Rhinovirus mRNA vaccine

Abstract

The present invention provides methods for identifying the amino acid sequence of a native polyprotein from a group A or C rhinovirus that can be used as an immunogen capable of eliciting an immune response against rhinoviruses from multiple serotypes within the same group. The present invention also provides immunogenic compositions comprising at least one mRNA comprising a non-native, optimized nucleic acid encoding a polyprotein identified by this method.

Description

[Technical Field]

[0001] Related Applications This application claims priority to European Patent Application No. 22315341.2, filed December 20, 2022, and European Patent Application No. 23306405.4, filed August 22, 2023; the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This specification references a Sequence Listing (submitted electronically as an .xml file entitled "PAT22129_Sequence_Listing" on December 20, 2023). The .xml file was created on December 18, 2023 and is 144 KB in size. The entire contents of the Sequence Listing are incorporated herein by reference.

[0003] The present invention relates to messenger RNA (mRNA)-based rhinovirus vaccines. The vaccines are specifically designed to elicit effective immune responses against multiple rhinovirus serotypes of the same group, particularly rhinovirus group A or C. The selected immunogens encoded by the mRNAs are likely to be native rhinovirus polyproteins that contain highly conserved and T-cell epitope-rich regions. [Background technology]

[0004] Rhinoviruses are small, non-enveloped, positive-strand RNA viruses belonging to the Picornaviridae family. They are characterized into three groups, A, B, and C, with 81, 33, and 56 serotypes described to date, respectively. The large number of serotypes is a major factor in the large genetic and antigenic variation observed among rhinoviruses.

[0005] The rhinovirus genome encodes a single polyprotein containing both structural and nonstructural proteins. Protease-dependent cleavage of the polyprotein generates precursor proteins P1, P2, and P3, which are similarly further cleaved into four structural (capsid) proteins, VP1, VP2, VP3, and VP4, and seven nonstructural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D, respectively. VP2 and VP4 result from cleavage of the intermediate polyprotein, VP0. Figure 1 provides a schematic diagram of the domain structure of rhinovirus mRNA.

[0006] Human rhinoviruses (HRVs) are the leading cause of the common cold, accounting for two-thirds of annual cases. Transmission occurs through direct contact with respiratory secretions and is associated with upper and lower respiratory tract infections. Currently, there are no approved antiviral therapies for the prevention or treatment of rhinovirus infections. Human challenge studies have shown that pre-loading antibodies reduce viral load and disease manifestations (Barclay et al., Epidemiol Infect. 1989 Dec;103(3):659-669; Alper et al., Clin Infect Dis. 1998 Jul;27(1):119-128; Touabi et al., Viruses. 2021 Feb;13(3):360). Symptom severity also correlates with T helper type 1 (T) responses to experimental rhinovirus infection. H 1) and has also been reported to be inversely correlated with interferon-gamma (IFNγ) responses (Parry et al., J Allergy Clin Immunol. 2000 Apr;105(4):692-698; Gern et al., Am J Respir Crit Care Med. 2000 Dec;162(6):2226-2231; Message et al., Proc Natl Acad Sci USA. 2008 Sept;105(36):13562-13567).

[0007] In healthy individuals, effective T HIn contrast, individuals suffering from respiratory conditions such as chronic obstructive pulmonary disease (COPD) and asthma lack type 2 helper T cells (T H 2) Due to the increased likelihood of mounting a response, HRV infection may result in severe illness associated with the virus. H The induction of 2 responses, combined with a delayed IFN response, may contribute to asthma exacerbation through mucus hypersecretion and allergic inflammation.

[0008] The lack of cross-protection from natural infection represents a clinical challenge that highlights the need for suitable vaccine strategies. Rhinovirus infections are usually mild in healthy individuals, but repeated infections that cause severe symptoms of the common cold can impose a significant economic burden on society in terms of lost work days. Various approaches exist for rhinovirus vaccine development. One approach is the induction of broadly neutralizing antibodies (Katpally et al., J Virol. 2009 Jul;83(14):7040-7048). Clinical data support the notion that T cells may prevent the development of symptomatic respiratory disease when antibody-mediated defenses against rhinovirus infection are circumvented, for example, because a patient's immune system has not previously been exposed to a particular serotype (Parry et al., J Allergy Clin Immunol. 2000 Apr;105(4):692-698; Gern et al., Am J Respir Crit Care Med. 2000 Dec;162(6):2226-2231; Message et al., Proc Natl Acad Sci USA. 2008 Sept;105(36):13562-13567).

[0009] Previous studies have shown that the structural protein VP4 contains conserved T cell epitopes across rhinovirus A and C subtypes, and targeting these conserved T cell epitopes through peptide-based approaches has been proposed (Gomez-Perozanz et al., Cells. 2021 Sept;10(9);2284). Unlike more established vaccine technologies, the efficacy of peptide-based approaches requires further preclinical and clinical validation. Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there remains a need for vaccines against rhinovirus infections, particularly for patients suffering from respiratory diseases such as COPD and / or asthma, that are effective in preventing or reducing complications associated with rhinovirus infections. [Means for solving the problem]

[0011] The present invention is based on the identification of conserved regions within the complete rhinovirus polyprotein of rhinovirus group A and rhinovirus group C strains that may be suitable as immunogens for eliciting immune responses against multiple rhinovirus serotypes of the same group. Such regions can be used to identify T cell epitope-rich regions to ensure they provide broad coverage of MHC-I and MHC-II alleles to elicit T cell responses in a large portion of the human population.

[0012] The inventors used a computational approach to identify conserved regions in the amino acid sequences of complete rhinovirus A and C polyproteins. This approach involved grouping rhinovirus sequences from groups A and C into phylogenetic clusters and identifying sequences that could cover at least two of these clusters to ensure that a selected immunogen would be capable of eliciting an immune response against multiple rhinovirus serotypes. While not wishing to be bound by any particular theory, the inventors believe that a native rhinovirus polyprotein that has at least 80% average identity (and optionally median identity) with amino acid sequences of rhinoviruses from at least two phylogenetic clusters would be suitable for providing broad protection.

[0013] The inventors analyzed published amino acid sequences encoding all or at least portions of the complete rhinovirus polyprotein. They focused on sequences of at least 800 amino acids, because this is the approximate length of the complete VP capsid region of the polyprotein. Because the VP proteins are exposed on the viral surface, they should represent most, if not all, of the differences between the various rhinovirus serotypes.

[0014] In particular, the present invention relates to a method for identifying rhinovirus polyproteins for use as immunogens capable of eliciting an immune response against rhinoviruses from multiple serotypes within a group, the method comprising the following steps: (a) obtaining a plurality of amino acid sequences from a database containing amino acid sequences from natural rhinovirus isolates; (b) removing amino acid sequences shorter than 800 amino acids from the plurality of amino acid sequences obtained in step (a); (c) assigning the amino acid sequences remaining after step (b) to different phylogenetic clusters; (d) aligning the amino acid sequences to determine a consensus amino acid sequence for the complete rhinovirus polyprotein for one or more phylogenetic clusters identified in step (c); (e) aligning the consensus amino acid sequence obtained in step (c) with the complete polyprotein of a natural rhinovirus isolate; and (f) selecting a rhinovirus polyprotein as an immunogen having an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the corresponding amino acid sequences of rhinoviruses from at least two phylogenetic clusters identified in step (c).

[0015] In some embodiments, the rhinovirus polyprotein selected in step (f) is VP0 polyprotein. In some embodiments, the rhinovirus polyprotein selected in step (f) is P2 polyprotein. In some embodiments, the group is rhinovirus group A. In some embodiments, the group is rhinovirus group C.

[0016] In some embodiments, one or more phylogenetic clusters comprise at least 5 different serotypes, hi some embodiments, one or more phylogenetic clusters comprise at least 10, 15, 20, or 25 different serotypes.

[0017] In some embodiments, the plurality of amino acid sequences obtained in step (a) is greater than 400 (eg, 500, 600, 700, or 800).

[0018] In some embodiments, determining a consensus sequence in step (d) comprises selecting the most frequent amino acid at each position. In some embodiments, determining a consensus sequence in step (d) comprises generating gaps when the sum of amino acids for a given position is less than 50% of the number of sequences obtained. In some embodiments, determining a consensus sequence in step (d) comprises selecting the most frequent amino acid when the sum of amino acids for a given position is greater than or equal to 50% of the number of sequences obtained.

[0019] Without wishing to be bound by any particular theory, the inventors believe that the immunogen selected in step (f) of the method can effectively induce a desired immune response, including, for example, an effective T cell response, when the immunogen is administered to a subject (e.g., a subject in need of immunization) in the form of at least one messenger RNA (mRNA) encoding the immunogen. Accordingly, in some embodiments, the method further comprises generating an optimized nucleic acid sequence encoding the rhinovirus polyprotein selected in step (f).

[0020] The present invention further relates to immunogenic compositions (e.g., vaccines) comprising non-naturally occurring mRNA encoding an immunogen identified by the methods of the present invention and, optionally, a carrier (e.g., lipid nanoparticles encapsulating the mRNA) or adjuvant. The inventors believe that immunogenic compositions comprising non-naturally occurring mRNA encoding a native rhinovirus protein or polyprotein are more effective at eliciting immune responses against multiple rhinovirus serotypes of the same group than other types of vaccines. In some embodiments, to achieve efficient transcription and expression of the mRNA, the nucleic acid encoding the immunogen is optimized (e.g., expression-optimized) to generate a non-naturally occurring optimized nucleic acid sequence. Sometimes, the rhinovirus-derived polyprotein itself can interfere with efficient expression. Therefore, to ensure efficient expression of the immunogen encoded by the mRNA, one or more amino acid substitutions can be introduced into the native amino acid sequence of the polyprotein selected as the immunogen. In one embodiment, a single amino acid substitution is introduced into the native amino acid sequence of the polyprotein selected as the immunogen.

[0021] Accordingly, the present invention also provides immunogenic compositions (e.g., vaccines) comprising at least one messenger RNA (mRNA) comprising a non-native, optimized nucleic acid sequence encoding a polyprotein from a Group A or Group C rhinovirus, wherein the polyprotein (a) has an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) with the amino acid sequences of corresponding polyproteins from at least two phylogenetic clusters of rhinoviruses of the same group; and (b) is naturally occurring except for one or more optional amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions). In one embodiment, the polyprotein (a) has an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) with the amino acid sequences of corresponding polyproteins from at least two phylogenetic clusters of rhinoviruses of the same group; and (b) is naturally occurring except for an optional single amino acid substitution. The amino acid sequence of the polyprotein is obtained or obtainable by the methods described herein for identifying rhinovirus polyproteins for use as immunogens.

[0022] In some embodiments, the amino acid sequence of the polyprotein has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequences of corresponding polyproteins from at least three, e.g., four, phylogenetic clusters of rhinoviruses of the same group.

[0023] In some embodiments, the polyprotein is a VP0 polyprotein comprising proteins VP2 and VP4. In some embodiments, the VP0 polyprotein is derived from a group C rhinovirus. In some embodiments, the group C rhinovirus is of serotype 11, 17, or 34. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of a rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:1. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of a rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:2. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of a rhinovirus C serotype 34 VP0 polyprotein set forth in SEQ ID NO:3.

[0024] In some embodiments, the VP0 polyprotein is from a group A rhinovirus. In some embodiments, the group A rhinovirus is of serotype 21 or 90. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:4. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:5.

[0025] In some embodiments, the polyprotein is a P2 polyprotein comprising proteins 2A, 2B, and 2C. In some embodiments, a single amino acid substitution is in the 2A protein that reduces or eliminates the proteolytic activity of the P2 polyprotein. In some embodiments, the single amino acid substitution is a C>A or C>S substitution in the catalytic triad of the active site of the 2A protein.

[0026] In some embodiments, the P2 polyprotein is derived from a Group A rhinovirus. In some embodiments, the Group A rhinovirus is of serotype 21 or 57. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO:6. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 57 P2 polyprotein set forth in SEQ ID NO:7.

[0027] In some embodiments, the P2 polyprotein is derived from a group C rhinovirus. In some embodiments, the group C rhinovirus is of serotype 11 or 17. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus C serotype 11 P2 polyprotein set forth in SEQ ID NO: 8. In some embodiments, the polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus C serotype 17 P2 polyprotein set forth in SEQ ID NO: 9.

[0028] In some embodiments, the immunogenic composition (e.g., vaccine) further comprises a second non-naturally occurring, optimized nucleic acid sequence encoding an additional polyprotein from a group A or C rhinovirus, wherein the additional polyprotein is distinct from the polyprotein. In some embodiments, the first and second nucleic acid sequences are part of the same mRNA. In some embodiments, the mRNA encodes a fusion protein comprising the polyprotein and the additional polyprotein. In some embodiments, the first and second nucleic acid sequences are encoded by separate non-naturally occurring mRNAs.

[0029] Combining multiple different polyproteins can be advantageous to extend the protection provided by the immunogenic composition, for example to induce an immune response against multiple rhinoviruses that are phylogenetically more distant from each other (e.g., multiple serotypes within the same group) or against multiple rhinoviruses from different groups (e.g., groups A and C). Accordingly, the present invention also provides immunogenic compositions comprising at least one messenger RNA (mRNA) comprising: (i) a first non-naturally occurring, optimized nucleic acid sequence encoding a first polyprotein from a Group A or Group C rhinovirus; and (ii) a second non-naturally occurring, optimized nucleic acid sequence encoding a second polyprotein from a Group A or Group C rhinovirus, wherein the second polyprotein is different from the first polyprotein, and wherein each of the first and second polyproteins (a) has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequence of a corresponding polyprotein from at least two phylogenetic clusters of rhinoviruses of the same group; and (b) an amino acid sequence that is natural except for one or more optional amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions). In one embodiment, the polyprotein (a) has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequences of corresponding polyproteins from at least two phylogenetic clusters of rhinoviruses of the same group; and (b) an amino acid sequence that is naturally occurring except for an optional single amino acid substitution. In some embodiments, the amino acid sequence of each of the first and second polyproteins has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequences of corresponding polyproteins from at least three, e.g., four, phylogenetic clusters of rhinoviruses of the same group.The amino acid sequences of the first and second polyproteins are obtained or obtainable by the methods for identifying rhinovirus polyproteins for use as immunogens described herein.

[0030] In some embodiments, the first polyprotein is a VP0 polyprotein comprising proteins VP2 and VP4. In some embodiments, the VP0 polyprotein is derived from a group A rhinovirus. In some embodiments, the group A rhinovirus is of serotype 21 or 90. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:4. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:5.

[0031] In some embodiments, the second polyprotein is a P2 polyprotein comprising proteins 2A, 2B, and 2C. In some embodiments, a single amino acid substitution is in the 2A protein that reduces or eliminates the proteolytic activity of the P2 polyprotein. In some embodiments, the single amino acid substitution is a C>A or C>S substitution in the catalytic triad of the active site of the 2A protein.

[0032] In some embodiments, the P2 polyprotein is derived from a group A rhinovirus. In some embodiments, the group A rhinovirus is of serotype 21 or 57. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO:6. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 57 P2 polyprotein set forth in SEQ ID NO:7.

[0033] The inventors demonstrate herein that the T cell epitope-rich regions in the VP0 and P2 polyproteins of serogroup 21 group A rhinoviruses cover the majority (>97%) of the MHC-I and MHC-II alleles present in the human population. Thus, in some embodiments, the immunogenic compositions of the invention are capable of eliciting a T cell response in at least 95% of a human population. In some embodiments, the immunogenic compositions are capable of eliciting a T cell response in at least 96%, at least 97%, at least 98%, or at least 99% of a human population. In some embodiments, the VP0 and P2 polyproteins comprise T cell epitope-rich regions that cover at least 95% of the MHC class I alleles in Table 4 and / or 95% of the MHC-II alleles in Table 5. In some embodiments, the T cell epitope-rich region covers at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class I alleles of Table 4 and / or at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class II alleles of Table 5.

[0034] VP proteins from different groups of rhinoviruses can vary greatly. For example, group A rhinoviruses primarily bind to the intercellular adhesion molecule 1 (ICAM-1) receptor. A few utilize the low-density lipoprotein receptor (LDLR) for binding. In contrast, certain variants of cadherin-related family member 3 (CDHR3) are the primary receptor for group C rhinoviruses. Thus, an immunogenic composition capable of inducing an immune response against multiple serogroups can include a VP0 polyprotein from a group A rhinovirus and a VP0 polyprotein from a group C rhinovirus.

[0035] Thus, in some embodiments, the second polyprotein is a VP0 polyprotein from a group C rhinovirus. In some embodiments, the group C rhinovirus is of serotype 11, 17, or 34. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:1. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:2. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 34 VP0 polyprotein set forth in SEQ ID NO:3.

[0036] Based on the inventors' analysis, inducing an immune response to multiple phylogenetically distant serogroups of the rhinovirus group can be improved by including one or more non-naturally occurring mRNAs encoding multiple different VP0 and / or P2 polyproteins from the same group.

[0037] Thus, in some embodiments, the first polyprotein is a VP0 polyprotein and the second polyprotein is a VP0 polyprotein, and the two phylogenetic clusters referred to in option (a) are different for the first polyprotein and the second polyprotein. In some embodiments, the first polyprotein and the second polyprotein are derived from rhinovirus C. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:1, and the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:2.

[0038] In some embodiments, the first polyprotein is a P2 polyprotein and the second polyprotein is a P2 polyprotein, and the two phylogenetic clusters referred to in option (a) are different for the first polyprotein and the second polyprotein. In some embodiments, the first polyprotein and the second polyprotein are derived from rhinovirus C. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus C serotype 11 P2 polyprotein set forth in SEQ ID NO:8, and the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus C serotype 17 P2 polyprotein set forth in SEQ ID NO:9.

[0039] In some embodiments, the first and second nucleic acid sequences are part of the same mRNA. In some embodiments, the mRNA encodes a fusion protein comprising the first polyprotein and the second polyprotein.

[0040] In some embodiments, the first and second nucleic acid sequences are encoded by distinct non-naturally occurring mRNAs.

[0041] In some embodiments, the immunogenic composition further comprises a third non-native optimized nucleic acid sequence encoding a third polyprotein from a Group A or Group C rhinovirus, wherein the third polyprotein is different from the first and second polyproteins.

[0042] Accordingly, the present invention also provides immunogenic compositions comprising at least one messenger RNA (mRNA) (e.g., one, two, or three mRNAs) comprising: (i) a first non-naturally occurring, optimized nucleic acid sequence encoding a first polyprotein from a Group A or C rhinovirus; (ii) a second non-naturally occurring, optimized nucleic acid sequence encoding a second polyprotein from a Group A or C rhinovirus; and (iii) a third non-naturally occurring, optimized nucleic acid sequence encoding a third polyprotein from a Group A or C rhinovirus; The second and third polyproteins are different from one another, and each of the first, second, and third polyproteins (a) has an average identity (and optionally a median identity) of at least 80%, 85%, 90%, 95%, or 99% with the amino acid sequence of a corresponding polyprotein from at least two phylogenetic clusters of rhinoviruses of the same group; and (b) has an amino acid sequence that is naturally occurring except for one or more optional amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions). In one embodiment, the polyproteins (a) have an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequence of a corresponding polyprotein from at least two phylogenetic clusters of rhinoviruses of the same group; and (b) has an amino acid sequence that is naturally occurring except for an optional single amino acid substitution. The amino acid sequences of the first, second, and third polyproteins are obtained or obtainable by the methods for identifying rhinovirus polyproteins for use as immunogens described herein.

[0043] For example, in some embodiments, it may be desirable to combine polyproteins from group A rhinoviruses (e.g., VP0 polyprotein and P2 polyprotein) with a rhinovirus C polyprotein (e.g., VP0 polyprotein), or to combine a group A rhinovirus polyprotein (e.g., VP0 polyprotein) with a rhinovirus C polyprotein (e.g., two VP0 polyproteins) to elicit an immune response against as many group A and C rhinoviruses as possible while minimizing the number of mRNAs included in the immunogenic composition.

[0044] Thus, in some embodiments, the first polyprotein is a VP0 polyprotein comprising proteins VP2 and VP4. In some embodiments, the VP0 polyprotein is derived from a group A rhinovirus. In some embodiments, the group A rhinovirus is of serotype 21 or 90. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to, or is identical to, the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:4. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to, or is identical to, the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:5.

[0045] In some embodiments, the second polyprotein is a P2 polyprotein comprising proteins 2A, 2B, and 2C. In some embodiments, a single amino acid substitution is in the 2A protein that reduces or eliminates the proteolytic activity of the P2 polyprotein. In some embodiments, the single amino acid substitution is a C>A substitution or a C>S substitution in the catalytic triad of the active site of the 2A protein.

[0046] In some embodiments, the P2 polyprotein is derived from a group A rhinovirus. In some embodiments, the group A rhinovirus is of serotype 21 or 57. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO:6. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 57 P2 polyprotein set forth in SEQ ID NO:7.

[0047] In some embodiments, the third polyprotein is a VP0 polyprotein from a group C rhinovirus. In some embodiments, the group C rhinovirus is of serotype 11, 17, or 34. In some embodiments, the third polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:1. In some embodiments, the third polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:2. In some embodiments, the third polyprotein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to or identical to the amino acid sequence of the rhinovirus C serotype 34 VP0 polyprotein set forth in SEQ ID NO:3.

[0048] In some embodiments, at least one mRNA encodes a fusion protein comprising a first polyprotein (e.g., a rhinovirus A VP0 polyprotein), a second polyprotein (e.g., a rhinovirus A P2 polyprotein), and optionally a third polyprotein (e.g., a rhinovirus C VP0 polyprotein).

[0049] In some embodiments, the first, second, and third nucleic acid sequences are encoded by distinct non-naturally occurring mRNAs.

[0050] In some embodiments, the immunogenic composition is capable of eliciting a T cell response in at least 95% of a human population. In some embodiments, the immunogenic composition is capable of eliciting a T cell response in at least 96%, at least 97%, at least 98%, or at least 99% of a human population. In some embodiments, the VP0 polyprotein and P2 polyprotein in the immunogenic composition comprise T cell epitope-rich regions that cover at least 95% of the MHC class I alleles of Table 4 and / or 95% of the MHC-II alleles of Table 5. In some embodiments, the T cell epitope-rich regions cover at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class I alleles of Table 4 and / or at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class II alleles of Table 5.

[0051] In some embodiments, the first polyprotein is a VP0 polyprotein from a group A rhinovirus. In some embodiments, the group A rhinovirus is of serotype 21 or 90. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:4. In some embodiments, the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:5.

[0052] In some embodiments, the second polyprotein is a first VP0 polyprotein from a group C rhinovirus. In some embodiments, the third polyprotein is a second VP0 polyprotein from a group C rhinovirus that is different from the second polyprotein. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO: 1. In some embodiments, the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO: 2.

[0053] In some embodiments, at least one mRNA encodes a fusion protein comprising a first polyprotein (e.g., a rhinovirus A VP0 polyprotein), a second polyprotein (e.g., a first rhinovirus C VP0 polyprotein), and a third polyprotein (e.g., a second rhinovirus C VP0 polyprotein). In some embodiments, the first, second, and third nucleic acid sequences are encoded by separate non-naturally occurring mRNAs.

[0054] In some embodiments, the immunogenic composition is capable of eliciting a T cell response in at least 95% of a human population, hi some embodiments, the immunogenic composition is capable of eliciting a T cell response in at least 96%, at least 97%, at least 98%, or at least 99% of a human population.

[0055] The first, second, and (optional) third nucleic acid sequences are typically optimized to (a) improve the yield of full-length mRNA during in vitro synthesis, and / or (b) maximize expression of the encoded polypeptide following delivery of the mRNA to a target cell in vivo.

[0056] In some embodiments, the mRNA comprises a 5' untranslated region. In one embodiment, the 5' untranslated region comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the mRNA comprises a 3' untranslated region. In one embodiment, the 3' untranslated region comprises the nucleotide sequence of SEQ ID NO: 11, 12, or 13. In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the mRNA comprises a 5' cap and a 3' tail.

[0057] In some embodiments, the mRNA comprises a polyadenylation (polyA) sequence. In certain embodiments, the mRNA comprises a polyA sequence comprising at least 90 nucleotides. In certain embodiments, the mRNA comprises a polyA sequence comprising about 200 nucleotides. In some embodiments, the mRNA comprises N-1-methylpseudouridine instead of uridine.

[0058] In some embodiments, the immunogenic composition further comprises a plurality of lipid nanoparticles (LNPs) encapsulating the mRNA. In some embodiments, the lipid components of the LNPs comprise or consist of cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally sterol-based lipids. In some embodiments, the cationic lipids are selected from the group consisting of cKK-E12, cKK-E10, HGT5000, HGT5001, ICE, HGT4001, HGT4002, HGT4003, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, OF-Deg-Lin, OF-02, GL-TES-SA-DMP-E18-2, GL-TES-SA-DME-E18-2, SY-3-E14-DMAPr, TL1 -10D-DMA, HEP-E3-E10, HEP-E4-E10, RL3-DMA-07D, RL2-DMP-07D, cHse-E-3-E10, cHse-E-3-E12, cDD-TE-4-E12, SI-4-E14-DMAPr, TL-1-12D-DMA, SY-010, SY-011, 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate (ALC-0315) and heptadecane- In some embodiments, the non-cationic lipid is selected from DSPC (1,2-diastereoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), 9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102). In some embodiments, the non-cationic lipid is selected from DSPC (1,2-diastereoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE ... Selected from 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, DOPC (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)).In some embodiments, the PEG-modified lipid is selected from DMG-PEG-2K and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, the sterol lipid is cholesterol.

[0059] In certain embodiments, the cationic lipid is selected from cKK E10 and ALC-0315. In certain embodiments, the pegylated lipid is selected from DMG-PEG2K or ALC-0159. In certain embodiments, the non-cationic lipid is selected from DOPE or DSPC.

[0060] In some embodiments, the present invention also relates to a vaccine comprising an immunogenic composition disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0061] The present invention also relates to a method for inducing an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic or vaccine composition of the present invention.

[0062] The present invention also relates to a method for alleviating or preventing one or more symptoms associated with a rhinovirus infection in a subject, the method comprising administering to the subject an effective amount of an immunogenic or vaccine composition of the present invention.

[0063] The present invention also relates to a method for reducing the severity of or preventing a rhinovirus infection in a subject, which method comprises administering to the subject an effective amount of an immunogenic or vaccine composition of the present invention.

[0064] In some embodiments, administering the immunogenic or vaccine composition enhances or converts an existing rhinovirus T cell response to a T cell response. H 1 Reorient towards a response.

[0065] In some embodiments, the subject has asthma or chronic obstructive pulmonary disease (COPD). In some embodiments, administration of the immunogenic or vaccine composition reduces or prevents exacerbations associated with rhinovirus infection in a subject with asthma or COPD.

[0066] In some embodiments, administration of the immunogenic or vaccine composition induces intracellular antibodies against one or more nonstructural polypeptides encoded by one or more mRNAs.

[0067] In some embodiments, the subject is over the age of 40. In some embodiments, the subject is over the age of 65.

[0068] In some embodiments, the immunogenic or vaccine composition is administered intramuscularly.

[0069] In some embodiments, the immunogenic or vaccine composition is administered once to a subject.

[0070] In some embodiments, the immunogenic or vaccine composition is administered to a subject multiple times. In some embodiments, the immunogenic or vaccine composition is administered at least two times. In some embodiments, the second administration, or any subsequent administration, occurs about one year or more after the first administration.

[0071] In some embodiments, the immunogenic or vaccine composition is administered once a year, hi some embodiments, the immunogenic composition is administered twice, five years apart.

[0072] In some embodiments, administration of the immunogenic or vaccine composition provides immunity against rhinovirus infection caused by group A, group B, and / or group C strains. In some embodiments, immunity is provided against multiple serotypes of the same group. In some embodiments, immunity is provided against multiple serotypes of different groups.

[0073] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings, in which: [Brief explanation of the drawings]

[0074] [Figure 1] Figure 1 is a schematic diagram of the domain structure of rhinovirus RNA encoding a polyprotein containing the structural (capsid) protein P1 and the nonstructural proteins P2 and P3. The P1 polyprotein is further processed to VP0, VP3, and VP1, and VP0 is further cleaved to VP4 and VP2. The P2 polyprotein is further processed to P2A, P2B, and P2C. The P3 polyprotein is further processed to P3A, P3B, P3C, and P3D. The positive-sense RNA encoding the polyprotein is flanked by a 5'-UTR and a poly(A)(AAAAn) tail, as shown. [Figure 2] Figure 2 shows a schematic representation of sequence conservation across the rhinovirus polyprotein. Peaks identify the percentage of sequence conservation across the rhinovirus A polyprotein. Lightly lined boxes identify regions of greater than 50 consecutive amino acids with at least 80% sequence conservation, and conserved regions are identified by residue number within the consensus sequence of the rhinovirus A polyprotein. For each residue, the percentage of sequence conservation was calculated using a sliding 15-amino acid sequence window centered at the position indicated in the diagram. Bold-lined boxes refer to the P2 nonstructural protein and VP0 structural protein, which have been identified as containing higher sequence conservation and contain regions with at least 95% sequence conservation, indicated by dashed lines. Sequence conservation was assessed using a sliding 15-amino acid window. [Figure 3A]Figures 3A–F show phylogenetic clustering of rhinovirus A serotypes based on the amino acid sequences of the complete rhinovirus A polyprotein (Figures 3A and 3B), VP0 polyprotein (Figures 3C and 3D), or P2 polyprotein (Figures 3E and 3F), respectively. Sequences shorter than 800 amino acids or containing X stretches longer than 10 amino acids were excluded from the analysis. Regardless of which polyprotein was selected for analysis, multiple phylogenetic clusters were identified and are shown as gray-shaded outlined regions. Each cluster is assigned a number. Phylogenetic analysis based on the amino acid sequences of the complete rhinovirus A polyprotein and P2 polyprotein yielded nearly identical clusters 1–4, as shown. For the VP0 polyprotein, only three clusters were identified. Cluster 1 was highly similar to cluster 1 for the complete polyprotein and P2 polyprotein. The other two clusters primarily contained serotypes from clusters 2 and 3 or clusters 3 and 4 for the complete polyprotein and P2 polyprotein, respectively, as shown. The in silico consensus sequences identified for each polyprotein are shown in the central area where the phylogenetic clusters branch (marked with white arrows). In Figure 3A, the branches of the phylogenetic tree containing the native polyproteins identified as the best and second-best matches to the consensus polyproteins (GenBank IDs FJ445121.1 and JN562727.1, respectively) are indicated by black arrowheads. In Figure 3C, the branches of the phylogenetic tree containing the native VP0 polyproteins identified as the best and second-best matches to the consensus VP0 polyproteins (GenBank IDs FJ445121.1 and FJ445167.1, respectively) are indicated by black arrowheads. In Figure 3E, the tree branches containing the native P2 polyproteins identified as the best and second best matches to the consensus sequence P2 polyprotein (GenBank IDs FJ445121.1 and KY369874.1, respectively) are indicated by black arrowheads. Tree branches further to the consensus sequence are indicated by regions outlined with dashed lines.Figures 3B, 3D, and 3F show schematic diagrams of the phylogenetic trees in Figures 3A, 3C, and 3E, respectively. The number of serotypes is shown for the gray-shaded and dashed-outlined regions representing phylogenetic clusters. Figures 3B, 3D, and 3F also include boxes listing all serotypes within each cluster / region, as indicated. The best and second-best matches to the consensus sequence polyprotein are indicated by black arrowheads. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 4] Figure 4A is a schematic diagram of the structural and nonstructural polypeptides encoded by the rhinovirus A polyprotein, and Figure 4B shows the location of published T cell epitopes. [Figure 5] 1 is an illustration of the location of human MHC class I and MHC class II epitopes along the VP0 polyprotein of rhinovirus A serotype 21 (GenBank ID FJ445121.1), corresponding to SEQ ID NO: 4. The amino acid sequence of the VP4 polypeptide is shown in black, the amino acid sequence of the VP2 polypeptide is shown in gray, and the locations of the MHC class I and MHC class II epitopes are shown schematically below the amino acid sequences. [Figure 6] Figures 6A and 6B illustrate predicted human MHC class I and MHC class II T cell epitopes across the entire length of the complete rhinovirus A polyprotein. Figure 6A is a schematic diagram of the structural and nonstructural polypeptides encoded by the rhinovirus A polyprotein. Figure 6B shows predicted MHC class I epitopes with percentile ranks <1% and indicates the likely locations of such epitopes within the polyprotein. Figure 6C shows predicted MHC class II epitopes with percentile ranks <3% and indicates the likely locations of such epitopes within the polyprotein. [Figure 7]The maps in Figures 7A-D show predicted T cell epitopes on the P2 polyprotein of rhinovirus A serotype 21 (GenBank ID FJ445121.1). Figure 7A provides a heat map of sequence conservation based on a previously generated consensus sequence of the rhinovirus A polyprotein. Darker regions indicate higher sequence conservation compared to lighter regions. Figure 7B shows the locations of six published MHC class I epitopes. Figures 7C and 7D show percentile ranks for predicted MHC class I and class II epitopes, respectively, indicating the potential locations of such epitopes within the polyprotein. Regions of high sequence conservation around residues 80-120, 250-280, and 380-450 are indicated by thin black boxes. [Figure 8] Figures 8A-D show predicted T cell epitopes on the VP0 polyprotein of rhinovirus A serotype 21 (GenBank ID FJ445121.1). Figure 8A provides a heat map of sequence conservation based on a previously generated consensus sequence of the rhinovirus A polyprotein. Darker regions indicate higher sequence conservation compared to lighter regions. Figure 8B shows the locations of three published MHC class-II epitopes and six published MHC class-I epitopes. Figures 8C and 8D show percentile ranks for predicted MHC class-I and class-II epitopes, respectively, indicating the potential locations of such epitopes within the polyprotein. Regions of high sequence conservation around residues 1-100, 150-200, and 229-299 are indicated by thin black boxes. [Figure 9]Figure 9 illustrates the expression of FLAG-tagged rhinovirus polyproteins in HeLa cells after transfection with the mRNAs encoding them. Mock-transfected cells were included as a control. Proteins encoded by each mRNA were FLAG-tagged. Cell lysates were probed with an anti-FLAG tag antibody (MAB8529) and visualized by Western blot. The first lane is a molecular weight ladder. The molecular weight of each band is indicated on the left side of the figure. The proteins encoded by the mRNAs used for transfection are indicated at the bottom of the figure: VP0 polyprotein (lanes 2 and 3), P2 polyprotein (lanes 4 and 5), P2-VP0 fusion protein (lanes 6 and 7), VP0 polyprotein with a secretory signal sequence (lanes 8 and 9), P2 polyprotein with a secretory signal sequence (lanes 10 and 11), and P2-VP0 fusion protein with a secretory signal sequence (lanes 12 and 13). Detection of proteins encoded by the mRNAs at the expected molecular weights is indicated by white dashed boxes. Non-specific bands can be observed at approximately 125 kDa and 50 kDa. Lysates of cells transfected with mRNA encoding the P2 polyprotein or with a fusion protein containing the P2 polyprotein showed much lower expression levels. [Figure 10A]Figures 10A–F show the phylogenetic clustering of rhinovirus C serotypes based on the amino acid sequences of the complete rhinovirus C polyprotein (Figures 10A and 10B), VP0 polyprotein (Figures 10C and 10D), or P2 polyprotein (Figures 10E and 10F), respectively. Sequences shorter than 800 amino acids or containing X stretches longer than 10 were excluded from the analysis. Regardless of whether the complete polyprotein or the VP0 polyprotein was selected for analysis, four phylogenetic clusters, labeled 1a, 1b, 2a, and 2b, containing 13, 16, 5, and 9 serotypes, respectively, were identified. These are indicated by the gray-shaded outlined regions. The in silico consensus sequences identified for each polyprotein are indicated at the center points (marked by white arrowheads) where the phylogenetic clusters branch. In Figure 10A, the phylogenetic tree branch containing the native polyprotein identified as the best match for the polyproteins of clusters 1a and 1b (GenBank ID: MZ153245.1) and 2a and 2b (GenBank ID: MZ268692.1), respectively, is indicated by a black arrowhead. In Figure 10C, the phylogenetic tree branch containing the native VP0 polyprotein identified as the best match for the polyproteins of all four clusters (GenBank ID: MZ322913.1) is indicated by a black arrowhead. Additionally, the phylogenetic tree branch containing the native VP0 polyprotein identified as the best match for the polyproteins of clusters 1a and 1b (GenBank ID: MZ153277.1) and 2a and 2b (GenBank ID: MZ268689.1), respectively, is indicated by an arrowhead. In Figure 10E, the branches of the phylogenetic tree containing the native P2 polyproteins identified as best matches to the polyproteins of clusters 1a and 1b (GenBank ID: MZ153245.1) and 2a and 2b (GenBank ID: OK254863.1), respectively, are indicated by black arrowheads. Figures 10B, 10D, and 10F show schematic diagrams of the phylogenetic trees of Figures 10A, 10C, and 10E, respectively. The number of serotypes is shown for the gray-shaded outlined and dashed outlined regions representing phylogenetic clusters.Figures 10B, 10D, and 10F also contain boxes listing all serotypes within each cluster / region, as indicated. The best match to the consensus sequence polyprotein from each cluster is indicated by a black arrowhead. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above. [Figure 11A]Figures 11A and 11B show that immunogenic compositions of the invention containing mRNA encoding precursors of the native rhinovirus VP0 polyprotein, VP4, and VP2 capsid proteins are effective in eliciting effective T cell responses in vivo against corresponding polyproteins from other phylogenetic clusters of rhinoviruses. C57BL / 6 mice were immunized twice, 3 weeks apart, with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein of rhinovirus A serotype A21, (ii) recombinant VP0 polyprotein from rhinovirus A serotype A16 formulated with the TH1 adjuvant SPA09, or (iii) empty LNPs, as indicated by the dark gray, light gray, and white boxes, respectively, at the bottom of the graph next to the mouse icon. Two weeks after immunization, spleens were harvested, and splenocytes were stimulated in vitro with overlapping peptide libraries covering the full-length VP0 polyprotein of rhinovirus A serotype 21 or corresponding peptide libraries from the VP0 polyproteins of rhinovirus A serotypes 1b and 8, respectively, as indicated. Figure 11A shows the percentage of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD4+ T cells after peptide stimulation (specific frequencies among parental CD4+ T cell populations after media background subtraction). The percentage of polyfunctional CD4+ T cells after stimulation with either serotype 21 or 1b VP0 peptides was significantly higher than that after stimulation with serotype 8 VP0 peptide (p<0.001; indicated as **). Figure 11B shows the percentage of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD8+ T cells after peptide stimulation (specific frequencies among parental CD8+ T populations after media background subtraction). No statistically significant differences were observed among the three peptide treatment groups (indicated by "ns"). In Figures 11A and 11B, the percentage of polyfunctional CD4+ or CD8+ T cells for each animal per experimental group (n=6) is represented by filled circles (serotype 21 VP0 peptide), squares (serotype 1b VP0 peptide), and triangles (serotype 8 VP0 peptide), respectively. Brackets indicate the experimental groups compared for statistical analysis. [Figure 11B]Figures 11A and 11B show that immunogenic compositions of the invention containing mRNA encoding precursors of the native rhinovirus VP0 polyprotein, VP4, and VP2 capsid proteins are effective in eliciting effective T cell responses in vivo against corresponding polyproteins from other phylogenetic clusters of rhinoviruses. C57BL / 6 mice were immunized twice, 3 weeks apart, with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein of rhinovirus A serotype A21, (ii) recombinant VP0 polyprotein from rhinovirus A serotype A16 formulated with the TH1 adjuvant SPA09, or (iii) empty LNPs, as indicated by the dark gray, light gray, and white boxes, respectively, at the bottom of the graph next to the mouse icon. Two weeks after immunization, spleens were harvested, and splenocytes were stimulated in vitro with overlapping peptide libraries covering the full-length VP0 polyprotein of rhinovirus A serotype 21 or corresponding peptide libraries from the VP0 polyproteins of rhinovirus A serotypes 1b and 8, respectively, as indicated. Figure 11A shows the percentage of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD4+ T cells after peptide stimulation (specific frequencies among parental CD4+ T cell populations after media background subtraction). The percentage of polyfunctional CD4+ T cells after stimulation with either serotype 21 or 1b VP0 peptides was significantly higher than that after stimulation with serotype 8 VP0 peptide (p<0.001; indicated as **). Figure 11B shows the percentage of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD8+ T cells after peptide stimulation (specific frequencies among parental CD8+ T populations after media background subtraction). No statistically significant differences were observed among the three peptide treatment groups (indicated by "ns"). In Figures 11A and 11B, the percentage of polyfunctional CD4+ or CD8+ T cells for each animal per experimental group (n=6) is represented by filled circles (serotype 21 VP0 peptide), squares (serotype 1b VP0 peptide), and triangles (serotype 8 VP0 peptide), respectively. Brackets indicate the experimental groups compared for statistical analysis. [Figure 12A]Figures 12A, 12B, and 12C show that immunization with an immunogenic composition of the invention containing mRNA encoding a native rhinovirus polyprotein induces specific cross-reactive polyfunctional CD4+ (TH1) and CD8+ T cells. T cell responses after immunization were assessed by intracellular cytokine staining (ICS). Figures 12A and 12B show the induction of specific polyfunctional IFN-γ-, IL-2-, and TNF-α-positive CD3+CD4+ and CD3+CD8+ cells, respectively. Figure 12C shows a TH1-type response, as indicated by the absence of IL-5-positive CD3+CD4+ cells. Mice were immunized twice, 3 weeks apart, with lipid nanoparticles (LNPs) encapsulating mRNA encoding VP0 polyprotein (VP0), VP0 polyprotein with an HA secretory signal (HA-SS VP0), P2 polyprotein (P2), or P2 polyprotein with an HA secretory signal (HA-SS P2) (n = 6 mice per group). The polyprotein encoded by the mRNA was derived from rhinovirus A serotype 21. Immunization with empty LNP served as a negative control. Immunization with the adjuvanted recombinant VP0 polyprotein of rhinovirus A serotype 16 (adj.rec.protein VP0 RV-16) served as a positive control. The percentages of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD3+CD4+ and CD3+CD8+ cells, as well as the percentage of IL-5-positive CD3+CD4+ cells, were assessed after in vitro stimulation of splenocytes with overlapping peptide libraries representing the VP0 or P2 polyproteins of rhinovirus A serotype A21 (VP0 A21 or P2A21), rhinovirus A serotype 1b (VP0 A1b or P2A1b), and rhinovirus A serotype A8 (VP0 A8 or P2A8), respectively, as indicated in the figures. Data are plotted as individual values ​​of the specified percentage between parental populations after background subtraction of the medium (VP0 A21 - filled circles; VP0 A1b - filled squares; VP0 A8 - filled triangles), and bars represent the mean + 95% confidence interval (CI). Statistical significance is indicated by * (p < 0.1), ** (p < 0.01), and *** (p < 0.001). Brackets indicate the experimental groups compared for statistical analysis.The data in Figures 12A, 12B and 12C demonstrate that the immunogenic compositions of the invention induce effective TH1-directed immune responses against multiple group A rhinoviruses representing different serotypes and phylogenetic clusters. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 13A] Figures 13A and 13B show that immunization with an immunogenic composition of the invention containing mRNA encoding a P2 polyprotein with a single amino acid substitution in the 2A protein that abolished its proteolytic activity was more effective in inducing robust cross-reactive polyfunctional CD4+ (TH1) T cell responses. Figures 13A and 13B show the induction of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD3+CD4+ and CD3+CD8+ cells, respectively. Mice were immunized twice, 3 weeks apart, with lipid nanoparticles (LNPs) encapsulating 2 μg of mRNA encoding wild-type rhinovirus A serotype 21 P2 polyprotein (wild-type P2) or the corresponding P2 polyprotein with a single amino acid substitution in the 2A protein that abolished its proteolytic activity (mutant P2). Immunization with empty LNPs served as a negative control. As shown in the figures, the percentages of specific polyfunctional IFN-γ-, IL-2-, and TNF-α-positive CD3+CD4+ cells (Figure 13A) and CD3+CD8+ cells (Figure 13B) were assessed after in vitro stimulation of splenocytes with overlapping peptide libraries derived from the P2 polyproteins of rhinovirus A serotype 21 (P2 A21), rhinovirus A serotype 1b (P2 1b), and rhinovirus A serotype 8 (P2 8), respectively. Data are plotted as individual values ​​of the percentage of specific polyfunctional CD4+ or CD8+ T cells among the parent populations after background subtraction of the medium (P2 A21—filled circles; P2 1b—filled triangles; P2 8—filled diamonds), and bars represent the mean + 95% confidence interval (CI). No statistical differences were observed between wild-type and mutant P2 in CD4+ or CD8+ T cells. [Figure 13B]Figures 13A and 13B show that immunization with an immunogenic composition of the invention containing mRNA encoding a P2 polyprotein with a single amino acid substitution in the 2A protein that abolished its proteolytic activity was more effective in inducing robust cross-reactive polyfunctional CD4+ (TH1) T cell responses. Figures 13A and 13B show the induction of specific polyfunctional IFN-γ, IL-2, and TNF-α-positive CD3+CD4+ and CD3+CD8+ cells, respectively. Mice were immunized twice, 3 weeks apart, with lipid nanoparticles (LNPs) encapsulating 2 μg of mRNA encoding wild-type rhinovirus A serotype 21 P2 polyprotein (wild-type P2) or the corresponding P2 polyprotein with a single amino acid substitution in the 2A protein that abolished its proteolytic activity (mutant P2). Immunization with empty LNPs served as a negative control. As shown in the figures, the percentages of specific polyfunctional IFN-γ-, IL-2-, and TNF-α-positive CD3+CD4+ cells (Figure 13A) and CD3+CD8+ cells (Figure 13B) were assessed after in vitro stimulation of splenocytes with overlapping peptide libraries derived from the P2 polyproteins of rhinovirus A serotype 21 (P2 A21), rhinovirus A serotype 1b (P2 1b), and rhinovirus A serotype 8 (P2 8), respectively. Data are plotted as individual values ​​of the percentage of specific polyfunctional CD4+ or CD8+ T cells among the parent populations after background subtraction of the medium (P2 A21—filled circles; P2 1b—filled triangles; P2 8—filled diamonds), and bars represent the mean + 95% confidence interval (CI). No statistical differences were observed between wild-type and mutant P2 in CD4+ or CD8+ T cells. [Figure 14]Figure 1 shows the number of immunogen-specific IFN-γ-secreting cells after immunization with the immunogenic composition of the present invention, as assessed by ELISPOT assay. Mice were immunized twice, 3 weeks apart, with lipid nanoparticles (LNPs) encapsulating mRNAs encoding VP0 polyprotein (VP0), VP0 polyprotein with an HA secretory signal (HA-SS VP0), P2-VP0 fusion protein (fused P2-VP0), P2-VP0 fusion protein with an HA secretory signal (fused P2-VP0 HA-SS), P2 polyprotein (P2), and P2 polyprotein with an HA secretory signal (HA-SS P2) (n = 6 mice per group). The polyproteins encoded by the mRNAs were derived from rhinovirus A serotype 21. Immunization with empty LNPs served as a negative control. Immunization with adjuvanted recombinant VP0 polyprotein of rhinovirus A serotype 16 (adj.rec.protein VP0 RV-16) served as a positive control. The figure shows the number of spot-forming cells per 10 splenocytes on a log10 scale after in vitro stimulation of splenocytes with overlapping peptide libraries representing the VP0 or P2 polyprotein of rhinovirus A serotype 21 (VP0 A21 or P2 A21). Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Statistical significance is indicated by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. [Figure 15A]Figures 15A and 15B show the dose-dependent induction of anti-VP2 IgG antibodies and anti-VP4 IgG antibodies, respectively, after immunization with the immunogenic compositions of the present invention, as assessed by ELISA assay. Mice (n = 6 per group) were immunized twice, 3 weeks apart, with either 0.2 μg or 2 μg of mRNA, as indicated. The mRNAs were encapsulated in lipid nanoparticles (LNPs) encoding VP0 polyprotein (VP0) and VP0 polyprotein with an HA secretion signal (HA-SS VP0), respectively, as indicated. The VP0 polyprotein encoded by the mRNA was derived from rhinovirus A serotype 21. Immunization with empty LNPs served as a negative control. Immunization with adjuvanted recombinant VP0 polyprotein of rhinovirus A serotype 16 (adj.rec.protein VP0 RV-16) served as a positive control. The assay threshold (detection limit) is indicated by the dashed line. At the 2 μg dose, immunization with mRNA encoding HA-SS-VP0 induced antibody titers comparable to those induced by the adjuvanted protein-based vaccine used as a positive control. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Antibody titers were calculated as the reciprocal dilution that gave an optical density (OD) of 1. Statistical significance is indicated by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. [Figure 15B]Figures 15A and 15B show the dose-dependent induction of anti-VP2 IgG antibodies and anti-VP4 IgG antibodies, respectively, after immunization with the immunogenic compositions of the present invention, as assessed by ELISA assay. Mice (n = 6 per group) were immunized twice, 3 weeks apart, with either 0.2 μg or 2 μg of mRNA, as indicated. The mRNAs were encapsulated in lipid nanoparticles (LNPs) encoding VP0 polyprotein (VP0) and VP0 polyprotein with an HA secretion signal (HA-SS VP0), respectively, as indicated. The VP0 polyprotein encoded by the mRNA was derived from rhinovirus A serotype 21. Immunization with empty LNPs served as a negative control. Immunization with adjuvanted recombinant VP0 polyprotein of rhinovirus A serotype 16 (adj.rec.protein VP0 RV-16) served as a positive control. The assay threshold (detection limit) is indicated by the dashed line. At the 2 μg dose, immunization with mRNA encoding HA-SS-VP0 induced antibody titers comparable to those induced by the adjuvanted protein-based vaccine used as a positive control. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Antibody titers were calculated as the reciprocal dilution that gave an optical density (OD) of 1. Statistical significance is indicated by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. [Figure 16A]Figures 16A and 16B show that immunization with the immunogenic composition of the present invention subsequently induces IgG antibodies capable of binding to multiple group A rhinoviruses representing different serotypes and phylogenetic clusters. IgG antibody binding to whole virus was assessed by ELISA assay. Figures 16A and 16B show anti-virion IgG titers against group A rhinovirus serotypes 21 and 1b, respectively. Mice were immunized twice, 3 weeks apart, with either 0.2 μg or 2 μg of mRNA (n=6 per group). The mRNAs were encapsulated in lipid nanoparticles (LNPs) and encoded the VP0 polyprotein (VP0) and the VP0 polyprotein with an HA secretion signal (HA-SS VP0), respectively, as indicated in the figure. The VP0 polyprotein encoded by the mRNA was derived from rhinovirus A serotype 21. Immunization with empty LNPs served as a negative control. Immunization with adjuvanted recombinant VP0 polyprotein of rhinovirus A serotype 16 (adj.rec.protein VP0 RV-16) served as a positive control. The assay threshold (detection limit) is indicated by the dashed line. At a 2 μg dose, immunization with mRNA encoding HA-SS-VP0 induced antibody titers against both serotype 21 and 1b virions that were comparable to those induced by adjuvanted protein-based vaccines. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Antibody titers were calculated as the reciprocal dilution that gave an optical density (OD) of 1. Statistical significance is indicated by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. [Figure 16B]Figures 16A and 16B show that immunization with the immunogenic composition of the present invention subsequently induces IgG antibodies capable of binding to multiple group A rhinoviruses representing different serotypes and phylogenetic clusters. IgG antibody binding to whole virus was assessed by ELISA assay. Figures 16A and 16B show anti-virion IgG titers against group A rhinovirus serotypes 21 and 1b, respectively. Mice were immunized twice, 3 weeks apart, with either 0.2 μg or 2 μg of mRNA (n=6 per group). The mRNAs were encapsulated in lipid nanoparticles (LNPs) and encoded the VP0 polyprotein (VP0) and the VP0 polyprotein with an HA secretion signal (HA-SS VP0), respectively, as indicated in the figure. The VP0 polyprotein encoded by the mRNA was derived from rhinovirus A serotype 21. Immunization with empty LNPs served as a negative control. Immunization with adjuvanted recombinant VP0 polyprotein of rhinovirus A serotype 16 (adj.rec.protein VP0 RV-16) served as a positive control. The assay threshold (detection limit) is indicated by the dashed line. At a 2 μg dose, immunization with mRNA encoding HA-SS-VP0 induced antibody titers against both serotype 21 and 1b virions that were comparable to those induced by adjuvanted protein-based vaccines. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Antibody titers were calculated as the reciprocal dilution that gave an optical density (OD) of 1. Statistical significance is indicated by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. [Figure 17]Figures 17A-F show CD4 T1 responses in human peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with overlapping peptide libraries representing the VP0 polyproteins of various rhinovirus A serotypes. PBMCs isolated from healthy human volunteers were stimulated with overlapping peptide libraries representing the VP0 polyproteins of rhinovirus A serotypes A21 (VP0 A21), 1b (VP0 A1b), and 8 (VP0 A8), respectively, as indicated. Cell culture medium was used as a negative control. Figures 17A-17F show the percentages of human CD4+ T cells secreting IFN-γ, IL-2, TNF-α, MIP-1β, IL-4, and IL-17A, respectively, after peptide stimulation, as determined by intracellular cytokine staining (ICS). Data are plotted as individual values ​​(open circles for negative control group; filled circles for experimental group), and lines indicate corresponding samples. Statistical significance is represented by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups that were compared for statistical analysis. Cumulatively, the data show that natural rhinovirus infection induces a significant CD4+ TH1 response to VP0 from group A in healthy humans. [Figure 17-1] Same as above. [Figure 17-2] Same as above. [Figure 18]Figures 18A-D show low or rare CD8+ T cell responses in human PBMCs after in vitro stimulation with overlapping peptide libraries representing the VP0 polyproteins of various rhinovirus A serotypes. PBMCs isolated from healthy human volunteers were stimulated with overlapping peptide libraries representing the VP0 polyproteins of rhinovirus A serotype 21 (VP0 A21), rhinovirus A serotype 1b (VP0 A1b), and rhinovirus A serotype 8 (VP0 A8), respectively, as indicated. Cell culture medium was used as a negative control. Figures 18A-18D show the percentage of human CD8+ T cells secreting IFN-γ, IL-2, TNF-α, and MIP-1β, respectively, after peptide stimulation, as determined by intracellular cytokine staining (ICS). Data are plotted as individual values ​​(open circles for negative control groups and filled circles for experimental groups), and lines indicate corresponding samples. Statistical significance is indicated by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. Data in Figures 18A-D show low or rare VP0-specific CD8+ T cell responses to Group A in healthy humans. [Figure 18-1] Same as above. [Figure 19]Figures 19A–F show CD4+ TH1 responses in human PBMCs after in vitro stimulation with overlapping peptide libraries representing various VP0 polyproteins of rhinovirus C serotypes. PBMCs isolated from healthy human volunteers were stimulated with overlapping peptide libraries representing the VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as indicated. Cell culture medium was used as a negative control. Figures 19A–19F show the percentages of CD4+ T cells secreting IFN-γ, IL-2, TNF-α, MIP-1β, IL-4, and IL-17A, respectively, after peptide stimulation, as determined by intracellular cytokine staining (ICS). Data are plotted as individual values ​​(open circles for negative control group; filled circles for experimental group), and lines indicate matching samples. Statistical significance is represented by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. Cumulatively, the data in Figures 19A-F show that natural rhinovirus infection induces significant CD4+ TH1 responses to VP0 from group C in healthy humans. [Figure 19-1] Same as above. [Figure 19-2] Same as above. [Figure 20]Figures 20A–D show the lack of CD8+ T cell responses in human PBMCs after in vitro stimulation with overlapping peptide libraries representing the VP0 polyproteins of various rhinovirus C serotypes. PBMCs isolated from healthy human volunteers were stimulated with overlapping peptide libraries representing the VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as indicated. Cell culture medium was used as a negative control. Figures 20A–D show the percentage of CD8+ T cells secreting IFN-γ, IL-2, TNF-α, and MIP-1β, respectively, after peptide stimulation, as determined by intracellular cytokine staining (ICS). Data are plotted as individual values ​​(open circles for negative control group; filled circles for experimental group), and lines indicate corresponding samples. Statistical significance is represented by * (p<0.1), ** (p<0.01), and *** (p<0.001). Brackets indicate the experimental groups compared for statistical analysis. The data in Figures 20A-D show no statistically significant VP0-specific CD8+ T cell responses to Group C in healthy humans. [Figure 20-1] Same as above. [Figure 21A]Figures 21A and 21B show that immunization with an immunogenic composition of the invention containing mRNA encoding the native rhinovirus C VP0 polyprotein is effective in eliciting antigen-specific CD4+ and CD8+ T cells. C57BL / 6 mice were immunized twice, 3 weeks apart, with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein or (ii) empty LNPs (negative control), as indicated by the dark and light gray boxes, respectively, at the bottom of the graph next to the mouse icon. Spleens were harvested from immunized mice 2 weeks after the final injection. The induction of antigen-specific CD4+ and CD8+ T cells was assessed by intracellular cytokine staining (ICS) after in vitro stimulation of splenocytes with overlapping peptide libraries covering the full-length VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as indicated. CD4+ and CD8+ cells were identified as antigen-specific T cells if they stained positive for any combination of IFN-γ, IL-2, and / or TNF-α, including single-, double-, or triple-positive staining. Data are presented as antigen-specific CD4+ or CD8+ T cells between the parent populations after media background subtraction. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Negative control data are shown only for T cells from mock-treated mice stimulated with a pool of peptides derived from the VP0 polyprotein of rhinovirus C serotype 34. Statistical significance relative to the corresponding negative control (i.e., T cells from mock-treated mice stimulated with the relevant overlapping peptide library) is indicated by * (p<0.1), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 21B]Figures 21A and 21B show that immunization with an immunogenic composition of the invention containing mRNA encoding the native rhinovirus C VP0 polyprotein is effective in eliciting antigen-specific CD4+ and CD8+ T cells. C57BL / 6 mice were immunized twice, 3 weeks apart, with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein or (ii) empty LNPs (negative control), as indicated by the dark and light gray boxes, respectively, at the bottom of the graph next to the mouse icon. Spleens were harvested from immunized mice 2 weeks after the final injection. The induction of antigen-specific CD4+ and CD8+ T cells was assessed by intracellular cytokine staining (ICS) after in vitro stimulation of splenocytes with overlapping peptide libraries covering the full-length VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as indicated. CD4+ and CD8+ cells were identified as antigen-specific T cells if they stained positive for any combination of IFN-γ, IL-2, and / or TNF-α, including single-, double-, or triple-positive staining. Data are presented as antigen-specific CD4+ or CD8+ T cells between the parent populations after media background subtraction. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Negative control data are shown only for T cells from mock-treated mice stimulated with a pool of peptides derived from the VP0 polyprotein of rhinovirus C serotype 34. Statistical significance relative to the corresponding negative control (i.e., T cells from mock-treated mice stimulated with the relevant overlapping peptide library) is indicated by * (p<0.1), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 22A]Figures 22A and 22B show that immunization with an immunogenic composition of the invention comprising mRNA encoding the native rhinovirus C VP0 polyprotein induces cross-reactive polyfunctional CD4+ and CD8+ T cells. Spleens were collected from C57BL / 6 mice immunized with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein or (ii) empty LNPs (negative control), as indicated by the dark and light gray boxes, respectively, at the bottom of the graph next to the mouse icon. Intracellular cytokine staining (ICS) was used to determine the percentages of IFN-γ, IL-2, and TNF-α-positive CD3+CD4+ and CD3+CD8+ cells after in vitro stimulation with overlapping peptide libraries covering the full-length VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as indicated. Data are shown as antigen-specific CD4+ or CD8+ T cells within the parent population after media background subtraction. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Negative control data are shown only for T cells from mock-treated mice stimulated with a pool of peptides derived from the VP0 polyprotein of rhinovirus C serotype 34. Statistical significance relative to the corresponding negative control (i.e., T cells from mock-treated mice stimulated with the relevant overlapping peptide library) is indicated by * (p<0.1), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 22B]Figures 22A and 22B show that immunization with an immunogenic composition of the invention comprising mRNA encoding the native rhinovirus C VP0 polyprotein induces cross-reactive polyfunctional CD4+ and CD8+ T cells. Spleens were collected from C57BL / 6 mice immunized with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein or (ii) empty LNPs (negative control), as indicated by the dark and light gray boxes, respectively, at the bottom of the graph next to the mouse icon. Intracellular cytokine staining (ICS) was used to determine the percentages of IFN-γ, IL-2, and TNF-α-positive CD3+CD4+ and CD3+CD8+ cells after in vitro stimulation with overlapping peptide libraries covering the full-length VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as indicated. Data are shown as antigen-specific CD4+ or CD8+ T cells within the parent population after media background subtraction. Data are plotted as individual values ​​(filled circles), and bars represent the mean + 95% confidence interval (CI). Negative control data are shown only for T cells from mock-treated mice stimulated with a pool of peptides derived from the VP0 polyprotein of rhinovirus C serotype 34. Statistical significance relative to the corresponding negative control (i.e., T cells from mock-treated mice stimulated with the relevant overlapping peptide library) is indicated by * (p<0.1), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 23]Figure 23 shows that immunization with an immunogenic composition of the invention comprising mRNA encoding the native rhinovirus C VP0 polyprotein induces a TH1-directed CD4+ T cell response. C57BL / 6 mice were immunized twice, 3 weeks apart, with (i) lipid nanoparticles (LNPs) encapsulating mRNA encoding the VP0 polyprotein or (ii) empty LNPs (negative control), as indicated by the dark and light gray boxes, respectively, at the bottom of the graph next to the mouse icon. Using intracellular cytokine staining (ICS), only a small percentage of CD4+ cells producing the TH2 cytokine IL-5 was detected after in vitro stimulation with overlapping peptide libraries covering the full-length VP0 polyproteins of rhinovirus C serotypes 34 (VP0 C34), 11 (VP0 C11), 07 (VP0 C07), 01 (VP0 C01), 17 (VP0 C17), 41 (VP0 C41), and 53 (VP0 C53), respectively, as shown in the figure. Data are shown as specific IL-5-secreting CD4+ T cells within the parent population after media background subtraction. Data are plotted as individual values ​​(black circles), and bars represent the mean + 95% confidence interval (CI). Negative control data are shown for T cells from mock-treated mice stimulated with a pool of peptides derived from the VP0 polyprotein of rhinovirus C serotype 34. Statistical significance relative to the corresponding negative control (i.e., T cells from sham-treated mice stimulated with peptides from the corresponding peptide pool) is indicated by * (p<0.1), ** (p<0.01), *** (p<0.001) and **** (p<0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0075] definition To facilitate understanding of the present invention, certain terms are first defined below. Further definitions for these and other terms are set forth throughout the specification.

[0076] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0077] Unless specifically stated or clear from context, as used herein, the term "or" is understood to be inclusive, including both "or" and "and."

[0078] As used herein, the term "mRNA" refers to a polyribonucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA can contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, optionally purified, transcribed in vitro, or chemically synthesized. Where appropriate, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. A typical mRNA includes a 5' cap, a 5' untranslated region (5' UTR), a protein coding region, a 3' untranslated region (3' UTR), and a 3' tail. In some embodiments, the tail structure is a poly(C) tail. More typically, the tail structure is a poly(A) tail.

[0079] When used herein to describe a rhinovirus polypeptide, protein, or polyprotein, the term "native" refers to the amino acid sequence of the polypeptide, protein, or polyprotein present in a rhinovirus isolate. In some embodiments, the rhinovirus polypeptides, proteins, or polyproteins disclosed herein contain a single amino acid substitution relative to the native amino acid sequence of the rhinovirus isolate to make the protein more suitable for use in the immunogenic compositions of the invention. For example, the inventors have found that expression of the P2 polyprotein of the invention can result in eIF4g cleavage, which can lead to global translational repression. Thus, the P2 polyprotein encoded by the mRNA of the invention is typically modified to produce a 2A protein with reduced or no proteolytic activity, e.g., by substituting serine or alanine for the cysteine ​​of the 2A protein that acts as a nucleophile in the catalytic triad. While not wishing to be bound by any particular theory, the inventors believe that the immunogenic or antigenic function of such modified polypeptides, proteins, or polyproteins is essentially identical to that of the native version.

[0080] As used herein, the term "sequence optimized" is used to indicate that a nucleotide sequence has been modified compared to a native or wild-type nucleic acid. Such modifications can include, for example, codon optimization and the use of 5' and 3' UTRs not normally associated with that native or wild-type nucleic acid. As used herein, the terms "codon optimization" and "codon-optimized" refer to the modification of the codon composition of a native or wild-type nucleic acid encoding a peptide, polypeptide, or protein, which does not change its amino acid sequence, thereby improving protein expression of the nucleic acid. In the context of the present invention, "codon optimization" can also refer to the process of using filters to remove suboptimal nucleotide sequences from a list of nucleotide sequences, such as filtering by guanine-cytosine (GC) content, codon adaptation index (CAI), the presence of destabilizing nucleic acid sequences or motifs, and / or the presence of pause sites and / or termination signals, thereby arriving at one or more optimized nucleotide sequences.

[0081] As used herein, the term "template DNA" (or "DNA template") refers to a DNA molecule containing a nucleic acid sequence encoding an RNA transcript to be synthesized by in vitro transcription. The template DNA is used as a template for in vitro transcription to produce an RNA transcript encoded by the template DNA. Template DNA contains all elements necessary for in vitro transcription, in particular, a promoter element for binding a DNA-dependent RNA polymerase, such as T3, T7, or SP6 RNA polymerase, operably linked to a DNA sequence encoding the desired mRNA transcript. Furthermore, template DNA may contain primer binding sites 5' and / or 3' of the DNA sequence encoding the mRNA transcript for determining the identity of the DNA sequence encoding the mRNA transcript, for example, by PCR or DNA sequencing. In the context of the present invention, "template DNA" may be a linear or circular DNA molecule. As used herein, the term "template DNA" may refer to a DNA vector, such as a plasmid DNA, containing a nucleic acid sequence encoding the desired RNA transcript.

[0082] As used herein, the term "localization sequence" refers to an amino acid sequence that promotes transcytosis of a linked polypeptide across an epithelium. A localization sequence can be linked to the carboxy terminus (C-terminus) of a polypeptide. A localization sequence can be linked to a polypeptide by a linker sequence. A localization sequence can also be exogenous to a polypeptide. For example, a localization sequence can promote transport of a linked polypeptide across a layer of airway epithelial cells so that a polypeptide comprising one of these sequences can be more effectively delivered to the airway or lung lumen.

[0083] As used herein, the term "adjuvant" refers to a substance or combination of substances that can be used to enhance the immune response to an antigen.

[0084] As used herein, the term "immunogen" or "immunogenic" refers to a compound, composition, or substance that, under suitable conditions, is capable of stimulating an immune response in a subject, such as the production of antibodies, a T cell response, or both, including compositions that are injected or absorbed into an animal. As used herein, the term "immunogenic composition" refers to a composition that generates an immune response that may or may not be a protective immune response. As used herein, "immunize" means to induce a protective immune response in a subject against an infectious disease (e.g., rhinovirus infection).

[0085] As used herein, the term "vaccine composition" or "vaccine" refers to a composition that generates a protective immune response in a subject. As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (prevents infection or prevents the development of a disease associated with the infection) or alleviates the symptoms of infection (e.g., infection with a rhinovirus). Vaccines can induce both prophylactic (preventive) and therapeutic responses.

[0086] As used herein, the term "subject" refers to a mammal, such as a human or other animal. Typically, the subject is a human. The subject may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and elderly subjects.

[0087] 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 application belongs and as commonly used in the art to which this application belongs. Publications and other reference materials mentioned herein to describe the background of the invention or to provide additional details regarding its practice are hereby incorporated by reference.

[0088] Summary Various embodiments of the methods, processes, steps, functions, and / or operations described herein may be performed by or using a computer, processor, etc. For example, a computer, etc. may be programmed to perform some or all of the methods, processes, steps, functions, and / or operations described herein. The computer may be controlled by a software program made up of program instructions such as source code, object code, executable code, etc. The software may be embodied on a computer-readable medium, which may include RAM (random access memory), ROM (read-only memory), EPROM (erasable programmable ROM), EEPROM (electronically erasable programmable ROM), magnetic disks, optical disks, solid-state disks, etc.

[0089] The rhinovirus mRNA genome encodes a polyprotein containing both structural and nonstructural rhinovirus polypeptides (Figure 1). We aligned a 539-amino acid sequence from various rhinovirus A serotypes and a 463-amino acid sequence from various rhinovirus C serotypes to identify regions of high sequence conservation. These regions were identified in the structural polypeptides VP4 and VP2, which are derived from the natural precursor VP0 polyprotein. We also identified regions of high sequence conservation in the nonstructural polypeptides P2A, P2B, and P2C, which are derived from the natural precursor P2 polyprotein.

[0090] The inventors believe that immunogenic compositions comprising non-natural mRNA encoding natural rhinovirus polyproteins will be more effective than other types of vaccines in eliciting immune responses against multiple rhinovirus serotypes of the same group.

[0091] Thus, the present invention provides anti-rhinovirus vaccines (particularly mRNA-based vaccines) that contain one or more non-naturally occurring mRNAs encoding one or more naturally occurring rhinovirus polyproteins that are highly conserved among multiple rhinoviruses of the same subgroup. The vaccines disclosed herein are designed to be capable of eliciting an effective immune response, such as an effective T cell response, against multiple rhinovirus serotypes, either from the same or multiple rhinovirus groups.

[0092] The inventors have discovered that structural polypeptides located within the precursor VP0 polyprotein of rhinovirus polyproteins (i.e., VP4 and VP2) and nonstructural polypeptides located within the precursor P2 polyprotein (i.e., 2A, 2B, and 2C) are particularly enriched in conserved T cell epitope-rich regions. Inclusion of these polyproteins or the mRNAs encoding them in vaccination approaches is expected to generate particularly potent T cell responses against multiple serotypes of group A and group C rhinoviruses. For example, the combination of VP0 and P2 polyproteins as a fusion protein (or the mRNA encoding it) is expected to elicit T cell responses in at least 95% of the human population.

[0093] Furthermore, the inventors' findings open up the opportunity to combine mRNAs encoding the VP0 polyprotein and / or P2 polyprotein from group A and group C rhinoviruses to provide combination vaccines that can induce effective immune responses against multiple serotypes from each group.

[0094] Identification of native rhinovirus polyproteins suitable as immunogens Without wishing to be bound by any particular theory, the inventors believe that immunogenic compositions containing non-natural mRNA encoding native rhinovirus proteins or polyproteins are more effective at eliciting immune responses against multiple rhinovirus serotypes of the same group than other types of vaccines, particularly vaccines that generate only a limited selection of rhinovirus-derived T cell epitopes containing peptides or polypeptides. This belief is based, in part, on the discovery that predicted T cell epitopes can elicit T cell responses only in carriers of specific HLA alleles. Therefore, to achieve broad coverage in a large proportion of the population, immunogenic compositions ideally induce the expression of multiple T cell epitopes to ensure that T cell responses are elicited in the majority of recipients.

[0095] Furthermore, native rhinovirus proteins or polyproteins are expected to be processed by immune cells in a manner that reflects the natural infection process. Because the antigen design approach disclosed herein is based on in silico prediction of T cell epitopes, the use of native proteins reduces the risks inherent in such approaches. In particular, some of the predicted T cell epitopes may not be generated by immune cells in vivo, whereas the use of native proteins (or mRNA encoding them) ensures the generation of multiple T cell epitopes following administration of the immunogenic composition.

[0096] Thus, the present invention provides one or more non-naturally occurring mRNAs encoding one or more non-structural rhinovirus polypeptides comprising one or more T-cell epitope-rich regions and / or one or more structural rhinovirus polypeptides comprising one or more T-cell epitope-rich regions, relative to the naturally occurring rhinovirus polyproteins in which they were identified.

[0097] In certain embodiments, immunogenic compositions (e.g., vaccines) of the invention comprise non-native mRNA encoding a non-structural rhinovirus protein or polyprotein, which is natural except for, for example, one or more optional amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions), which may be introduced, for example, to improve expression (e.g., through the provision of a non-native signal sequence and / or the removal of proteolytic activity). In some embodiments, the non-structural rhinovirus protein is or includes one of P2A, P2B, and P2C. In one particular embodiment, the non-structural rhinovirus protein is the P2 polyprotein. In some embodiments, immunogenic compositions (e.g., vaccines) of the invention comprise mRNA encoding multiple non-structural rhinovirus proteins or polyproteins (e.g., multiple P2 polyproteins from rhinovirus A and / or C).

[0098] In other specific embodiments, non-native mRNA encoding one or more structural rhinovirus polypeptides comprising one or more T-cell epitope-rich regions described herein is provided as a rhinovirus protein or polyprotein, typically a native rhinovirus protein or polyprotein. In certain embodiments, immunogenic compositions (e.g., vaccines) of the invention comprise non-native mRNA encoding a structural rhinovirus protein or polyprotein, which is native except for one or more optional amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions), which may be introduced, for example, to improve expression (e.g., through the provision of a non-native signal sequence and / or removal of proteolytic activity). In some embodiments, the structural rhinovirus protein is or comprises one of VP2 and VP4. In one specific embodiment, the structural rhinovirus protein is the VP0 polyprotein. In some embodiments, immunogenic compositions (e.g., vaccines) of the invention comprise mRNA encoding multiple structural rhinovirus proteins or polyproteins (e.g., multiple VP0 polyproteins from rhinovirus A and / or C).

[0099] In some embodiments, it is advantageous to provide mRNA encoding a non-natural fusion protein of two or more native rhinovirus proteins or polyproteins, e.g., a fusion protein comprising one or more native structural rhinovirus proteins (or polyproteins) and one or more non-structural rhinovirus proteins (or polyproteins). For example, the native structural proteins can be selected from VP2 and VP4. The non-native non-structural proteins can be selected from P2A, P2B, and P2C. The one or more structural proteins and one or more non-structural proteins can be arranged in any order in the fusion protein. More typically, the one or more structural proteins and one or more non-structural proteins are arranged similarly to a native polyprotein. For example, P2A, P2B, and P2C can be arranged as in the native P2 polyprotein. Similarly, VP4 and VP2 can be arranged as in the native VP0 polyprotein. The VP0 polyprotein and P2 polyprotein can be arranged in either of two possible combinations. In one embodiment, the fusion protein is VP0-P2 (i.e., VP0 is in the N-terminal position). In another embodiment, the fusion protein is P2-VP0 (ie, P2 is in the N-terminal position).

[0100] In some embodiments, the fusion protein comprises multiple native structural or non-structural rhinovirus proteins (or polyproteins), such as multiple VP0 polyproteins or multiple P2 polyproteins from different serotypes of rhinovirus A and / or C.

[0101] Accurately predicting T cell epitope-rich regions across multiple rhinovirus serotypes can be difficult. Therefore, the inventors' bioinformatics approach focuses, at least in part, on identifying regions in rhinovirus polyproteins that are highly conserved across multiple rhinovirus polyproteins from different serotypes. In particular, the present invention provides methods for identifying rhinovirus polyproteins for use as immunogens capable of eliciting an immune response against rhinoviruses from multiple serotypes within a group. The method according to the present invention comprises the following steps: (a) obtaining a plurality of amino acid sequences from a database comprising amino acid sequences from natural rhinovirus isolates; (b) removing amino acid sequences shorter than 800 amino acids from the plurality of amino acid sequences obtained in step (a); (c) assigning the amino acid sequences remaining after step (b) to different phylogenetic clusters; (d) aligning the amino acid sequences to determine a consensus amino acid sequence for the complete rhinovirus polyprotein for one or more phylogenetic clusters identified in step (c); (e) aligning the consensus amino acid sequence obtained in step (c) with the complete polyprotein of the natural rhinovirus isolate; and (f) selecting a rhinovirus polyprotein as an immunogen having an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the corresponding amino acid sequences of rhinoviruses from at least two phylogenetic clusters identified in step (c). For example, a suitable algorithm for performing the alignment in step (d) is MAFFT (Katoh & Toh, Bioinformatics 2010;26(15):1899-900, hereby incorporated by reference).

[0102] The inventors used this computational approach to identify conserved regions in the amino acid sequences of complete rhinovirus A and C polyproteins. The inventors believe that native rhinovirus polyproteins that have an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to amino acid sequences of rhinoviruses from at least two phylogenetic clusters are capable of eliciting an immune response against substantially all serotypes in the at least two phylogenetic clusters, e.g., by inducing an effective T cell response. Highly conserved regions that are particularly suitable for practicing the present invention have an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to amino acid sequences of rhinoviruses from multiple phylogenetic clusters.

[0103] In particular, the inventors have identified naturally occurring VP0 polyproteins and P2 polyproteins having amino acid sequences that have an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequences of VP0 polyproteins or P2 polyproteins from at least two phylogenetic clusters of rhinovirus group A or C. More generally, these naturally occurring VP0 polyproteins and P2 polyproteins have amino acid sequences that have an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequences of VP0 polyproteins or P2 polyproteins from at least three, e.g., four, phylogenetic clusters of rhinovirus group A or C.

[0104] For example, an exemplary naturally occurring rhinovirus A VP0 polyprotein has an amino acid sequence having at least about 80% average identity (and optionally median identity) with the amino acid sequences of VP0 polyproteins from at least three phylogenetic clusters. An exemplary naturally occurring rhinovirus A P2 polyprotein has an amino acid sequence having at least about 80% average identity (and optionally median identity) with the amino acid sequences of P2 polyproteins from at least four phylogenetic clusters. An exemplary naturally occurring rhinovirus C VP0 polyprotein has an amino acid sequence having at least about 80% average identity (and optionally median identity) with the amino acid sequences of VP0 polyproteins from at least four phylogenetic clusters. An exemplary naturally occurring rhinovirus P2 polyprotein has an amino acid sequence having at least about 80% average identity (and optionally median identity) with the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters.

[0105] Phylogenetic clusters can be identified using a suitable algorithm, for example based on maximum likelihood. Maximum likelihood phylogenetic trees can be calculated for large alignments using algorithms such as FastTree2 (Price et al., PLoS ONE 2010;5(3):e9490, incorporated herein by reference). Another suitable algorithm is PhyML (Guindon et al. Nucleic Acids Res. 2005;33(Web Server issue):W557-9, incorporated herein by reference). Both FastTree and PhyML provide nearly identical phylogenetic trees and can therefore be used interchangeably to perform the computational methods described herein.

[0106] Phylogenetic clusters typically represent at least 5 different serogroups of rhinovirus group A or C. In some embodiments, phylogenetic clusters represent at least 10 different serogroups of rhinovirus group A or C. In some embodiments, phylogenetic clusters represent 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different serogroups of rhinovirus group A or C.

[0107] For example, rhinovirus group A can be divided into four phylogenetic clusters (referred to herein as clusters 1-4) based on the amino acid sequence of the complete polyprotein. These four phylogenetic clusters correspond to 20, 5, 28, and 3 serotypes, respectively. Cluster 1 includes serotypes 9, 13, 15, 19, 22, 32, 38, 41, 43, 57, 60, 61, 64, 67, 73, 74, 75, 82, 94, and 96. Cluster 2 includes serotypes 2, 23, 30, 39, and 49. Cluster 3 includes serotypes 10, 11, 18, 21, 24, 25, 29, 31, 33, 34, 40, 44, 47, 50, 54, 55, 56, 57, 59, 62, 63, 66, 76, 77, 85, 90, 98, and 100. Cluster 4 includes serotypes 1, 16 and 81. Corresponding clusters can also be identified when the phylogenetic analysis is restricted to the amino acid sequence of the nonstructural P2 polyprotein.

[0108] In some embodiments, the VP0 polyprotein, the P2 polyprotein, or both, of human rhinovirus A serotype 21 strain (GenBank ID: FJ445121.1, strain name: ATCC VR-1131) are capable of eliciting an immune response (e.g., a protective immune response) against at least rhinovirus A serotypes in cluster 3. In other embodiments, the VP0 polyprotein, the P2 polyprotein, or both, of human rhinovirus A serotype 21 strain (GenBank ID: FJ445121.1, strain name: ATCC VR-1131) are capable of eliciting an immune response (e.g., a protective immune response) against at least rhinovirus A serotypes in clusters 2 and 3. In a further embodiment, the VP0 polyprotein, the P2 polyprotein, or both of human rhinovirus A serotype 21 strain (GenBank ID: FJ445121.1, strain name: ATCC VR-1131) are capable of eliciting an immune response (e.g., a protective immune response) against rhinovirus A serotypes of clusters 1 to 4.

[0109] Based on the amino acid sequence of the complete polyprotein, rhinovirus group C can be divided into two phylogenetic clusters (referred to herein as 1ab and 2ab), corresponding to 29 and 14 serotypes, respectively. Each of these clusters can be further subdivided into four phylogenetic clusters (referred to herein as 1a, 1b, 2a, and 2b), corresponding to 13, 16, 5, and 9 serotypes, respectively. Cluster 1a includes rhinovirus C serotypes 8, 12, 15, 16, 17, 23, 25, 28, 30, 31, 41, 42, and 56. Cluster 1b includes rhinovirus C serotypes 2, 4, 9, 19, 26, 33, 35, 36, 40, 47, 48, 49, 50, 51, 53, and 55. Cluster 2a includes rhinovirus C serotypes 5, 11, 34, 45, and 54. Cluster 2b includes rhinovirus C serotypes 1, 3, 6, 7, 22, 32, 39, 43, and 57. These four clusters are also identified when the phylogenetic analysis is restricted to the amino acid sequence of the structural VP0 polyprotein. Corresponding clusters can also be identified when the phylogenetic analysis is restricted to the amino acid sequence of the nonstructural P2 polyprotein.

[0110] In some embodiments, the VP0 polyprotein of human rhinovirus C serotype 34 strain (GenBank ID: MZ322913.1, strain name: 7H8M5V) is capable of eliciting an immune response (e.g., a protective immune response) against rhinovirus C serotypes in clusters 1a, 1b, 2a, and 2b. In some embodiments, the P2 polyprotein of human rhinovirus C serotype 17 strain (GenBank ID: MZ153245.1, strain name: RvC17 / USA / 2021 / RCC55) is capable of eliciting an immune response (e.g., a protective immune response) against at least rhinovirus C serotypes in clusters 1a and 1b. In some embodiments, the VP0 polyprotein of human rhinovirus C serotype 17 strain (GenBank ID: MZ153277.1, strain name: RvC17 / USA / 2021 / 368038-4) is capable of eliciting an immune response (e.g., a protective immune response) against at least clusters 1a and 1b of rhinovirus C serotypes. In some embodiments, the P2 polyprotein of human rhinovirus C serotype 11 strain (GenBank ID: OK254863.1, strain name: RvC11 / USA / 2021 / L2PJH9) is capable of eliciting an immune response (e.g., a protective immune response) against at least clusters 2a and 2b of rhinovirus C serotypes. In some embodiments, the VP0 polyprotein of human rhinovirus C serotype 11 strain (GenBank ID: MZ268689.1, isolate: 469843) is capable of eliciting an immune response (e.g., a protective immune response) against at least cluster 2a and 2b rhinovirus C serotypes.

[0111] In some embodiments, the invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus A VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus A. In some embodiments, the invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus A VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity and median identity to the amino acid sequences of VP0 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus A.

[0112] In some embodiments, the present invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus C. In some embodiments, the present invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity and median identity to the amino acid sequences of VP0 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus C.

[0113] In some embodiments, the present invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus A P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus A. In some embodiments, the present invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus A P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity and median identity to the amino acid sequences of P2 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus A.

[0114] In some embodiments, the present invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus A P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus C. In some embodiments, the present invention provides immunogenic compositions comprising at least one non-naturally occurring messenger RNA (mRNA) encoding a rhinovirus C P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity and median identity to the amino acid sequences of P2 polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of rhinovirus C.

[0115] Tables 2 and 3 provide exemplary amino acid sequences of naturally occurring rhinovirus A and C proteins and polyproteins that may be encoded by one or more mRNAs as described herein. An exemplary rhinovirus A sequence is derived from human rhinovirus A serotype 21 strain (GenBank ID: FJ445121.1, strain name: ATCC VR-1131). In some embodiments, amino acid sequences derived from other naturally occurring rhinovirus A proteins or polyproteins may be used in the practice of the present invention. For example, human rhinovirus A serotype 24, 57, or 90 strains (e.g., GenBank IDs: JN562727.1, strain name: HRV-A24_p1025_sR2625_2009; KY369874.1, strain name: SC9723; and FJ445167.1, strain name: ATCC VR-1291; respectively) may be suitable substitutes for the aforementioned human rhinovirus A serotype 21.

[0116] For example, because circulating rhinovirus A strains naturally mutate, repeating the analysis provided in Example 1 herein can yield polyproteins from different rhinovirus A strains that better match the polyproteins of circulating rhinovirus A strains. As described above, polyproteins can be selected based on having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) with the amino acid sequences of polyproteins from at least two (e.g., at least three or at least four) phylogenetic clusters of circulating rhinoviruses of group A.

[0117] Thus, in some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO: 4, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acids comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO: 4.

[0118] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of a rhinovirus A serotype 90 VP0 polyprotein, such as set forth in SEQ ID NO: 5, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acid comprises an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of a rhinovirus A serotype 90 VP0 polyprotein, such as set forth in SEQ ID NO: 5.

[0119] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO: 6, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acids comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO: 6.

[0120] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating mRNA, etc. by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of rhinovirus A serotype 57 P2 polyprotein, such as set forth in SEQ ID NO: 7, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acid comprises an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of rhinovirus A serotype 57 P2 polyprotein, such as set forth in SEQ ID NO: 7.

[0121] Exemplary VP0 rhinovirus C sequences are derived from serotypes 11, 17, and 34 of human rhinovirus C (GenBank ID: MZ268689.1, isolate: 469843, GenBank ID: MZ153277.1, strain name: RvC17 / USA / 2021 / 368038, and GenBank ID: MZ322913.1, isolate: 7H8M5V, respectively). Exemplary P2 rhinovirus C sequences are derived from serotypes 11 and 17 of human rhinovirus C (GenBank ID: OK254863.1, strain name: RvC11 / USA / 2021 / L2PJH9, and GenBank ID: MZ153245.1, strain name: RvC17 / USA / 2021 / RCC55, respectively). In some embodiments, amino acid sequences derived from other naturally occurring rhinovirus C proteins or polyproteins can be used in the practice of the invention. Because circulating rhinovirus C strains naturally mutate, different rhinovirus C strains that better match circulating rhinovirus C strains can be obtained by repeating the analysis provided in Example 8 of the present application.

[0122] Thus, in some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of a rhinovirus C serotype 34 VP0 polyprotein, such as set forth in SEQ ID NO: 3, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acids comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of a rhinovirus C serotype 34 VP0 polyprotein, such as set forth in SEQ ID NO: 3.

[0123] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of rhinovirus C serotype 11 VP0 polyprotein, such as set forth in SEQ ID NO: 1, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acid comprises an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of rhinovirus C serotype 11 VP0 polyprotein, such as set forth in SEQ ID NO: 1.

[0124] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of a rhinovirus C serotype 17 VP0 polyprotein, such as set forth in SEQ ID NO: 2, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acid comprises an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of a rhinovirus C serotype 17 VP0 polyprotein, such as set forth in SEQ ID NO: 2.

[0125] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of rhinovirus C serotype 11 P2 polyprotein, such as set forth in SEQ ID NO: 8, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acids comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of rhinovirus C serotype 11 P2 polyprotein, such as set forth in SEQ ID NO: 8.

[0126] In some embodiments, the invention provides non-naturally occurring nucleic acids (e.g., mRNA or DNA templates for generating, e.g., mRNA by in vitro transcription) that comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of rhinovirus C serotype 17 P2 polyprotein, such as set forth in SEQ ID NO: 9, so long as the sequence meets the stringent selection criteria of the methods for identifying rhinovirus polyproteins for use as immunogens disclosed herein. In some embodiments, the non-naturally occurring nucleic acids comprise an optimized nucleotide sequence that encodes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of rhinovirus C serotype 17 P2 polyprotein, such as set forth in SEQ ID NO: 9.

[0127] secretory signal sequence In some embodiments of the present invention, the rhinovirus polypeptides, proteins, and polyproteins described herein are operably linked to a non-native secretory signal sequence from a different virus. Such sequences have been shown to increase secretion from mammalian cells into the surrounding extracellular space. Typically, the secretory signal sequence is derived from a virus capable of infecting human cells. While not wishing to be bound by any particular theory, the inventors hypothesize that fusing the rhinovirus polypeptides, proteins, and polyproteins described herein to such non-native viral secretory signal sequences will enhance the immunogenicity and therefore the efficacy of the immunogenic compositions described herein.

[0128] In some embodiments, secretory signal sequences for use with the present invention are derived from influenza secretory signal sequence, SARS CoV-2 secretory signal sequence, varicella zoster virus (VZV) secretory signal sequence, measles secretory signal sequence, rubella secretory signal sequence, mumps secretory signal sequence, Ebola secretory signal sequence, and smallpox secretory signal sequence.

[0129] In specific embodiments, secretory signal sequences for use with the present invention are selected from influenza hemagglutinin (HA) secretory signal sequence, SARS CoV-2 spike protein secretory signal sequence, VZV gB secretory signal sequence, VZV gE secretory signal sequence, VZV gI secretory signal sequence, VZV gK secretory signal sequence, measles F protein secretory signal sequence, rubella E1 protein secretory signal sequence, rubella E2 protein secretory signal sequence, mumps F protein secretory signal sequence, Ebola GP protein secretory signal sequence, and smallpox 6 kDa IC protein secretory signal sequence. These secretory signal sequences are derived from viruses that have previously been administered to humans as part of immunogenic compositions and are therefore considered safe for use in humans.

[0130] In some embodiments, the secretory signal sequence is selected from Table 1.

[0131] [Table 1]

[0132] In specific embodiments, secretory signal sequences for use with the rhinovirus polypeptides, proteins, and polyproteins described herein comprise an HA secretory signal sequence from influenza A or influenza B. In one specific embodiment, the secretory signal sequence is derived from the HA secretory signal sequence of an influenza A virus (e.g., an H1N1 subtype, such as A / California / 7 / 2009).

[0133] Without wishing to be bound by any particular theory, the inventors hypothesize that fusing a nonstructural rhinovirus polypeptide that is not normally secreted (such as P2) to a secretory signal sequence further enhances the immune response (e.g., T cell response) to the polypeptide.

[0134] Exemplary Rhinovirus A Polyproteins The inventors found that the amino acid sequence of the polyprotein of human rhinovirus A serotype 21 strain (GenBank ID: FJ445121.1, strain name: ATCC VR-1131) is most similar to a consensus sequence generated from 539 individual rhinovirus A polyprotein sequences. Without wishing to be bound by any particular theory, the inventors hypothesize that the polyprotein sequence of this particular rhinovirus A serotype may be used to construct an immunogenic composition that is effective in inducing an immune response against multiple rhinovirus A serotypes.

[0135] Thus, in a specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 6, or a non-proteolytic version thereof. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO: 4.

[0136] In some embodiments, the rhinovirus A VP0 polyprotein or the rhinovirus A P2 polyprotein is operably linked to a non-native secretory signal sequence from an influenza A virus to increase secretion of the protein encoded by the mRNA. Thus, in specific embodiments, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO:30, or a non-proteolytic version thereof. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO:31, or a non-proteolytic version thereof. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO:32. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO:33.

[0137] Combined, the P2 polyprotein and VP0 polyprotein from rhinovirus A serotype 21 strains are predicted to cover 97-99% of all human MHC-I and MHC-II alleles worldwide. Thus, in a further specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 6 or 30, or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO: 4 or 32. In a specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 30, or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO: 32. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 6, or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO: 4. In some embodiments, immunogenic compositions of the invention comprise a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 30, or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO: 4. In other embodiments, immunogenic compositions of the invention comprise a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 6, or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO: 32.

[0138] Even broader protection may be achieved by further including in the immunogenic compositions of the invention an mRNA encoding a polyprotein from a phylogenetically distant serogroup. Rhinovirus A serotype 8 (GenBank ID: FJ445113.1, strain name: ATCC VR-1118) is derived from a serogroup that is phylogenetically distant compared to rhinovirus A serotype 8. Inclusion of the polyprotein P2 of rhinovirus A serotype 8 may be beneficial in broadening the efficacy of the immunogenic compositions of the invention. Thus, in certain embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO: 80 or a non-proteolytic version thereof (e.g., having the amino acid sequence of SEQ ID NO: 81). In some embodiments, the rhinovirus A P2 polyprotein is operably linked to a non-native secretory signal sequence from an influenza A virus to increase secretion of the protein encoded by the mRNA. Thus, in some embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA encoding a rhinovirus P2 polyprotein having the amino acid sequence of SEQ ID NO: 82 or a non-proteolytic version thereof.

[0139] For example, to provide immunity against infection caused by multiple rhinovirus A serotypes, an immunogenic composition may include one or more non-naturally occurring mRNAs encoding one or more native polyproteins of rhinovirus A serotype 21 and one or more non-naturally occurring mRNAs encoding one or more native polyproteins of rhinovirus A serotype 8.

[0140] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A serotype 21 VP0 polyprotein having the amino acid sequence of SEQ ID NO:4, a second non-naturally occurring mRNA encoding a rhinovirus A serotype 21 P2 polyprotein having the amino acid sequence of SEQ ID NO:6 or a non-proteolytic version thereof (e.g., SEQ ID NO:77), and a third non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO:80 or a non-proteolytic version thereof (e.g., SEQ ID NO:81).

[0141] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A serotype 21 VP0 polyprotein having the amino acid sequence of SEQ ID NO: 32, a second non-naturally occurring mRNA encoding a rhinovirus A serotype 21 P2 polyprotein having the amino acid sequence of SEQ ID NO: 6 or a non-proteolytic version thereof (e.g., SEQ ID NO: 77), and a third non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO: 80 or a non-proteolytic version thereof (e.g., SEQ ID NO: 81).

[0142] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A serotype 21 VP0 polyprotein having the amino acid sequence of SEQ ID NO: 32, a second non-naturally occurring mRNA encoding a rhinovirus A serotype 21 P2 polyprotein having the amino acid sequence of SEQ ID NO: 30 or a non-proteolytic version thereof, and a third non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO: 82 or a non-proteolytic version thereof.

[0143] In Table 2, the rhinovirus A P2 polypeptide is in bold and the rhinovirus A VP0 polypeptide is underlined. The secretory signal sequence is shown in italics. VP0 polyproteins containing a secretory signal sequence from influenza virus A contain a sequence DTL between the secretory signal and the VP0 polyprotein. DTL corresponds to the first three amino acids at the N-terminus of the mature influenza HA polypeptide from which the exemplary secretory signal is derived.

[0144] [Table 2]

[0145] [Table 3]

[0146] [Table 4]

[0147] [Table 5]

[0148] [Table 6]

[0149] [Table 7]

[0150] The active site of P2A in rhinoviruses is highly conserved. In rhinovirus A, the active site consists of a catalytic triad consisting of, for example, a cysteine ​​(C) residue at position 106 (Cys106), a histidine (H) residue at position 18 (His18), and an aspartic acid (D) residue at position 35 (Asp35), although the exact number of residues can vary between serotypes. For example, in rhinovirus A serotype 21, the catalytic triad is formed by Cys106, His18, and Asp35. In rhinovirus A serotype 8, the catalytic triad is formed by Cys107, His18, and Asp36. Nonproteolytic versions of P2A can be generated by mutating one or more of these sites, for example, Cys106 or Cys107, respectively.

[0151] In some embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA encoding a fusion protein comprising a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO:6 or a non-proteolytic version thereof (e.g., SEQ ID NO:77) and a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO:4. In some embodiments, the fusion is P2-VP0 (i.e., P2 is at the N-terminus). In alternative embodiments, the fusion is VP0-P2 (i.e., VP0 is at the N-terminus). In either configuration, the rhinovirus A P2 polyprotein can be a non-proteolytic version. Providing both the P2 polyprotein and the VP0 polyprotein in a single mRNA is advantageous because it simplifies the production of the immunogenic composition. For example, only a single mRNA needs to be produced by in vitro transcription. Similarly, when mRNA is encapsulated in lipid nanoparticles, only a single mRNA needs to be encapsulated during production.

[0152] In some embodiments, the fusion protein can include a secretory signal sequence, such as that shown for the amino acids set forth in SEQ ID NOs:30 and 32. Typically, the secretory signal sequence is located at the N-terminus of the fusion protein. Thus, in some embodiments, the fusion protein includes a rhinovirus A VP0 polyprotein having the amino acid sequence of SEQ ID NO:4 and a rhinovirus A P2 polyprotein having the amino acid sequence of SEQ ID NO:6 or a non-proteolytic version thereof (e.g., SEQ ID NO:77). In other embodiments, the rhinovirus A P2 polyprotein has the amino acid sequence of SEQ ID NO:6 or a non-proteolytic version thereof (e.g., SEQ ID NO:77), and the rhinovirus A VP0 polyprotein has the amino acid sequence of SEQ ID NO:4.

[0153] In a specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a P2-VP0 fusion protein having the amino acid sequence of SEQ ID NO: 34. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a P2-VP0 fusion protein with an N-terminal influenza virus A-derived secretory signal sequence having the amino acid sequence of SEQ ID NO: 35. In some embodiments, the exemplified fusion protein comprises a non-proteolytic version of P2.

[0154] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a fusion protein comprising a rhinovirus A serotype 21 P2 polyprotein having the amino acid sequence of SEQ ID NO:6, or a non-proteolytic version thereof (e.g., SEQ ID NO:77), and a rhinovirus A serotype 21 VP0 polyprotein having the amino acid sequence of SEQ ID NO:4, and a second non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO:80, or a non-proteolytic version thereof (e.g., SEQ ID NO:81). In some embodiments, the fusion is P2-VP0 (i.e., P2 is at the N-terminus). In alternative embodiments, the fusion is VP0-P2 (i.e., VP0 is at the N-terminus).

[0155] In some embodiments, the fusion protein may include a secretory signal sequence as shown for the amino acids set forth in SEQ ID NOs: 30 and 32. Thus, in some embodiments, an immunogenic composition comprises a first non-naturally occurring mRNA encoding a fusion protein comprising a rhinovirus A serotype 21 P2 polyprotein having the amino acid sequence of SEQ ID NO: 30, or a non-proteolytic version thereof, and a rhinovirus A serotype 21 VP0 polyprotein having the amino acid sequence of SEQ ID NO: 4, and a second non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO: 80, or a non-proteolytic version thereof (e.g., SEQ ID NO: 81). In an alternative embodiment, the immunogenic composition comprises a first non-naturally occurring mRNA encoding a fusion protein comprising a rhinovirus A serotype 21 VP0 polyprotein having the amino acid sequence of SEQ ID NO: 32 and a rhinovirus A serotype 21 P2 polyprotein having the amino acid sequence of SEQ ID NO: 6 or a non-proteolytic version thereof (e.g., SEQ ID NO: 77), and a second non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO: 80 or a non-proteolytic version thereof (e.g., SEQ ID NO: 81).

[0156] In certain embodiments, immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a P2-VP0 fusion protein having the amino acid sequence of SEQ ID NO:34 and a second non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO:80 or a non-proteolytic version thereof (e.g., SEQ ID NO:81). In some embodiments, exemplary fusion proteins comprise a non-proteolytic version of P2. In alternative embodiments, immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a P2-VP0 fusion protein having the amino acid sequence of SEQ ID NO:35 and a second non-naturally occurring mRNA encoding a rhinovirus A serotype 8 P2 polyprotein having the amino acid sequence of SEQ ID NO:80 or a non-proteolytic version thereof (e.g., SEQ ID NO:81). In some embodiments, exemplary fusion proteins comprise a non-proteolytic version of P2.

[0157] Exemplary Rhinovirus C Polyproteins The inventors have found that the amino acid sequence of the VP0 polyprotein of human rhinovirus C serotype 34, having the amino acid sequence set forth in SEQ ID NO:3, is most similar to a single VP0 consensus sequence compiled from 463 individual rhinovirus C polyprotein sequences. Without wishing to be bound by any particular theory, the inventors hypothesize that the VP0 polyprotein of this particular rhinovirus C serotype (or the mRNA encoding it) may be used to construct immunogenic compositions effective in eliciting immune responses against multiple rhinovirus C serotypes.

[0158] Thus, in certain embodiments, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO:3.

[0159] Our analysis revealed that group C rhinoviruses can be divided into two large phylogenetic clusters, 1ab and 2ab, each of which can be further divided into two phylogenetic clusters, 1a and 1b, and 2a and 2b, respectively. Separate consensus sequences were determined in silico for each of the two larger phylogenetic clusters. We also found that the amino acid sequence of the VP0 polyprotein of human rhinovirus C serotype 17, having the amino acid sequence set forth in SEQ ID NO:2, is most similar to the VP0 consensus sequence determined for phylogenetic cluster 1ab. The amino acid sequence of the VP0 polyprotein of human rhinovirus C serotype 11, having the amino acid sequence set forth in SEQ ID NO:1, is most similar to the VP0 consensus sequence determined for phylogenetic cluster 2ab. Without wishing to be bound by any particular theory, we hypothesize that the VP0 polyproteins of these two rhinovirus C serotypes can also be used to construct immunogenic compositions effective in eliciting immune responses against multiple rhinovirus C serotypes.

[0160] Thus, in certain embodiments, the immunogenic compositions of the invention comprise one or more non-naturally occurring mRNAs encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO:2 and a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO:1.

[0161] Considering the phylogenetic diversity of group C rhinoviruses, nonstructural rhinovirus C polypeptide sequences can be used to construct immunogenic compositions capable of eliciting effective immune responses against as many rhinovirus C serotypes as possible. The inventors have found that the amino acid sequence of the P2 polyprotein of human rhinovirus C serotype 17, having the amino acid sequence set forth in SEQ ID NO:9, is most similar to the P2 consensus sequence determined for phylogenetic cluster 1ab. The amino acid sequence of the P2 polyprotein of human rhinovirus C serotype 11, having the amino acid sequence set forth in SEQ ID NO:8, is most similar to the P2 consensus sequence determined for phylogenetic cluster 2ab.

[0162] Thus, in certain embodiments, the immunogenic compositions of the invention comprise one or more non-naturally occurring mRNAs encoding a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO:9 or a non-proteolytic version thereof and a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO:8 or a non-proteolytic version thereof.

[0163] At the same time, it can be expected that the identified VP0 polyprotein and P2 polyprotein may be even more effective in eliciting a comprehensive immune response against group C rhinoviruses. Thus, in one embodiment, the immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a VP0 polyprotein having the amino acid sequence of SEQ ID NO:3, a non-naturally occurring mRNA encoding a P2 polyprotein having the amino acid sequence of SEQ ID NO:9 or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a P2 polyprotein having the amino acid sequence of SEQ ID NO:8 or a non-proteolytic version thereof. In another embodiment, the immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO:2, a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO:1, a non-naturally occurring mRNA encoding a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO:9 or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO:8 or a non-proteolytic version thereof.

[0164] In some embodiments, the rhinovirus C VP0 polyprotein and / or the rhinovirus C P2 polyprotein are operatively linked to a non-native secretory signal sequence from the influenza A virus to increase secretion of the proteins encoded by the mRNA. Thus, in specific embodiments, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO: 36 or a non-proteolytic version thereof. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO: 37. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO: 38. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO: 39. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO: 40.

[0165] Thus, in a further specific embodiment, the immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO: 8, 9, 36 or 37 or a non-proteolytic version thereof, and a non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO: 1, 2, 3, 38, 39 or 40.

[0166] In Table 3, the rhinovirus C VP0 polypeptide is underlined and the rhinovirus C P2 polypeptide is shown in bold. The secretory signal sequence is shown in italics. VP0 polyproteins containing a secretory signal sequence from influenza virus A contain a sequence DTL between the secretory signal and the VP0 polyprotein. DTL corresponds to the first three amino acids at the N-terminus of the mature influenza HA polypeptide from which the exemplary secretory signal is derived.

[0167] [Table 8]

[0168] [Table 9]

[0169] [Table 10]

[0170] [Table 11]

[0171] [Table 12]

[0172] The active site of P2A in rhinoviruses is highly conserved. In rhinovirus C, the active site consists of the catalytic triad: a cysteine ​​(C) residue at position 105 (Cys105), a histidine (H) residue at position 18 (His18), and an aspartic acid (D) residue at position 34 (Asp34). Nonproteolytic versions of P2A can be generated by mutating one or more of these sites, for example, Cys105.

[0173] In some embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA encoding a fusion protein comprising a P2 polyprotein having the amino acid sequence of SEQ ID NO:8 or 9, or a non-proteolytic version thereof, and a VP0 polyprotein having the amino acid sequence of SEQ ID NO:1, 2, or 3. In some embodiments, the fusion is P2-VP0 (i.e., P2 is at the N-terminus). In alternative embodiments, the fusion is VP0-P2 (i.e., VP0 is at the N-terminus). In either configuration, the rhinovirus A P2 polyprotein can be a non-proteolytic version. Providing both the P2 polyprotein and the VP0 polyprotein in a single mRNA is advantageous because it simplifies the production of the immunogenic composition. For example, only a single mRNA needs to be produced by in vitro transcription. Similarly, when mRNA is encapsulated in lipid nanoparticles, only a single mRNA needs to be encapsulated during production.

[0174] In some embodiments, the fusion protein can include a secretory signal sequence, such as that shown for the amino acids set forth in SEQ ID NOs: 36, 37, 38, 39, and 40. Typically, the secretory signal sequence is located at the N-terminus of the fusion protein. Thus, in some embodiments, the fusion protein comprises a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO: 38, 39, or 40, and a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO: 36 or 37, or a non-proteolytic version thereof. In other embodiments, the fusion protein comprises a rhinovirus C P2 polyprotein having the amino acid sequence of SEQ ID NO: 36 or 37, or a non-proteolytic version thereof, and a rhinovirus C VP0 polyprotein having the amino acid sequence of SEQ ID NO: 38, 39, or 40.

[0175] In a specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a P2-VP0 fusion protein having the amino acid sequence of SEQ ID NO:41. In another specific embodiment, an immunogenic composition of the invention comprises an mRNA encoding a P2-VP0 fusion protein with an N-terminal influenza virus A-derived secretory signal sequence having the amino acid sequence of SEQ ID NO:42. In some embodiments, the exemplified fusion protein comprises a non-proteolytic version of P2. In a specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a P2-VP0 fusion protein with an N-terminal influenza virus A-derived secretory signal sequence having the amino acid sequence of SEQ ID NO:43. In another specific embodiment, an immunogenic composition of the invention comprises a non-naturally occurring mRNA encoding a P2-VP0 fusion protein with an N-terminal influenza virus A-derived secretory signal sequence having the amino acid sequence of SEQ ID NO:44.

[0176] coverage To provide immunity against infection caused by multiple rhinovirus serotypes, an immunogenic composition can include one or more non-naturally occurring mRNAs encoding a first naturally occurring rhinovirus polyprotein and one or more non-naturally occurring mRNAs encoding a second naturally occurring rhinovirus polyprotein.

[0177] For example, the first naturally occurring rhinovirus polyprotein can be a structural rhinovirus polyprotein (e.g., VP0) and the second naturally occurring rhinovirus polyprotein can be a nonstructural rhinovirus polyprotein (e.g., P2). In some embodiments, the structural rhinovirus polyprotein and the nonstructural rhinovirus polyprotein are derived from the same group of rhinovirus (e.g., Group A or Group C). In some embodiments, the structural rhinovirus polyprotein and the nonstructural rhinovirus polyprotein are derived from the same rhinovirus (i.e., the same strain). In some embodiments, the structural rhinovirus polyprotein and the nonstructural rhinovirus polyprotein are derived from different rhinoviruses (i.e., different strains). In some embodiments, the structural rhinovirus polyprotein and the nonstructural rhinovirus polyprotein are derived from different groups of rhinovirus (e.g., Group A and Group C).

[0178] In some embodiments, the first naturally occurring rhinovirus polyprotein is a structural rhinovirus polyprotein (e.g., VP0) from a first rhinovirus, and the second naturally occurring rhinovirus polyprotein is a structural rhinovirus polyprotein (e.g., VP0) from a second rhinovirus. The first and second rhinoviruses can be from the same group of rhinoviruses (e.g., group A or group C). For example, the first structural polyprotein (e.g., VP0) can be from a first rhinovirus A, and the second structural polyprotein (e.g., VP0) can be from a second rhinovirus A. In some embodiments, the first structural polyprotein (e.g., VP0) can be from a first rhinovirus C, and the second structural polyprotein (e.g., VP0) can be from a second rhinovirus C. In some embodiments, the first and second rhinoviruses can be from different groups of rhinoviruses (e.g., groups A and C). In some embodiments, the first native rhinovirus polyprotein is a nonstructural rhinovirus polyprotein (e.g., P2) from a first rhinovirus, and the second native rhinovirus polyprotein is a nonstructural rhinovirus polyprotein (e.g., P2) from a second rhinovirus. The first and second rhinoviruses can be derived from the same group of rhinoviruses (e.g., group A or group C). For example, the first nonstructural polyprotein (e.g., P2) can be derived from a first rhinovirus A, and the second nonstructural polyprotein (e.g., P2) can be derived from a second rhinovirus A. In some embodiments, the first nonstructural polyprotein (e.g., P2) can be derived from a first rhinovirus C, and the second nonstructural polyprotein (e.g., P2) can be derived from a second rhinovirus C. In some embodiments, the first and second rhinoviruses are derived from different groups of rhinoviruses (e.g., groups A and C).

[0179] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to (and optionally median identical to) the amino acid sequence of a VP0 polyprotein from at least two phylogenetic clusters of rhinovirus A; and a second non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to (and optionally median identical to) the amino acid sequence of a VP0 polyprotein from at least two phylogenetic clusters of rhinovirus C.

[0180] In some embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA comprising a first nucleic acid sequence encoding a rhinovirus A VP0 polyprotein having an amino acid sequence that has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus A; and a second nucleic acid sequence encoding a rhinovirus C VP0 polyprotein, the rhinovirus C VP0 polyprotein having an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C.

[0181] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a first rhinovirus C VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C; and a second non-naturally occurring mRNA encoding a second rhinovirus C VP0 polyprotein, the second rhinovirus C VP0 polyprotein having an amino acid sequence that is different from the first rhinovirus C VP0 polyprotein and that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C. The two phylogenetic clusters comprising VP0 polyproteins having amino acid sequences with an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequence of the VP0 polyprotein are different from the two phylogenetic clusters comprising VP0 polyproteins having amino acid sequences with an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) with the amino acid sequence of the first rhinovirus C VP0 polyprotein.

[0182] In some embodiments, the immunogenic compositions of the invention comprise a first nucleic acid sequence encoding a first rhinovirus C VP0 polyprotein, the first nucleic acid sequence having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C; and a second nucleic acid sequence encoding a second rhinovirus C VP0 polyprotein, the second rhinovirus C VP0 polyprotein being different from the first rhinovirus C VP0 polyprotein and having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C. The two phylogenetic clusters comprising VP0 polyproteins having amino acid sequences with an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequence of the VP0 polyprotein are distinct from the two phylogenetic clusters comprising VP0 polyproteins having amino acid sequences with an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequence of the first rhinovirus C VP0 polyprotein.

[0183] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein having an amino acid sequence that has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus A; and a second non-naturally occurring mRNA encoding a rhinovirus A P2 polyprotein having an amino acid sequence that has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus A.

[0184] In some embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA comprising: a first nucleic acid sequence encoding a rhinovirus A VP0 polyprotein having an amino acid sequence that has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus A; and a second nucleic acid sequence encoding a rhinovirus A P2 polyprotein having an amino acid sequence that has an average identity (and optionally a median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus A.

[0185] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to (and optionally median identical to) the amino acid sequence of a VP0 polyprotein from at least two phylogenetic clusters of rhinovirus C; and a second non-naturally occurring mRNA encoding a rhinovirus C P2 polyprotein having an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to (and optionally median identical to) the amino acid sequence of a P2 polyprotein from at least two phylogenetic clusters of rhinovirus C.

[0186] In some embodiments, the immunogenic compositions of the invention comprise a non-naturally occurring mRNA comprising a first nucleic acid sequence encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C; and a second nucleic acid sequence encoding a rhinovirus C P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus C.

[0187] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C; a second non-naturally occurring mRNA encoding a first rhinovirus C P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus C; and a second non-naturally occurring mRNA encoding a first rhinovirus C P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus C. and a third non-naturally occurring mRNA encoding a P2 polyprotein, wherein the two phylogenetic clusters containing P2 polyproteins having amino acid sequences with an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequence of the second rhinovirus C P2 polyprotein are distinct from the two phylogenetic clusters containing P2 polyproteins having amino acid sequences with an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequence of the first rhinovirus C P2 polyprotein.

[0188] In some embodiments, the immunogenic compositions of the invention comprise a first nucleic acid sequence encoding a rhinovirus C VP0 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of VP0 polyproteins from at least two phylogenetic clusters of rhinovirus C; a second nucleic acid sequence encoding a first rhinovirus C P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus C; and a second nucleic acid sequence encoding a first rhinovirus C P2 polyprotein having an amino acid sequence that has at least 80% (e.g., at least 85%, at least 90%, or at least 95%) average identity (and optionally median identity) to the amino acid sequences of P2 polyproteins from at least two phylogenetic clusters of rhinovirus C. and a third nucleic acid sequence encoding a P2 polyprotein, wherein two phylogenetic clusters comprising a P2 polyprotein having an amino acid sequence that has an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequence of the second rhinovirus C P2 polyprotein are distinct from two phylogenetic clusters comprising a P2 polyprotein having an amino acid sequence that has an average identity (and optionally median identity) of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) to the amino acid sequence of the first rhinovirus C P2 polyprotein.

[0189] Rhinovirus polypeptides containing T-cell epitope-rich regions Up to one-third of all rhinovirus infections are asymptomatic in both healthy and asthmatic subjects. In asthmatic subjects, symptomatic infections can lead to worsening of asthma symptoms and are thought to cause 20-30% of asthma exacerbations. Previous studies suggest that CD4+ and CD8+ T cells capable of recognizing rhinovirus-associated antigens are present in the circulation of healthy subjects. These T cells may be involved in immune surveillance and can rapidly induce adaptive immune responses after rhinovirus infection.

[0190] It has been hypothesized that the rapid adaptive immune response results from the engagement of effector memory T cells that are cross-reactive with T cell epitopes conserved across multiple rhinoviruses. These memory T cells are formed after previous infection with different rhinovirus strains.

[0191] The inventors have demonstrated for the first time that the P2 polyprotein of rhinovirus A contains a conserved region that is rich in T cell epitopes. Thus, without wishing to be bound by any particular theory, the P2 polyprotein may be capable of inducing an immune response, and in particular a T cell response, that is broadly protective against infection with multiple rhinoviruses.

[0192] Specifically, the inventors have discovered that the nonstructural rhinovirus polypeptides P2A, P2B, and P2C that make up the P2 polyprotein contain regions of high sequence conservation. A region of high sequence conservation comprises one or more stretches of at least 30 contiguous amino acids that share at least 80% sequence identity among at least 20 (e.g., at least 30, 40, or 50) rhinovirus serotypes. In some embodiments, a region of high sequence conservation comprises one or more stretches of at least 50 contiguous amino acids that share at least 80% sequence identity among at least 20 (e.g., at least 30, 40, or 50) rhinovirus serotypes.

[0193] Based on our analysis, the rhinovirus P2 polyprotein contains regions with high sequence conservation across various serotypes, such as residues 80-120, 250-280, and approximately 380-450 of the rhinovirus A VP0 polyprotein, respectively. These conserved regions are also rich in T cell epitopes. Therefore, without wishing to be bound by any particular theory, rhinovirus A proteins P2A, P2B, and P2C, or polypeptides derived therefrom containing one or more of these conserved T cell-rich regions, may be particularly effective in inducing T cell responses against multiple rhinovirus serotypes (particularly multiple rhinovirus A serotypes). For example, a nonstructural rhinovirus polypeptide of the invention may contain residues 80-120, 250-280, and 380-450 of the rhinovirus A P2 polyprotein (e.g., the P2 polyprotein encoded by SEQ ID NO: 6).

[0194] Based on the inventors' analysis, rhinovirus VP0 polyproteins also contain regions of high sequence conservation across various serotypes, e.g., around residues 1-200 and 220-300 of the rhinovirus A VP0 polyprotein, respectively. These conserved regions are also rich in T cell epitopes. Thus, without wishing to be bound by any particular theory, the VP0 polyprotein may also be capable of inducing an immune response, and in particular a T cell response, that is broadly protective against infection with multiple rhinoviruses.

[0195] Specifically, the N-terminal 30 residues of structural rhinovirus polypeptides, particularly VP4 and VP2 of the rhinovirus A VP0 polyprotein, show particularly high sequence conservation. Furthermore, residues 1-100 of VP0, which comprise a portion of the structural capsid polypeptide, are also enriched in T cell epitopes. Additionally, the inventors have identified the region encompassing residues 150-200, particularly residues 220-300, as being enriched in T cell epitopes. Thus, without wishing to be bound by any particular theory, rhinovirus proteins VP4 and VP2, or polypeptides derived therefrom that contain one or more of these T cell-rich regions, may be particularly effective in inducing T cell responses against multiple rhinovirus serotypes (particularly multiple rhinovirus A serotypes). For example, structural rhinovirus polypeptides of the invention may comprise residues 1-200 and residues 220-300 of the rhinovirus A VP0 polyprotein (e.g., the VP0 polyprotein encoded by SEQ ID NO:4).

[0196] The inventors used a computational approach to identify conserved regions of the complete rhinovirus polyprotein and predicted both MHC class I and class II T cell epitopes. Using this approach, the inventors discovered several conserved regions containing stretches of at least 30 contiguous amino acids with at least 80% sequence identity among rhinoviruses belonging to the same group. Indeed, in some instances, the conserved stretches with at least 80% sequence identity are longer (e.g., about 50 or about 100 amino acids in length). The inventors also identified stretches of at least 40 contiguous amino acids within these conserved regions with 90% sequence identity among rhinoviruses of the same group. In some conserved regions, shorter stretches of at least 30 contiguous amino acids within these conserved regions shared at least 95% sequence identity among rhinoviruses of the same group.

[0197] Notably, the inventors have found that several of the conserved T cell epitope-rich regions are located in nonstructural polypeptides, particularly within the precursor P2 polyprotein. To the inventors' knowledge, these nonstructural polypeptides, including one or more T cell epitope-rich regions, particularly 2A, 2B, and 2C, provided as polyproteins, have not previously been used in vaccination approaches against rhinoviruses. While not wishing to be bound by any particular theory, the inventors believe that these nonstructural polypeptides may be particularly effective in eliciting immune responses against multiple rhinovirus serotypes and potentially rhinoviruses from different groups because they have not been exposed to the same evolutionary pressures as the structural polypeptides that form rhinovirus capsids and may have given rise to the many known rhinovirus serotypes.

[0198] Furthermore, the inventors have found that structural polypeptides located within the VP0 region of the complete rhinovirus polyprotein (i.e., VP4 and VP2) are particularly enriched in conserved T cell epitope-rich regions. Therefore, inclusion of these structural polypeptides in immunogenic compositions is expected to elicit particularly potent T cell responses against multiple rhinovirus serotypes. Indeed, the inventors' computational analysis suggests that the combination of the P2 and VP0 polyproteins can elicit T cell responses in at least 95% of the human population.

[0199] Without wishing to be bound by any particular theory, the inventors believe that one or more mRNAs encoding one or more nonstructural rhinovirus polypeptides containing one or more T cell epitope-rich regions, alone or in combination with one or more mRNAs encoding one or more structural rhinovirus polypeptides containing one or more T cell epitope-rich regions, provide particularly effective immunogenic compositions because the one or more mRNAs are expressed intracellularly and thus can mimic virus-infected cells in vivo in a subject. Thus, in certain embodiments, the present invention specifically relates to immunogenic compositions comprising one or more mRNAs encoding one or more nonstructural rhinovirus polypeptides containing one or more T cell epitope-rich regions. In exemplary embodiments, the one or more T cell epitope-rich regions of the one or more nonstructural rhinovirus polypeptides comprise a class I T cell epitope and / or a class II T cell epitope.

[0200] In certain embodiments, the one or more non-naturally occurring mRNAs encoding one or more nonstructural rhinovirus polypeptides encode a first naturally occurring rhinovirus protein or polyprotein. In certain embodiments, the first naturally occurring rhinovirus protein or polyprotein comprises at least one of rhinovirus proteins 2A, 2B, and 2C. In some embodiments, the first naturally occurring rhinovirus protein or polyprotein is a polyprotein comprising rhinovirus proteins 2A, 2B, and 2C.

[0201] In some embodiments, one or more T cell epitopes of one or more nonstructural rhinovirus polypeptides are located in conserved regions comprising one or more stretches of at least 30 contiguous amino acids that have at least 80% sequence identity with a corresponding stretch of the rhinovirus A serotype 21 polyprotein, such as set forth in SEQ ID NO: 45. In some embodiments, one or more T cell epitopes are located in conserved regions comprising one or more stretches of at least 50 contiguous amino acids that have at least 80% sequence identity with a corresponding stretch of the rhinovirus A serotype 21 polyprotein, such as set forth in SEQ ID NO: 45. In certain embodiments, one or more T cell epitopes are located within one or more regions corresponding to residues 80-120, 250-280, and 380-450, respectively, of the rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO: 6.

[0202] In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO:6. In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of a rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO:6. In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO:6. In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of a rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO:6. In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of a rhinovirus A serotype 21 P2 polyprotein, such as set forth in SEQ ID NO:6. In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO: 6. In some embodiments, the first protein or polyprotein has or comprises an amino acid sequence that is identical to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO: 6.

[0203] Many T cell epitopes are located within structural rhinovirus polypeptides. The inclusion of one or more mRNAs encoding one or more structural rhinovirus polypeptides containing one or more T cell epitope-rich regions can make an immunogenic composition more effective in inducing a broad T cell response against rhinovirus. Thus, in some embodiments, one or more mRNAs further encode one or more structural rhinovirus polypeptides containing one or more T cell epitope-rich regions. In some embodiments, one or more T cell epitope-rich regions of one or more nonstructural rhinovirus polypeptides comprise a class I T cell epitope and / or a class II T cell epitope.

[0204] In some embodiments, the immunogenic compositions of the invention comprise T H 1-Directed T cell response (e.g. T H In some embodiments, the immunogenic compositions of the invention are capable of inducing polyreactive T cells (e.g., CD4+ T cells that express IFN-γ, IL-2, and TNF-α). In certain embodiments, the T cell response is cross-reactive to multiple rhinoviruses of the same group (e.g., group A or group C). In some embodiments, the immunogenic compositions of the invention are capable of eliciting effective T cell responses in the absence of adjuvant.

[0205] In certain embodiments, the one or more non-native mRNAs encoding one or more structural rhinovirus polypeptides encode a second naturally occurring rhinovirus protein or polyprotein. In certain embodiments, the second naturally occurring rhinovirus protein or polyprotein comprises at least one of the rhinovirus proteins VP4 and VP2. In some embodiments, the second naturally occurring rhinovirus protein or polyprotein is a polyprotein comprising the rhinovirus proteins VP4 and VP2.

[0206] In some embodiments, one or more T cell epitopes of one or more structural rhinovirus polypeptides are located in a conserved region comprising one or more stretches of at least 30 contiguous amino acids that have at least 80% sequence identity with a corresponding stretch of a rhinovirus A serotype 21 polyprotein, such as set forth in SEQ ID NO: 45. In some embodiments, one or more T cell epitopes are located in a conserved region comprising one or more stretches of at least 50 contiguous amino acids that have at least 80% sequence identity with a corresponding stretch of a rhinovirus A serotype 21 polyprotein, such as set forth in SEQ ID NO: 45. In certain embodiments, one or more T cell epitope-rich regions are located within one or more regions corresponding to residues 1-100, 150-200, and 220-300, respectively, of a rhinovirus A serotype 21 polyprotein, VP0, such as set forth in SEQ ID NO: 4.

[0207] In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO:4. In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO:4. In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO:4. In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO:4. In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein, such as set forth in SEQ ID NO:4. In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein as set forth in SEQ ID NO: 4. In some embodiments, the second protein or polyprotein has or comprises an amino acid sequence that is identical to the amino acid sequence of a rhinovirus A serotype 21 VP0 polyprotein as set forth in SEQ ID NO: 4.

[0208] In some embodiments, the separate non-naturally occurring mRNA molecules encode one or more nonstructural polypeptides and one or more structural polypeptides.

[0209] In some embodiments, the same mRNA molecule encodes one or more nonstructural polypeptides and one or more structural polypeptides. In some embodiments, the mRNA molecule encoding one or more nonstructural polypeptides and one or more structural polypeptides encodes a fusion protein. In some embodiments, the fusion protein comprises a native polyprotein comprising one or more nonstructural polypeptides and a native polyprotein comprising one or more structural polypeptides. In some embodiments, the polyprotein comprising one or more nonstructural polypeptides comprises at least one of rhinovirus proteins 2A, 2B, and 2C. In some embodiments, the polyprotein comprising one or more nonstructural polypeptides comprises rhinovirus proteins 2A, 2B, and 2C. In some embodiments, the polyprotein comprising one or more structural polypeptides comprises rhinovirus proteins VP4 and VP2.

[0210] In some embodiments, the fusion protein has or comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion protein has or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion protein has or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion protein has or comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion protein has or comprises an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion protein has or comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion protein has or comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 34.

[0211] MHC-I / MHC-II allele coverage The polyproteins disclosed herein have been particularly selected because the multiple T cell epitope-rich regions they contain provide broad coverage of MHC-I and MHC-II alleles. Published T cell epitope sequences identified for rhinoviruses are publicly available on the IEDB website (http: / / www.iedb.org / ). The IEDB website also indicates whether the epitope type is B cell, T cell MHC-I, or T cell MHC-II.

[0212] The IEDB alleles are designed to cover 97% of the world's human population MHC-I alleles and 99% of the world's human population MHC-II alleles. The MHC-I and MHC-II alleles that provide 97% and 99% coverage of the world's human population are provided in Tables 3 and 4, respectively.

[0213] [Table 13]

[0214] [Table 14]

[0215] Without wishing to be bound by any particular theory, an immunogenic composition comprising T cell epitopes for all of the MHC-I and / or MHC-II alleles identified in Tables 3 and 4 should provide coverage of a large portion of the human population. Thus, the composition should induce an immune response, and in particular a T cell response, that is broadly protective against infection with multiple rhinoviruses for a large portion of the human population. As shown in the Examples, the immunogenic compositions disclosed herein comprise non-naturally occurring mRNAs encoding both structural and non-structural rhinovirus polypeptides that contain multiple T cell epitope-rich regions that are capable of collectively eliciting a T cell response in at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) of the human population. In particular, the structural and non-structural rhinovirus polypeptides encoded by these mRNAs contain T cell epitope-rich regions that cover at least 95% (at least 96%, at least 97%, at least 98%, or at least 99%) of the MHC class I alleles in Table 4 and at least 95% (at least 96%, at least 97%, at least 98%, or at least 99%) of the MHC-II alleles in Table 5.

[0216] In some embodiments, the one or more T cell epitope-rich regions elicit a T cell response in at least 95% of a human population. In some embodiments, the one or more T cell epitope-rich regions elicit a T cell response in at least 96% of a human population. In some embodiments, the one or more T cell epitope-rich regions elicit a T cell response in at least 97% of a human population. In some embodiments, the one or more T cell epitope-rich regions elicit a T cell response in at least 98% of a human population. In some embodiments, the one or more T cell epitope-rich regions elicit a T cell response in at least 99% of a human population.

[0217] In some embodiments, the one or more T cell epitope-rich regions cover at least 95% of the MHC class I alleles of Table 4 and / or 95% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 95% of the MHC class I alleles of Table 4 and at least 95% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 96% of the MHC class I alleles of Table 4 and / or 96% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 95% of the MHC class I alleles of Table 4 and at least 96% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 97% of the MHC class I alleles of Table 4 and / or 97% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 97% of the MHC class I alleles of Table 4 and at least 97% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 98% of the MHC class I alleles of Table 4 and / or 98% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 98% of the MHC class I alleles of Table 4 and at least 98% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 99% of the MHC class I alleles of Table 4 and / or 99% of the MHC-II alleles of Table 5. In some embodiments, the one or more T cell epitope-rich regions cover at least 99% of the MHC class I alleles of Table 4 and at least 99% of the MHC-II alleles of Table 5.

[0218] Generation of optimized nucleotide sequences The present invention also provides sequence-optimized mRNAs encoding one or more nonstructural rhinovirus polypeptides containing one or more T cell epitope-rich regions and / or one or more structural rhinovirus polypeptides containing one or more T cell epitope-rich regions. These mRNAs are modified relative to their native counterparts to (a) improve the yield of full-length mRNA during in vitro synthesis and (b) maximize expression of the encoded polypeptides after delivery of the mRNA to target cells in vivo. Sequence motifs that promote rapid degradation of the mRNA in target cells have also been removed.

[0219] The process for generating optimized nucleotide sequences may include first creating a list of codon-optimized sequences and then applying three filters to the list. Specifically, it is subjected to a motif selection filter, a guanine-cytosine (GC) content analysis filter, and a codon adaptation index (CAI) analysis filter, resulting in an updated list of optimized nucleotide sequences. The updated list now includes nucleotide sequences containing features predicted to interfere with efficient transcription and / or translation of the encoded polypeptide.

[0220] Codon optimization The genetic code has 64 possible codons. Each codon contains a sequence of three nucleotides. The frequency of usage for each codon in the protein-coding region of the genome can be calculated by determining the number of instances in which a particular codon appears in the protein-coding region of the genome, and then dividing the resulting value by the total number of codons that code for the same amino acid in the protein-coding region of the genome.

[0221] Codon usage tables contain experimentally derived data regarding the frequency with which each codon is used to encode a particular amino acid for the particular biological source for which the table was generated. This information is expressed for each codon as a percentage (0-100%) or fraction (0-1) of the frequency with which that codon is used to encode a particular amino acid compared to the total number of times that codon encodes that amino acid.

[0222] Codon usage tables are stored in publicly available databases such as the Codon Usage Database (Nakamura et al. (2000) Nucleic Acids Research 28(1):292; available online at https: / / www.kazusa.or.jp / codon / ) and the High-performance Integrated Virtual Environment - codon Usage Tables (HIVE-CUTs) database (Athey et al., (2017), BMC Bioinformatics 18(1):391; available online at http: / / hive.biochemistry.gwu.edu / review / codon).

[0223] During the first stage of codon optimization, codons with codon usage frequencies below a threshold frequency (e.g., 10%) are excluded from the first codon usage table, which reflects the frequency of each codon in a given organism (e.g., a mammal or human). The codon usage frequencies of codons that were not excluded in the first stage are normalized to create a normalized codon usage table. An optimized nucleotide sequence encoding a target amino acid sequence is generated by selecting a codon for each amino acid in the amino acid sequence based on the usage frequencies of one or more codons associated with the given amino acid in the normalized codon usage table. The probability of selecting a particular codon for a given amino acid is equal to the usage frequency associated with the codon associated with this amino acid in the normalized codon usage table.

[0224] The codon-optimized sequences of the present invention are generated by a computer-implemented method for generating optimized nucleotide sequences. The method includes: (i) receiving an amino acid sequence encoding a peptide, polypeptide, or protein; (ii) receiving a first codon usage table, the first codon usage table including a list of amino acids, each amino acid in the table associated with at least one codon, each codon associated with a usage frequency; (iii) excluding codons associated with a usage frequency below a threshold frequency from the codon usage table; (iv) creating a normalized codon usage table by normalizing the usage frequencies of codons not excluded in step (iii); and (v) generating an optimized nucleotide sequence encoding the amino acid sequence by selecting a codon for each amino acid in the amino acid sequence based on the usage frequency of one or more codons associated with the amino acid in the normalized codon usage table. The threshold frequency can be in the range of 5% to 30%, particularly 5%, 10%, 15%, 20%, 25%, or 30%. In the context of the present invention, the threshold frequency is typically 10%.

[0225] The step of generating a normalized codon usage table includes: (a) distributing the usage frequency of each codon associated with the first amino acid and removed in step (iii) among the remaining codons associated with the first amino acid; and (b) repeating step (a) for each amino acid to generate a normalized codon usage table. In some embodiments, the usage frequency of the removed codon is distributed evenly among the remaining codons. In some embodiments, the usage frequency of the removed codon is distributed among the remaining codons in proportion to the usage frequency of each remaining codon. "Distribution" in this context may be defined as taking the combined magnitude of the usage frequencies of the removed codons associated with a particular amino acid and assigning a portion of this combined frequency to each of the remaining codons that encode the particular amino acid.

[0226] The step of selecting a codon for each amino acid includes: (a) identifying, in a normalized codon usage table, one or more codons associated with a first amino acid in the amino acid sequence; (b) selecting a codon associated with the first amino acid, wherein the probability of selecting a particular codon is equal to the frequency of usage associated with the codon associated with the first amino acid in the normalized codon usage table; and (c) repeating steps (a) and (b) until a codon has been selected for each amino acid in the amino acid sequence.

[0227] The step of generating an optimized nucleotide sequence by selecting a codon for each amino acid in the amino acid sequence (step (V) of the above method) is carried out n times to generate a list of optimized nucleotide sequences.

[0228] Motif Screening A motif screening filter is applied to the list of optimized nucleotide sequences, and optimized nucleotide sequences encoding any known negative cis-regulatory elements and negative repeat elements are removed from the list to generate an updated list.

[0229] For each optimized nucleotide sequence in the list, it is also determined whether it contains a termination signal. Any nucleotide sequence containing one or more termination signals is removed from the list generating an updated list. In some embodiments, the termination signal has the following nucleotide sequence: 5'-X1ATCTX2TX3-3' (where X1, X2, and X3 are independently selected from A, C, T, or G). In some embodiments, the termination signal has one of the following nucleotide sequences: TATCTGTT; and / or TTTTTT; and / or AAGCTT; and / or GAAGAGC; and / or TCTAGA. In an exemplary embodiment, the termination signal has the following nucleotide sequence: 5'-X1AUCUX2UX3-3' (where X1, X2, and X3 are independently selected from A, C, U, or G). In certain embodiments, the termination signal has one of the following nucleotide sequences: UAUCUGUU; and / or UUUUUU; and / or AAGCUU; and / or GAAGAGC; and / or UCUAGA.

[0230] Guanine-cytosine (GC) content The method further includes determining the guanine-cytosine (GC) content of each optimized nucleotide sequence in the updated list of optimized nucleotide sequences. The GC content of a sequence is the percentage of bases in a nucleotide sequence that are guanine or cytosine. The list of optimized nucleotide sequences is further updated by removing a nucleotide sequence from the list if the GC content of the nucleotide sequence is outside a predetermined GC content range.

[0231] Determining the GC content of each of the optimized nucleotide sequences includes, for each nucleotide sequence, determining the GC content of one or more additional portions of the nucleotide sequence, where the additional portions are non-overlapping with each other and with the first portion; and updating the list of optimized sequences includes removing a nucleotide sequence if the GC content of any portion is outside a predetermined GC content range; optionally, determining the GC content of the nucleotide sequence is stopped if the GC content of any portion is determined to be outside the predetermined GC content range. In some embodiments, the first portion and / or one or more additional portions of the nucleotide sequence comprise a predetermined number of nucleotides, optionally, the predetermined number of nucleotides is in the range of 5 to 300 nucleotides, or 10 to 200 nucleotides, or 15 to 100 nucleotides, or 20 to 50 nucleotides. In the context of the present invention, the predetermined number of nucleotides is typically 30 nucleotides. The predetermined GC content range may be 15% to 75%, or 40% to 60%, or 30% to 70%. In the context of the present invention, the predetermined GC content range is typically 30% to 70%.

[0232] A suitable GC content filter in accordance with the present invention may first analyze the first 30 nucleotides of an optimized nucleotide sequence, i.e., nucleotides 1-30 of the optimized nucleotide sequence. The analysis may include determining the number of nucleotides in a portion that are either G or C, and determining the GC content of the portion may include dividing the number of G or C nucleotides in the portion by the total number of nucleotides in the portion. The result of this analysis provides a value representing the proportion of nucleotides in the portion that are G or C, which may be a percentage, e.g., 50%, or a decimal, e.g., 0.5. If the GC content of the first portion is outside a predetermined GC content range, the optimized nucleotide sequence may be removed from the list of optimized nucleotide sequences.

[0233] If the GC content of the first portion is within the predetermined GC content range, the GC content filter may then analyze a second portion of the optimized nucleotide sequence. In this example, this may be the second 30 nucleotides of the optimized nucleotide sequence, i.e., nucleotides 31 through 60. The portion analysis may be repeated for each portion until either: if a portion is found that has a GC content that is outside the predetermined GC content range, the optimized nucleotide sequence may be removed from the list; or, if the entire optimized nucleotide sequence has been analyzed and no such portion is found, the GC content filter may keep the optimized nucleotide sequence on the list and move on to the next optimized nucleotide sequence in the list.

[0234] Codon Adaptation Index (CAI) The method further includes determining a codon adaptation index for each optimized nucleotide sequence in the latest updated list of optimized nucleotide sequences. The codon adaptation index of a sequence is a measure of codon usage bias and can be a value between 0 and 1. The latest updated list of optimized nucleotide sequences is further updated by removing a nucleotide sequence if its codon adaptation index is less than or equal to a predetermined codon adaptation index threshold. The codon adaptation index threshold can be 0.7, 0.75, 0.8, 0.85, or 0.9. The inventors have found that optimized nucleotide sequences having a codon adaptation index of 0.8 or greater result in very high protein yields. Therefore, in the context of the present invention, the codon adaptation index threshold is typically 0.8.

[0235] The codon adaptation index can be calculated for each optimized nucleotide sequence in any manner apparent to one of skill in the art, for example, as described in "The codon adaptation index—a measure of directional synonymous codon usage bias, and its potential applications" (Sharp and Li, 1987. Nucleic Acids Research 15(3): pp. 1281-1295); available online at: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC340524 / .

[0236] The implementation of the codon adaptation index calculation may involve a method similar to the following: For each amino acid in a sequence, the weight of each codon in the sequence is calculated as a function of its relative adaptability (w i The relative fitness can be expressed by a parameter called the codon f i the observed frequency of the synonymous codon f for that amino acid and the maximum frequency of j The codon adaptation index of a sequence can then be calculated as the geometric mean of the weights associated with each codon across the length of the sequence (measured in codons).The reference sequence set used to calculate the codon adaptation index can be the same reference sequence set from which the codon usage table used in the method of the present invention is derived.

[0237] Exemplary Optimized Nucleotide Sequences Exemplary optimized nucleotide sequences encoding the nonstructural and structural rhinovirus polyproteins shown in Table 6 were generated according to the methods described herein.

[0238] In a specific embodiment, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 46 or 47. In another specific embodiment, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 48 or 49.

[0239] In some embodiments, the VP0 polyprotein or the P2 polyprotein is operably linked to a non-native secretory signal sequence from the influenza A virus to increase secretion of the protein encoded by the mRNA. Thus, in specific embodiments, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 50 or 51. In another specific embodiment, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 52 or 53.

[0240] Together, the P2 polyprotein and VP0 polyprotein from rhinovirus A serotype 21 strains are predicted to provide T cell epitopes that cover 97-99% of all human MHC-I and MHC-II alleles worldwide. Thus, in a further specific embodiment, the immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 46, 47, 50, or 51, and an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 48, 49, 52, or 53. In a specific embodiment, the immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 50 or 51, and an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 52 or 53. In another specific embodiment, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 46 or 47, and an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 48 or 49. In some embodiments, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 50 or 51, and an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 48 or 49. In other embodiments, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a P2 polyprotein having the nucleic acid sequence of SEQ ID NO: 46 or 47, and an mRNA comprising an optimized nucleotide sequence encoding a VP0 polyprotein having the nucleic acid sequence of SEQ ID NO: 52 or 53.

[0241] In Table 6, the optimized nucleotide sequence encoding the VP0 polyprotein is underlined, and the optimized nucleotide sequence encoding the P2 polyprotein is shown in bold. The secretory signal sequence is shown in italics. The VP0 polyprotein containing the secretory signal sequence contains the nucleic acid sequence GATACTCTG, which encodes the amino acid DTL, between the secretory signal sequence and the VP0 polyprotein.

[0242] [Table 15]

[0243] [Table 16]

[0244] [Table 17]

[0245] [Table 18]

[0246] [Table 19]

[0247] [Table 20]

[0248] [Table 21]

[0249] [Table 22]

[0250] In some embodiments, the immunogenic compositions of the present invention comprise an mRNA containing an optimized nucleotide sequence encoding a fusion protein comprising the P2 polyprotein and the VP0 polyprotein. Providing an mRNA containing an optimized nucleotide sequence encoding the fusion protein is convenient because it simplifies the production of the immunogenic composition. For example, only a single mRNA needs to be produced by in vitro transcription. Similarly, when mRNA is encapsulated in lipid nanoparticles, only a single mRNA needs to be encapsulated during production.

[0251] In some embodiments, the fusion protein is P2-VP0 (i.e., P2 is at the N-terminus). In some embodiments, the P2-VP0 fusion protein has the nucleic acid sequence of SEQ ID NO: 54 or 55. In alternative embodiments, the fusion protein is VP0-P2 (i.e., VP0 is at the N-terminus). In some embodiments, the VP0-P2 fusion protein has the nucleic acid sequence of SEQ ID NO: 56 or 57.

[0252] In some embodiments, the fusion protein is operably linked to a non-native secretory signal sequence from the influenza A virus to increase secretion of the protein encoded by the mRNA. Typically, the secretory signal sequence is located at the N-terminus of the fusion protein. Thus, in some embodiments, the fusion protein comprises a P2 polyprotein having the amino acid sequence of SEQ ID NO:30 at its N-terminus and a VP0 polyprotein having the amino acid sequence of SEQ ID NO:4 at its C-terminus. Thus, in a specific embodiment, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a fusion protein having the nucleic acid sequence of SEQ ID NO:58 or 59. In other embodiments, the fusion protein comprises a VP0 polyprotein having the amino acid sequence of SEQ ID NO:32 at its N-terminus and a P2 polyprotein having the amino acid sequence of SEQ ID NO:6 at its C-terminus. Thus, in a specific embodiment, an immunogenic composition of the invention comprises an mRNA comprising an optimized nucleotide sequence encoding a fusion protein having the nucleic acid sequence of SEQ ID NO:60 or 61.

[0253] Further rhinovirus nucleotide sequences Methods other than those described herein for optimizing nucleotide sequences for use with mRNA therapy are known to those of skill in the art. These methods may result in deviations from the nucleotide sequences encoding the rhinovirus polypeptides, proteins, or polyproteins of the invention. The present disclosure also encompasses such variant nucleotide sequences.

[0254] Thus, in some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:46 or 47 and encodes an amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:46 or 47 and encodes an amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:46 or 47 and encodes an amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:46 or 47 and encodes an amino acid sequence as set forth in SEQ ID NO:2. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:46 or 47 and encodes an amino acid sequence as set forth in SEQ ID NO:6.

[0255] In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:48 or 49 and encodes the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:48 or 49 and encodes the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:48 or 49 and encodes the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:48 or 49 and encodes the amino acid sequence as set forth in SEQ ID NO:4.

[0256] In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 80% identical to the P2-VP0 fusion nucleotide sequence of SEQ ID NO:54, 55, 62, or 63 and encodes an amino acid sequence as set forth in SEQ ID NO:34. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 85% identical to the P2-VP0 fusion nucleotide sequence of SEQ ID NO:54, 55, 62, or 63 and encodes an amino acid sequence as set forth in SEQ ID NO:34. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 90% identical to the P2-VP0 fusion nucleotide sequence of SEQ ID NO:54, 55, 62, or 63 and encodes an amino acid sequence as set forth in SEQ ID NO:34. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 95% identical to the P2-VP0 fusion nucleotide sequence of SEQ ID NO:54, 55, 62, or 63 and encodes an amino acid sequence as set forth in SEQ ID NO:34. In some embodiments, an optimized nucleotide sequence for use in the immunogenic compositions of the invention comprises a nucleic acid sequence that is at least 99% identical to the P2-VP0 fusion nucleotide sequence of SEQ ID NO: 54, 55, 62, or 63 and encodes an amino acid sequence as set forth in SEQ ID NO: 34.

[0257] In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 80% identical to the VP0-P2 fusion nucleotide sequence of SEQ ID NO: 56, 57, 64, or 65 and encodes an amino acid sequence as set forth in SEQ ID NO: 66. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 85% identical to the VP0-P2 fusion nucleotide sequence of SEQ ID NO: 56, 57, 64, or 65 and encodes an amino acid sequence as set forth in SEQ ID NO: 66. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 90% identical to the VP0-P2 fusion nucleotide sequence of SEQ ID NO: 56, 57, 64, or 65 and encodes an amino acid sequence as set forth in SEQ ID NO: 66. In some embodiments, an optimized nucleotide sequence for use in an immunogenic composition of the invention comprises a nucleic acid sequence that is at least 95% identical to the VP0-P2 fusion nucleotide sequence of SEQ ID NO: 56, 57, 64, or 65 and encodes an amino acid sequence as set forth in SEQ ID NO: 66. In some embodiments, an optimized nucleotide sequence for use in the immunogenic compositions of the invention comprises a nucleic acid sequence that is at least 99% identical to the VP0-P2 fusion nucleotide sequence of SEQ ID NO: 56, 57, 64, or 65 and encodes an amino acid sequence as set forth in SEQ ID NO: 66.

[0258] mRNA mRNA structural elements A typical mRNA according to the present invention comprises a 5' cap, a 5' untranslated region (5' UTR), a protein coding region, a 3' untranslated region (3' UTR), and a 3' tail.

[0259] 5' Cap In certain embodiments, the mRNA of the invention comprises a 5' cap having the following structure: [ka] .

[0260] Typically, a 5' cap and / or 3' tail can be added after mRNA synthesis. The presence of a cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" helps protect the mRNA from exonuclease degradation. Alternatively, 5' cap and / or 3' tail sequences can be included in the DNA template sequence used in the in vitro transcription reaction.

[0261] A 5' cap can be added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, generating a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, mG(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0262] 3' tail In one specific embodiment, the tail structure of the mRNA comprises a poly(A) tail. In another specific embodiment, the tail structure of the mRNA comprises a poly(C) tail. In some embodiments, the tail structure comprises at least 50 adenosine or cytosine nucleotides. In typical embodiments, the tail structure is about 100-500 nucleotides in length. For example, a tail structure (e.g., a poly(A) tail) of 100-250 nucleotides in length may be particularly useful for therapeutic uses of the mRNA.

[0263] The poly(A) or poly(C) tail on the 3' end of an mRNA typically comprises at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, or at least 500 adenosine or cytosine nucleotides, respectively. In some embodiments, the tail structure comprises a combination of poly(A) and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides, hi some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0264] 5'UTR and 3'UTR In some embodiments, the mRNA comprising an optimized nucleotide sequence encoding one more nonstructural rhinovirus polyproteins or proteins and / or a polypeptide comprising one or more structural rhinovirus polyproteins or proteins also contains 5' and 3' untranslated region (UTR) sequences. In some embodiments, the mRNA comprises a 5' untranslated region (5'UTR) that differs from the native 5'UTR in a native mRNA encoding a rhinovirus polyprotein. In certain embodiments, the 5'UTR has the nucleotide sequence of SEQ ID NO: 10.

[0265] In some embodiments, the mRNA comprises a 3' untranslated region (3'UTR) that differs from the native 3'UTR in a native mRNA encoding a rhinovirus polyprotein. In certain embodiments, the 3'UTR has the nucleotide sequence of SEQ ID NO: 11, 12, or 13.

[0266] Exemplary 5' and 3' UTR sequences are shown in Table 7 below.

[0267] [Table 23]

[0268] Typically, from 5' to 3', the mRNA according to the present invention comprises a 5' cap as shown in paragraph

[0258] , a 5' UTR as set forth in SEQ ID NO: 10, an optimized nucleotide sequence of the present invention, a 3' UTR as set forth in SEQ ID NO: 11, 12 or 13, and a poly(A) tail of 100 to 250 nucleotides in length.

[0269] nucleotide In some embodiments, the mRNA comprises or consists of natural nucleosides (or unmodified nucleosides; i.e., adenosine, guanosine, cytidine, and uridine). In some embodiments, the mRNA comprises one or more modified nucleosides, such as nucleoside analogs (e.g., adenosine analogs, guanosine analogs, cytidine analogs, or uridine analogs). The presence of one or more nucleoside analogs (e.g., N-1-methylpseudouridine) may render the mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only natural nucleosides.

[0270] Thus, in some embodiments, the mRNA comprises both unmodified and modified nucleosides. In some embodiments, one or more modified nucleosides are nucleoside analogs. In some embodiments, one or more modified nucleosides comprise at least one modification selected from a modified sugar and a modified nucleobase. In some embodiments, the mRNA comprises one or more modified internucleoside linkages.

[0271] In some embodiments, the one or more modified nucleosides are nucleoside analogs selected from the group consisting of 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine.

[0272] For example, U.S. Patent No. 8,278,036 and WO 2011 / 012316 include discussions of 5-methyl-cytidine, pseudouridine, and 2-thio-uridine and their incorporation into mRNA. In some embodiments, the mRNA can be RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of such modified RNAs are disclosed in U.S. Patent Application Publication No. 2012 / 0195936 and WO 2011 / 012316, both of which are incorporated herein by reference in their entireties.

[0273] mRNA containing N-1-methylpseudouridine instead of uridine has been found to be particularly suitable for use in immunogenic compositions. Thus, in certain embodiments, the mRNA contains unmodified nucleosides (adenosine, guanosine, cytidine) and modified nucleosides (N-1-methylpseudouridine). In certain embodiments, all uridines in the mRNA are replaced with pseudouridines, such as methylpseudouridines, such as N-1-methylpseudouridine.

[0274] In vitro transcription The mRNA of the present invention can be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Patent Application Publication No. 2018 / 0258423 and WO 2018 / 157153 and can be used to practice the present invention, all of which are incorporated herein by reference. For example, the mRNA of the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template or DNA vector containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0275] For the preparation of mRNA by IVT, a DNA template or DNA vector can be transcribed in vitro. A suitable DNA template or DNA vector typically contains a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired nucleotide sequence for the desired mRNA and a termination signal (terminator).

[0276] In one aspect, the present invention provides a DNA vector encoding an mRNA comprising an optimized nucleotide sequence described herein. In some embodiments, the DNA vector further comprises a promoter and / or a terminator. In one embodiment, the promoter is an SP6 RNA polymerase promoter. In another embodiment, the promoter is a T7 RNA polymerase promoter. In some embodiments, the RNA polymerase promoter is operably linked to the optimized nucleotide sequence. In some embodiments, the nucleic acid is linear or circular.

[0277] Post-synthesis purification Various methods can be used to purify mRNA after synthesis. In some embodiments, mRNA is purified using tangential flow filtration (TFF). Suitable purification methods include those described in U.S. Patent Application Publication Nos. 2016 / 0040154, 2015 / 0376220, 2018 / 0251755, 2018 / 0251754, WO 2020 / 097509 filed November 8, 2019, and WO 2020 / 232371 filed May 15, 2020, all of which are incorporated herein by reference and may be used to practice the present invention. Because purity requirements for mRNA products are more stringent for therapeutic applications, it may be advantageous to purify the mRNA of the present invention, which may be included in a pharmaceutical composition in some embodiments of the present invention.

[0278] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing. In some embodiments, the mRNA is purified either before, after, or both before and after capping and tailing by centrifugation. In some embodiments, the mRNA is purified either before, after, or both before and after capping and tailing by filtration. In some embodiments, the mRNA is purified either before, after, or both before and after capping and tailing by tangential flow filtration (TFF).

[0279] Lipid nanoparticles (LNPs) Lipid nanoparticles (LNPs) encapsulating the mRNA of the present invention are also provided. In some embodiments, lipid nanoparticles suitable for use with the present invention comprise one or more cationic lipids, one or more non-cationic lipids (e.g., DOPE and / or cholesterol), and one or more PEG-modified lipids (e.g., DMG-PEG2K).

[0280] Typical lipid nanoparticles for use in the present invention are composed of four lipid components: a cationic lipid (e.g., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE), a cholesterol-based lipid (e.g., cholesterol), and a PEG-modified lipid (e.g., DMG-PEG2K). In certain embodiments, the non-cationic lipid is DOPE. The molar ratio of cationic lipid to non-cationic lipid to cholesterol to PEG-modified lipid is typically between about 30-60:25-35:20-30:1-15, respectively. Exemplary LNPs for use with the immunogenic compositions of the present invention may be composed of a cationic lipid selected from cKK-E12, cKK-E10, OF-Deg-Lin, and OF-02; a non-cationic lipid selected from DOPE and DEPE; a cholesterol-based lipid such as cholesterol; and a PEG-modified lipid such as DMG-PEG-2K.

[0281] In some embodiments, the lipid nanoparticles comprise three or fewer distinct lipid components. Exemplary lipid nanoparticles are composed of three lipid components: a cationic lipid (e.g., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE), and a PEG-modified lipid (e.g., DMG-PEG2K). In certain embodiments, the three distinct lipid components are HGT4002, DOPE, and DMG-PEG2K. In one exemplary embodiment, HGT4002, DOPE, and DMG-PEG2K are present in a molar ratio of about 60:35:5, respectively. Such LNPs may be particularly suitable for aerosol delivery of mRNA of the present invention.

[0282] Lipid nanoparticles for use in the present invention can be prepared by a variety of techniques currently known in the art, such as those described in U.S. Patent Application Publication Nos. 2011 / 0244026, 2016 / 0038432, 2018 / 0153822, 2018 / 0125989, and WO 2021 / 016430, filed July 23, 2020, all of which are incorporated herein by reference.

[0283] Lipid nanoparticle formulations In some embodiments, the majority of the LNPs in a composition of the invention, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNPs in the compositions of the invention have a size of about 150 nm or less (e.g., about 145 nm or less, about 140 nm or less, about 135 nm or less, about 130 nm or less, about 125 nm or less, about 120 nm or less, about 115 nm or less, about 110 nm or less, about 105 nm or less, about 100 nm or less, about 95 nm or less, about 90 nm or less, about 85 nm or less, or about 80 nm or less).

[0284] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs in compositions provided herein have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm). In some embodiments, the LNPs have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm). Compositions having LNPs with an average size of about 50-70 nm (e.g., 55-65 nm) may be particularly suitable for pulmonary delivery via nebulization.

[0285] In some embodiments, the dispersity, or molecular size heterogeneity measure (PDI), of the LNPs in the pharmaceutical compositions provided by the present disclosure is less than about 0.5. In some embodiments, the LNPs have a PDI less than about 0.5. In some embodiments, the LNPs have a PDI less than about 0.4. In some embodiments, the LNPs have a PDI less than about 0.3. In some embodiments, the LNPs have a PDI less than about 0.28. In some embodiments, the LNPs have a PDI less than about 0.25. In some embodiments, the LNPs have a PDI less than about 0.23. In some embodiments, the LNPs have a PDI less than about 0.20. In some embodiments, the LNPs have a PDI less than about 0.18. In some embodiments, the LNPs have a PDI less than about 0.16. In some embodiments, the LNPs have a PDI less than about 0.14. In some embodiments, the LNPs have a PDI less than about 0.12. In some embodiments, the LNPs have a PDI less than about 0.10. In some embodiments, the LNPs have a PDI of less than about 0.08.

[0286] In some embodiments, the LNPs have an encapsulation efficiency of greater than about 80%. In some embodiments, the LNPs have an encapsulation efficiency of greater than about 85%. In some embodiments, the LNPs have an encapsulation efficiency of greater than about 90%. In some embodiments, the LNPs have an encapsulation efficiency of greater than about 92%. In some embodiments, the LNPs have an encapsulation efficiency of greater than about 95%. In some embodiments, the LNPs have an encapsulation efficiency of greater than about 98%. In some embodiments, the LNPs have an encapsulation efficiency of greater than about 99%. Typically, LNPs for use with the compositions of the invention have an encapsulation efficiency of at least 90%-95%.

[0287] cationic lipids Various cationic lipids suitable for use in LNPs are known in the art. These include, for example, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DLinKC2DMA, DLin-KC2-DM, and C12-200. Exemplary cationic lipids suitable for use in the LNPs, compositions, pharmaceutical compositions, and methods of the invention are described herein, including, for example, cationic lipids such as those described in WO 2010 / 144740, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having the following compound structure: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate: [ka] and pharmaceutically acceptable salts thereof.

[0288] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include ionizable cationic lipids such as those described in WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein R and R are each independently hydrogen, an optionally substituted, undefined saturated or unsaturated C-C 20 Alkyl and optionally substituted, unspecified saturated or unsaturated C-C 20 acyl; L and L are each independently selected from the group consisting of hydrogen, optionally substituted C-C 30Alkyl, optionally substituted, unsaturated C-C 30 Alkenyl, and optionally substituted C-C 30 alkynyl; m and o are each independently selected from the group consisting of 0 and any positive integer (e.g., m is 3); and n is 0 or any positive integer (e.g., n is 1). In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention include a cationic lipid having the following compound structure: (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (“HGT5000”) [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having the following compound structure: (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (“HGT5001”) [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having the following compound structure: (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (“HGT5002”) [ka] and pharmaceutically acceptable salts thereof.

[0289] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include those cationic lipids described as amino alcohol lipidoids in WO 2010 / 053572, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0290] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2016 / 118725, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0291] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2016 / 118724, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0292] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0293] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, L Each instance of is independently an optionally substituted C-C 40 In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0294] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; and each R A are independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen; each R Bare independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention include a cationic lipid, "Target 23," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0295] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0296] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2020 / 097384, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, 1 and R 2 is independently H or a C1-C6 aliphatic; each m is independently an integer having a value from 1 to 4; each A is independently a covalent bond or arylene; each L 1 are independently an ester, thioester, disulfide, or anhydride group; each L 2 independently, C2-C 10 is aliphatic; each X 1 is independently H or OH; each R 3 independently, C6-C 20 In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0297] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In some embodiments, the cationic lipids of the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0298] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0299] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2017 / 004143, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0300] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-;L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 Alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 chloroalkenylene; R a is H or C1-C 12 alkyl; R 1 and R 2are independently C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and;R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2.

[0301] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0302] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids having the following formula: [ka] and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n Q is selected from -OR, -OH, -O(CH2) n and a heterocycle; n is 1, 2, or 3. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0303] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids such as those described in WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0304] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions, and methods of the invention include those cationic lipids described in WO 2022 / 066678, filed September 22, 2021, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having the following compound structure: [ka] (GL-TES-SA-DME-E18-2) and pharmaceutically acceptable salts thereof.

[0305] In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] (GL-TES-SA-DMP-E18-2) and pharmaceutically acceptable salts thereof.

[0306] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cleavable cationic lipids such as those described in WO 2021 / 202694, filed March 31, 2021, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids having the following compound structure: [ka] (SY-3-E14-DMAPr) and pharmaceutically acceptable salts thereof.

[0307] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cleavable cationic lipids such as those described in WO 2022 / 066916, filed September 23, 2021, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention include cationic lipids having the following compound structure: [ka] (HEP-E3-E10) and pharmaceutically acceptable salts thereof.

[0308] In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] (HEP-E4-E10) and pharmaceutically acceptable salts thereof.

[0309] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include those cationic lipids described in WO 2020 / 257716, filed June 19, 2020, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having a compound structure according to the following formula: [ka] or a pharmaceutically acceptable salt thereof, 2 , R 3 and R 4 each of which is independently C6-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 alkynyl; L 1 is C1-C 30 Alkylene; C2-C 30Alkenylene; or C2-C 30 alkynylene, and B 1 is an ionizable nitrogen-containing group. 1 is C1-C 10 In embodiments, L is alkylene. 1 is unsubstituted C1-C 10 In embodiments, L is alkylene. 1 is (CH2)2, (CH2)3, (CH2)4 or (CH2)5. In an embodiment, L 1 is (CH2), (CH2)6, (CH2)7, (CH2)8, (CH2)9 or (CH2) 10 In an embodiment, B 1 is independently NH, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl. 1 teeth, [ka] In an embodiment, B 1 teeth, [ka] In an embodiment, B 1 teeth, [ka] In an embodiment, R 2 , R 3 and R 4 each independently being an unsubstituted straight chain C-C 22 Alkyl, unsubstituted straight chain C6-C 22 Alkenyl, unsubstituted straight chain C6-C 22 Alkynyl, unsubstituted branched C6-C 22 Alkyl, unsubstituted branched C6-C 22 Alkenyl or unsubstituted branched C-C 22 In embodiments, R is alkynyl. 2 , R 3 and R 4 Each of 22In embodiments, R 2 , R 3 and R 4 Each of the is -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , -C 17 H 35 , -C 18 H 37 , -C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49 or -C 25 H 51 In an embodiment, R 2 , R 3 and R 4 each independently represents -O(CO)R 5 or -C(O)OR 5 C6-C substituted by 12 alkyl, where R 5 is unsubstituted C6-C 14 In an embodiment, R 2 , R 3 and R 4 Each of 22 In embodiments, R is an alkenyl. 2 , R 3 and R 4are -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8CH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18 CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH( CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2) 11 CH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0310] In an embodiment, the C6-C 22 Alkenyl is monoalkenyl, dienyl, or trienyl. In embodiments, R 2 , R 3 and R 4 Each of the [ka] In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0311] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions and methods of the invention include cleavable cationic lipids such as those described in WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; and R2 is selected from the group consisting of the following two formulae: [ka] and R3 and R4 each independently represent an optionally substituted saturated or unsaturated C6-C 20 Alkyl and optionally substituted, unspecified saturated or unsaturated C-C 20 acyl; n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid "HGT4001" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention include a cationic lipid, "HGT4002," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention include a cationic lipid, "HGT4003," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention include a cationic lipid, "HGT4004," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention include a cationic lipid, "HGT4005," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0312] Other suitable cationic lipids for use in the LNPs, compositions, pharmaceutical compositions, and methods of the invention include cleavable cationic lipids such as those described in WO 2019 / 222424, which is incorporated herein by reference. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having any of the general formulas or structures (1a)-(21a), (1b)-(21b), and (22)-(237) described in WO 2019 / 222424. In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise a cationic lipid having a structure according to Formula (I'): [ka] (In the formula, R X are independently -H, -L 1 -R 1 or -L 5A -L 5B -B'; L 1 , L 2 and L 3 each independently represents a covalent bond, —C(O)—, —C(O)O—, —C(O)S—, or —C(O)NR L - and; Each L 4A and L 5A are independently —C(O)—, —C(O)O—, or —C(O)NR L - and; Each L 4B and L 5B are independently C1-C 20 Alkylene; C2-C 20 Alkenylene; or C2-C 20 is alkynylene; Each B and B' is NR 4 R 5 or a 5-10 membered nitrogen-containing heteroaryl; Each R 1 , R 2 and R 3 independently, C6-C 30 Alkyl, C6-C 30Alkenyl or C6-C 30 is alkynyl; Each R 4 and R 5 are independently hydrogen, C1-C 10 Alkyl; C2-C 10 Alkenyl; or C2-C 10 is alkynyl; Each R L are independently hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 alkynyl).

[0313] In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise the following compound structure: [ka] The cationic lipid is compound (139) of WO 2019 / 222424, which has the formula:

[0314] In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid that is RL3-DMA-07D, having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0315] In some embodiments, the LNPs, compositions, pharmaceutical compositions and methods of the invention comprise a cationic lipid that is RL2-DMP-07D, having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0316] In some embodiments, the LNPs, compositions, pharmaceutical compositions, and methods of the invention comprise the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA") (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference). Other cationic lipids suitable for the LNPs, compositions, pharmaceutical compositions, and methods of the invention include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium ("DOSPA") (Behr et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference). Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0317] Further exemplary cationic lipids suitable for the LNPs, compositions, pharmaceutical compositions, and methods of the invention also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-di Methylammonium chloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholest-5-ene-3- beta-oxy)-3'-oxapentoxy)-3-dimethyl-1,1-(cis,cis-9',1-2'-octadecadienoxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA");(2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA(2R)”); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA(2R)”); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see WO 2010 / 042877; Semple et al., Nature Biotech. 28:172-176 (2010), incorporated herein by reference). (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); WO 2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole, dialkylamino, or guanidinium moiety.

[0318] In some embodiments, the one or more cationic lipids suitable for the LNPs, compositions, pharmaceutical compositions, and methods of the invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0319] In some embodiments, the LNPs, compositions, and pharmaceutical compositions of the invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the LNPs, compositions, and pharmaceutical compositions, e.g., lipid nanoparticles, measured by weight. In some embodiments, the LNPs, compositions, and pharmaceutical compositions of the invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the LNPs, compositions, and pharmaceutical compositions, e.g., lipid nanoparticles, measured as mol% of the total lipid content. In some embodiments, the LNPs, compositions, and pharmaceutical compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the LNPs, compositions, and pharmaceutical compositions, e.g., lipid nanoparticles, measured by weight. In some embodiments, the LNPs, compositions, and pharmaceutical compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the LNPs, compositions, and pharmaceutical compositions, e.g., lipid nanoparticles, measured by mol %.

[0320] Non-cationic lipids In some embodiments, the lipid nanoparticles contain one or more non-cationic lipids. As used herein, the term "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the term "anionic lipid" refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-ol. Examples of lipid nanoparticles suitable for use with the present invention include, but are not limited to, carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. In some embodiments, lipid nanoparticles suitable for use with the present invention comprise DOPE as a non-cationic lipid component. In other embodiments, lipid nanoparticles suitable for use with the present invention comprise DEPE as a non-cationic lipid component.

[0321] In some embodiments, the non-cationic lipid is a neutral lipid, i.e., a lipid that carries no net electrical charge under the conditions in which the LMP, composition, or pharmaceutical composition is formulated and / or administered.

[0322] Cholesterol-based lipids In some embodiments, lipid nanoparticles comprise one or more cholesterol-based lipids.For example, suitable cholesterol-based cationic lipids include DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al. Biochem. Biophys. Res. Comm. 179,280 (1991); Wolf et al. BioTechniques 23,139 (1997); U.S. Patent No. 5,744,335; all of which are incorporated herein by reference), or imidazole cholesterol ester (ICE) as disclosed in International Publication No. 2011 / 068810 (incorporated herein by reference), which has the following structure: [ka]

[0323] In some embodiments, the cholesterol-based lipid is cholesterol.

[0324] PEG modified lipid In some embodiments, the lipid nanoparticles comprise one or more PEGylated lipids.

[0325] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER) comprising N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), alone or preferably in combination with other lipid pharmaceutical compositions, including transfer vehicles (e.g., lipid nanoparticles), is also contemplated by the present invention.

[0326] Contemplated PEG-modified lipids include C6-C 20These include, but are not limited to, polyethylene glycol chains of 5 kD or less covalently attached to lipids with long alkyl chains. In some embodiments, the PEG-modified lipid or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components can prevent complex aggregation, prolong the circulatory life, and also provide a means for increasing the delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237; incorporated herein by reference), or they can be selected to be rapidly exchanged from pharmaceutical compositions in vivo (see U.S. Pat. No. 5,885,613; incorporated herein by reference). Particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 Lipid nanoparticles suitable for use with the present invention typically comprise a PEG-modified lipid, such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0327] In some embodiments, one or more PEG-modified lipids account for about 4% of total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids account for about 5% of total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids account for about 6% of total lipids by molar ratio. For certain applications, such as pulmonary delivery, lipid nanoparticles in which PEG-modified lipid components account for about 5% of total lipids by molar ratio have been found to be particularly suitable.

[0328] Exemplary Lipid Formulations Exemplary LNPs for use with the present invention may be composed of one of the following combinations of cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally cholesterol: cKK-E12, DOPE, cholesterol, and DMG-PEG2K; cKK-E10, DOPE, cholesterol, and DMG-PEG2K; OF-Deg-Lin, DOPE, cholesterol, and DMG-PEG2K; OF-02, DOPE, cholesterol, and DMG-PEG2K; GL-HEPES-E3-E12-DS-4-E10, DOPE, cholesterol, and DMG-PEG2K; C12-200, DOPE, cholesterol, and DMG-PEG2K; HGT4003, DOPE, cholesterol, and DMG-PEG2K; ICE, DOPE, cholesterol, and DMG-PEG2K; HGT4001, DOPE, cholesterol, and DMG-PEG2K; HGT4002, DOPE, cholesterol, and DMG-PEG2K; TL1-01D- DMA, DOPE, cholesterol, and DMG-PEG2K; TL1-04D-DMA, DOPE, cholesterol, and DMG-PEG2K; TL1-08D-DMA, DOPE, cholesterol, and DMG-PEG2K; TL1-10D-DMA, DOPE, cholesterol, and DMG-PEG2K; ICE, DOPE, and DMG-PEG2K; HGT4001, DOPE, and DMG-PEG2K; HGT4002, DOPE, and DMG-PEG2K; SY-3-E14-D MAPr, DOPE, cholesterol, and DMG-PEG2K; RL3-DMA-07D, DOPE, cholesterol, and DMG-PEG2K; RL2-DMP-07D, DOPE, cholesterol, and DMG-PEG2K; cHse-E-3-E10, DOPE, cholesterol, and DMG-PEG2K; cHse-E-3-E12, DOPE, cholesterol, and DMG-PEG2K; or cDD-TE-4-E12, DOPE, cholesterol, and DMG-PEG2K. In certain embodiments, LNPs may be composed of SY-3-E14-DMAPr, DOPE, cholesterol, and DMG-PEG2K. In other specific embodiments, LNPs may be composed of RL3-DMA-07D, DOPE, cholesterol, and DMG-PEG2K.In yet other specific embodiments, the LNPs may be composed of RL2-DMP-07D, DOPE, cholesterol, and DMG-PEG2K. In yet other specific embodiments, the LNPs may be composed of cHse-E-3-E10, DOPE, cholesterol, and DMG-PEG2K. In yet other specific embodiments, the LNPs may be composed of cHse-E-3-E12, DOPE, cholesterol, and DMG-PEG2K. In yet other specific embodiments, the LNPs may be composed of cDD-TE-4-E12, DOPE, cholesterol, and DMG-PEG2K.

[0329] In some embodiments, the cationic lipid (e.g., cKK-E12, cKK-E10, OF-Deg-Lin, OF-02, GL-HEPES-E3-E12-DS-4-E10, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, ICE, HGT4001, and / or HGT4002) comprises about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the lipid nanoparticles by molar ratio. In some embodiments, the percentage of cationic lipid (e.g., cKK-E12, cKK-E10, OF-Deg-Lin, OF-02, GL-HEPES-E3-E12-DS-4-E10, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, ICE, HGT4001 and / or HGT4002) is about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, or about 60% or more of the lipid nanoparticle by molar ratio.

[0330] In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids can be between about 30:60:25:35:20:30:1:15. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 40:30:20:10. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 40:30:25:5. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 40:32:25:3. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 50:25:20:5.

[0331] In certain embodiments, the LNPs comprise a 35% to 55% molar ratio of a cationic lipid (e.g., OF-02, GL-HEPES-E3-E12-DS-4-E10, or cKK-E10); a 5% to 40% molar ratio of a non-cationic lipid (e.g., DOPE); a 20% to 45% molar ratio of a cholesterol-based lipid (e.g., cholesterol); and a 1% to 2% molar ratio of a PEG-modified lipid (e.g., DMG-PEG2K).

[0332] In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is 40:30:28.5:1.5. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is 46.3:9.4:42.7:1.6. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is 50:10:38.5:1.5.

[0333] In some embodiments, the LNPs comprise a 40% molar ratio of OF-02, GL-HEPES-E3-E12-DS-4-E10, or c-KK-E10; a 30% molar ratio of DOPE; a 28.5% molar ratio of cholesterol; and a 1.5% molar ratio of DMG-PEG2K. In some embodiments, the LNPs comprise a 46.3% molar ratio of ALC-0315; a 9.4% molar ratio of DSPC; a 42.7% molar ratio of cholesterol; and a 1.6% molar ratio of ALC-0159. In some embodiments, the LNPs comprise a 50% molar ratio of SM-102; a 10% molar ratio of DSPC; a 38.5% molar ratio of cholesterol; and a 1.5% molar ratio of DMG-PEG2K. Such lipid nanoparticles are particularly suitable for delivery of mRNA via intramuscular administration.

[0334] In typical ternary lipid nanoparticles suitable for use with the present invention, the molar ratio of cationic lipid to non-cationic lipid to PEG-modified lipid can be about 55-65:30-40:1-15, respectively. In some embodiments, a molar ratio of cationic lipid (e.g., sterol-based lipid) to non-cationic lipid (e.g., DOPE or DEPE) to PEG-modified lipid (e.g., DMG-PEG2K) of 60:35:5 is particularly suitable for delivery of lipid nanoparticles, for example, via nebulization.

[0335] polymer In some embodiments, suitable LNP delivery vehicles are formulated using polymers as carriers, either alone or in combination with other carriers, including various lipids, as described herein. Thus, in some embodiments, LNP, as used herein, also encompasses nanoparticles comprising polymers. Suitable polymers can include, for example, polyacrylate, polyalkylcyanoacrylate, polylactide, polylactide-polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethyleneimine (PEI). When PEI is present, it can be branched PEI with a molecular weight ranging from 10 to 40 kDa, for example, 25 kDa branched PEI (Sigma #408727).

[0336] composition Compositions (e.g., immunogenic compositions or vaccines) of the invention may include one or more non-naturally occurring mRNAs encoding polyproteins of different serotypes of rhinovirus A and / or C and may be capable of eliciting an immune response against a wide variety of serotypes of rhinovirus A and / or C.

[0337] In some embodiments, immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus A and a second non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus C. In some embodiments, immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus C and a second non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus A.

[0338] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus A and a second non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus C.

[0339] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus A and a second non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus C.

[0340] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus A, a second non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus A, and a third non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus C.

[0341] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus A, a second non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus A, a third non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus C, and a fourth non-naturally occurring mRNA encoding at least one P2 polyprotein of rhinovirus C.

[0342] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a first VP0 polyprotein of rhinovirus C, a second non-naturally occurring mRNA encoding a first P2 polyprotein of rhinovirus C, a third non-naturally occurring mRNA encoding a second VP0 polyprotein of rhinovirus C, and a fourth non-naturally occurring mRNA encoding a second P2 polyprotein from rhinovirus C.

[0343] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a first VP0 polyprotein of rhinovirus C, a second non-naturally occurring mRNA encoding a first P2 polyprotein of rhinovirus C, a third non-naturally occurring mRNA encoding a second VP0 polyprotein of rhinovirus C, a fourth non-naturally occurring mRNA encoding a second P2 polyprotein from rhinovirus C, a fifth non-naturally occurring mRNA encoding a VP0 polyprotein of rhinovirus A, and a sixth non-naturally occurring mRNA encoding a P2 polyprotein of rhinovirus A.

[0344] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a VP0 polyprotein of rhinovirus A and a P2 polyprotein of rhinovirus A (e.g., as a fusion protein), and a second non-naturally occurring mRNA encoding at least one VP0 polyprotein of rhinovirus C.

[0345] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a first VP0 polyprotein of rhinovirus C and a first P2 polyprotein of rhinovirus C (e.g., as a fusion protein), and a second non-naturally occurring mRNA encoding a second VP0 polyprotein of rhinovirus C and a second P2 polyprotein from rhinovirus C (e.g., as a fusion protein).

[0346] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein and a rhinovirus A P2 polyprotein (e.g., as a fusion protein), and a second non-naturally occurring mRNA encoding a rhinovirus C VP0 polyprotein and a rhinovirus C P2 polyprotein (e.g., as a fusion protein).

[0347] In some embodiments, the immunogenic compositions of the invention comprise a first non-naturally occurring mRNA encoding a rhinovirus A VP0 polyprotein and a rhinovirus A P2 polyprotein (e.g., as a fusion protein), a second non-naturally occurring mRNA encoding a rhinovirus C first VP0 polyprotein and a rhinovirus C first P2 polyprotein (e.g., as a fusion protein), and a third non-naturally occurring mRNA encoding a rhinovirus C second VP0 polyprotein and a rhinovirus C second P2 polyprotein (e.g., as a fusion protein).

[0348] In some embodiments, the immunogenic compositions of the invention comprise non-naturally occurring mRNAs encoding a VP0 polyprotein of rhinovirus A, a first VP0 polyprotein of rhinovirus C, and a second VP0 polyprotein of rhinovirus C (e.g., as a fusion protein).

[0349] mRNA concentration The present invention provides compositions comprising mRNA of the present invention. In some embodiments, compositions according to the present invention comprise mRNA of the present invention at a concentration ranging from about 0.5 mg / mL to about 1.0 mg / mL. In some embodiments, the mRNA is at a concentration of at least 0.5 mg / mL. In some embodiments, the mRNA is at a concentration of at least 0.6 mg / mL. In some embodiments, the mRNA is at a concentration of at least 0.7 mg / mL. In some embodiments, the mRNA is at a concentration of at least 0.8 mg / mL. In some embodiments, the mRNA is at a concentration of at least 0.9 mg / mL. In some embodiments, the mRNA is at a concentration of at least 1.0 mg / mL. In an exemplary embodiment, the mRNA is at a concentration of about 0.6 mg / mL to about 0.8 mg / mL.

[0350] Pharmaceutically Acceptable Carriers and Excipients Typically, the mRNA in the composition is encapsulated in LNP. To stabilize the mRNA or the LNP encapsulating it, or to enhance the in vivo expression of the mRNA, the composition of the present invention can be formulated with one or more carriers, stabilizing agents, or other excipients. Such compositions can be pharmaceutical compositions, and therefore, they can contain one or more pharmaceutically acceptable excipients. The one or more pharmaceutically acceptable excipients can be selected from buffers, sugars, salts, surfactants, or combinations thereof.

[0351] In some embodiments, the pharmaceutical composition is formulated with a diluent. In some embodiments, the diluent is selected from the group consisting of ethylene glycol, glycerol, propylene glycol, sucrose, trehalose, or combinations thereof. In some embodiments, the formulation comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% diluent.

[0352] In some embodiments, the LNPs are suspended in an aqueous solution comprising a disaccharide. Suitable disaccharides for use with the present invention include trehalose and sucrose. For example, in some embodiments, the LNPs are suspended in an aqueous solution comprising trehalose, e.g., 10% (w / v) trehalose, in water. In other embodiments, the LNPs are suspended in an aqueous solution comprising sucrose, e.g., 10% (w / v) sucrose in water.

[0353] In some embodiments, the aqueous solution further comprises a buffer, a salt, a surfactant, or a combination thereof.

[0354] In some embodiments, the salt is selected from the group consisting of NaCl, KCl, and CaCl. Thus, in some embodiments, the salt is NaCl. In some embodiments, the salt is KCl. In some embodiments, the salt is CaCl.

[0355] In some embodiments, the buffer is selected from the group consisting of a phosphate buffer, a citrate buffer, an imidazole buffer, a histidine buffer, and a Good's buffer. Thus, in some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is an imidazole buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a Good's buffer. In some embodiments, the Good's buffer is a Tris buffer or a HEPES buffer.

[0356] In certain embodiments, the buffer is a phosphate buffer (e.g., a citrate-phosphate buffer), a Tris buffer (e.g., Tris-HCl), or an imidazole buffer. In some embodiments, the buffer is or comprises an acetate buffer.

[0357] In some embodiments, the compositions comprise a buffer and a salt (typically in addition to a suitable diluent such as a disaccharide or, optionally, propylene glycol), in which the total concentration of the buffer and salt is selected from about 40 mM Tris buffer and about 75-125 mM NaCl, about 50 mM Tris buffer and about 50 mM-100 mM NaCl, about 100 mM Tris buffer and about 100 mM-200 mM NaCl and about 40 mM imidazole, or about 100 mM-125 mM NaCl and about 50 mM imidazole and 75 mM-100 mM NaCl.

[0358] In some embodiments, the composition comprises a buffer (e.g., phosphate or Tris), a salt (e.g., KCl or NaCl, or both), and a sugar (e.g., a disaccharide such as sucrose or trehalose). In certain embodiments, the composition is an aqueous solution (e.g., including water for injection) comprising a buffer, a salt, and a sugar. Additional excipients may include NaOH or HCl (e.g., to adjust the pH of the composition).

[0359] Adjuvants In various embodiments, the immunogenic compositions (e.g., vaccines) described herein further comprise an adjuvant. Adjuvants can include suspensions of minerals (e.g., alum, aluminum salts (e.g., aluminum hydroxide / aluminum oxyhydroxide (AlOOH), aluminum phosphate (AlPO), aluminum hydroxyphosphate sulfate (AAHS), and / or aluminum potassium sulfate)) to which the antigen is adsorbed; or water-in-oil emulsions in which an antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), which may include killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant). In some embodiments, the adjuvant is squalene-based. Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199, which are incorporated herein by reference).Adjuvants can also include biological molecules such as lipids and costimulatory molecules. Exemplary biological adjuvants include AS04 (Didierlaurent et al., J Immunol. 2009;183(10):6186-97, incorporated herein by reference), IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41 BBL.

[0360] In certain embodiments, the immunogenic compositions (e.g., vaccines) of the invention do not include an adjuvant. For example, the compositions may include one or more non-naturally occurring mRNAs, e.g., encapsulated in one or more lipid nanoparticles without a separate adjuvant component.

[0361] Therapeutically effective amount: The mRNA according to the present invention is provided in a therapeutically effective amount in the pharmaceutical composition (for example, immunogenic composition or vaccine) provided herein.As used herein, the term "therapeutically effective amount" is mainly determined based on the total amount of therapeutic agent contained in the pharmaceutical composition of the present invention.Generally, a therapeutically effective amount is sufficient to achieve meaningful benefit to the subject.

[0362] Packaging Immunogenic compositions (e.g., vaccines) of the invention can be packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or mucosal (e.g., nasopharyngeal, pulmonary, or intranasal) administration. Vaccine compositions can be in the form of, for example, a lyophilized, ready-to-use formulation that requires reconstitution with a physiological buffer (e.g., PBS) immediately prior to use. In some embodiments, immunogenic compositions (e.g., vaccines) of the invention are provided in the form of an aqueous or frozen aqueous solution and can be administered directly to a subject without reconstitution (after thawing if previously frozen).

[0363] Thus, the present disclosure provides articles of manufacture, such as kits, that provide the immunogenic compositions (e.g., vaccines) of the invention in a single container, or that provide the compositions (e.g., vaccines) in one container and a physiological buffer for reconstitution in another container. The containers may contain single-use doses or multi-use doses. The containers may be pre-treated glass vials or ampoules. The articles of manufacture may also include instructions for use.

[0364] In certain embodiments, the immunogenic compositions (e.g., vaccines) of the present invention are provided for use by intramuscular injection. The compositions can be injected into a subject, for example, into the deltoid muscle of the subject's upper arm. In some embodiments, the immunogenic compositions (e.g., vaccines) are provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the immunogenic compositions (e.g., vaccines) are provided for use by mucosal administration (e.g., as an intranasal spray). In some embodiments, the immunogenic compositions (e.g., vaccines) are provided for use by inhalation (e.g., for pulmonary delivery) and are provided in a pre-filled pump, aerosol generator, or inhaler.

[0365] In certain embodiments, the immunogenic composition (e.g., vaccine) is provided for cutaneous injection, e.g., into the epidermis, dermis, or subcutaneously of the skin. In some embodiments, the composition is provided in a device suitable for cutaneous injection, such as a needle (e.g., an epidermal, dermal, or hypodermic needle), a needle-free device, a microneedle device, or a microprojection array device. Examples of microneedle or microprojection array devices suitable for cutaneous injection are described in U.S. Patent Application Publication Nos. 20230270842A1, 20220339416A1, 20210085598A1, 20200246450A1, 20220143376A1, 20180264244A1, 20180263641A1, and 20110245776A1.

[0366] therapeutic use In some embodiments, the invention provides methods for eliciting an immune response in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein. In some embodiments, the invention provides immunogenic compositions comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein for use in eliciting an immune response in a subject. In some embodiments, the invention provides use of a composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein in a method of manufacturing a medicament, wherein the composition is for eliciting an immune response in a subject (e.g., formulated for eliciting an immune response).

[0367] In some embodiments, the invention provides methods for reducing or preventing one or more symptoms associated with rhinovirus infection in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein. In some embodiments, the invention provides immunogenic compositions comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein for use in reducing or preventing one or more symptoms associated with rhinovirus infection in a subject. In some embodiments, the invention provides use of a composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein in a method of manufacturing a medicament, wherein the composition is for reducing or preventing one or more symptoms associated with rhinovirus infection in a subject (e.g., formulated for the purpose).

[0368] In some embodiments, the invention provides methods for reducing the severity of a rhinovirus infection in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein. In some embodiments, the invention provides immunogenic compositions comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein for use in reducing the severity of a rhinovirus infection in a subject. In some embodiments, the invention provides use of a composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein in a method of manufacturing a medicament, wherein the composition is for (e.g., formulated for) reducing the severity of a rhinovirus infection in a subject.

[0369] In some embodiments, the invention provides methods for preventing rhinovirus infection in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein. In some embodiments, the invention provides immunogenic compositions comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein for use in preventing rhinovirus infection in a subject. In some embodiments, the invention provides use of a composition comprising one or more non-naturally occurring messenger RNAs (mRNAs) encoding one or more non-structural and / or one or more structural rhinovirus polypeptides as described herein in a method for the manufacture of a medicament, wherein the composition is for (e.g., formulated to) prevent rhinovirus infection in a subject.

[0370] In some embodiments, administration of the immunogenic compositions of the invention enhances or converts an existing rhinovirus T cell response to T H In some embodiments, the present invention provides a method for enhancing or redirecting an existing rhinovirus T cell response in a subject toward a T cell response. H In some embodiments, the present invention provides a composition for use in enhancing or redirecting an existing rhinovirus T cell response in a subject, the composition comprising an effective amount of the immunogenic composition of the invention. H The present invention provides a method for producing a composition for redirecting a response towards a human immunodeficiency virus (HIV)-1 (HIV-1) antigen, the composition being an immunogenic composition of the invention.

[0371] In some embodiments of the invention, administration of the immunogenic composition induces intracellular antibodies against one or more nonstructural polypeptides encoded by the mRNA. In some embodiments, the invention provides a composition for use in inducing intracellular antibodies against one or more nonstructural polypeptides encoded by the mRNA in a subject, the composition comprising an effective amount of the immunogenic composition of the invention. In some embodiments, the invention provides a method for producing a composition for inducing intracellular antibodies against one or more nonstructural polypeptides encoded by the mRNA in a subject, the composition being an immunogenic composition of the invention.

[0372] In some embodiments of the invention, administration of the immunogenic composition provides immunity against rhinovirus infection caused by group A, group B, and / or group C strains. In some embodiments, administration provides immunity against infection caused by multiple rhinovirus serotypes. In some embodiments, the multiple serotypes are from the same group of rhinovirus (e.g., group A or group C). For example, in some embodiments, immunity is provided against one or more rhinovirus A serotypes. In some embodiments, immunity is provided against about 20 or more, about 30 or more, about 40 or more, or about 50 or more rhinovirus A serotypes. In some embodiments, immunity is provided against one or more rhinovirus A serotypes and one or more rhinovirus C serotypes.

[0373] In some embodiments, the immunogenic compositions of the invention are administered prophylactically.

[0374] In an alternative embodiment, the immunogenic compositions of the invention are administered after rhinovirus symptoms and / or confirmation that the subject has a rhinovirus infection.

[0375] In some embodiments, compositions of the invention are formulated for parenteral administration, such as intramuscular, intravenous, subcutaneous, intraperitoneal, or intradermal administration. In some embodiments, compositions of the invention are formu...

Claims

1. 1. A method for identifying rhinovirus polyproteins for use as immunogens capable of eliciting an immune response against rhinoviruses from multiple serotypes within a group, comprising: a. obtaining a plurality of amino acid sequences from a database containing amino acid sequences from natural rhinovirus isolates; b. removing amino acid sequences shorter than 800 amino acids from the plurality of amino acid sequences obtained in step (a); c. assigning the amino acid sequences remaining after step (b) to different phylogenetic clusters; d. aligning the amino acid sequences to determine a consensus amino acid sequence for the complete rhinovirus polyprotein for one or more phylogenetic clusters identified in step (c); e. aligning the consensus amino acid sequence obtained in step (c) with the complete polyprotein of a naturally occurring rhinovirus isolate; f. Selecting a rhinovirus polyprotein as an immunogen that has an average identity of at least 80% with the corresponding amino acid sequence of a rhinovirus from at least two phylogenetic clusters identified in step (c). A method comprising:

2. 2. The method of claim 1, wherein the rhinovirus polyprotein selected in step (f) is VP0 polyprotein.

3. 2. The method of claim 1, wherein the rhinovirus polyprotein selected in step (f) is the P2 polyprotein.

4. The method of any one of claims 1 to 3, wherein the group is rhinovirus group A.

5. The method according to any one of claims 1 to 3, wherein the group is rhinovirus group C.

6. The method of any one of claims 1 to 3, wherein the one or more phylogenetic clusters each comprise at least five different serotypes.

7. 7. The method of claim 6, wherein the one or more phylogenetic clusters each comprise at least 10, 15, 20, or 25 different serotypes.

8. 8. The method of claim 1, wherein the plurality of amino acid sequences obtained in step (a) is greater than 400.

9. 9. The method of claim 1, wherein determining the consensus sequence in step (d) comprises selecting the most frequent amino acid at each position.

10. 10. The method of claim 1, wherein the determination of the consensus sequence in step (d) comprises generating a gap if the sum of the amino acids for a given position is less than 50% of the number of sequences obtained.

11. 10. The method of any one of claims 1 to 9, wherein determining the consensus sequence in step (d) comprises selecting the most frequent amino acid when the sum of the amino acids for a given position is greater than or equal to 50% of the number of sequences obtained.

12. 12. The method of any one of claims 1 to 11, further comprising generating an optimized nucleic acid sequence encoding the rhinovirus polyprotein selected in step (f).

13. 1. An immunogenic composition comprising at least one messenger RNA (mRNA) comprising a non-native, optimized nucleic acid sequence encoding a polyprotein from a group A or C rhinovirus, wherein the polyprotein comprises: a. has an average identity of at least 80% with the amino acid sequence of corresponding polyproteins from at least two phylogenetic clusters of rhinoviruses of the same group; b. Naturally occurring except for an optional single amino acid substitution; An immunogenic composition comprising an amino acid sequence.

14. The immunogenic composition of claim 13, wherein the amino acid sequence of the polyprotein has an average identity of at least 80% with the amino acid sequences of corresponding polyproteins from at least three, for example four, phylogenetic clusters of rhinoviruses of the same group.

15. The immunogenic composition of claim 13 or 14, wherein the polyprotein is the VP0 polyprotein comprising proteins VP2 and VP4.

16. The immunogenic composition of claim 15, wherein the VP0 polyprotein is derived from a group C rhinovirus.

17. The immunogenic composition of claim 16, wherein the group C rhinovirus is serotype 11, 17, or 34.

18. 18. The immunogenic composition of any one of claims 13 to 17, wherein the polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:

1.

19. 18. The immunogenic composition of any one of claims 13 to 17, wherein the polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:

2.

20. 18. The immunogenic composition of any one of claims 13 to 17, wherein the polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 34 VP0 polyprotein set forth in SEQ ID NO:

3.

21. The immunogenic composition of claim 15, wherein the VP0 polyprotein is derived from a group A rhinovirus.

22. 22. The immunogenic composition of claim 21, wherein the group A rhinovirus is serotype 21 or 90.

23. 23. The immunogenic composition of claim 21 or 22, wherein the polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:

4.

24. 23. The immunogenic composition of claim 21 or 22, wherein the polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:

5.

25. 15. The immunogenic composition of claim 13 or 14, wherein the polyprotein is a P2 polyprotein comprising proteins 2A, 2B and 2C.

26. 26. The immunogenic composition of claim 25, wherein the single amino acid substitution is in the 2A protein and reduces or eliminates the proteolytic activity of the P2 polyprotein.

27. 27. The immunogenic composition of claim 26, wherein the single amino acid substitution is a C>A or C>S substitution in the catalytic triad of the active site of the 2A protein.

28. The immunogenic composition according to any one of claims 25 to 27, wherein the P2 polyprotein is derived from a group A rhinovirus.

29. 29. The immunogenic composition of claim 28, wherein the group A rhinovirus is serotype 21 or 57.

30. 30. The immunogenic composition of claim 29, wherein the polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO:

6.

31. 30. The immunogenic composition of claim 29, wherein the polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 57 P2 polyprotein set forth in SEQ ID NO:

7.

32. The immunogenic composition according to any one of claims 25 to 27, wherein the P2 polyprotein is derived from a group C rhinovirus.

33. 33. The immunogenic composition of claim 32, wherein the group C rhinovirus is of serotype 11 or 17.

34. 34. The immunogenic composition of claim 33, wherein the polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus C serotype 11 P2 polyprotein set forth in SEQ ID NO:

8.

35. 34. The immunogenic composition of claim 33, wherein the polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus C serotype 17 P2 polyprotein set forth in SEQ ID NO:

9.

36. 36. The immunogenic composition of any one of claims 1 to 35, further comprising a second non-native optimized nucleic acid sequence encoding an additional polyprotein from a group A or C rhinovirus, wherein the additional polyprotein is distinct from the polyprotein.

37. 37. The immunogenic composition of claim 36, wherein the first and second non-naturally occurring optimized nucleic acid sequences are part of the same mRNA.

38. The immunogenic composition of claim 37, wherein the mRNA encodes a fusion protein comprising the polyprotein and an additional polyprotein.

39. 38. The immunogenic composition of claim 37, wherein the first and second non-naturally occurring optimized nucleic acid sequences are encoded by distinct mRNAs.

40. 1. An immunogenic composition comprising at least one messenger RNA (mRNA), (i) a first non-native optimized nucleic acid sequence encoding a first polyprotein from a Group A or Group C rhinovirus; (ii) a second non-native optimized nucleic acid sequence encoding a second polyprotein from a Group A or Group C rhinovirus; and Including, the second polyprotein is different from the first polyprotein, and each of the first polyprotein and the second polyprotein is a. have an average identity of at least 80% with the amino acid sequence of corresponding polyproteins from at least two phylogenetic clusters of rhinoviruses of the same group; and b. Naturally occurring except for an optional single amino acid substitution; An immunogenic composition comprising an amino acid sequence.

41. The immunogenic composition of claim 40, wherein the amino acid sequence of each of the first and second polyproteins has an average identity of at least 80% with the amino acid sequences of corresponding polyproteins from at least three, for example four, phylogenetic clusters of rhinoviruses of the same group.

42. 40. The immunogenic composition of claim 40 or 39, wherein the first polyprotein is a VP0 polyprotein comprising proteins VP2 and VP4.

43. 43. The immunogenic composition of claim 42, wherein the VP0 polyprotein is derived from a group A rhinovirus.

44. 44. The immunogenic composition of claim 43, wherein the group A rhinovirus is of serotype 21 or 90.

45. 44. The immunogenic composition of claim 42 or 43, wherein the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:

4.

46. 44. The immunogenic composition of claim 42 or 43, wherein the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:

5.

47. The immunogenic composition of any one of claims 40 to 46, wherein the second polyprotein is the P2 polyprotein comprising proteins 2A, 2B and 2C.

48. 48. The immunogenic composition of claim 47, wherein the single amino acid substitution in the 2A protein reduces or eliminates the proteolytic activity of the P2 polyprotein.

49. 49. The immunogenic composition of claim 48, wherein the single amino acid substitution is a C>A substitution or a C>S substitution in the catalytic triad of the active site of the 2A protein.

50. The immunogenic composition of any one of claims 47 to 49, wherein the P2 polyprotein is derived from a group A rhinovirus.

51. 51. The immunogenic composition of claim 50, wherein the group A rhinovirus is of serotype 21 or 57.

52. 52. The immunogenic composition of any one of claims 47 to 51, wherein the second polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO:

6.

53. 52. The immunogenic composition of any one of claims 47 to 51, wherein the second polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 57 P2 polyprotein set forth in SEQ ID NO:

7.

54. The immunogenic composition of any one of claims 40 to 46, wherein the second polyprotein is the VP0 polyprotein from a group C rhinovirus.

55. 55. The immunogenic composition of claim 54, wherein the group C rhinovirus is of serotype 11, 17, or 34.

56. 56. The immunogenic composition of claim 54 or 55, wherein the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:

1.

57. 56. The immunogenic composition of claim 54 or 55, wherein the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:

2.

58. 56. The immunogenic composition of claim 54 or 55, wherein the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 34 VP0 polyprotein set forth in SEQ ID NO:

3.

59. The immunogenic composition of claim 40 or 41, wherein the first polyprotein is a VP0 polyprotein and the second polyprotein is a VP0 polyprotein, and the two phylogenetic clusters mentioned in option a are different for the first polyprotein and the second polyprotein.

60. 60. The immunogenic composition of claim 59, wherein the first and second polyproteins are derived from rhinovirus C.

61. 61. The immunogenic composition of claim 59 or 60, wherein the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO: 1, and the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:

2.

62. 42. The immunogenic composition of claim 40 or 41, wherein the first polyprotein is P2 polyprotein and the second polyprotein is P2 polyprotein, and the two phylogenetic clusters mentioned in option a are different for the first polyprotein and the second polyprotein.

63. 63. The immunogenic composition of claim 62, wherein the first and second polyproteins are derived from rhinovirus C.

64. 64. The immunogenic composition of claim 62 or 63, wherein the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 11 P2 polyprotein set forth in SEQ ID NO: 8, and the second polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 17 P2 polyprotein set forth in SEQ ID NO:

9.

65. 65. The immunogenic composition of any one of claims 40 to 64, wherein the first and second nucleic acid sequences are part of the same mRNA.

66. 66. The immunogenic composition of claim 65, wherein the mRNA encodes a fusion protein comprising the first polyprotein and the second polyprotein.

67. 67. The immunogenic composition of any one of claims 40 to 66, wherein the first and second nucleic acid sequences are encoded by separate mRNAs.

68. 68. The immunogenic composition of any one of claims 40 to 67, further comprising a third non-native optimized nucleic acid sequence encoding a third polyprotein from a group A or group C rhinovirus, wherein the third polyprotein is different from the first and second polyproteins.

69. The immunogenic composition of any one of claims 40 to 68, wherein the composition is capable of eliciting a T cell response in at least 95% of the human population.

70. 70. The immunogenic composition of claim 69, wherein the composition is capable of eliciting a T cell response in at least 96%, at least 97%, at least 98%, or at least 99% of the human population.

71. 54. The immunogenic composition of any one of claims 47 to 53, wherein the VP0 polyprotein and the P2 polyprotein comprise T cell epitope-rich regions that cover at least 95% of the MHC class I alleles of Table 4 and / or 95% of the MHC-II alleles of Table 5.

72. 72. The immunogenic composition of claim 71, wherein the T-cell epitope-rich regions cover at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class-I alleles of Table 4 and / or at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class-II alleles of Table 5.

73. 1. An immunogenic composition comprising at least one messenger RNA (mRNA), (i) a first non-native optimized nucleic acid sequence encoding a first polyprotein from a Group A or Group C rhinovirus; (ii) a second non-native optimized nucleic acid sequence encoding a second polyprotein from a group A or C rhinovirus; and (iii) a third non-native optimized nucleic acid sequence encoding a third polyprotein from a Group A or Group C rhinovirus; Including; The first, second, and third polyproteins are different from one another, and each of the first, second, and third polyproteins comprises: a. has an average identity of at least 80% with the amino acid sequence of corresponding polyproteins from at least two phylogenetic clusters of rhinoviruses of the same group; b. Naturally occurring except for an optional single amino acid substitution; An immunogenic composition comprising an amino acid sequence.

74. 74. The immunogenic composition of claim 73, wherein the first polyprotein is a VP0 polyprotein comprising proteins VP2 and VP4.

75. 75. The immunogenic composition of claim 74, wherein the VP0 polyprotein is derived from a group A rhinovirus.

76. 76. The immunogenic composition of claim 75, wherein the group A rhinovirus is of serotype 21 or 90.

77. 77. The immunogenic composition of claim 75 or 76, wherein the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 21 VP0 polyprotein set forth in SEQ ID NO:

4.

78. 77. The immunogenic composition of claim 75 or 76, wherein the first polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus A serotype 90 VP0 polyprotein set forth in SEQ ID NO:

5.

79. 79. The immunogenic composition of any one of claims 73 to 78, wherein the second polyprotein is the P2 polyprotein comprising proteins 2A, 2B and 2C.

80. 80. The immunogenic composition of claim 79, wherein the single amino acid substitution is in the 2A protein and reduces or eliminates the proteolytic activity of the P2 polyprotein.

81. 81. The immunogenic composition of claim 80, wherein the single amino acid substitution is a C>A substitution or a C>S substitution in the catalytic triad of the active site of the 2A protein.

82. The immunogenic composition of any one of claims 79 to 81, wherein the P2 polyprotein is derived from a group A rhinovirus.

83. 83. The immunogenic composition of claim 82, wherein the group A rhinovirus is of serotype 21 or 57.

84. 84. The immunogenic composition of any one of claims 79 to 83, wherein the second polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 21 P2 polyprotein set forth in SEQ ID NO:

6.

85. 84. The immunogenic composition of any one of claims 79 to 83, wherein the second polyprotein has an amino acid sequence that is at least 80% identical, or identical except for an optional single amino acid substitution, to the amino acid sequence of the rhinovirus A serotype 57 P2 polyprotein set forth in SEQ ID NO:

7.

86. 86. The immunogenic composition of any one of claims 73 to 85, wherein the at least one mRNA encodes a fusion protein comprising the first polyprotein and the second polyprotein, and optionally the third polyprotein.

87. 86. The immunogenic composition of any one of claims 73 to 85, wherein the first, second and third non-naturally occurring optimized nucleic acid sequences are encoded by distinct mRNAs.

88. 88. The immunogenic composition of any one of claims 73 to 87, wherein the third polyprotein is the VP0 polyprotein from a group C rhinovirus.

89. 89. The immunogenic composition of claim 88, wherein the group C rhinovirus is of serotype 11, 17, or 34.

90. 90. The immunogenic composition of claim 88 or 89, wherein the third polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 11 VP0 polyprotein set forth in SEQ ID NO:

1.

91. 90. The immunogenic composition of claim 88 or 89, wherein the third polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 17 VP0 polyprotein set forth in SEQ ID NO:

2.

92. 90. The immunogenic composition of claim 88 or 89, wherein the third polyprotein has an amino acid sequence that is at least 80% identical or identical to the amino acid sequence of the rhinovirus C serotype 34 VP0 polyprotein set forth in SEQ ID NO:

3.

93. The immunogenic composition of any one of claims 73 to 92, wherein the composition is capable of eliciting a T cell response in at least 95% of the human population.

94. 94. The immunogenic composition of claim 93, wherein the composition is capable of eliciting a T cell response in at least 96%, at least 97%, at least 98%, or at least 99% of the human population.

95. 95. The immunogenic composition of any one of claims 73 to 94, wherein the VP0 polyprotein and the P2 polyprotein comprise T cell epitope-rich regions that cover at least 95% of the MHC class I alleles of Table 4 and / or 95% of the MHC-II alleles of Table 5.

96. 96. The immunogenic composition of claim 95, wherein the T-cell epitope-rich regions cover at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class-I alleles in Table 4 and / or at least 96%, at least 97%, at least 98%, or at least 99% of the MHC class-II alleles in Table 5.

97. 97. The immunogenic composition of any one of claims 1 to 96, wherein the first, second and (optional) third non-naturally occurring optimized nucleic acid sequences are optimized to (a) improve the yield of full-length mRNA during in vitro synthesis, and / or (b) maximize expression of the encoded polypeptide following delivery of the mRNA to a target cell in vivo.

98. 98. The immunogenic composition of any one of claims 1 to 97, wherein the at least one mRNA or the at least two mRNAs comprise a 5' untranslated region (UTR).

99. 99. The immunogenic composition of claim 98, wherein the 5'UTR has the nucleotide sequence set forth in SEQ ID NO:

10.

100. 100. The immunogenic composition of any one of claims 1 to 99, wherein the at least one mRNA or the at least two mRNAs further comprise a 3' untranslated region (UTR).

101. The immunogenic composition of claim 100, wherein the 3'UTR has the nucleotide sequence set forth in SEQ ID NO: 11, 12 or 13.

102. The immunogenic composition of any one of claims 1 to 101, wherein the at least one mRNA or the at least two mRNAs comprise a 5' cap.

103. The immunogenic composition of any one of claims 1 to 102, wherein the at least one mRNA or the at least two mRNAs comprise a polyadenylation (polyA) sequence comprising at least 90 nucleotides.

104. The immunogenic composition of any one of claims 1 to 103, wherein said at least one mRNA or said at least two mRNAs contain N-1-methylpseudouridine instead of uridine.

105. The immunogenic composition of any one of claims 1 to 104, further comprising a plurality of lipid nanoparticles (LNPs) encapsulating the at least one mRNA or the at least two mRNAs.

106. The immunogenic composition of claim 105, wherein the lipid component of the LNP comprises or consists of cationic lipids, non-cationic lipids, PEG-modified lipids, and optionally sterol-based lipids.

107. a. The cationic lipid is cKK-E12, cKK-E10, HGT5000, HGT5001, ICE, HGT4001, HGT4002, HGT4003, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, OF-Deg-Lin, OF-02, GL-TES-SA-DMP-E18-2, GL-TES-SA-DME-E18-2, SY-3-E14-DMAPr, TL1-10D -DMA, HEP-E3-E10, HEP-E4-E10, RL3-DMA-07D, RL2-DMP-07D, cHse-E-3-E10, cHse-E-3-E12, cDD-TE-4-E12, SI-4-E14-DMAPr, TL-1-12D-DMA, SY-010, SY-011, and 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate (ALC-0315); b. the non-cationic lipid is selected from DSPC (1,2-diasteroyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, DOPC (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)); c. the PEG-modified lipid is selected from DMG-PEG-2K and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and / or d. The sterol lipid is cholesterol; The immunogenic composition of claim 106.

108. The immunogenic composition of any one of claims 105 to 107, wherein the cationic lipid is selected from cKK-E10 and ALC-0315.

109. The immunogenic composition of any one of claims 105 to 108, wherein the pegylated lipid is selected from DMG-PEG2K or ALC-0159.

110. The immunogenic composition of any one of claims 105 to 109, wherein the non-cationic lipid is selected from DOPE or DSPC.

111. A vaccine composition comprising the immunogenic composition of any one of claims 1 to 110 and a pharmaceutically acceptable carrier.

112. 112. A method for inducing an immune response in a subject, the method comprising administering to the subject an effective amount of the immunogenic composition of any one of claims 1 to 110 or the vaccine composition of claim 111.

113. A method for reducing or preventing one or more symptoms associated with a rhinovirus infection in a subject, the method comprising administering to the subject an effective amount of an immunogenic composition described in any one of claims 1 to 110 or a vaccine composition described in claim 111.

114. A method for reducing or preventing the severity of a rhinovirus infection in a subject, comprising administering to the subject an effective amount of an immunogenic composition described in any one of claims 1 to 110 or a vaccine composition described in claim 111.

115. administering the vaccine composition enhances or converts the pre-existing rhinovirus T cell response to T cells. Ｈ 115. The method of any one of claims 112 to 114, wherein the method redirects the data towards one response.

116. 116. The method of any one of claims 112 to 115, wherein the subject suffers from asthma or chronic obstructive pulmonary disease (COPD).

117. 117. The method of claim 116, wherein administration of the vaccine composition reduces or prevents exacerbated symptoms associated with asthma or COPD.

118. 118. The method of any one of claims 112 to 117, wherein the subject is 40 years of age or older.

119. 119. The method of any one of claims 112 to 118, wherein the subject is 65 years of age or older.

120. 120. The method of any one of claims 112 to 119, wherein the immunogenic or vaccine composition is administered intramuscularly.

121. 121. The method of any one of claims 112 to 120, wherein the immunogenic or vaccine composition is administered to the subject once.

122. 122. The method of any one of claims 112 to 121, wherein the immunogenic or vaccine composition is administered to the subject multiple times.

123. 123. The method of claim 122, wherein the immunogenic composition or the vaccine composition is administered at least twice.

124. 124. The method of claim 122 or 123, wherein the second administration, or any subsequent administration, occurs about one year or more after the first administration.

125. 123. The method of claim 122, wherein the immunogenic or vaccine composition is administered once a year.

126. 123. The method of claim 122, wherein the immunogenic composition or the vaccine composition is administered twice, five years apart.

127. 127. The method of any one of claims 112 to 126, wherein administration of the immunogenic composition or the vaccine composition provides immunity against rhinovirus infection caused by group A and / or group C strains.

128. 128. The method of claim 127, wherein immunity is provided against multiple serotypes of the same rhinovirus group.

129. 129. The method of claim 128, wherein immunity is provided against multiple serotypes of different rhinovirus groups.

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