H5) Influenza A vims; or (b) the influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the influenza A vims is a hemagglutinin 13 (Hl 3).

[0173] In any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises,Attorney Docket No. MSZ0003PCTconsists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B virus, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A virus, or a HA globular head domain of an first influenza B vims (e.g., the same influenza B vims as the HA stalk domain), wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, 120 loop, 150 loop, 160 loop, and 190 helix.

[0174] In any aspect or embodiment described herein, the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, 120 loop, 150 loop, 160 loop, and 190 helix.

[0175] In any aspect or embodiment described herein, the HA globular head domain is one described in the Example. In any aspect or embodiment described herein, the HA stalk domain is one described in the Examples.

[0176] In any aspect or embodiment described herein, at least one of (1) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims; (2) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acidAttorney Docket No. MSZ0003PCTsequence of a HA globular head domain of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; or (3) a combination thereof.

[0177] In any aspect or embodiment described herein, at least one of (1) the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus; (2) the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; or (3) a combination thereof.

[0178] In any aspect or embodiment described herein, at least one of (1) the first influenza A virus is an influenza A virus that originates from a different species than the first influenza B virus; (2) the second influenza A vims is an influenza A vims that originates from a different species than the second influenza B vims; (3) the first influenza A vims is an avian influenza A vims; (4) the second influenza A vims is an avian influenza A vims; or (5) a combination thereof.

[0179] In any aspect or embodiment described herein, at least one of (1) the first influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims; (2) the second influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims; (3) the first influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims, a hemagglutinin 5 (H5) Influenza A vims, or a subtype thereof; (4) the second influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims, a hemagglutinin 5 (H5) Influenza A vims, or a subtype thereof; or (5) a combination thereof.

[0180] In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide (e.g., a first chimeric influenza vims HA polypeptide and / or a chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, the amino acid sequence of the HA globular head domain of an HA of an influenza A vims subtype H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17, or H18 (preferably H13 (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6) or H5 (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP)). In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide (e.g., a first chimeric influenza vims HA polypeptide and / or a chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of the HA of the influenza B vims, wherein one or more antigenicAttorney Docket No. MSZ0003PCTsites have been replaced with antigenic sites of the HA globular head domain of an HA of an influenza A virus subtype H2, H5, H6, H8, H9, Hll, H12, H13, H16, H17, or H18 (preferably H13 (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6) or H5 (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP)).

[0181] In any aspect or embodiment described herein, (1) at least one of the first influenza B virus is B / Brisbane / 20 / 2008 Influenza vims and the first Influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims; the second influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the second influenza A vims is a hemagglutinin 5 (H5) Influenza A vims; or a combination thereof; or (2) at least one of the first influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the first influenza A vims is a hemagglutinin 5 (H5) Influenza A vims or a subtype thereof; the second influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the second influenza A vims is a hemagglutinin 13 (H13) Influenza A vims or a subtype thereof; or a combination thereof.

[0182] In any aspect or embodiment described herein, (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the second nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof (e.g., wherein the first chimeric influenza vims HA polypeptide and the second chimeric influenza vims HA polypeptide have different amino acid sequence or SEQ ID NOs).

[0183] In any aspect or embodiment described herein, (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0184] In any aspect or embodiment described herein, (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5Attorney Docket No. MSZ0003PCTor 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0185] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0186] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0187] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0188] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0189] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptideAttorney Docket No. MSZ0003PCTcomprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0190] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0191] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0192] In any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

[0193] In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide described herein (e.g., the first chimeric Influenza vims HA polypeptide and / or the second chimeric Influenza vims HA polypeptide) further comprises, consists essentially of, or consists of, an influenza vims HA transmembrane domain (e.g., a HA transmembrane domain of the influenza B vims). In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) further comprises, consists essentially of, or consists of, an influenza vims HA cytoplasmic domain (e.g., a HA cytoplasmic domain of the Influenza B vims). In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide (e.g., the first chimericAttorney Docket No. MSZ0003PCTinfluenza virus HA polypeptide and / or the second chimeric Influenza virus HA polypeptide) further comprises, consists essentially of, or consists of, an influenza virus HA transmembrane domain (e.g., a HA transmembrane domain of the influenza B virus) and an influenza virus HA cytoplasmic domain (e.g., a HA cytoplasmic domain of the Influenza B virus). In any aspect or embodiment described herein, the influenza vims HA transmembrane domain and the influenza vims HA cytoplasmic domain are from the same Influenza vims HA as the Influenza vims HA stalk domain. By way of example, in any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of the influenza B vims and a HA cytoplasmic domain of the Influenza B vims. For example, in any aspect or embodiment described herein, (1) the first chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B vims (e.g., the first Influenza B vims) and a HA cytoplasmic domain of an influenza B vims (e.g., the first Influenza B vims); (2) the second chimeric influenza vims HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B vims (e.g., the second Influenza B vims) and a HA cytoplasmic domain of an influenza B vims (e.g., the second Influenza B vims); or (3) a combination thereof.

[0194] In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:1. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric Influenza vims HA polypeptide) comprises, consists essentially or, or consists of, an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 5. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the secondAttorney Docket No. MSZ0003PCTchimeric influenza virus HA polypeptide) comprises, consists essentially of, or consists of, an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

[0195] In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza virus HA polypeptide) comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO:1. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide of the present disclosure (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric Influenza vims HA polypeptide) comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric Influenza vims HA polypeptide) comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7.

[0196] A full-length influenza HA typically comprises an HA1 domain an HA2 domain. The HA stalk domain is formed by two segments of the HA1 domain and most of the HA2 domain (e.g., the portion of the HA2 domain that does not include the transmembrane and cytoplasmic domains). The two segments of the HA1 domain are separated, in primary sequence, by the globular head domain. In any aspect or embodiment described herein, the globular head domain comprises the amino acid residues between the residues designated Apand Aq. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) maintain such a stmcture. That is, in any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) comprise, consists essentially of, or consists of, a stable stalk stmcture composed of an HA1 domain and an HA2 domain, and a globular head domain separating the two segments of the HA1 domain (in primary sequence), wherein said globular head domain is heterologous to the stalk domain formed by the other segments of the HA1 domain and the HA2 domain.Attorney Docket No. MSZ0003PCT

[0197] Typically, an HA stalk domain comprises an N-terminal HA1 stem segment, a C-temiinal HA1 stem segment, and a portion of an HA2 domain (i.e., a portion of an HA2 domain that does not include the transmembrane and cytoplasmic domains; sometimes referred to as an “HA2 stem domain”). One skilled in the art will understand that the exact location of the C-terminus of the HA stalk domain is determined according to the hydrophobicity of the HA2 domain of the particular influenza A virus HA strain and can be identified using programs such as, e.g., the TMHMM server (www.cbs.dtu.dk / services / TMHMM / ; see, e.g., Cuthbertson et al., 2005, Protein Eng Des Sei, 18(6):295-308) hydrophobicity prediction, or uniprot. In specific embodiments, the HA2 stem domain is from the same influenza vims strain or subtype as the portion of the HA stem domain of the HA1 subunit.

[0198] Typically, an influenza A vims HA1 N-terminal stem segment corresponds to a polypeptide consisting of the N-terminal amino acid of a mature HA1 (i.e., an HA1 lacking a signal peptide) through the cysteine residue located in sequence at approximately the 52ndresidue of the HA1 according to H3 numbering. This cysteine residue, termed Apherein, is generally capable of forming a disulfide bridge with a cysteine residue in the C-terminal stem segment of HA 1.

[0199] Typically, an Influenza A vims HA1 C-terminal stem segment corresponds to a polypeptide consisting of the cysteine residue located in the amino acid sequence at approximately the 277th residue of a mature HA1 (i.e., an HA1 lacking a signal peptide) according to H3 numbering through the C-terminal amino acid of the HA1. This cysteine residue, termed Aq herein, is generally capable of forming a disulfide bridge with cysteine residue Ap in the N-terminal stem segment of HA1.

[0200] Those of skill in the art will recognize that an influenza A vims HA globular head domain typically comprises the amino acid residues intervening the cysteine (Cys) that corresponds to amino acid position 52 of an influenza A vims hemagglutinin HA1 domain according to H3 numbering and the cysteine (Cys) that corresponds to amino acid position 277 of an influenza A vims hemagglutinin HA1 domain according to H3 numbering.

[0201] In any aspect or embodiment describe herein, a chimeric influenza vims HA polypeptide provided herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) comprises, consists essentially of, or consists of, an HA stalk domain and an HA globular head domain, wherein the HA head domain is heterologous to the HA stalk domain, and wherein the chimeric HA has a primary stmcture of, in the following order: an HA 1 N-terminal stem segment, an HA globular headAttorney Docket No. MSZ0003PCTdomain, an HA1 C-terminal stem segment, and a portion of the HA2 domain (e.g., the HA2 domain lacking the transmembrane and cytoplasmic domains), optionally further comprising, consisting essentially of, or consisting of, the transmembrane domain and / or cytoplasmic portion of the HA2 domain. The primary sequence of a chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) might be formed by a single polypeptide, or it might be formed by multiple polypeptides. Typically, a single polypeptide is expressed by any technique deemed suitable by one of skill in the art.

[0202] In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) comprises, consists essentially of, or consists of, an HA globular head domain (e.g., a portion of the HA1 domain) and an HA stalk domain, wherein the HA stalk domain comprises, consists essentially of, or consists of, an HA1 N-tenuinal stem segment, an HA1 C-terminal stem segment, and HA2 stem domain.

[0203] In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) further comprises, consists essentially of, or consists of, a signal peptide. Typically, the signal peptide is cleaved during or after polypeptide expression and translation to yield a mature chimeric influenza virus HA polypeptide. In any aspect or embodiment described herein, provided herein are mature chimeric influenza virus HA polypeptides that lack a signal peptide. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) further comprises, consists essentially of, or consists of, a signal peptide, wherein the signal peptide might be based on any influenza virus signal peptide known to those of skill in the art. The signal peptide of an influenza vims HA may be determined using software, such as, e.g., signalP 5.0. In any aspect or embodiment described herein, the signal peptides are based on influenza B virus HA signal peptides. In any aspect or embodiment described herein, the signal peptides are based on the signal peptide of an influenza B vims HA selected from B / Brisbane / 20 / 2008 Influenza vims HA and B / Phuket / 3073 / 2013 Influenza vims HA. In any aspect or embodiment described herein, the signal peptides are based on influenza A vims HA signal peptides. In any aspect or embodiment described herein, the signal peptides are based on the signal peptide of an influenza A vims HA selected from the group consisting of Hl, H2, H3, H4, H5, H6, H7, H8,Attorney Docket No. MSZ0003PCTH9, H10, Hll, H12, H13, H14, H15, H16, H17, and H18. In any aspect or embodiment described herein, the signal peptide comprises, consists essentially of, or consists of, the amino acid sequence of the signal peptide of the HA of influenza virus A / Hong Kong / 4801 / 14. In any aspect or embodiment described herein, the signal peptide comprises, consists essentially of, or consists of, the amino acid sequence of the signal peptide of the HA of influenza virus A / California / 04 / 09. In any aspect or embodiment described herein, the signal peptide comprises, consists essentially of, or consists of, the amino acid sequence of the signal peptide of the HA of influenza vims A / Wedge-tailed shearwater / Westem Australia / 2576 / 1979 or A / Duck / Czechslovakia / 1956. In any aspect or embodiment described herein, the signal peptide comprises, consists essentially of, or consists of, the amino acid sequence of the signal peptide of the HA of influenza virus A / Vietnam / 1203 / 04 or influenza virus A / mallard / Sweden / 24 / 02. In any aspect or embodiment described herein, the signal peptide might be any signal peptide deemed useful to one of skill in the art.

[0204] In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) are capable of forming a three dimensional structure that is similar to the three dimensional structure of a native influenza HA. Structural similarity might be evaluated based on any technique deemed suitable by those of skill in the art. For instance, reaction, e.g. under non-denaturing conditions, of a chimeric HA with a neutralizing antibody or antiserum that recognizes a native influenza HA might indicate structural similarity. Useful neutralizing antibodies or antisera are described in, e.g. Sui, et al., 2009, Nat. Struct. Mol. Biol. 16(3):265-273, Ekiert et al., February 26, 2009, Science [DOI: 10.1126 / science.1171491], and Kashyap et al., 2008, Proc. Natl. Acad. Sci. USA 105(16):5986-5991, the contents of which are hereby incorporated by reference in their entireties. In any aspect or embodiment described herein, the antibody or antiserum is an antibody or antiserum that reacts with a non-contiguous epitope (i.e., not contiguous in primary sequence) that is formed by the tertiary or quaternary structure of a HA.

[0205] In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) is monomeric. In any aspect or embodiment described herein, the chimeric Influenza virus HA polypeptide described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) forms a multimeric complex. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide described herein (e.g., the first chimericAttorney Docket No. MSZ0003PCTinfluenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) forms a trimeric complex. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) is in a pre-fusion conformation. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) is in a post-fusion conformation. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptides provided herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) are in a mix of pre-fusion and post-fusion conformations.

[0206] In any aspect or embodiment described herein, a chimeric influenza virus HA polypeptide described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) retains one, two, or more, or all of the functions of a wild-type influenza virus HA. In any aspect or embodiment described herein, non-limiting examples of functions of a wild-type influenza virus HA include fusogenic activity, receptor binding activity, budding, particle formation, or a combination thereof. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) has fusogenic activity. Assays known to one skilled in the art can be utilized the assess the fusogenic activity of a chimeric influenza virus HA polypeptide described herein, such as, for example, immunofluorescence assays and pseudotyped virus-like-particle assays. In any aspect or embodiment described herein, the chimeric influenza virus HA polypeptide described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) does not retain one, two, or more, or all of the functions of a wild-type influenza virus HA. In any aspect or embodiment described herein, the chimeric influenza vims HA polypeptide described herein (e.g., the first chimeric influenza vims HA polypeptide and / or the second chimeric influenza vims HA polypeptide) does not have fusogenic activity.Lipid Nanoparticles

[0207] In one aspect, provided herein are lipid nanoparticles comprising, consisting essentially of, or consisting of, a nucleoside-modified RNA molecule described herein. In any aspect or embodiment described herein, the lipid nanoparticle may comprise, consists essentially of, or consists of, any lipid capable of forming a particle to which the one or moreAttorney Docket No. MSZ0003PCTnucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In any aspect or embodiment described herein, provided herein is a lipid nanoparticle comprising, consists essentially of, or consists of, a nucleoside-modified RNA molecule described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) and one or more lipids. In any aspect or embodiment described herein, the one or more lipids includes at least one of a cationic lipid, anionic lipid, neutral lipid, Zwitterionic lipid, or combinations thereof. In any aspect or embodiment described herein, the one or more lipids includes an ionizable lipid. In any aspect or embodiment described herein, the one or more lipids includes a cationic ionizable lipid. In any aspect or embodiment described herein, the one or more lipids is one or more described in Hou et al., 2021, Nature Reviews Materials 6: 1078-1094; Tenchov et al, 2021, ACS Nano 15 (11): 16982-17015; Jung et al., 2022, Theranostics 12(17): 7509-7531;Swingle et al., 2021, Trends in Molecular Medicine 27(6): 616-617. For example, any aspect or embodiment described herein, the one or more lipids may include one or more lipids depicted below and described in Hou et al., 2021, Nature Reviews Materials 6: 1078-1094, which is reproduced below and incorporated herein by reference. In any aspect or embodiment described herein, the lipid nanoparticle further comprises, consists essentially of, or consists of, phospholipids (e.g., phosphatidylcholine and phosphatidylethanolamine), cholesterol, and / or polyethylene glycol (PEG)-functionalized lipids (PEG-lipids) (e.g., polyethylene glycol-lipid). In any aspect or embodiment described herein, provided herein is a lipid nanoparticle comprising, consisting essentially of, or consists of, a nucleoside-modified RNA molecule described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide), one or more cationic lipids, and one or more stabilizing lipids, such as, e.g., neutral lipids and pegylated lipids. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule described herein (e.g., the first chimeric influenza virus HA polypeptide and / or the second chimeric influenza virus HA polypeptide) is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.Figure 2 from Hou et al., 2021, Nature Reviews Materials 6: 1078-1094.Attorney Docket No. MSZ0003PCT3060il0, tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2,5-dihydro-lH-imidazole-2-carboxylate: ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6, 1 -diy l)bi s( 2-Attorney Docket No. MSZ0003PCThexyldecanoate); ALC-0159, 2- [(polyethylene glycol)-2000]-N, N-ditetradecylacetamide; flsitosterol. (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro- lH-cyclopenta[a]phenanthren-3-ol; BAME-016B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate; BHEM-Cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4,7,8,9,10, 11, 12, 13, 14, 15, 16,17-tetradecahydro- lH-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N, N-bis(2-hydroxyethyl)-N-methylethan-l -aminium bromide; Cl 2- 200, 1, 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin- l-yl)ethyl)azanediyl) bis(dodecan-2-ol): cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione; DC-Cholesterol, 3 |3-[N-(N', N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2- (spenninecarboxamido)ethyl]-N, N-dimethyl-l-propanaminium trifluoroacetate; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOTMA, l,2-di-O-octadecenyl-3-trimethylammonium-propane; DSPC, l,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl) 9, 9', 9", 9'", 9'"', 9"'"- ((((benzene-1,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3, 1 -diyl)) tris(azanetriyl))hexanonanoate; Lipid H (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino) octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, 1-diyl))bis(azanetriyl))tetrakis(ethane-2, 1 -diyl) (9Z,9'Z,9" Z,9"'Z, 12Z, 12'Z, 12" Z, 12"'Z)-tetrakis (octadeca-9,12-dienoate); PEG2000-DMG, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; TT3, Nl, N3, N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide.

[0208] In any aspect or embodiment described herein, lipid or hydrocarbyl compounds useful in lipid nanoparticle formulations of the present disclosure include those disclosed International Patent Application No. WO 2017 / 004143 Al, which is incorporated by reference herein in its entirety for all purposes, and include, for example:Attorney Docket No. MSZ0003PCTAttorney Docket No. MSZ0003PCTAttorney Docket No. MSZ0003PCTAttorney Docket No. MSZ0003PCTAttorney Docket No. MSZ0003PCTAttorney Docket No. MSZ0003PCTAttorney Docket No. MSZ0003PCT

[0209] As used herein, unless the context implies otherwise, the term “cationic lipid” refers to a lipid that is positively charged or is capable of being positively charged under physiologically relevant conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Additional, exemplary cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle underAttorney Docket No. MSZ0003PCTdifferent pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (see, e.g., Semple, S. C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (see, e.g., Hafez, I. M., et al., Gene Ther 8:1188-1196 (2001)) critical to the intracellular delivery of nucleic acids.

[0210] In any aspect or embodiment described herein, the cationic lipid comprises a mixture of any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. In any aspect or embodiment described herein, such lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA); N, N-distearyl-N, N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP); 3-(N — (N', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N, N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used, including, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3 -phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N. Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA).

[0211] As used herein, unless the context implies otherwise, the term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.Pegylated lipids are known in the art and include, in any aspect or embodiment describedAttorney Docket No. MSZ0003PCTherein, l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0212] As used herein, unless the context implies otherwise, the term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, in any aspect or embodiment described herein, such lipids include, but are not limited to, phosphotidy Icholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethanolamines such as l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0213] As used herein, unless the context implies otherwise, the term “charged lipid” refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g. pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include, any aspect or embodiment described herein, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Chol).

[0214] As used herein, unless the context implies otherwise, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.

[0215] As used herein, unless the context implies otherwise, the term “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA as described herein, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, a second nucleoside-modified RNA molecule, or a first nucleoside-modified RNA molecule and a second nucleoside-modifiedAttorney Docket No. MSZ0003PCTRNA molecule), with full encapsulation, partial encapsulation, or both. In any aspect or embodiment described herein, the nucleic acid (e.g., mRNA as described herein, a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, a second nucleoside-modified RNA molecule, or a first nucleoside-modified RNA molecule and a second nucleoside-modified RNA molecule) is fully encapsulated in the lipid nanoparticle.

[0216] In any aspect or embodiment described herein, a lipid nanoparticle comprises at least one of (4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), (polyethylene glycol)-2000]-N, N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol. In specific embodiments, a lipid nanoparticle comprises 0.43 mg (4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), 0.05 mg 2[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide, 0.09 mg 1,2-distearoyl-sn-glycero-3phosphocholine, and 0.2 mg cholesterol, or a combination thereof.

[0217] In any aspect or embodiment described herein, a lipid nanoparticle comprises at least one of an ionizable lipid SM-102 (CAS Reg. No. 2089251-47-6); cholesterol; 1,2-distearoyl-snglycero-3 phosphocholine (DSPC); and 1 monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000-DMG), or a combination thereof.

[0218] In any aspect or embodiment described herein, a nucleoside-modified RNA molecule (e.g., the nucleoside-modified RNA molecule of the present disclosure, the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure) is encapsulated in a lipid nanoparticle is not significantly (e.g., less than 10% of the nucleoside-modified RNA molecule encapsulated in the lipid nanoparticle) degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. In any aspect or embodiment described herein, less than 10% of the nucleoside-modified RNA molecule (e.g., the nucleoside-modified RNA molecule of the present disclosure, the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure) encapsulated in the lipid nanoparticle is degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Exemplary assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.Attorney Docket No. MSZ0003PCT

[0219] In any aspect or embodiment described herein, a lipid nanoparticle or a plurality of lipid nanoparticles comprise an ionizable cationic, a modified-nucleoside RNA molecule described herein (e.g., the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure), and one or more of neutral lipids, steroids, and polymer conjugated lipids.

[0220] In any aspect or embodiment described herein, the neutral lipid is selected from at least one of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, or a combination thereof. In any aspect or embodiment described herein, the neutral lipid is DSPC. In any aspect or embodiment described herein, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1.

[0221] In any aspect or embodiment described herein, a lipid nanoparticle or a plurality of lipid nanoparticles further comprises a steroid or steroid analogue. In any aspect or embodiment described herein, the steroid or steroid analogue is cholesterol. In any aspect or embodiment described herein, the molar ratio of a cationic ionizable lipid to cholesterol ranges from about 2: 1 to 1: 1.

[0222] In any aspect or embodiment described herein, the polymer conjugated lipid is a pegylated lipid. For example, in any aspect or embodiment described herein, pegylated lipid includes a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(oj-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as o>-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(m-methoxy(polyethoxy)ethyl)carbamate. In any aspect or embodiment described herein, the molar ratio of an ionizable cationic lipid to the pegylated lipid ranges from about 100:1 to about 25:1.

[0223] In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 1000 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 750 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticlesAttorney Docket No. MSZ0003PCTranges from about 1 nm to about 500 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 400 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 300 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 200 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 150 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 nm to about 100 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 10 nm to about 100 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 10 nm to about 50 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 25 to about 70 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 35 to about 60 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 40 nm to about 50 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 50 nm to about 100 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 10 nm to about 150 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles ranges from about 1 to about 5 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm,Attorney Docket No. MSZ0003PCTabout 26 nm, about 27 nm, about 28 nm, about 29 nm, or about 30 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 39 nm, or about 40 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 41 nm, about 42 nm, about 43 nm, about 44 nm, about 45 nm, about 46 nm, about 47 nm, about 48 nm, about 49 nm, or about 50 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, or about 60 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, or about 70 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 71 nm, about 72 nm, about 73 nm, about 74 nm, about 75 nm, about 76 nm, about 77 nm, about 78 nm, about 79 nm, or about 80 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 88 nm, about 88 nm, about 89 nm, or about 90 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 80 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, or about 100 nm. In any aspect or embodiment described herein, the diameter of a lipid nanoparticle or the average or mean diameter of a plurality of lipid nanoparticles is about 125 nm, about 130 nm, about 140 nm, or about 150 nm.

[0224] In any aspect or embodiment described herein, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60Attorney Docket No. MSZ0003PCTnm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially nontoxic. In any aspect or embodiment described herein, the nucleoside-modified RNA molecule (e.g., the nucleoside-modified RNA molecule of the present disclosure, the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure), when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

[0225] In any aspect or embodiment described herein, a lipid nanoparticle or plurality of lipid nanoparticles described herein may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of nanoparticles. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure the zeta potential of nanoparticles. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, and Worcestershire, UK) may also be used to measure multiple characteristics of nanoparticles, such as particle size, polydispersity index, and zeta potential.

[0226] In any aspect or embodiment described herein, a plurality of lipid nanoparticles as described herein may be relatively homogenous. In any aspect or embodiment described herein, a polydispersity index (PDI) may be used to indicate the homogeneity of a plurality of lipid nanoparticles, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. In any aspect or embodiment described herein, a plurality of lipid nanoparticles described herein may have a polydispersity index of from about 0 to about 0.25 (e.g,, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25). In any aspect or embodiment described herein, the poly dispersity index of the plurality of lipid nanoparticles is from about 0.01 to about 0.20. In any aspect or embodiment described herein, the polydispersity index of the plurality of lipid nanoparticles is from about 0.02 to about 0.06. In any aspect or embodiment described herein, the poly dispersity index of the plurality of lipid nanoparticles is from about 0.10 to about 0.20. In any aspect or embodiment described herein, 50% or more of the lipid nanoparticles in a plurality of lipid nanoparticles have about the same PDI, size distribution, size shape, surface characteristics and / or inner structure. In any aspect or embodimentAttorney Docket No. MSZ0003PCTdescribed herein, 60% or more of the nanoparticles in a plurality of lipid nanoparticles have about the same PDI, size distribution, size shape, surface characteristics and / or inner structure. In any aspect or embodiment described herein, 70% or more of the lipid nanoparticles in a plurality of lipid nanoparticles have about the same PDI, size distribution, size shape, surface characteristics and / or inner structure. In any aspect or embodiment described herein, 80% or more of the lipid nanoparticles in a plurality of lipid nanoparticles have about the same PDI, size distribution, size shape, surface characteristics and / or inner structure. In any aspect or embodiment described herein, 90% or more of the lipid nanoparticles in a plurality of lipid nanoparticles have about the same PDI, size distribution, size shape, surface characteristics and / or inner structure. In any aspect or embodiment described herein, the PDI is measured by dynamic light scattering using Zetasizer NanoZS or as described in the Examples.

[0227] The efficiency of encapsulation of a nucleoside-modified RNA molecule (e.g., the nucleoside-modified RNA molecule of the present disclosure, the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure) describes the amount of the nucleoside-modified RNA molecule that is encapsulated or otherwise associated with a lipoid nanoparticle after preparation, relative to the initial amount provided. In any aspect or embodiment described herein, the encapsulation efficiency is desirably high e.g., close to 100 %). The encapsulation efficiency may be measured, for example, by comparing the amount of nucleoside-modified RNA molecule in a solution containing the lipid nanoparticle before and after breaking up the nanoparticle with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free nucleoside-modified RNA molecule in a solution. In any aspect or embodiment described herein, encapsulation efficiency is determined using a Ribogreen assay or as described in the Examples. For the lipid nanoparticles described herein, in any aspect or embodiment described herein, the encapsulation efficiency of a nucleoside-modified RNA molecule (e.g., the nucleoside-modified RNA molecule of the present disclosure, the first nucleoside-modified ribonucleic acid (RNA) molecule of the present disclosure, the second nucleoside-modified ribonucleic acid (RNA) molecule of the present disclosure, or the first nucleoside-modified ribonucleic acid (RNA) molecule of the present disclosure and the second nucleoside-modified ribonucleic acid (RNA) molecule of the present disclosure) may be at least 50 % (for example 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %,Attorney Docket No. MSZ0003PCT92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100 %). In any aspect or embodiment described herein, the encapsulation efficiency is at least 70 %. In any aspect or embodiment described herein, the encapsulation efficiency is at least 75 %. In any aspect or embodiment described herein, the encapsulation efficiency is at least 80 %. In some embodiments, the encapsulation efficiency is at least 85 %. In any aspect or embodiment described herein, the encapsulation efficiency is at least 90 %. In any aspect or embodiment described herein, the encapsulation efficiency is at least about 95%. In any aspect or embodiment described herein, the encapsulation efficiency is about 80%, about 85%, or about 90%. In any aspect or embodiment described herein, the encapsulation efficiency is about 95%.

[0228] The zeta potential of a lipid nanoparticle or plurality of lipid nanoparticles may be used to indicate the electrokinetic potential of the nanoparticles. For example, the zeta potential may describe the surface charge of a lipid nanoparticle. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In any aspect or embodiment described herein, the zeta potential of a lipid nanoparticle is from about -10 mV to about +20 mV, from about -10 mV to about +15mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0229] In any aspect or embodiment described herein, a lipid nanoparticle or plurality of lipid nanoparticles has an apparent acid dissociation constant (pKa) of about 5 to about 8. In any aspect or embodiment described herein, a lipid nanoparticle or plurality of lipid nanoparticles has an apparent acid dissociation constant (pKa) of about 6 to about 7. In any aspect or embodiment described herein, a lipid nanoparticle or plurality of lipid nanoparticles has an apparent acid dissociation constant (pKa) of about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8. The apparent pKa may be measured by techniques known to one of skill in the art. For example, in any aspect or embodiment described herein, acid-base titration and 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS) fluorescent methods may be used to determine the apparent pKa.Attorney Docket No. MSZ0003PCT

[0230] In any aspect or embodiment described herein, the lipid nanoparticles have an average hydrodynamic diameter of about 80 nm with a polydispersity index of about 0.02 to about 0.06 as measured by dynamic light scattering using, e.g., a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, United Kingdom).

[0231] Another aspect of the present disclosure relates to a lipid nanoparticle composition (e.g., any composition described herein may be a lipid nanoparticle composition) comprising the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure), and a lipid component. In any aspect or embodiment described herein, the lipid component comprises a cationic lipid. In any aspect or embodiment described herein, the lipid component comprises a ionizable lipid. In any aspect or embodiment described herein, the lipid component comprises a cationic ionizable lipid. In any aspect or embodiment described herein, the lipid nanoparticle composition further comprises phosphatidylcholine (e.g., distearoylphosphatidylcholine), cholesterol, and polyethylene glycol-lipid (e.g., polyethylene glycol-conjugated lipid). For example, in any aspect or embodiment described herein, the lipid nanoparticle composition comprising the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure), and a lipid component, wherein the lipid component comprises an ionizable and / or cationic lipid (e.g., a cationic ionizable lipid or an ionizable lipid), phosphatidylcholine (e.g., distearoylphosphatidylcholine), cholesterol, and polyethylene glycol-lipid (e.g., polyethylene glycol-conjugated lipid). Thus, in any aspect or embodiment described herein, the lipid nanoparticle composition comprising the nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure), and a lipid component, wherein the lipid component comprises an ionizable and / or cationic lipid, distearoylphosphatidylcholine, cholesterol, and polyethylene glycol-conjugated lipid.Attorney Docket No. MSZ0003PCT

[0232] In any aspect or embodiment described herein, the lipid-to-drug (e.g., the nucleoside-modified RNA molecule) ratio is about 10:1 to about 40:1 weight / weight (w / w), about 15:1 to about 25:1 w / w, or about 17:1 to about 23:1 w / w. For example, in any aspect or embodiment described herein, the lipid-to-drug (e.g., the nucleoside-modified RNA molecule) ratio is about 10:1 to about 40:1 w / w, about 10:1 to about 35:1 w / w, about 10:1 to about 30:1 w / w, about 10:1 to about 25:1 w / w, about 10:1 to about 20:1 w / w, about 10:1 to about 15:1 w / w, about 15:1 to about 40:1 w / w, about 15:1 to about 35:1 w / w, about 15:1 to about 30:1 w / w, about 15:1 to about 25:1 w / w, about 15:1 to about 20:1 w / w, 20:1 to about 40:1 w / w, about 20:1 to about 35:1 w / w, about 20:1 to about 30:1 w / w, about 20:1 to about 25:1 w / w, about 25:1 to about 40:1 w / w, about 25:1 to about 35:1 w / w, about 25:1 to about 30:1 w / w, about 30:1 to about 40:1 w / w, about 30:1 to about 35:1 w / w, about 35:1 to about 40: 1 w / w, or about 20: 1 w / w.

[0233] In any aspect or embodiment described herein, the dose of the lipid nanoparticle composition is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the nucleoside-modified RNA molecule (e.g., the first nucleoside-modified RNA molecule of the present disclosure, the second nucleoside-modified RNA molecule of the present disclosure, or the first nucleoside-modified RNA molecule of the present disclosure and the second nucleoside-modified RNA molecule of the present disclosure).

[0234] In any aspect or embodiment described herein, the lipid nanoparticles having phospholipid, which is selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Di ether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-Attorney Docket No. MSZ0003PCTphosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0235] In any aspect or embodiment described herein, the lipid component of a nanoparticle may include one or more structural lipids. Structural lipids can be selected from, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In any aspect or embodiment described herein, the structural lipid is cholesterol. In any aspect or embodiment described herein, the structural lipid includes cholesterol and a corticosteroid (such as, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In any aspect or embodiment described herein, a lipid component includes cholesterol.

[0236] In any aspect or embodiment described herein, the lipid component of a nanoparticle may include one or more PEG or PEG-modified lipids. Such lipids may be alternately referred to as PEGylated lipids. For example, a PEG lipid is a lipid modified with polyethylene glycol. By way of further example, a PEG lipid may be selected from the nonlimiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, DMG-PEG (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol), obtainable from Avanti Polar Lipids, Alabaster, Ala.), DMG-PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In any aspect or embodiment described herein, a lipid component includes DMG-PEG. In any aspect or embodiment described herein, a lipid component includes DMG-PEG2000.

[0237] In any aspect or embodiment described herein, cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), l,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), l,2-dilinolenyloxy-N, N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are DPPC, DOPC and dodecylphosphocholine. In any aspect or embodiment described herein, the lipids can be saturated or unsaturated. The use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail.Attorney Docket No. MSZ0003PCT

[0238] In any aspect or embodiment described herein, cationic, ionizable or cationisable lipids also include, but are not limited to, DSDMA, N, N-dioleyl-N, N- dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N, N, N- trimethylammonium chloride and 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(l-(2,3- dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N, N- dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N, N-dimethylaminopropane (y-DLenDMA), 98N12-5, 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxy-propane (DLin-DAC), 1,2-Dilinoleyoxy-3 -morpholinopropane (DLin-MA), 1,2-Dilinoleoyl- 3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylamino- propane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. CI), ICE (Imidazol-based), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3] -dioxolane) HGT4003, 1,2- Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. CI), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N, N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N, N-Dioleylamino)-l,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-Dilinoleyl-4-dimethylaminomethyl- [1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N, N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12- dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl- 4-(dimethylamino)butanoate (MC3), ALNY-100 ((3aR,5s,6aS)-N, N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12- dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), 1,1 ’-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin- 1 -yl)ethylazanediyl)didodecan-2-ol (C 12-200), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin- K-DMA), NC98-5 (4,7, 13-tris(3-oxo-3-(undecylamino)propyl)-Nl, N16-diundecyl-4,7,10,13-tetraazahexadecane- 1, 16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9.28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3- DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N, N-dimethylpropan-1 -amine (MC3 Ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-Attorney Docket No. MSZ0003PCT19-yloxy)-N, N-dimethylbutan-l-amine (MC4 Ether), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3phospho-ethanolamine (DOPE), from GIBCO / BRL, Grand Island, N. Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECT AM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspemiine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing.

[0239] In any aspect or embodiment described herein, the LNPs comprise a lipid-conjugate, a cationic lipid, a steroid and a neutral lipid. For example, in any aspect or embodiment described herein, the cationic lipid is preferably ionizable or cationisable (e.g., it becomes protonated as the pH is lowered below the pKaof the ionizable group of the lipid, but is progressively more neutral at higher pH values). In any aspect or embodiment described herein, when positively charged, the lipid is then able to associate with negatively charged nucleic acids. In any aspect or embodiment described herein, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid. For example, in any aspect or embodiment described herein, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable cationic lipid. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 5-25% non-cationic lipid. For example, in any aspect or embodiment described herein, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15- 20%, or 20-25% non-cationic lipid. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 25-55% sterol. For example, in any aspect or embodiment described herein, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 0.5-15% polymerAttorney Docket No. MSZ0003PCTconjugated lipid of the disclosure. For example, in any aspect or embodiment described herein, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1- 5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% polymer conjugated lipid of the disclosure. In any aspect or embodiment described herein, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non- cationic lipid, 25-55% sterol, and 0.5-15% polymer conjugated lipid of the disclosure. For example, in any aspect or embodiment described herein, the cationic lipid of an LNP may be cationisable (e.g., it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values). At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0240] In any aspect or embodiment described herein, liposomes can be formed from a single lipid or from a mixture of lipids. For example, in any aspect or embodiment described herein, a mixture may comprise (i) a mixture of anionic lipids, (ii) a mixture of cationic lipids, (iii) a mixture of zwitterionic lipids, (iv) a mixture of anionic lipids and cationic lipids, (v) a mixture of anionic lipids and zwitterionic lipids, (vi) a mixture of zwitterionic lipids and cationic lipids, or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids. Similarly, in any aspect or embodiment described herein, a mixture may comprise both saturated and unsaturated lipids. For example, in any aspect or embodiment described herein, a mixture may comprise DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and / or DMG (anionic, saturated). In any aspect or embodiment described herein, where a mixture of lipids is used, not all of the component lipids in the mixture need to be amphiphilic (e.g., one or more amphiphilic lipids can be mixed with cholesterol).Compositions

[0241] In one aspect, provided herein is a composition comprising a RNA molecule described herein (e.g., a nucleoside-modified RNA molecule described herein, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule). A further aspect of the present disclosure provides a composition comprising, consisting essentially of, or consisting of, the nucleoside-modified RNA molecule (e.g., a first nucleoside-modified RNA molecule and / or a second nucleoside-modified ribonucleic acid (RNA) molecule) of the present disclosure, and a pharmaceutically acceptable carrier. Thus, for example, the composition comprises, consists essentially of, or consists of, the firstAttorney Docket No. MSZ0003PCTnucleoside-modified RNA molecule of the present disclosure and / or a second nucleoside-modified RNA molecule) of the present disclosure, and a pharmaceutically acceptable carrier. In any aspect or embodiment described herein, the composition further comprises a lipid component. For example, in any aspect or embodiment described herein, the lipid component is part of a lipid nanoparticle, such as described herein.

[0242] In any aspect or embodiment described herein, the composition is monovalent. In any aspect or embodiment described herein, the composition is divalent or trivalent. In any aspect or embodiment described herein, the composition is multivalent. In any aspect or embodiment described herein, provided herein is a composition comprising two, three, or more RNA molecules described herein (e.g., two, three, or more nucleoside-modified RNA molecules (e.g., a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) described herein). In any aspect or embodiment described herein, provided herein is a composition comprising a nucleoside-modified RNA molecule described herein (e.g., a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, provided herein is a composition comprising two RNA molecules (e.g., nucleoside-modified RNA molecules), wherein one RNA molecule (e.g., a nucleoside-modified RNA molecule) is translated into a first chimeric influenza vims HA polypeptide of the present disclosure, and the other RNA molecule (e.g., a nucleoside-modified RNA molecule) is translated into a second chimeric influenza vims HA polypeptide of the present disclosure. In any aspect or embodiment described herein, provided herein is a composition comprising three RNA molecules (e.g., nucleoside-modified RNA molecules), wherein one RNA molecule (e.g., a nucleoside-modified RNA molecule) is translated into a first chimeric influenza vims HA polypeptide of the present disclosure, the second RNA molecule (e.g., a nucleoside-modified RNA molecule) is translated into a second chimeric influenza vims HA polypeptide of the present disclosure, and the third RNA molecule (e.g., a nucleoside-modified RNA molecule) is translated into a nucleoside-modified RNA molecule of the present disclosure. In any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of theAttorney Docket No. MSZ0003PCTfirst influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7 (preferably, SEQ ID NO: 5 or 7). In some embodiments, the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the second chimeric influenza vims HA polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7 (preferably, SEQ ID NO: 1 or 3). In any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and the second chimeric influenza vims HA polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 3. For example, in any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and the second chimeric influenza vims HA polypeptide comprises the amino acid sequence of SEQ ID NO: 1. By way of further example, in any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and the second chimeric influenza vims HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, the RNA molecule (e.g., nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) is complexed with peptides containing strings of positively charged amino acids, such as lysine and arginine, to form a protamine-RNA complex. In any aspect or embodiment describedAttorney Docket No. MSZ0003PCTherein, the RNA molecule (e.g., nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) is associated or encapsulated in a polymer (e.g., a cationic polymer, such as, e.g., polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer and polysaccharides). In any aspect or embodiment described herein, the RNA molecule (e.g., nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) is associated or encapsulated in an emulsion (e.g., a cationic nanoemulsion). In any aspect or embodiment described herein, the RNA molecule (e.g., nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) is associated or encapsulated in a liposome. In any aspect or embodiment described herein, approximately 80% to approximately 95% of the RNA molecule (e.g., nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) are encapsulated in a polymer, an emulsion, or a liposome. In any aspect or embodiment described herein, approximately 80% to approximately 95% of the RNA molecule (e.g., nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) are complexed with peptides in a protamine-RNA complex. In any aspect or embodiment described herein, the composition further comprises a pharmaceutically acceptable carrier.

[0243] In any aspect or embodiment described herein, provided herein is a lipid nanoparticle composition comprising an RNA molecule (e.g., a nucleoside-modified RNA molecule, a first nucleoside-modified RNA molecule, and / or a second nucleoside-modified RNA molecule) described herein and a lipid component. In any aspect or embodiment described herein, provided herein is a lipid nanoparticle composition comprising a nucleoside-modified RNA molecule described herein (e.g., a first nucleoside-modified RNA molecule and / or a second nucleoside-modified RNA molecule) and a lipid component. In any aspect or embodiment described herein, provided herein is a lipid nanoparticle composition comprising, consisting essentially of, or consists of, two nucleoside-modified RNA molecules and a lipid component, wherein one nucleoside-modified RNA molecule is translated into a first chimeric influenza virus HA polypeptide of the present disclosure, and the other nucleoside-modified RNA molecule is translated into a second chimeric influenza virus HA polypeptide of the present disclosure. In any aspect or embodiment described herein, provided herein is a composition comprising three nucleoside-modified RNA molecules and a lipid component, wherein one nucleoside-modified RNA molecule is translated into a first chimeric influenza virus HA polypeptide described herein, the secondAttorney Docket No. MSZ0003PCTnucleoside-modified RNA molecule is translated into a second chimeric influenza virus HA polypeptide described herein, and the third nucleoside-modified RNA molecule is translated into a nucleoside-modified RNA molecule described herein. In any aspect or embodiment described herein, the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B virus, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7 (preferably, SEQ ID NO: 5 or 7). In some embodiments, the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine. In any aspect or embodiment described herein, the second chimeric influenza vims HA polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7 (preferably, SEQ ID NO: 1 or 3). In any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and the second chimeric influenza vims HA polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 3. For example, in any aspect or embodiment described herein, the first chimeric influenza vims HA polypeptide comprises, consists essentially of, orAttorney Docket No. MSZ0003PCTconsists of, the amino acid sequence of SEQ ID NO: 5, and the second chimeric influenza virus HA polypeptide comprises the amino acid sequence of SEQ ID NO: 1. By way of further example, in any aspect or embodiment described herein, the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and the second chimeric influenza virus HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In any aspect or embodiment described herein, the composition further comprises a pharmaceutically acceptable carrier. In any aspect or embodiment described herein, the lipid component comprises one or more lipids, such as described herein (e.g., in the Examples). In any aspect or embodiment described herein, the lipid component comprises a cationic lipid. In any aspect or embodiment described herein, provided herein is a composition comprising a lipid nanoparticle(s) described herein (e.g., in the Examples). In any aspect or embodiment described herein, the lipid nanoparticle composition further comprises a pharmaceutically acceptable diluent, adjuvant, or excipient.

[0244] As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. In specific embodiments, the pharmaceutically acceptable carrier is a diluent, excipient, or vehicle. For example, the pharmaceutically acceptable carrier is a diluent (e.g., saline). Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin. The formulation should suit the mode of administration.

[0245] In any aspect or embodiment described herein, compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, in any aspect or embodiment described herein, a composition may be formulated to be suitable for parenteral, oral, intradermal, intranasal, transdermal, intranodal, or intraperitoneal administration. In any aspect or embodiment described herein, a composition described herein may be formulated for intravenous, subcutaneous, intramuscular, topical, intranasal, intradermal, transdermal, or pulmonary administration. In any aspect or embodimentAttorney Docket No. MSZ0003PCTdescribed herein, a composition described herein may be formulated for intramuscular administration.

[0246] In any aspect or embodiment described herein, provided herein is a composition described in the Examples.

[0247] In any aspect or embodiment described herein, a composition is prepared, packaged, and / or sold as a single unit dose of a nucleoside-modified RNA molecule, or a plurality of single unit doses of a nucleoside-modified RNA molecule.

[0248] A composition described herein may be used to induce an immune response to one, two, or multiple (e.g., 3, 4, 5, 6, or more) influenza viruses (e.g., influenza B viruses, influenza A viruses, group 1 influenza A viruses, group 2 influenza A viruses, or a combination thereof) in a subject (e.g., human subject). In any aspect or embodiment described herein, a composition described herein may be used to induce a cross-reactive immune response to two or more (e.g., 3, 4, 5, 6, or more) influenza viruses (e.g., influenza B viruses, influenza A viruses, group 1 influenza A viruses, group 2 influenza A viruses, or a combination thereof) in a subject (e.g., human subject). A composition described herein may be used to induce an immune response that provides protection (e.g., full or partial protection) against one, two, or multiple (e.g., 3, 4, 5, 6, or more) influenza viruses (e.g., an influenza B virus, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject (e.g., human subject). In any aspect or embodiment described herein, the immune response induced provides protection (e.g., full or partial protection) against a homologous influenza B vims, a homologous influenza A vims, a homologous group 1 influenza A vims, a homologous group 2 influenza A vims, or a combination thereof. In any aspect or embodiment described herein, the immune response induced provides protection (e.g., full or partial protection) against a heterotypic influenza B vims, a heterotypic influenza A vims, a heterotypic group 1 influenza A vims, a heterotypic group 2 influenza A vims, or a combination thereof. A composition described herein may be used to immunize a subject (e.g., human subject) against influenza vims (e.g., an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof). A composition described herein may also be used to prevent an Influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject (e.g., human subject). In any aspect or embodiment described herein, a composition described herein may be used in a method described herein.Attorney Docket No. MSZ0003PCT

[0249] In any aspect or embodiment described herein, a composition described herein is a vaccine.

[0250] The compositions described herein can be included in a container, pack, or dispenser together with instructions for administration. Another aspect of the present disclosure provides a kit comprising a container containing the nucleoside-modified RNA molecule of the present disclosure, the composition of the present disclosure, the first composition of the present disclosure, or the second composition of the present disclosure. A further aspect of the present disclosure provides a kit comprising a first container containing the first composition of the present disclosure and a second container containing the second composition of the present disclosure. Another aspect of the present disclosure provides a kit comprising two or more containers, wherein each container comprises a different nucleoside-modified mRNA molecule described herein and / or composition described herein.

[0251] In any aspect or embodiment described herein, provided herein is a composition (e.g., a pharmaceutical composition) comprising an antibody (ies) that binds to influenza virus HA (e.g., an influenza B virus, an influenza A virus, a group 1 influenza A virus, a group 2 influenza A vims, or a combination thereof), which was generated using an composition comprising a nucleoside-modified RNA molecule described herein. The composition may further comprise a pharmaceutically acceptable carrier. The antibody may be polyclonal or monoclonal. In any aspect or embodiment described herein, the antibody is human or humanized. The antibody(ies) may be used to passively immunize a subject e.g., a human subject).

[0252] An additional aspect of the present disclosure relates to a composition comprising the nucleoside-modified RNA molecule of the present disclosure, and a pharmaceutically acceptable carrier.Prophylactic Uses

[0253] In one aspect, provided herein are methods of preventing influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject, methods of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof), and methods of inducing an immune response to influenza vimses (e.g., a cross-reactive immune response to plurality of influenza A vims strains or subtypes (e.g., group 1 or group 2 influenza A vimses), influenza B vims strains or subtypes, or a combination thereof) using a composition described herein.Attorney Docket No. MSZ0003PCT

[0254] In any aspect or embodiment described herein, provided herein is a method of preventing influenza virus disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject, comprising administering to the subject a composition described herein. In any aspect or embodiment described herein, provided herein is a method of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group I influenza A vims, a group 2 influenza A vims, or a combination thereof), the method comprising administering to the subject a composition described herein. In any aspect or embodiment described herein, provided herein is a method of inducing immune response to Influenza vimses (e.g., influenza B vimses, influenza A vimses, group 1 influenza A vimses, group 2 influenza A vimses, or a combination thereof) in a subject, the method comprising administering to the subject a composition described herein. In any aspect or embodiment described herein, provided herein is a method of inducing a cross-reactive immune response to two or more influenza vimses (e.g., influenza B vimses, influenza A vimses, group 1 influenza A vimses, group 2 influenza A vimses, or a combination thereof) in a subject, the method comprising administering to the subject a composition described herein. The composition may be administered by any route of administration. For example, in any aspect or embodiment described herein, the composition may be administered to the subject by parenteral, oral, intradermal, intranasal, transdermal, intranodal, or intraperitoneal administration. In any aspect or embodiment described herein, the composition may be administered to the subject by intravenous, subcutaneous, intramuscular, topical, intranasal, intradermal, transdermal, or pulmonary administration. In any aspect or embodiment described herein, the composition is administered intramuscularly to the subject.

[0255] In any aspect or embodiment described herein, provided herein is a method of preventing influenza vims disease (e.g., influenza vims disease caused by (e.g., an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof)) in a subject, the method comprising sequentially administering to the subject two or more compositions described herein. In any aspect or embodiment described herein, provided herein is a method of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof), the method comprising sequentially administering to the subject two or more compositions described herein. In any aspect or embodiment described herein, provided herein is a method ofAttorney Docket No. MSZ0003PCTinducing immune response to influenza viruses (e.g., influenza B viruses, an influenza A virus, group 1 influenza A virus, group 2 influenza A viruses, or a combination thereof) in a subject, the method comprising sequentially administering to the subject two or more compositions described herein. In any aspect or embodiment described herein, provided herein is a method of inducing a cross-reactive immune response to two or more influenza viruses (e.g., influenza B viruses, influenza A viruses, group 1 influenza A viruses, group 2 influenza A viruses, or a combination thereof) in a subject, the method comprising sequentially administering to the subject two or more compositions described herein.

[0256] Thus, for example, an aspect of the present disclosure provides a method for inducing an immune response against influenza virus hemagglutinin (HA) in a subject or preventing influenza virus symptoms (e.g., disease) in a subject, the method comprising: (a) administering to the subject a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza virus HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B virus, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (b) administering to the subject a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain,Attorney Docket No. MSZ0003PCTsubtype, or group than the first influenza A virus, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0257] By way of further example, another aspect of the present disclosure provides a method for inducing an immune response against influenza virus hemagglutinin (HA) in a subject or preventing influenza virus symptoms (e.g., disease) in a subject, the method comprising: (a) administering to the subject a does of a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza virus HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B virus, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A virus, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (b) administering to the subject a dose of a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza virus HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B virus of a different strain, subtype, or group than the first influenza B virus, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0258] In any aspect or embodiment described herein, a composition is administered to a subject about 21 days to about 60 days apart from another composition. In any aspect or embodiment described herein, a composition is administered to the subject from about 21 days to about 30 days (e.g., about 29 days) apart from another composition. In any aspect or embodiment described herein, a composition is administered to the subject about 21 days toAttorney Docket No. MSZ0003PCTabout 28 days apart from another composition. The compositions may be administered by any route of administration. For example, the composition may be administered to the subject by parenteral, oral, intradermal, intranasal, transdermal, intranodal, or intraperitoneal administration. In any aspect or embodiment described herein, the composition may be administered to the subject by intravenous, subcutaneous, intramuscular, topical, intranasal, intradermal, transdermal, or pulmonary administration. In any aspect or embodiment described herein, the compositions are administered intramuscularly to the subject. In some embodiments, two or more compositions are administered by the same or different routes of administration. In any aspect or embodiment described herein, the compositions are monovalent. In some embodiments, the compositions are divalent or trivalent. In any aspect or embodiment described herein, the dose of a composition administered to the subject is a dose described herein.

[0259] In any aspect or embodiment described herein, provided herein is a method of preventing influenza virus disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject, the method comprising administering to the subject a dose of a composition described herein. In any aspect or embodiment described herein, provided herein is a method of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof), the method comprising administering to the subject a dose of a composition described herein. In any aspect or embodiment described herein, provided herein is a method of inducing immune response to influenza vimses (e.g., influenza B viruses, influenza A vimses, group 1 influenza A vimses, group 2 influenza A vimses, or a combination thereof) in a subject, the method comprising administering to the subject a dose of a composition described herein. In any aspect or embodiment described herein, provided herein is a method of inducing a cross-reactive immune response to two or more influenza vimses (e.g., influenza B vimses, influenza A vimses, group 1 influenza A vimses, group 2 influenza A vimses, or a combination thereof) in a subject, the method comprising administering to the subject a dose of a composition described herein. The composition may be administered by any route of administration. For example, in any aspect or embodiment described herein, the composition may be administered to the subject by parenteral, oral, intradermal, intranasal, transdermal, intranodal, or intraperitoneal administration. In any aspect or embodiment described herein, the composition may be administered to the subject by intravenous, subcutaneous,Attorney Docket No. MSZ0003PCTintramuscular, topical, intranasal, intradermal, transdermal, or pulmonary administration. In any aspect or embodiment described herein, the composition is administered intramuscularly to the subject.

[0260] While not intending to be bound by any particular theory of operation, it is believed that sequential immunization with nucleoside-modified RNA molecules encoding chimeric influenza virus HA polypeptides described herein are useful for presenting one or more relatively conserved antigenic regions to a subject’s immune system in order to generate an immune response that is capable of cross-reacting with a plurality of influenza virus strains and subtypes. Accordingly, in any aspect or embodiment described herein, provided herein are methods of inducing an immune response in a subject (e.g., human subject) that is capable of cross-reacting with antigenically distant influenza virus strains and subtypes (e.g., antigenically distant influenza B viruses, influenza A viruses, group 1 influenza A viruses, group 2 influenza A viruses, or a combination thereof), the method comprising sequentially administering to the subject two compositions described herein, wherein one composition comprises a first nucleoside-modified RNA molecule described herein, and the second composition comprises a second nucleoside-modified RNA molecule described herein. In any aspect or embodiment described herein, provided herein are methods of inducing an immune response in a subject (e.g., human subject) that is capable of cross-reacting with a plurality of Influenza virus strains and subtypes (e.g., antigenically distant influenza virus strains and subtypes, such as antigenically distant influenza B viruses, influenza A viruses, group 1 influenza A viruses, group 2 influenza A viruses, or a combination thereof), the method comprising sequentially administering to the subject two compositions described herein, wherein one composition comprises a first nucleoside-modified RNA molecule described herein, and the second composition comprises a second nucleoside-modified RNA molecule described herein.

[0261] In any aspect or embodiment described herein, the first composition and second composition are administered to a subject using techniques known in the art, such as, e.g., electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, lipid nanoparticles, biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)), or the use of TransIT®-mRNA transfection Kit (Minis, Madison WI). In any aspect or embodiment described herein, theAttorney Docket No. MSZ0003PCTfirst composition and / or second composition are lipid nanoparticle compositions described herein. In any aspect or embodiment described herein, the first composition and second composition are administered to the subject from about 3 weeks to about 9 months apart from each other. In any aspect or embodiment described herein, the first composition and second composition are administered to the subject from about 21 days to about 60 days apart from each. In any aspect or embodiment described herein, the first composition and second composition are administered to the subject from about 21 days to about 30 days (e.g., about 29 days) apart from each. In any aspect or embodiment described herein, the first composition and second composition are administered to the subject from about 21 days to about 28 days apart from each. The compositions may be administered by any route of administration. For example, in any aspect or embodiment described herein, the composition may be administered to the subject by parenteral, oral, intradermal, intranasal, transdermal, intranodal, or intraperitoneal administration. In any aspect or embodiment described herein, the composition may be administered to the subject by intravenous, subcutaneous, intramuscular, topical, intranasal, intradermal, transdermal, or pulmonary administration. In any aspect or embodiment described herein, each composition is administered to the subject intramuscularly. In some embodiments, the first composition and / or second composition is administered to a subject at a dose disclosed herein. In any aspect or embodiment described herein, the subject is administered a third composition comprising a nucleoside-modified RNA molecule.

[0262] In any aspect or embodiment described herein, provided herein are methods of preventing influenza virus disease (e.g., influenza virus disease caused by an influenza B virus, an influenza A virus, a group 1 influenza A virus, a group 2 influenza A virus, or a combination thereof) in a subject, methods of immunizing a subject against influenza virus disease (e.g., influenza virus disease caused by an influenza B virus, an influenza A virus, a group 1 influenza A virus, a group 2 influenza A virus, or a combination thereof), and / or inducing an immune response against influenza virus (e.g., an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereol) using a method or regimen described in the Examples.

[0263] In any aspect or embodiment described herein, the methods of preventing of influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject and / or methods of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1Attorney Docket No. MSZ0003PCTinfluenza A virus, a group 2 influenza A virus, or a combination thereof) induce antibodies (e.g., IgG) cross-reactive with a plurality of influenza A viruses, influenza B viruses, group 1 influenza A viruses, group 2 influenza A viruses, or a combination thereof. In any aspect or embodiment described herein, the methods of preventing of influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject and / or methods of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) induce antibodies (e.g., IgG) cross-reactive with a plurality of influenza viruses (e.g., an influenza B vims(es), an influenza A vims(es), a group 1 influenza A virus(es), a group 2 influenza A vims(es), or a combination thereof). In any aspect or embodiment described herein, the methods of preventing of influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) in a subject and / or methods of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims, an influenza A vims, a group 1 influenza A vims, a group 2 influenza A vims, or a combination thereof) induce antibodies (e.g., IgG) cross-reactive with a plurality of influenza vims strains and subtypes (e.g., antigenically distant influenza B vimses, influenza A vimses, group 1 influenza A vimses, group 2 influenza A vimses, or a combination thereof). In any aspect or embodiment described herein, the methods of preventing of influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A vims(es), a group 2 influenza A vims(es), or a combination thereof) in a subject and / or methods of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A virus(es), a group 2 influenza A vims(es), or a combination thereof) induce antibodies (e.g., IgG) cross-reactive with a plurality of influenza vims strains and subtypes (e.g., antigenically distant influenza B vimses, influenza A vimses, group 1 influenza A vimses, group 2 influenza A vimses, or a combination thereof).

[0264] In any aspect or embodiment described herein, the methods for preventing an influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A vims(es), a group 2 influenza A vims(es), or a combination thereof) and / or immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1Attorney Docket No. MSZ0003PCTinfluenza A virus(es), a group 2 influenza A virus(es). or a combination thereof) provided herein result in a reduction in the replication of the influenza virus (e.g., an influenza B vims(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) in the subject as measured by in vivo and in vitro assays known to those of skill in the art and described herein. In any aspect or embodiment described herein, the replication of the influenza virus (e.g., an influenza B vims(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) is reduced by approximately 1 log or more, approximately 2 logs or more, approximately 3 logs or more, approximately 4 logs or more, approximately 5 logs or more, approximately 6 logs or more, approximately 7 logs or more, approximately 8 logs or more, approximately 9 logs or more, approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.

[0265] In any aspect or embodiment described herein, the methods of preventing an influenza vims disease (e.g., influenza vims disease caused by an influenza B virus(es), an influenza A vims(es), a group 1 influenza A vims(es), a group 2 influenza A virus(es), or a combination thereof) in a subject results in one, two, or more of the following: prevents the onset of one or more symptoms of the disease, reduces the number of symptoms of the disease, reduces the severity of the symptoms of the disease, and / or reduces the length of the disease. Symptoms of influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A vims(es), a group 2 influenza A vims(es), or a combination thereof) include fever, chills, cough, sore throat, mnny or stuffy nose, muscle or body aches, headaches, fatigue (tiredness), vomiting and diarrhea.

[0266] In any aspect or embodiment described herein, a method of immunizing a subject against influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A virus(es), a group 2 influenza A vims(es), or a combination thereof) comprises the methodology set forth in the Examples. In any aspect or embodiment described herein, a method for preventing an influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A virus(es), a group 2 influenza A vims(es), or a combination thereof) in a subject comprises the methodology set forth in the Examples. In any aspect orAttorney Docket No. MSZ0003PCTembodiment described herein, a method of immunizing a subject against influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) comprises an immunization regimen set forth in the Examples, using the same chimeric HA polypeptides or other chimeric HA polypeptides described herein. In any aspect or embodiment described herein, a method for preventing an influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) in a subject comprises an immunization regimen set forth in the Examples, using the same chimeric HA polypeptides or other chimeric HA polypeptides described herein.

[0267] In any aspect or embodiment described herein, the methods for preventing an influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) and / or immunizing a subject against influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an Influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) provided herein provide at least partial protection against influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an influenza A vims(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof).

[0268] A composition described herein (e.g., a lipid nanoparticle composition described herein) may be administered to a subject by a variety of routes of administration. The routes of administration may include, in any aspect or embodiment described herein, intranasal, intratracheal, oral, topical, intradermal, intramuscular, intraperitoneal, transdermal, intravenous, conjunctival and subcutaneous routes. In any aspect or embodiment described herein, a composition described herein is administered to a subject intramuscularly.

[0269] In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 300 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 250 pg (e.g., about 1 pg to about 250 pg, about 1 pg to about 225 pg, about 1Attorney Docket No. MSZ0003PCTpg to about 200 pg, about 1 pg to about 175 pg, about 1 pg to about 150 jag, about 1 jag to about 125 jag, about 1 jag to about 100 jag, about 1 jag to about 75 jag, about 1 jag to about 50 pg, about 1 pg to about 25 pg, about 25 pg to about 250 pg, about 25 pg to about 225 pg, about 25 pg to about 200 pg, about 25 pg to about 175 pg, about 25 pg to about 150 pg, about 25 pg to about 125 pg, about 25 pg to about 100 pg, about 25 pg to about 75 pg, about 25 pg to about 50 pg, about 50 pg to about 250 pg, about 50 pg to about 225 pg, about 50 pg to about 200 pg, about 50 pg to about 175 pg, about 50 pg to about 150 pg, about 50 pg to about 125 pg, about 50 pg to about 100 pg, about 50 pg to about 75 pg, about 75 pg to about 250 pg, about 75 pg to about 225 pg, about 75 pg to about 200 pg, about 75 pg to about 175 pg, about 75 pg to about 150 pg, about 75 pg to about 125 pg, about 75 pg to about 100 pg, about 100 pg to about 250 pg, about 100 pg to about 225 pg, about 100 pg to about 200 pg, about 100 pg to about 175 pg, about 100 pg to about 150 pg, about 100 pg to about 125 pg, about 125 pg to about 250 pg, about 125 pg to about 225 pg, about 125 pg to about 200 pg, about 125 pg to about 175 pg, about 125 pg to about 150 pg, about 150 pg to about 250 pg, about 150 pg to about 225 pg, about 150 pg to about 200 pg, about 150 pg to about 175 pg, about 175 pg to about 250 pg, about 175 pg to about 225 pg, about 175 pg to about 200 pg, about 200 pg to about 250 pg, about 200 pg to about 225 pg, or about 225 pg to about 250 pg) of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 200 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 150 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 100 pg of a nucleoside-modifiedAttorney Docket No. MSZ0003PCTRNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 75 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 50 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 1 pg to about 30 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 10 pg to about 100 pg (e.g., about 10 pg to about 100 pg, about 10 pg to about 90 pg, about 10 pg to about 80 pg, about 10 pg to about 70 pg, about 10 pg to about 60 pg, about 10 pg to about 50 pg, about 10 pg to about 40 pg, about 10 pg to about 30 pg, about 10 pg to about 20 pg, about 20 pg to about 100 pg, about 20 pg to about 90 pg, about 20 pg to about 80 pg, about 20 pg to about 70 pg, about 20 pg to about 60 pg, about 20 pg to about 50 pg, about 20 pg to about 40 pg, about 20 pg to about 30 pg, about 30 pg to about 100 pg, about 30 pg to about 90 pg, about 30 pg to about 80 pg, about 30 pg to about 70 pg, about 30 pg to about 60 pg, about 30 pg to about 50 pg, about 30 pg to about 40 pg, about 40 pg to about 100 pg, about 40 pg to about 90 pg, about 40 pg to about 80 pg, about 40 pg to about 70 pg, about 40 pg to about 60 pg, about 40 pg to about 50 pg, about 50 pg to about 100 pg, about 50 pg to about 90 pg, about 50 pg to about 80 pg, about 50 pg to about 70 pg, about 50 pg to about 60 pg, about 60 pg to about 100 pg, about 60 pg to about 90 pg, about 60 pg to about 80 pg, about 60 pg to about 70 pg, about 70 pg toAttorney Docket No. MSZ0003PCTabout 100 pg, about 70 pg to about 90 jag, about 70 jag to about 80 jag, about 80 jag to about 100 |ag, about 80 jag to about 90 jag, or about 90 |ag to about 100 pg) of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 10 pg to about 75 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 10 pg to about 50 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 10 pg to about 30 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 10 pg, about 20 pg, or about 30 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 40 pg, about 50 pg, or about 60 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g.,Attorney Docket No. MSZ0003PCTa lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 70 pg, about 80 pg, about 90 pg, or about 100 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 10 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In some embodiments, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 30 pg of a nucleoside-modified RNA molecule. In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is about 100 pg of a nucleoside-modified RNA molecule of the present disclosure (e.g., the first nucleoside-modified RNA molecule, the second nucleoside-modified RNA molecule, or the first nucleoside-modified RNA molecule and the second nucleoside-modified RNA molecule). In any aspect or embodiment described herein, the dose of a composition described herein (e.g., a lipid nanoparticle composition described herein) administered to a subject in a method disclosed herein is dose described in the Examples.

[0270] In any aspect or embodiment described herein, the subject is a human. In any aspect or embodiment described herein, the subject is a human infant. In any aspect or embodiment described herein, the subject is a human child. In any aspect or embodiment described herein, the subject is a human adult. In any aspect or embodiment described herein, the subject is an elderly human. In any aspect or embodiment described herein, the subject is naive. In any aspect or embodiment described herein, the subject has not been previously vaccinated for influenza. In any aspect or embodiment described herein, the subject has been previously vaccinated for influenza. In any aspect or embodiment described herein, the subject has a condition that makes them more vulnerable to influenza disease (e.g., severe influenza disease).

[0271] In any aspect or embodiment described herein, provided herein is a method for producing an antibody(ies) to influenza virus, the method comprising administering to a subject (e.g., a human subject or non-human subject) a composition described herein. In anyAttorney Docket No. MSZ0003PCTaspect or embodiment described herein, provided herein is a method for producing an antibody(ies) to influenza virus HA, the method comprising administering to a subject (e.g., a human subject or non-human subject) a composition described herein. In any aspect or embodiment described herein, provided herein is a method for producing an antibody(ies) to influenza virus, the method comprising administering to a subject (e.g., a human subject or non-human subject) two or more compositions described herein. In any aspect or embodiment described herein, provided herein is a methods for producing an antibody(ies) to influenza virus HA, the method comprising administering to a subject (e.g., a human subject or non-human subject) two or more compositions described herein. In any aspect or embodiment described herein, provided herein is a method for producing an antibody(ies) to influenza virus HA, the method comprising administering to a subject (e.g., a human subject or non-human subject) a composition described herein in a regimen described herein (e.g., in this section or the Examples). In any aspect or embodiment described herein, the antibody(ies) is isolated from the subject using a technique(s) known to one of skill in the art. In any aspect or embodiment described herein, the antibody(ies) is optimized using a technique(s) known to one of skill in the art. In any aspect or embodiment described herein, the antibody(ies) is humanized using a technique(s) known to one of skill in the art. In any aspect or embodiment described herein, the antibody(ies) is human because it was induced in a non-human animal capable of producing human antibodies.

[0272] In any aspect or embodiment described herein, hybridomas are produced using B cells expressing an antibody produced by a method described herein.

[0273] In another aspect, provided herein is an antibody(ies) induced by a method described herein. In any aspect or embodiment described herein, provided herein is a pharmaceutical composition comprising an antibody described herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The antibody or a pharmaceutical composition thereof may be used in a method of treating and / or preventing influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) in a subject (e.g., human subject), and / or in a method for detecting influenza virus or HA, and / or diagnosing influenza vims infection. The antibody may be a monoclonal or polyclonal antibody. The antibody may be a human or humanized antibody.

[0274] In any aspect or embodiment described herein, provided herein are method of treating and / or preventing influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A vims(es), a group 2Attorney Docket No. MSZ0003PCTinfluenza A virus(es), or a combination thereof) in a subject (e.g., a human subject), the method comprising administering to the subject an antibody(ies) produced by methods described herein, or a pharmaceutical composition comprising the antibody(ies). In any aspect or embodiment described herein, provided herein are method of treating and / or preventing influenza virus disease (e.g., influenza virus disease caused by an influenza B virus(es), an influenza A virus(es), a group 1 influenza A virus(es), a group 2 influenza A virus(es), or a combination thereof) in a subject (e.g., a human subject), comprising administering to the subject an antibody(ies) produced by methods described herein in admixture with a pharmaceutically acceptable carrier. The antibody may be a monoclonal or polyclonal antibody. In any aspect or embodiment described herein, the antibody(ies) administered to the subject has been humanized. In any aspect or embodiment described herein, the antibody(ies) is administered to the subject are human. In any aspect or embodiment described herein, the antibody(ies) administered to the subject have one, two, or more, or all of the characteristics of the antibodies described in the Examples. In any aspect or embodiment described herein, the subject is a human. In any aspect or embodiment described herein, the subject is a human infant. In any aspect or embodiment described herein, the subject is a human child. In any aspect or embodiment described herein, the subject is a human adult. In any aspect or embodiment described herein, the subject is an elderly human.

[0275] In any aspect or embodiment described herein, the antibody(ies) or pharmaceutical composition is administered may be administered to a subject by a variety of routes. For example, in any aspect or embodiment described herein, the antibody(ies) or pharmaceutical composition may be administered parenterally. In any aspect or embodiment described herein, the antibody(ies) or pharmaceutical composition is administered intramuscularly. In any aspect or embodiment described herein, the antibody(ies) or pharmaceutical composition is administered subcutaneously. In any aspect or embodiment described herein, the antibody(ies) or pharmaceutical composition is administered intravenously.

[0276] In any aspect or embodiment described herein, the antibody(ies) or pharmaceutical composition is administered to a subject at a dose of 5 mg / kg to 50 mg / kg. In any aspect or embodiment described herein, the average weight of a human adult subject is 60 kg to 80 kg. In any aspect or embodiment described herein, the average weight of a human child is 9 kg to 60 kg. In any aspect or embodiment described herein, the average weight of a human infant is 3 kg to 10 kg. In any aspect or embodiment described herein, theAttorney Docket No. MSZ0003PCTantibody(ies) or pharmaceutical composition is administered to a subject at a dose of 45 mg to 4,000 mg.

[0277] In any aspect or embodiment described herein, the methods of treating and / or preventing an influenza vims disease (e.g., influenza vims disease caused by an influenza B vims(es), an influenza A vims(es), a group 1 influenza A vims(es), a group 2 influenza A vims(es), or a combination thereof) in a subject results in one, two, or more of the following: prevents the onset of one or more symptoms of the disease, reduces the number of symptoms of the disease, reduces the severity of the symptoms of the disease, and / or reduces the length of the disease.

[0278] A further aspect of the present disclosure provides a composition comprising, consisting essentially of, or consisting of, the nucleoside-modified RNA molecule (e.g., a first nucleoside-modified ribonucleic acid (RNA) molecule and / or a second nucleoside-modified ribonucleic acid (RNA) molecule) of the present disclosure, and a pharmaceutically acceptable carrier. Thus, for example, the composition comprises, consists essentially of, or consists of, the first nucleoside-modified ribonucleic acid (RNA) molecule of the present disclosure and / or a second nucleoside-modified ribonucleic acid (RNA) molecule) of the present disclosure, and a pharmaceutically acceptable carrier

[0279] In any aspect or embodiment described herein, the composition further comprises a lipid component. For example, in any aspect or embodiment described herein, the lipid component is part of a lipid nanoparticle, such as described herein.

[0280] Another aspect of the present disclosure provides a method for inducing an immune response against influenza vims HA or preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising administering to the subject a dose of the nucleoside-modified RNA molecule of the present disclosure, or the composition of the present disclosure, wherein the method is effective for inducing an immune response against HA or preventing influenza vims symptoms in the subject.

[0281] In any aspect or embodiment described herein, the dose is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the nucleoside-modified RNA molecule.

[0282] In another aspect, provided herein are methods for inducing an immune response to influenza vims in a subject, the method comprising administering to the subject two or more compositions described herein. In another aspect, provided herein are methods for preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising administering to the subject two or more compositions described herein. In another aspect, provided herein are methods of immunizing a subject against influenza vims disease, theAttorney Docket No. MSZ0003PCTmethod comprising administering to the subject two or more compositions described herein. In any aspect or embodiment described herein, each composition comprises a different nucleoside-modified mRNA molecule described herein. In any aspect or embodiment described herein, each composition comprises a different nucleoside-modified mRNA molecule encoding a chimeric influenza vims HA polypeptide. In any aspect or embodiment described herein, the two or more compositions are administered intramuscularly to the subject. In any aspect or embodiment described herein, the subject is human.

[0283] An additional aspect of the present disclosure provides a method for inducing an immune response against influenza vims hemagglutinin (HA) in a subject or preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising: (a) administering to the subject a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (b) administering to the subject a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second Influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine, wherein the method is effective forAttorney Docket No. MSZ0003PCTinducing an immune response against HA or preventing influenza virus symptoms in the subject.

[0284] Yet another aspect of the present disclosure provides a method for inducing an immune response against influenza virus hemagglutinin (HA) in a subject or preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising: (a) administering to the subject a dose of a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein: (i) the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims, (ii) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, and (iii) the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (b) administering to the subject a dose of a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein: (i) the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims, (ii) the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second Influenza A vims of a different strain, subtype, or group than the first influenza A vims, and (iii) the second nucleoside-modified RNA molecule comprises at least one pseudouridine, wherein the method is effective for inducing an immune response against HA or preventing influenza vims symptoms in the subject.

[0285] In any aspect or embodiment described herein, at least one of (1) the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a HA of a first influenza A vims; (2) the second HAAttorney Docket No. MSZ0003PCTglobular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; or (3) a combination thereof.

[0286] In any aspect or embodiment described herein, at least one of (1) the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus; (2) the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; or (3) a combination thereof.

[0287] In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof. In any aspect or embodiment described herein, the antigenic sites comprise, consist essentially of, or consist of, 120 loop, 150 loop, 160 loop, and 190 helix.

[0288] In any aspect or embodiment described herein, at least one of (1 ) the first influenza A vims is an influenza A vims that originates from a different species than the first influenza B vims; (2) the second influenza A vims is an influenza A vims that originates from a different species than the second influenza B vims; (3) the first influenza A vims is an avian influenza A vims; (4) the second influenza A vims is an avian influenza A vims; or (5) a combination thereof.

[0289] In any aspect or embodiment described herein, at least one of (1) the first influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims: (2) the second influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims; (3) the first influenza A vims is a hemagglutinin 13 (H13) Influenza A vims (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6), a hemagglutinin 5 (H5) Influenza A vims (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP), or a subtype thereof; (4) the second influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6), a hemagglutinin 5 (H5) Influenza A vims (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP), or a subtype thereof; or (5) a combination thereof.

[0290] In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza vims HA polypeptide (e.g., a first chimeric influenza vims HA polypeptide and / or a chimeric influenza vims HA polypeptide) comprises, consistsAttorney Docket No. MSZ0003PCTessentially of, or consists of, the amino acid sequence of the HA globular head domain of an HA of an influenza A virus subtype H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17, or H18 (preferably H13 (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6) or H5 (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP)). In any aspect or embodiment described herein, the HA globular head domain of the chimeric influenza virus HA polypeptide (e.g., a first chimeric influenza virus HA polypeptide and / or a chimeric influenza virus HA polypeptide) comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of the HA of the influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of the HA globular head domain of an HA of an influenza A virus subtype H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17, or H18 (preferably H13 (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6) or H5 (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP)).

[0291] In any aspect or embodiment described herein, at least one of the first influenza B virus is B / Brisbane / 20 / 2008 Influenza virus and the first influenza A virus is a hemagglutinin 13 (H13) Influenza A virus (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6); the second influenza B virus is B / Phuket / 3073 / 2013 Influenza virus and the second influenza A virus is a hemagglutinin 5 (H5) Influenza A virus (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP); or a combination thereof.

[0292] In any aspect or embodiment described herein, at least one of the first influenza B virus is B / Phuket / 3073 / 2013 Influenza virus and the first influenza A virus is a hemagglutinin 5 (H5) Influenza A virus or a subtype thereof (e.g., A / Vietnam / 1203 / 04 H5N1-PR8-IBCDC-RG / GLP); the second influenza B virus is B / Brisbane / 20 / 2008 Influenza virus and the second influenza A virus is a hemagglutinin 13 (H13) Influenza A virus or a subtype thereof (e.g., A / black-headed gull / Sweden / 1 / 1999 H13N6); or a combination thereof.

[0293] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza virus HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B virus (e.g., the first influenza B virus) and a HA cytoplasmic domain of an influenza B virus (e.g., the first influenza B virus); (2) the second chimeric influenza virus HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B virus (e.g., the second influenza B virus) and a HA cytoplasmic domain of an influenza B virus (e.g., the second influenza B virus); or (3) a combination thereof.

[0294] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the aminoAttorney Docket No. MSZ0003PCTacid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the second nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide have different amino acid sequence or SEQ ID NOs). For example, in any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the second nucleoside-modified RNA molecule comprises at least one pseudouridine (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide have different amino acid sequence or SEQ ID NOs).

[0295] In any aspect or embodiment described herein, at least one of: ( 1 ) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide are transcribed from different nucleotide sequence or SEQ ID NOs). For example, in any aspect or embodiment described herein, (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide are transcribed from different nucleotide sequence or SEQ ID NOs).

[0296] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQAttorney Docket No. MSZ0003PCTID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine: or (3) a combination thereof. For example, in any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0297] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof. For example, in any aspect or embodiment described herein, (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine.

[0298] In any aspect or embodiment described herein, at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof. For example, in any aspect or embodiment described herein, (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; and (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modifie...

Claims

Attorney Docket No. MSZ0003PCTCLAIMSWhat Is Claimed Is:

1. A nucleoside-modified ribonucleic acid (RNA) molecule encoding a chimeric influenza virus hemagglutinin (HA) polypeptide comprising a HA globular head domain and a HA stalk domain, wherein:the HA stalk domain comprises an amino acid sequence of the HA stalk domain of an influenza B virus;the HA globular head domain comprises an amino acid sequence of (i) the HA globular head domain of an influenza A virus or (ii) a HA globular head domain of an influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A virus; andthe nucleoside-modified RNA molecule comprises at least one pseudouridine.

2. The nucleoside-modified RNA molecule of claim 1, wherein the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof.

3. The nucleoside-modified RNA molecule of claim 1 or 2, wherein the influenza A virus is an influenza A virus that originates from a different species than the influenza B virus.

4. The nucleoside-modified RNA molecule of any one of claims 1-3, wherein the influenza A vims is an avian influenza A vims.

5. The nucleoside-modified RNA molecule of any one of claims 1-4, wherein at least one of:the influenza B vims is B / Phuket / 3073 / 2013 Influenza vims or B / Brisbane / 20 / 2008 Influenza vims;the influenza A vims is a hemagglutinin 5 (H5) Influenza A vims, a hemagglutinin 13 (Hl 3) Influenza A vims, or subtype thereof; ora combination thereof.

6. The nucleoside-modified RNA molecule of any one of claims 1-5, wherein: the influenza B vims is B / Phuket / 3073 / 2013 Influenza vims and the influenza A vims is a hemagglutinin 5 (H5) Influenza A vims: orthe influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the influenza A vims is a hemagglutinin 13 (H13).Attorney Docket No. MSZ0003PCT7. The nucleoside-modified RNA molecule of any one of claims 1-5, wherein the chimeric influenza virus HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of the influenza B virus and a HA cytoplasmic domain of the influenza B virus.

8. The nucleoside-modified RNA molecule of any one of claims 1-6, wherein the HA globular head domain comprises the amino acid sequence of the HA globular head domain the influenza A virus.

9. The nucleoside-modified RNA molecule of any one of claims 1 -6, wherein the HA globular head domain comprises the amino acid sequence of the HA globular head domain of the influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of the influenza A virus.

10. The nucleoside-modified RNA molecule of any one of claims 1-6, wherein the nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine.

11. A nucleoside-modified RNA molecule encoding a chimeric influenza virus HA polypeptide comprising, consisting essentially of, or consisting of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the nucleoside-modified RNA molecule comprises at least one pseudouridine.

12. A composition comprising, consisting essentially of, or consisting of, the nucleoside-modified RNA molecule of any one of claims 1 to 11, and a pharmaceutically acceptable carrier.

13. The composition of claim 12, further comprising a lipid component (e.g., the lipid component is part of a lipid nanoparticle).

14. A method for inducing an immune response against influenza virus hemagglutinin (HA) or preventing influenza virus symptoms in a subject, the method comprising administering to the subject at least one dose of the nucleoside-modified RNA molecule of any one of claims 1 to 11, or the composition of claim 12 or 13, wherein the method is effective for inducing an immune response against HA or preventing influenza virus symptoms in the subject.

15. The method of claim 14, wherein the dose is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the nucleoside-modified RNA molecule.

16. A method for inducing an immune response against influenza virus hemagglutinin (HA) or preventing influenza virus symptoms (e.g., disease) in a subject, the method comprising:Attorney Docket No. MSZ0003PCT(a) administering to the subject a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenza virus HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein:the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims,the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, andthe first nucleoside-modified RNA molecule comprises at least one pseudouridine; and(b) administering to the subject a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein:the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims,the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, andthe second nucleoside-modified RNA molecule comprises at least one pseudouridine.

17. A method for inducing an immune response against influenza vims hemagglutinin (HA) or preventing influenza vims symptoms (e.g., disease) in a subject, the method comprising:(a) administering to the subject a dose of a first composition comprising a first nucleoside-modified ribonucleic acid (RNA) molecule encoding a first chimeric influenzaAttorney Docket No. MSZ0003PCTvirus HA polypeptide that comprises, consists essentially of, or consists of, a first HA globular head domain and a first HA stalk domain, wherein:the first HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of a first influenza B vims,the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A vims, or the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A vims, andthe first nucleoside-modified RNA molecule comprises at least one pseudouridine; and(b) administering to the subject a dose of a second composition comprising a second nucleoside-modified RNA molecule encoding a second chimeric influenza vims HA polypeptide that comprises, consists essentially of, or consists of, a second HA globular head domain and a second HA stalk domain, wherein:the second HA stalk domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA stalk domain of an influenza B vims of a different strain, subtype, or group than the first influenza B vims,the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first Influenza A vims, or the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B vims, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims, andthe second nucleoside-modified RNA molecule comprises at least one pseudouridine.

18. The method of claim 16 or 17, wherein at least one of:the first HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head of a first influenza A vims;the second HA globular head domain comprises, consists essentially of, or consists of, an amino acid sequence of a HA globular head domain of a second influenza A vims of a different strain, subtype, or group than the first influenza A vims; ora combination thereof;Attorney Docket No. MSZ0003PCT19. The method of any one of claims 16-18, wherein at least one of:the first globular HA head domain comprises an amino acid sequence of a HA globular head domain of the first influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a first influenza A virus;the second globular HA head domain comprises an amino acid sequence of a HA globular head domain of the second influenza B virus, wherein one or more antigenic sites have been replaced with antigenic sites of a second influenza A virus of a different strain, subtype, or group than the first influenza A virus; ora combination thereof.

20. The method of any one of claims 16-19, wherein the antigenic sites comprise, consist essentially of, or consist of, at least one of 120 loop, 150 loop, 160 loop, 190 helix, or a combination thereof.

21. The method of any one of claims 16-20, wherein at least one of:the first influenza A virus is an influenza A vims that originates from a different species than the first influenza B vims;the second influenza A vims is an influenza A vims that originates from a different species than the second influenza B vims;the first influenza A vims is an avian influenza A vims;the second influenza A vims is an avian influenza A vims; ora combination thereof.

22. The method of any one of claims 16-21, wherein at least one of:the first influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims;the second influenza B vims is B / Brisbane / 20 / 2008 Influenza vims or B / Phuket / 3073 / 2013 Influenza vims;the first influenza A vims is a hemagglutinin 13 (H13) Influenza A vims, a hemagglutinin 5 (H5) Influenza A vims, or a subtype thereof;the second influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims, a hemagglutinin 5 (H5) Influenza A vims, or a subtype thereof; ora combination thereof.

23. The method of any one of claims 16-22, wherein:at least one of the first influenza B vims is B / Brisbane / 20 / 2008 Influenza vims and the first Influenza A vims is a hemagglutinin 13 (Hl 3) Influenza A vims; the secondAttorney Docket No. MSZ0003PCTinfluenza B virus is B / Phuket / 3073 / 2013 Influenza virus and the second influenza A virus is a hemagglutinin 5 (H5) Influenza A virus: or a combination thereof; orat least one of the first influenza B virus is B / Phuket / 3073 / 2013 Influenza virus and the first influenza A virus is a hemagglutinin 5 (H5) Influenza A virus or a subtype thereof; the second influenza B virus is B / Brisbane / 20 / 2008 Influenza virus and the second influenza A virus is a hemagglutinin 13 (Hl 3) Influenza A virus or a subtype thereof; or a combination thereof.

24. The method of any one of claims 16-23, wherein at least one of:the first chimeric influenza virus HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B virus (e.g., the first influenza B virus) and a HA cytoplasmic domain of an influenza B virus (e.g., the first influenza B virus);the second chimeric influenza virus HA polypeptide further comprises, consists essentially of, or consists of, a HA transmembrane domain of an influenza B virus (e.g., the second influenza B virus) and a HA cytoplasmic domain of an influenza B virus (e.g., the second influenza B virus); ora combination thereof.

25. The method of any one of claims 16-24, wherein at least one of:the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine;the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, 3, 5, or 7, and wherein the second nucleoside-modified RNA molecule comprises at least one pseudouridine; ora combination thereof (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide have different amino acid sequence or SEQ ID NOs).

26. The method of any one of claims 16-25, wherein at least one of:the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine;the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, 4, 6, or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; orAttorney Docket No. MSZ0003PCTa combination thereof (e.g., wherein the first chimeric influenza virus HA polypeptide and the second chimeric influenza virus HA polypeptide are transcribed from different nucleotide sequence or SEQ ID NOs).

27. The method of any one of claims 16-24, wherein:(a) at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(b) at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1 or 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5 or 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(c) at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(d) at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(e) at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;Attorney Docket No. MSZ0003PCT(f) at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(g) at least one of (1) the first chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza virus HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(h) at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof;(i) at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 3, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 5, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof; or(j) at least one of (1) the first chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 1, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; (2) the second chimeric influenza vims HA polypeptide comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 7, and wherein the first nucleoside-modified RNA molecule comprises at least one pseudouridine; or (3) a combination thereof.

28. The method of any one of claims 16-27, wherein:(a) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6 or 8, wherein the transcription occurred in the presence of a modifiedAttorney Docket No. MSZ0003PCTnucleoside of uridine; (2) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2 or 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(b) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2 or 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6 or 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(c) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(d) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(e) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(f) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(g) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;Attorney Docket No. MSZ0003PCT(h) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof;(i) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 4, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 6, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof; or(j) at least one of (1) the first nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 2, wherein the transcription occurred in the presence of a modified nucleoside of uridine; (2) the second nucleoside-modified RNA molecules is transcribed from SEQ ID NO: 8, wherein the transcription occurred in the presence of a modified nucleoside of uridine; or (3) a combination thereof.

29. The method of any one of claims 16-28, wherein administering the second composition is performed after a period of time (e.g., about 25 days to about 90 days, about 40 days to about 75 days, about 45 days to about 65 days, or about 55 days to about 60 days) from administering the first composition.

30. The method of any one of claims 16-28, wherein the first composition and the second composition are co-administered (e.g., at the same time, in the same composition or within about from 1 to 24 hours).

31. The method of any one of claims 16-30, wherein at least one of (1) the dose of the first composition is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the first nucleoside-modified RNA molecule, (2) the dose of the second composition is about 10 pg to about 100 pg (e.g., about 30 pg to about 60 pg) of the second nucleoside-modified RNA molecule, or (3) a combination thereof.

32. The method of any one of claims 14-31, wherein the subject is human.

33. The method of any one of claims 14-32, further comprisingprior to administering the nucleoside-modified RNA molecule (e.g., claim 14 or 15), administering an influenza vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) HA antigen (e.g., a seasonal quadrivalent influenza vaccine); orAttorney Docket No. MSZ0003PCTprior to administering the first composition (e.g., claims 16-32), administering an influenza vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) HA antigen (e.g., a seasonal quadrivalent influenza vaccine).

34. A kit comprising:a container including the nucleoside-modified RNA molecule of any one of claims 1 to 11, the composition of claim 12 or 13, or the first composition or the second composition of any one of claims 16-33; ora first container including the first composition of any one of claims 16-33 and a second container including the second composition of any one of claims 16-33.

Images