Rhinovirus vaccine

Abstract

The present invention relates to immunogenic compositions, particularly immunogenic compositions for preventing, treating, or ameliorating human rhinovirus (RV) infection. The present invention particularly relates to RV VP0 peptides (or proteins) and polynucleotides encoding such peptides, and their use in immunogenic compositions to elicit an immune response and prevent rhinovirus infection.

Description

[Technical field]

[0001] The present invention relates to immunogenic compositions, particularly immunogenic compositions for preventing, treating, or ameliorating human rhinovirus (RV) infection. The present invention particularly relates to RV VP0 peptides (or proteins) and polynucleotides encoding such peptides, and their use in immunogenic compositions to elicit an immune response and prevent rhinovirus infection. [Background technology]

[0002] Rhinoviruses (RVs) are picornaviruses that are small, single-stranded RNA viruses with genome sizes of approximately 6.8–7.2 Kb [1]. RVs are the major cause of acute attacks (exacerbations) of pulmonary diseases, such as the common cold, viral wheezing illnesses of childhood, asthma, bronchiectasis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and chronic fibrosing lung diseases [2,3]. RVs cause approximately 75%–90% of such diseases.

[0003] Currently, there are no effective anti-RV treatments and anti-RV vaccines. The main obstacle in the development of an RV vaccine has been the circulation of numerous genetically and antigenically distinct strains. Recent studies have estimated that there are nearly 180 genetically distinct strains of RV in circulation [4]. These strains are classified into three species based on their genomic identity: RV-A, RV-B, and RV-C [1,5]. RV-A and RV-C are the most numerous species and the most important in terms of clinical disease. RV-A and RV-C are responsible for the majority of exacerbations of asthma and COPD and childhood wheezing diseases that result in hospitalization. However, RV-B is also associated with an increased risk of wheezing diseases [7]. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680 [Non-Patent Document 2] Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882 [Non-Patent Document 3] Bartlett et al., 2008, Nat. Med. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need to provide immunogenic compositions capable of protecting individuals against many, if not all, RV strains. [Means for solving the problem]

[0006] Using detailed bioinformatic analyses, we identified an RV VP0 protein from a single representative strain that, when used as a vaccine immunogen, surprisingly elicited cellular immunity against all other members of that RV species.

[0007] Thus, according to a first aspect of the present invention there is provided an immunogenic composition comprising at least one isolated Human Rhinovirus (RV) VP0 peptide or an isolated polynucleotide encoding said peptide, wherein the peptide is selected from the group consisting of RV-C VP0 peptide, RV-B VP0 peptide, RV-A28 VP0, and RV-A89 VP0, or a variant or fragment thereof.

[0008] As shown in the Examples, the inventors have surprisingly discovered that the novel rhinovirus immunogens can induce a broad range of cellular immune responses that cross-react with other RV strains of the same species. For example, as shown in Figures 4, 8, and 9, splenocytes from mice immunized with RV-B06 VP0 cross-react with VP0 peptides from other members of the RV-B species, and splenocytes from mice immunized with RV-C19 VP0 or RV-C24 VP0 cross-react with VP0 peptides from other members of the RV-C species, respectively. Furthermore, the inventors have demonstrated that immunization with mRNA encoding RV VP0 peptides induces a broad range of cellular immune responses. For example, as shown in Figure 15, the inventors have demonstrated that immunization of mice with RV-C24 VP0 mRNA induces cellular immunity that cross-reacts with other members of the RV C species. Thus, advantageously, the immunogenic composition according to the present invention overcomes the challenge of antigenic heterogeneity among RV species, which has significantly hindered the development of RV vaccines to date.

[0009] In a preferred embodiment, the immunogenic composition comprises the RV-C VP0 peptide.

[0010] In another preferred embodiment, the immunogenic composition comprises the RV-B VP0 peptide.

[0011] In another preferred embodiment, the immunogenic composition comprises RV-A28 VP0 and / or RV-A89 VP0.

[0012] Alternatively, in another embodiment, the immunogenic composition comprises at least two isolated human rhinovirus (RV) VP0 peptides, or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-C VP0 peptide, RV-B VP0 peptide, RV-A28 VP0, and RV-A89 VP0.

[0013] In another preferred embodiment, the immunogenic composition comprises at least three isolated human rhinovirus (RV) VP0 peptides, or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-C VP0 peptide, RV-B VP0 peptide, RV-A28 VP0, and RV-A89 VP0.

[0014] Preferably, the RV-C VP0 peptide is selected from the group consisting of RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or a variant or fragment thereof.

[0015] Preferably, the RV-B VP0 peptide is RV-B06 VP0.

[0016] Thus, in one embodiment, the immunogenic composition preferably comprises at least two isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-A28, RV-A89, RV-B06 VP0, RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

[0017] Alternatively, the immunogenic composition preferably comprises at least three isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-A28, RV-A89, RV-B06 VP0, RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

[0018] Alternatively, the immunogenic composition preferably comprises at least four isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-A28, RV-A89, RV-B06 VP0, RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

[0019] Alternatively, the immunogenic composition comprises at least five isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-A28, RV-A89, RV-B06 VP0, RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

[0020] Alternatively, the immunogenic composition comprises at least six isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-A28, RV-A89, RV-B06 VP0, RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

[0021] Alternatively, the immunogenic composition comprises at least seven isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding the peptides, wherein the peptides are selected from the group consisting of RV-A28, RV-A89, RV-B06 VP0, RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

[0022] In one embodiment, the immunogenic composition may further comprise an isolated RV-A16 VP0 peptide and / or an isolated RV-A29 VP0 peptide, or an isolated polynucleotide encoding those peptides, or a variant or fragment thereof.

[0023] In a preferred embodiment, the immunogenic composition according to the first aspect comprises three isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding said peptides, wherein the peptides are selected from the group consisting of RV-A16 VP0, RV-B06 VP0RV, and RV-C24 VP0, or variants or fragments thereof.

[0024] It is well known to those skilled in the art that there are three different species of rhinoviruses: rhinovirus A (RV-A), also called rhinovirus type A; rhinovirus B (RV-B), also called rhinovirus type B; and rhinovirus C (RV-C), also called rhinovirus type C. RVs are further classified into strains based on their nucleotide sequence homology. As used herein, the term "strain" refers to a subdivision within the rhinovirus species and is based on the VP1 gene sequence of the rhinovirus. For example, the terms RV-A16, RV-A28, RV-A29, RV-A89, RV-B06, RV-C07, RV-C19, RV-C24, and RV-C01 refer to strains of RV. RVs have been classified according to several other parameters, including receptor specificity and antiviral susceptibility.

[0025] RV has a 25 nm capsid with icosahedral symmetry, consisting of 60 copies each of four virally encoded proteins (VP1, VP2, VP3, and VP4), surrounding a single-stranded RNA genome of approximately 7,500 nucleotides. The RNA has positive polarity, is polyadenylated at its 3' end, and is covalently linked at its 5' end to a small protein, VPg. The primary translation product of this RNA is a single "large" polyprotein, which is split into three smaller polyproteins, designated P1, P2, and P3, which are subsequently processed by proteolytic cleavage to generate the mature viral proteins. The P1 polyprotein consists of four peptides (1A or VP4, 1B or VP2, 1C or VP3, and 1D or VP1), the P2 polyprotein consists of three peptides (2A, 2B, and 2C), and the P3 polyprotein consists of four peptides (3A, 3B, 3C, and 3D). 2A and 3C are viral proteases, and 3D corresponds to the viral RNA polymerase. The P1 polyprotein is the precursor that gives rise to the four structural proteins of the nucleocapsid. The P1 polyprotein is first cleaved to produce the VP0 polyprotein, which contains the amino acid sequences of the VP4 and VP2 peptides, the VP3 peptide, and the VP1 peptide. When the virus is assembled, the VP0 polyprotein is cleaved into the VP4 and VP2 peptides.

[0026] The term "VP0 polyprotein", "VP0 peptide", or "peptide 1AB" will be known to those skilled in the art to refer to a protein precursor derived from the RV P1 polyprotein and consisting of the amino acid sequence of the VP4 and VP2 peptides. The VP0 polyprotein is typically about 330 amino acids in length. The amino acid sequence of the VP0 polyprotein varies slightly depending on the strain or species of RV.

[0027] Thus, a fragment of a RV VP0 peptide may comprise or consist of the corresponding VP2 and / or VP4 peptide, i.e., a fragment of RV VP0 may have at least 1, 2, 3, 4, 5, 10, 20, 25, 30, 45, 50, 60, 70, 80, 90, or 100 fewer amino acids than the full-length RV VP0 described herein.

[0028] In one embodiment, the peptide, variant, or fragment thereof comprises or consists of at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 66 amino acids, at least 67 amino acids, at least 68 amino acids, or at least 69 amino acids.

[0029] In another embodiment, the peptide, variant, or fragment thereof comprises or consists of at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids, at least 230 amino acids, at least 240 amino acids, at least 250 amino acids, at least 255 amino acids, at least 260 amino acids, at least 261 amino acids, at least 262 amino acids, at least 263 amino acids, at least 264 amino acids, at least 265 amino acids, at least 266 amino acids, or at least 267 amino acids.

[0030] In another embodiment, the peptide, variant, or fragment thereof comprises or consists of at least 280 amino acids, at least 290 amino acids, at least 300 amino acids, at least 310 amino acids, at least 320 amino acids, or at least 330 amino acids.

[0031] As used herein, the term "isolated" means removed from its natural environment, i.e., from a rhinovirus or a cell infected with a rhinovirus. Typically, this refers to a peptide or nucleic acid that is substantially free of cellular material, bacterial material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized.

[0032] In one embodiment, at least two or more of the RV VP0 peptides may form a fusion peptide or protein. It is well known to those skilled in the art that a fusion peptide / protein is a peptide that contains all or part of the amino acid sequences of at least two or more individual peptide units linked together via a covalent linkage, for example via a peptide (amide) bond. The two or more peptides may be directly linked to each other or may be linked using a linker sequence. In another embodiment, at least three, four, or five or more of the RV VP0 peptides may form a fusion peptide. Alternatively, multiple RV VP0 peptides may be stored as individual peptides and used simultaneously, i.e., not fused to each other.

[0033] The present invention also relates to an immunogenic composition comprising an isolated polynucleotide encoding an isolated human rhinovirus (RV) VP0 peptide.

[0034] In a preferred embodiment, the polynucleotide comprises a nucleic acid sequence encoding a peptide. Preferably, the nucleic acid sequence is under the control of elements necessary for its expression in mammalian cells, particularly human cells. The nucleic acid sequence is incorporated into a plasmid, which can be further formulated in a delivery vehicle such as a liposome to facilitate introduction into a host cell.

[0035] As used herein, the term "nucleic acid" includes DNA and RNA, such as messenger RNA (mRNA) or self-amplifying RNA (saRNA), and may be double-stranded or single-stranded. Most preferably, the nucleic acid is mRNA.

[0036] As used herein, the expression "elements necessary for expression in mammalian cells" is understood to mean all elements that allow the transcription of DNA or DNA fragments into mRNA or saRNA and the translation of the latter into proteins inside mammalian cells, for example human cells. Typically, elements necessary for the expression of nucleic acids in mammalian cells include a promoter that is functional in the selected mammalian cell and may be constitutive or inducible, a ribosome binding site, an initiation codon (ATG) if necessary, a region encoding a signal peptide (e.g., a lipidation signal peptide), a stop codon, and a 3' terminal region (translation and / or transcription terminator). Other transcription control elements, such as enhancers, operators, and repressors, may also be operably associated with the polynucleotide to direct transcription and / or translation into the cell. The signal peptide coding region is preferably adjacent to the nucleic acid contained in the immunogenic composition of the invention and is placed in the correct reading frame. The signal peptide coding region may be homologous or heterologous to the DNA molecule encoding the mature or fusion peptide of the invention and may be specific to the secretion apparatus of the host used for expression. The open reading frame constituted by the nucleic acid contained in the immunogenic composition of the invention, alone or together with a signal peptide, is placed under the control of a promoter such that transcription and translation occur in the host system. Promoters (and regions encoding signal peptides) are widely known and available to those skilled in the art.

[0037] The nucleic acid sequences may be codon optimized to enhance transcription of DNA encoding the peptides and / or fusion peptides of the invention and / or to elongate translation of mRNA encoding the peptides and / or fusion peptides.

[0038] The immunogenic composition may comprise a concatemer of isolated polynucleotides encoding RV VP0 peptides. In a preferred embodiment, the polynucleotide encoding the RV VP0 peptide is an mRNA. Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide encoding at least two or more RV VP0 peptides. In another embodiment, the polynucleotide encodes at least three, four, or five or more of the RV VP0 peptides. Alternatively, multiple RV VP0 peptides may be encoded by multiple separate polynucleotides, but are used simultaneously, i.e., not fused together.

[0039] The inventors have found that VP0 peptides from RV-A and RV-B species induce stronger immune responses compared to VP0 peptides from the RV-C group. Thus, the inventors believe that concatemers in which the RV-C VP0 peptide is encoded first are beneficial because they allow more of the RV-C VP0 peptide to be expressed. Thus, in one embodiment, the nucleic acid sequence encoding the RV-C VP0 peptide is located 5' to the nucleic acid sequence encoding the RV-B VP0 peptide, the RV-A28 VP0 peptide, and / or the RV-A89 VP0 peptide.

[0040] Alternatively, in another embodiment, the nucleic acid sequences encoding the RV-A VP0 peptide, the RV-B VP0 peptide, and the RV-C VP0 peptide are arranged in any order.

[0041] In one preferred embodiment, the immunogenic composition comprises an isolated polynucleotide encoding an RV-C24 VP0 peptide, an isolated polynucleotide encoding an RV-A16 VP0 peptide, and an isolated polynucleotide encoding an RV-B06 VP0 peptide. Preferably, the polynucleotides encoding the RV VP0 peptides are separated by the 2A peptide coding sequence.

[0042] In one embodiment, the amino acid sequence of the RV-A16 VP0 peptide is provided herein as SEQ ID NO:1, as follows: [ka]

[0043] Thus, preferably, the RV-A16 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO:1 or a variant or fragment thereof.

[0044] The VP4 peptide of RV-A16 comprises amino acid residues 1-69 of SEQ ID NO: 1, and the VP2 peptide of RV-A16 comprises amino acid residues 70-330 of SEQ ID NO: 1. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-A16 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0045] In one embodiment, the RV-A16 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO:2 as follows: [ka]

[0046] Thus, preferably, the RV-A16 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 2, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 2, or a variant or fragment thereof.

[0047] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:2 is provided herein as SEQ ID NO:3, as follows: [ka]

[0048] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 3, or a variant or fragment thereof.

[0049] In one embodiment, the amino acid sequence of the RV-B06 VP0 peptide is provided herein as SEQ ID NO:4, as follows: [ka]

[0050] Thus, preferably, the RV-B06 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 4 or a variant or fragment thereof.

[0051] The VP4 peptide of RV-B06 comprises amino acid residues 1-69 of SEQ ID NO: 4, and the VP2 peptide of RV-B06 comprises amino acid residues 70-331 of SEQ ID NO: 4. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-B06 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0052] In one embodiment, the RV-B06 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO:5 as follows: [ka]

[0053] Thus, preferably, the RV-B06 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 5, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 5, or a variant or fragment thereof.

[0054] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:5 is provided herein as SEQ ID NO:6, as follows: [ka]

[0055] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 6, or a variant or fragment thereof.

[0056] In one embodiment, the amino acid sequence of the RV-C07 VP0 peptide is provided herein as SEQ ID NO:7, as follows: [ka]

[0057] Thus, preferably, the RV-C07 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 7 or a variant or fragment thereof.

[0058] The VP4 peptide of RV-C07 comprises amino acid residues 1-67 of SEQ ID NO: 7, and the VP2 peptide of RV-C07 comprises amino acid residues 68-330 of SEQ ID NO: 7. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-C07 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0059] In one embodiment, the RV-C07 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO:8 as follows: [ka]

[0060] Thus, preferably, the RV-C07 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 8, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 8, or a variant or fragment thereof.

[0061] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:8 is provided herein as SEQ ID NO:9, as follows. [ka]

[0062] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 9 or a variant or fragment thereof.

[0063] In one embodiment, the amino acid sequence of the RV-C19 VP0 peptide is provided herein as SEQ ID NO: 10, as follows: [ka]

[0064] Thus, preferably, the RV-C19 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 10 or a variant or fragment thereof.

[0065] The VP4 peptide of RV-C19 comprises amino acid residues 1-67 of SEQ ID NO: 10, and the VP2 peptide of RV-C19 comprises amino acid residues 68-330 of SEQ ID NO: 10. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-C19 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0066] In one embodiment, the RV-C19 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO:11 as follows: [ka]

[0067] Thus, preferably, the RV-C19 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 11, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 11, or a variant or fragment thereof.

[0068] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:11 is provided herein as SEQ ID NO:12, as follows: [ka]

[0069] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 12, or a variant or fragment thereof.

[0070] In one embodiment, the amino acid sequence of the RV-C24 VP0 peptide is provided herein as SEQ ID NO: 13, as follows: [ka]

[0071] Thus, preferably, the RV-C24 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 13 or a variant or fragment thereof.

[0072] The VP4 peptide of RV-C24 comprises amino acid residues 1-67 of SEQ ID NO: 13, and the VP2 peptide of RV-C24 comprises amino acid residues 68-328 of SEQ ID NO: 13. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-C24 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0073] In one embodiment, the RV-C24 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO: 14 as follows: [ka]

[0074] Thus, preferably, the RV-C24 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 14, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 14, or a variant or fragment thereof.

[0075] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:14 is provided herein as SEQ ID NO:15, as follows. [ka]

[0076] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 15 or a variant or fragment thereof.

[0077] In one embodiment, the amino acid sequence of the RV-C01 VP0 peptide is provided herein as SEQ ID NO: 16, as follows: [ka]

[0078] Thus, preferably, the RV-C01 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 16 or a variant or fragment thereof.

[0079] The VP4 peptide of RV-C01 comprises amino acid residues 1-67 of SEQ ID NO: 16, and the VP2 peptide of RV-C01 comprises amino acid residues 68-328 of SEQ ID NO: 16. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-C01 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0080] In one embodiment, the RV-C01 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO: 17 as follows: [ka]

[0081] Thus, preferably, the RV-C01 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 17, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 17, or a variant or fragment thereof.

[0082] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:17 is provided herein as SEQ ID NO:18, as follows. [ka]

[0083] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 18 or a variant or fragment thereof.

[0084] In one embodiment, the amino acid sequence of the RV-A28 VP0 peptide is provided herein as SEQ ID NO: 19, as follows: [ka]

[0085] Thus, preferably, the RV-A28 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 19 or a variant or fragment thereof.

[0086] The VP4 peptide of RV-A28 comprises amino acid residues 1-69 of SEQ ID NO: 19, and the VP2 peptide of RV-A28 comprises amino acid residues 70-332 of SEQ ID NO: 19. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-A28 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0087] In one embodiment, the RV-A28 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO: 20 as follows: [ka]

[0088] Thus, preferably, the RV-A28 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 20, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 20, or a variant or fragment thereof.

[0089] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:20 is provided herein as SEQ ID NO:21, as follows. [ka]

[0090] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 21 or a variant or fragment thereof.

[0091] In one embodiment, the amino acid sequence of the RV-A29 VP0 peptide is provided herein as SEQ ID NO:22, as follows: [ka]

[0092] Thus, preferably, the RV-A29 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 22 or a variant or fragment thereof.

[0093] The VP4 peptide of RV-A29 comprises amino acid residues 1-69 of SEQ ID NO: 22, and the VP2 peptide of RV-A29 comprises amino acid residues 70-335 of SEQ ID NO: 22. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-A29 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0094] In one embodiment, the RV-A29 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO:23 as follows: [ka]

[0095] Thus, preferably, the RV-A29 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 23, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 23, or a variant or fragment thereof.

[0096] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:23 is provided herein as SEQ ID NO:24, as follows. [ka]

[0097] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 24 or a variant or fragment thereof.

[0098] In one embodiment, the amino acid sequence of the RV-A89 VP0 peptide is provided herein as SEQ ID NO:25, as follows: [ka]

[0099] Thus, preferably, the RV-A89 VP0 peptide comprises an amino acid sequence substantially as set out in SEQ ID NO: 25 or a variant or fragment thereof.

[0100] The VP4 peptide of RV-A89 comprises amino acid residues 1-69 of SEQ ID NO: 25, and the VP2 peptide of RV-A89 comprises amino acid residues 70-336 of SEQ ID NO: 25. Thus, in embodiments where the immunogenic composition comprises a fragment of RV-A89 VP0, the fragment may comprise or consist of amino acids that form the VP2 and / or VP4 peptides.

[0101] In one embodiment, the RV-A89 VP0 peptide is encoded by the nucleotide sequence (DNA) of SEQ ID NO:26 as follows: [ka]

[0102] Thus, preferably, the RV-A89 VP0 peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 26, or a variant or fragment thereof. Alternatively, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence substantially as set out in SEQ ID NO: 26, or a variant or fragment thereof.

[0103] In one embodiment, an RNA sequence corresponding to the DNA sequence of SEQ ID NO:26 is provided herein as SEQ ID NO:27, as follows. [ka]

[0104] Thus, in one embodiment, the immunogenic composition comprises an isolated polynucleotide comprising a nucleotide sequence (RNA) substantially as set out in SEQ ID NO: 27 or a variant or fragment thereof.

[0105] The term "immunogenic composition" as used throughout refers to a composition of matter (intended for administration to a subject) that contains at least one antigen of a rhinovirus or induces the expression of at least one antigen (in the case of nucleic acid immunization) and has the capacity to induce an immune response in the subject to which it is administered. Such an immune response may be a cellular and / or antibody-mediated immune response directed at least to the antigen of the composition.

[0106] The immunogenic compositions described herein provide an effective means of vaccinating a subject against RV infection. Thus, in a preferred embodiment, the immunogenic composition is a vaccine.

[0107] The immunogenic composition may include a suitable adjuvant. In a preferred embodiment, the adjuvant is a Th1 adjuvant. Examples of Th1 adjuvants that promote Th1 immune responses include TLR-9 agonists, such as CpG oligonucleotides, or TLR-4 agonists. Alternatively, in another embodiment, the adjuvant is incomplete Freund's adjuvant (IFA).

[0108] Further examples of adjuvants may include aluminum salts, synthetic forms of DNA, carbohydrates, tablet binders, ion exchange resins, preservatives, polymers, emulsions, and / or lipids. Examples of adjuvants may include monosodium glutamate, sucrose, dextrose, aluminum bovine, human serum albumin, cytosine phosphoguanine, potassium phosphate, plasdone C, anhydrous lactose, cellulose, polacrilin potassium, glycerin, asparagine, citric acid, potassium phosphate, magnesium sulfate, ferric ammonium citrate, 2-phenoxyethanol, aluminum, beta propiolactone, bovine extract, DOPC, EDTA, formaldehyde, thimerosal, phenol, potassium aluminum sulfate, potassium glutamate, sodium borate, sodium metabisulfite, urea, PLGA, PVA, PLA, PVP, cyclodextrin-based stabilizers, oil-in-water emulsion adjuvants, and / or lipid-based adjuvants.

[0109] The immunogenic composition according to the first aspect is particularly suitable for the treatment or prophylaxis (ie vaccination) against rhinovirus infection.

[0110] Thus, in a second aspect of the invention there is provided an immunogenic composition according to the first aspect for use in therapy or prophylaxis.

[0111] In a third aspect, there is provided an immunogenic composition according to the first aspect for use in eliciting an immune response.

[0112] In a fourth aspect, there is provided a method of inducing an immune response in a subject, the method comprising administering a therapeutically effective amount of an immunogenic composition according to the first aspect to a subject in need thereof.

[0113] It will be understood that the uses and methods of the present invention include vaccination.

[0114] In a preferred embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, induces an immune response against rhinovirus infection.

[0115] Preferably, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, induces an immune response against at least one, at least two, or at least three species of rhinovirus, more particularly against RV-A, RV-B, and / or RV-C.

[0116] Advantageously, the immune response elicited by the immunogenic composition according to the first aspect is a specific cell-mediated immune response directed not only against the homologous strain of rhinovirus from which the immunogenic composition was derived, but also against other (heterologous) strains of rhinovirus within the same species of rhinovirus, and this can be extended to strains of rhinovirus within other groups of rhinovirus.

[0117] Thus, preferably, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, comprises RV-1A, RV-A1, RV-A10, RV-A100, RV-A101, RV-A103, RV-A106, RV-A107, RV-A108, RV-A11, RV-A12, RV-A13, RV-A15, RV-A16, RV-A18, RV-A19, RV-A1B, RV-A2, RV-A20, RV-A21, RV-A22, RV-A23, RV-A24, RV-A25, RV-A28, RV-A29, RV-A30, RV-A31 , RV-A32, RV-A33, RV-A34, RV-A36, RV-A38, RV-A39, RV-A40, RV-A41, RV-A43, RV-A44, RV-A45, RV-A 46, RV-A47, RV-A49, RV-A50, RV-A51, RV-A53, RV-A54, RV-A55, RV-A56, RV-A57, RV-A58, RV-A59, RV -A60, RV-A61, RV-A62, RV-A63, RV-A64, RV-A65, RV-A66, RV-A67, RV-A68, RV-A7, RV-A71, RV-A73, R In a preferred embodiment, the immunogenic composition for use according to the third aspect or the method according to the fourth aspect induces an immune response against all strains of RV-A.

[0118] Preferably, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, elicits an immune response against at least one strain of RV-B selected from the group consisting of RV-B103, RV-B102, RV-B101, RV-B100, RV-B27, RV-B26, RV-B4, RV-B97, RV-B35, RV-B84, RV-B93, RV-B92, RV-B91, RV-B17, RV-B5, RV-B42, RV-B6, RV-B37, RV-B48, RV-B69, RV-B52, RV-B72, RV-B3, RV-B14, RV-B99, RV-B86, RV-B83, RV-B79, and RV-B70. In a preferred embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, induces an immune response against all strains of RV-B.

[0119] Preferably, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, is selected from the group consisting of RV-C58, RV-C50, RV-C48, RV-C46, RV-C13, RV-C51, RV-C55, RV-C56, RV-C45, RV-C44, RV-C41, RV-C4, RV-C38, RV-C34, RV-C33, RV-C31, RV-C30, RV-C29, RV-C27, RV-C26, RV-C24, RV-C23, RV-C21, RV-C16, RV-C14, RV-C47, RV-C53 ...46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, RV-C46, In a preferred embodiment, the immunogenic composition for use according to the third aspect or the method according to the fourth aspect induces an immune response against at least one strain of RV-C selected from the group consisting of RV-C54, RV-C22, RV-C37, RV-C49, RV-C25, RV-C18, RV-C12, RV-C20, RV-C19, RV-C28, RV-C36, RV-C39, RV-C15, RV-C5, RV-C11, RV-C9, RV-C10, RV-C1, RV-C43, RV-C42, RV-C40, RV-C32, RV-C17, RV-C8, RV-C7, RV-C6, RV-C3, RV-C2, and RV-C35.

[0120] Furthermore, it is well known that rhinoviruses are a major cause of the common cold, viral wheezing diseases of childhood, and acute attacks (exacerbations) of lung diseases such as asthma, bronchiectasis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and chronic fibrosing lung disease.

[0121] Thus, in a preferred embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, induces an immune response against an acute attack of pulmonary disease, such as the common cold, a viral wheezing disease, asthma, an asthma exacerbation, bronchiectasis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and / or a chronic fibrosing pulmonary disease.

[0122] As used herein, the phrase "eliciting an immune response" can include eliciting a specific cell-mediated immune response. This refers to the development of a specific T-lymphocyte response in a subject after administration of an immunogenic composition. The two main cellular effectors of the specific T-lymphocyte response are helper T cells and cytotoxic T lymphocytes (CTLs).

[0123] CD4+ "helper" T cells or helper T cells are immune response mediators and play a key role in establishing and maximizing the potential of adaptive immune responses. Although these cells have some direct cytotoxic activity, they essentially "manage" the immune response by directing other cells involved in the protection of the organism against pathogens. Activation of naive helper T cells triggers their release of cytokines, which affect the activity of many cell types, such as B lymphocytes, CTLs, and the APCs (antigen presenting cells) that activate them. Helper T cells require a milder activation stimulus than cytotoxic T cells. Helper T cells may provide extra signals that "help" the activation of cytotoxic cells. Two types of effector CD4+ helper T cell responses can be elicited by professional APCs designated Th1 and Th2. Measurement of the cytokines associated with the Th1 or Th2 response will give a measure of the success of immunization. This can be achieved by specific ELISAs or ELISPOTs designed for the measurement of IFN-γ, IL-2 and other Th1 cytokines, or Th2 cytokines such as IL-4, IL-5, IL-13, among others.

[0124] As used herein, the phrase "helper T cell-mediated immune response" refers to an immune response in which CD4+ T cells or helper T cells are activated to secrete lymphokines and stimulate both cell-mediated and antibody-mediated branches of the immune system. As known to those skilled in the art, activation of helper T cells promotes lymphokine secretion, immunoglobulin isotype switching, affinity maturation of antibody responses, activation of macrophages, and / or enhanced activity of natural killer cells and cytotoxic T cells. Lymphokines are proteins secreted by lymphocytes that affect their own activity and / or the activity of other cells. Lymphokines include, but are not limited to, interleukins and cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, or IFN-γ.

[0125] It is well known to those skilled in the art that helper T cells differentiate into two major subtypes of cells known as Th1 and Th2 cells (also known as type 1 and type 2 helper T cells, respectively).

[0126] Moreover, it is well known to those skilled in the art that Th1 cells primarily secrete IL-2 and IFN-γ, which promote cellular immune responses by maximizing the killing efficiency of macrophages and the proliferation of cytotoxic CD8+ T cells. Furthermore, the type 1 cytokine IFN-γ increases the production of IL-12 by dendritic cells and macrophages, and through positive feedback, IL-12 stimulates the production of IFN-γ in helper T cells, thereby promoting a Th1 profile. IFN-γ also inhibits the production of cytokines such as IL-4, a key cytokine associated with type 2 responses, thereby acting to sustain its own response.

[0127] In contrast, Th2 cells primarily secrete IL-4, IL-5, and IL-13 to promote humoral immune responses by stimulating B cells to proliferate and induce antibody class switching in B cells. Type 2 responses further promote their own profile using two distinct cytokines: IL-4 acts on helper T cells to promote the production of Th2 cytokines (including itself), while IL-10 inhibits a variety of cytokines including IL-2 and IFN-γ in helper T cells and IL-12 in dendritic cells and macrophages.

[0128] Preferably, the cell-mediated immune response elicited by the immunogenic composition of the invention is primarily a Th1 cell-mediated immune response. In a preferred embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, elicits an immune response through the secretion of IFN-γ.

[0129] An important component of protective cellular immunity induced by natural infection with respiratory viruses is tissue-resident memory T cells (T RM) is the formation of these T RM The cells rapidly proliferate and clear subsequent infection. Thus, in another preferred embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, comprises the step of: RM ) to induce an immune response.

[0130] In another preferred embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, elicits an immune response through the production of RV VP0-specific IgG antibodies.

[0131] Alternatively, in another embodiment, the immunogenic composition for use according to the third aspect, or the method according to the fourth aspect, elicits a cytotoxic T cell immune response.

[0132] Cytotoxic T cells (also known as Tc, killer T cells, or cytotoxic T lymphocytes (CTLs)), which generally express the CD8 marker, are a subgroup of T cells and can also participate in T cell-mediated immune responses. They induce the death of cells infected with viruses (and other pathogens). These CTLs directly attack other cells that carry certain foreign or abnormal molecules on their surface. Such cellular cytotoxicity capacity can be detected using an in vitro cytolytic assay (chromium release assay). Thus, the induction of specific cellular immunity can be demonstrated by the presence of such cytotoxic T cells, where antigen-loaded target cells are lysed by specific CTLs generated in vivo after vaccination or infection.

[0133] Similar to helper T cells, CD8+ T cells contain distinct subsets, which have been designated Tc1 and Tc2, in analogy with the Th1 / Th2 nomenclature.

[0134] The Tc1 immune response involves specific IFN-γ-producing CD8+ T cells that become activated, proliferate, and produce IFN-γ upon specific antigenic stimulation. The levels of IFN-γ-producing CD8+ T cells can be measured by ELISPOT and flow cytometric measurement of intracellular IFN-γ in these cells.

[0135] Naive cytotoxic T cells are activated when their T cell receptors (TCRs) interact strongly with peptide-bound MHC class I molecules. This affinity depends on the type and orientation of the antigen / MHC complex and maintains the binding of the CTL to the infected cell. Once activated, CTLs acquire function through a process called clonal expansion, rapidly dividing to produce an army of "armed" effector cells. Activated CTLs then migrate throughout the body searching for cells bearing unique MHC class I+ peptides. This can be used to identify such CTLs in vitro by using peptide-MHC class I tetramers in a flow cytometry assay.

[0136] When effector CTLs are exposed to these infected cells, they release the cytotoxins perforin and granulysin that form pores in the plasma membrane of the target cells, allowing ions and water to enter the infected cells, causing them to rupture or lyse. CTLs release granzymes, serine proteases that enter the cells through the pores and induce apoptosis (cell death). The release of these molecules from CTLs can be used as a measure of successful induction of a cellular immune response after vaccination. This can be done by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay (ELISPOT), which can quantitatively measure CTLs. Since CTLs can also produce important cytokines such as IFN-γ, quantitative measurements of IFN-γ-producing CD8 cells can be achieved by ELISPOT and flow cytometric measurements of intracellular IFN-γ in these cells.

[0137] It will be appreciated that the immunogenic compositions according to the invention may be used in medicine as a monotherapy (i.e., use of the immunogenic composition alone) for vaccination against RV infection. Alternatively, the immunogenic compositions according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing RV infection.

[0138] The immunogenic composition of the present invention may be combined into a composition having several different forms, particularly depending on the manner in which the composition is to be used. That is, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomal suspension, polyplex, emulsion, lipid nanoparticles (e.g., containing peptides, DNA, or RNA on or encapsulated therein), or any other suitable form that may be administered to a person or animal in need of vaccination. The lipid nanoparticles may comprise one or more components selected from the group consisting of cationic lipids (preferably ionizable), phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids. It will be understood that the vehicle of the pharmaceutical according to the present invention should be well tolerated by the subject to which it is given.

[0139] The medicaments comprising the immunogenic compositions of the present invention may be used in several ways. For example, oral administration may be required, in which case the agents may be included in a composition that can be taken orally, for example in the form of a tablet, capsule, or liquid. The compositions comprising the agents and medicaments of the present invention may be administered by inhalation (e.g., intranasally). The compositions may be formulated for topical use. For example, a cream or ointment may be applied to the skin.

[0140] The immunogenic compositions of the present invention may be incorporated into a sustained or delayed release device. Such a device may be inserted, for example, on or under the skin, and release the drug over a period of weeks or even months. The device may be located at least near the treatment site. Such a device may be particularly advantageous when long-term treatment with the immunogenic composition is required, and when frequent administration (e.g., at least daily injections) is usually required.

[0141] However, in a preferred embodiment, the medicament according to the invention may be administered to a subject by injection into the bloodstream, muscle, skin, or directly into the site requiring treatment. Injection may be intravenous (bolus or infusion), or subcutaneous (bolus or infusion), or intradermal (bolus or infusion), or intramuscular (bolus or infusion).

[0142] It will be understood that the amount of immunogenic composition required will be determined by its biological activity and bioavailability, which will depend on the mode of administration, the physicochemical properties of the immunogenic composition, and whether it is used in monotherapy or combination therapy. The frequency of administration will also be influenced by the half-life of the active agent in the subject being treated. The optimal dosage to be administered may be determined by one skilled in the art and will vary depending on the specific immunogenic composition used, the strength of the composition, the mode of administration, and the type and progression of the viral infection. Additional factors depending on the particular subject being treated will result in the need to adjust the dosage, including the subject's age, weight, sex, diet, and timing of administration.

[0143] Generally, a daily dose of 0.001 μg / kg body weight to 100 mg / kg body weight of the immunogenic composition of the invention may be used for immunization depending on the agent used. More preferably, the daily dose of agent is 1 μg / kg body weight to 100 mg / kg body weight, more preferably 10 μg / kg to 10 mg / kg body weight, and most preferably about 100 μg / kg to 10 mg / kg body weight.

[0144] The daily dose may be given as a single administration (e.g. a single daily injection or inhalation of a nasal spray). Alternatively, the immunogenic composition may require administration two or more times during the day. By way of example, the immunogenic composition may be administered as an initial primer and a subsequent boost, or two boosts administered at weekly or monthly intervals. Preferably, the immunogenic composition is administered as an initial primer and a subsequent boost, which may be administered at intervals of 2-6 weeks. Preferably, subsequent boosts may be administered annually for susceptible patients, such as those with weakened immune systems. To formulate the specific formulation of the immunogenic composition according to the invention and the exact treatment regime (e.g. daily dose of drug and frequency of administration), known procedures (e.g. in vivo experiments, clinical trials, etc.), such as those conventionally employed by the pharmaceutical industry, may be used.

[0145] A "subject" may be a vertebrate, a mammal, or a livestock animal. Thus, the compositions and medicaments according to the invention may be used to treat any animal, such as livestock (e.g., horses), pets, or for other veterinary purposes. Most preferably, however, the subject is a human being.

[0146] A "therapeutically effective amount" of an immunogenic composition is any amount, which when administered to a subject, is the amount as described above necessary to ameliorate, prevent, or treat, preferably prophylactically, any given disease.

[0147] For example, the immunogenic composition of the present invention may be used in an amount of about 0.001 μg to about 1 mg, preferably about 0.001 μg to about 500 μg. The amount of the immunogenic composition is preferably about 0.01 μg to about 250 μg, most preferably about 0.1 μg to about 100 μg. Preferably, the immunogenic composition according to the present invention is administered in a dose of 1 to 50 μg.

[0148] The immunogenic compositions of the present invention may further comprise a pharma- ceutically acceptable vehicle. As referred to herein, a "pharma- ceutically acceptable vehicle" is any known compound or combination of known compounds known to those of skill in the art to be useful in formulating a pharmaceutical composition.

[0149] In one embodiment, the pharma- ceutically acceptable vehicle may be a solid and the composition may be in the form of a powder or tablet. A solid pharma- ceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, coatings, or tablet disintegrants. The vehicle may be an encapsulating agent. In powders, the vehicle is a finely divided solid mixed with a finely divided active agent according to the invention. In tablets, the active agent (e.g., an immunogenic composition according to the invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain up to 99% of the active agent. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0150] However, the pharmaceutical vehicle may be liquid and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The immunogenic composition according to the invention may be dissolved or suspended in a pharma- ceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture thereof, or a pharma- ceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical excipients, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity adjusters, stabilizers, or osmolality adjusters. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned excipients, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g. fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions may be a halogenated hydrocarbon or other pharma- ceutically acceptable propellant.

[0151] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized by, for example, subcutaneous, intradermal, intrathecal, epidural, intraperitoneal, intravenous, and especially intramuscular injection. The nucleic acid sequences or expression cassettes of the invention may be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injection medium.

[0152] The immunogenic compositions of the present invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydrides copolymerized with ethylene oxide), etc. The immunogenic compositions according to the present invention may also be orally administered in liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0153] It will be appreciated that the present invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, comprising substantially the amino acid or nucleic acid sequence of any of the sequences referred to herein, including variants and fragments thereof. The terms "substantially an amino acid / nucleotide / peptide sequence", "variant" and "fragment" may refer to a sequence having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, such as 40% identity with the sequences identified as SEQ ID NOs: 1-27.

[0154] Amino acid / polynucleotide / polypeptide sequences having sequence identity with any of the mentioned sequences of greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, even more preferably greater than 80% sequence identity are also envisaged. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the mentioned sequences, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity with any of the sequences mentioned herein.

[0155] Those skilled in the art will know how to calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences. To calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, and then the sequence identity value must be calculated. The identity percentage for two sequences may take different values ​​depending on (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison, and (ii) the parameters used by the alignment method, such as local vs. global alignment, the pair score matrix used (e.g. BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as functional forms and constants.

[0156] There are many different ways to calculate the percentage of identity between two sequences after alignment. For example, one may divide the number of identities by (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average length of the sequences, (iv) the number of ungapped positions, or (v) the number of equivalent positions excluding overhangs. It will be further recognized that the percentage of identity is also highly length dependent. Thus, the shorter the sequence pair, the higher the sequence identity that would be expected to occur by chance.

[0157] It will therefore be appreciated that accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is the preferred method for generating protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: gap opening penalty = 15.0, gap extension penalty = 6.66, matrix = identity. For protein alignments: gap opening penalty = 10.0, gap extension penalty = 0.2, matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, GAPDIST = 4. Those skilled in the art will be aware that these and other parameters may need to be varied for optimal sequence alignment.

[0158] Preferably, the calculation of the percentage identity between two amino acid / polynucleotide / polypeptide sequences may be calculated from the alignment as follows: (N / T)*100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions compared, including gaps, with or without overhangs. Preferably, the overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences includes (i) preparing a sequence alignment, for example using the ClustalW program with a suitable set of parameters as presented above, and (ii) substituting the values ​​of N and T into the following formula: sequence identity=(N / T)*100.

[0159] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash with 0.2xSSC / 0.1% SDS at about 20-65°C. Alternatively, a substantially similar polypeptide may differ from the sequence shown in, for example, the amino acid sequence SEQ ID NO: 1-27 by at least one, but less than 5, 10, 20, 50, or 100 amino acids.

[0160] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be varied or altered without substantially affecting the sequence of the protein encoded thereby to provide a functional variant thereof. A suitable nucleotide variant is one that has a sequence altered by the substitution of different codons that code for the same amino acid in the sequence, thereby resulting in a silent (synonymous) change. Another suitable variant is one that has a homologous nucleotide sequence but includes all or a portion of the sequence, altered by the substitution of different codons that code for amino acids with side chains that have similar biophysical properties to the amino acid it replaces, resulting in a conservative change. For example, small non-polar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. The positively charged (basic) amino acids include lysine, arginine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Thus, it will be recognized which amino acids may be replaced with amino acids having similar biophysical properties, and one of skill in the art will know the nucleotide sequences that code for these amino acids.

[0161] All of the features described in this specification (including any accompanying claims, summary, and drawings) and / or all of the steps of any method or process disclosed may be combined with any of the above embodiments in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0162] For a better understanding of the invention, and to show how an embodiment thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0163] [Figure 1] FIG. 1 shows a heatmap visualizing pairwise identity scores between 150 rhinovirus strain VP0 protein sequences calculated using the MUSCLE algorithm after hierarchical clustering, showing pairwise identity scores above (>59.9%, blue) or below (<59.9%, red) the threshold predicted to be required for cross-immunity. [Diagram 2] Circular tree clustering 150 rhinovirus strain VP0 protein sequences into three clusters: among the analyzed sequences, cluster 1 (red) contains all RV-A species VP0 sequences, cluster 2 (black) contains all RV-B species VP0 sequences, and cluster 3 (green) contains all RV-C species VP0 sequences. [Diagram 3]A set of histograms showing the number of IFN-γ producing splenocytes in mice immunized with RV-A16 VP0 protein after stimulation with RV-A16 VP0 (A) or peptide pools of the indicated RV-A (B) or RV-B / C (C) species. Mice were sacrificed 35-49 days after the first immunization, splenocytes were collected, stimulated as indicated, and IFN-γ producing splenocytes (per 106 splenocytes) were enumerated by ELISPOT. (A) and (B): n=24 mice. Data were pooled from three independent experiments. (C): n=5 mice. Data are presented as geometric mean ± 95% confidence interval. Significant differences were compared to irrelevant peptide using a linear mixed effects model. ***: p<0.0001; ns: not significant. [Figure 4] A set of histograms showing the number of IFN-γ producing splenocytes in mice immunized with RV-B06 VP0 protein after stimulation with RV-B06 VP0 (A) or peptide pools of the indicated RV-B (B) or RV-A / C species (C). Mice were sacrificed 35-49 days after the first immunization, splenocytes were collected, stimulated as indicated, and IFN-γ producing splenocytes (per 106 splenocytes) were counted by ELISPOT. (A) and (B): n=24 mice. Data were pooled from three independent experiments. (C): n=7 mice. Data are presented as geometric mean ± 95% confidence interval. Significant differences were compared to irrelevant peptide using a linear mixed effects model. ***: p<0.0001; **: p<0.001; *p<0.01; ns: not significant. [Diagram 5]A set of histograms showing the number of IFN-γ producing splenocytes in mice immunized with RV-C07 VP0 protein after stimulation with RV-C07 VP0 (A) or peptide pools of the indicated RV-C (B) or RV-A / B species (C). Mice were sacrificed 35-49 days after the first immunization, splenocytes were collected, stimulated as indicated, and IFN-γ producing splenocytes (per 106 splenocytes) were counted by ELISPOT. n=7 mice. Data were pooled from two independent experiments. Data are presented as geometric mean ± 95% confidence interval. Significant differences were compared with irrelevant peptide using a linear mixed effects model. ***: p<0.0001; **: p<0.01; *p<0.05; ns: not significant. [Figure 6] A set of dot plots showing the median fragment identity / similarity score for each RV-C strain VP0 sequence across all other RV-C VP0 strains plotted against the median sequence identity / similarity score for each strain relative to RV-C07 alone. (A) All C strains plotted as a single cluster. (B) The C strains were split into two clusters. [Figure 7] A set of histograms showing the number of IFN-γ producing splenocytes in mice immunized with RV-C01 VP0 protein after stimulation with RV-C01 VP0 (A) or peptide pools of the indicated RV-C (B) or RV-A / B species (C). Mice were sacrificed 35-49 days after the first immunization, splenocytes were collected, stimulated as indicated, and IFN-γ producing splenocytes (per 106 splenocytes) were enumerated by ELISPOT. n=7 mice. Data were pooled from two independent experiments. Data are presented as geometric mean ± 95% confidence interval. Significant differences were compared to irrelevant peptide using a linear mixed effects model. ***: p<0.0001; **: p<0.01; *p<0.05; ns: not significant. [Figure 8]A set of histograms showing the number of IFN-γ producing splenocytes in mice immunized with RV-C19 VP0 protein after stimulation with RV-19 VP0 (A) or peptide pools of the indicated RV-C (B) or RV-A / B (C) species. Mice were sacrificed 35-49 days after the first immunization, splenocytes were collected, stimulated as indicated, and IFN-γ producing splenocytes (per 106 splenocytes) were enumerated by ELISPOT. N=12 mice. Data were pooled from two independent experiments. Data are presented as geometric mean ± 95% confidence interval. Significant differences were compared to irrelevant peptide using a linear mixed effects model. ***: p<0.0001; **: p<0.001; *p<0.01; ns: not significant. [Figure 9] A set of histograms showing the number of IFN-γ producing splenocytes in mice immunized with RV-C24 VP0 protein after stimulation with RV-C24 VP0 (A) or peptide pools of the indicated RV-C (B) or RV-A / B (C) species. Mice were sacrificed 35-49 days after the first immunization, splenocytes were collected, stimulated as indicated, and IFN-γ producing splenocytes (per 106 splenocytes) were enumerated by ELISPOT. (A) and (B): n=26 mice. Data were pooled from two independent experiments. (C): n=7 mice. Data are presented as geometric mean ± 95% confidence interval. Significant differences were compared to irrelevant peptide using a linear mixed effects model. ***: p<0.0001. [Figure 10]FIG. 1 is a line graph showing anti-RV VP0 antibody titers from mice immunized twice with either VP0 protein of RV-B06, RV-C01, RV-C07, RV-C19, or RV-C24 or control (PBS or vaccine adjuvant alone). Sera were collected 42 days after the first immunization and immunogen-specific IgG2a titers were quantified by ELISA. Each curve represents anti-RV VP0 IgG2a titers specific for the VP0 protein with which the mice were immunized (n=7 mice / immunogen; mean±SEM). Sera from control immunized mice (adjuvant alone or PBS; n=7 per group) were pooled and each pooled serum was tested by ELISA specific for each VP0 (mean±SEM of responses across VP0 antigens). [Figure 11] A set of histograms showing the results of ELISPOT experiments analyzing the cellular immunity against other strains of RV induced in cynomolgus monkeys by immunization with rRV-C24 VP0 protein (FIG. 11B) and adjuvant alone (control, FIG. 11A). Bars represent the mean activity / 2×10 PBMC±SEM from three technical replicates. [Figure 12] Figure 12A shows the secretion of IFN-γ (Figure 12A) and IL-4 (Figure 12B) in response to stimulation with a peptide pool derived from RV C species in cynomolgus monkeys immunized with rRV-C24 VP0 protein or adjuvant alone (control). Bars represent the mean activity / 2x105 PBMC±SEM from three technical replicates. [Figure 13] 1 is a histogram showing the concentration of RV-C24 VP0-binding IgG quantified by ELISA in the plasma of cynomolgus monkeys at various time points after immunization with rRV-C24 VP0 protein. Bars represent the mean IgG concentration and dots represent the response of individual animals. Arrows indicate the time when animals were immunized. [Figure 14]Western blots of HEK293 lysates at various times after transfection with RV VP0 mRNA. The marker lane is designated M and labels the 38 kDa and 28 kDa bands. The predicted molecular weight of RV VP0 protein is approximately 36 kDa. Figure 14A is a Western blot of lysates of HEK293 cells transfected with RV-A16 VP0 mRNA 24, 48, 72, or 96 hours after transfection. A positive control of recombinant RV-A16 VP0 protein was loaded on the gel (labeled rRV-A16 VP0). Detection of RV-A16 VP0 protein was performed with a monoclonal anti-RV-A16 VP0 antibody. Figure 14B is a Western blot of lysates of HEK293 cells transfected with RV-B06 or RV-C24 VP0 mRNA at 24, 48, 72, or 96 hours post-transfection. Detection of RV-B06 and C24 VP0 proteins was performed with antisera from mice immunized with recombinant RV-B06 or recombinant RV-C24 VP0 proteins, respectively. [Figure 15] Histograms showing the results of an ELISPOT experiment analyzing the cellular immunity against other strains of RV in mice induced by immunization with RV-C24 VP0 mRNA. Bars represent the mean activity / 5x105 splenocytes±SEM from 6 animals. *p<0.05, ANOVA. [Figure 16] 1 is a histogram showing the concentration of RV-C24 VP0-binding IgG quantified by ELISA in the serum of mice at various time points after immunization with RV-C24 VP0 mRNA. Bars indicate mean IgG concentration ± SEM. Arrows indicate the time when animals were immunized. [Figure 17]17A-17B are histograms showing the results of ELISPOT experiments analyzing the cellular immunity induced by immunization of cynomolgus monkeys with mRNA concatemers consisting of RV-C24 / A16 / B06 VP0 mRNA in sequences separated by the 2A peptide coding sequence. Secretion of IFN-γ (FIG. 17A) or IL-4 (FIG. 17B) was quantified by ELISPOT. Bars represent the mean activity / 2×105 PBMC±SEM from three technical replicates. [Figure 18] 18A-C are histograms showing concentrations of RV-A16 (FIG. 18A), RV-B06 (FIG. 18B), and RV-C24 (FIG. 18C) VP0-binding IgG quantified by ELISA in plasma at various time points following immunization of cynomolgus monkeys with mRNA concatemers consisting of RV-C24 / A16 / B06 VP0 mRNAs in a sequence separated by the 2A peptide coding sequence. Arrows indicate the time when animals were immunized. Bars indicate IgG concentrations detected in a single animal. Each graph was generated using data from the same animal. [Figure 19] Histogram showing the concentration of RV-A01 RNA in the lungs of control and RV-A16 VP0 mRNA immunized mice as determined by RT-qPCR 1, 6, and 14 days after infection with RV-A01. Bars represent the mean RV-A01 RNA ± SEM from 8 animals. *p<0.05, ANOVA. [Figure 20] Histograms showing the results of ELISPOT experiments analyzing how mouse cellular immunity generated by immunization with RV-A16 VP0 mRNA responds to subsequent infection with the heterotypic RV-A01 in splenocytes harvested 14 days after infection with RV-A1. Bars represent the mean spot-forming units / 1x106 splenocytes±SEM from 8 animals. *p<0.05, ***p<0.001, ANOVA. [Figure 21] 1 is a histogram showing the results of an ELISPOT experiment analyzing cellular immunity from control mice whose spleens were also harvested 14 days after infection with RV-A01. Bars represent the mean spot-forming units / 1×10 splenocytes±SEM from eight animals. [Figure 22] Histograms showing the mean numbers of CD62- CD44+ effector CD4+ (FIG. 22A) and CD8+ (FIG. 22B) T cells in the lungs of RV-A16 VP0 mRNA immunized and control mice, respectively, after infection with RV-A1. Bars represent the mean cell numbers±SEM from 8 animals. **p<0.01, ***p<0.001, ANOVA. [Figure 23] A set of correlation plots plotting the frequency of Th1 cells against the frequency of Th2 cells for each mouse infected with RV-A1 after immunization with RV-A16 VP0 mRNA (Figure 23B) or control (Figure 23A). [Figure 24] A set of histograms showing the numbers of CD4+ (FIG. 24A) and CD8+ (FIG. 24B) TRM cells in the BAL of control and RV-A16 VP0 mRNA immunized mice at various time points after RV-A1 infection. Bars represent the mean cell number or mean cell frequency±SEM from eight animals. ****p<0.0001, ANOVA. [Diagram 25] A set of histograms showing the frequency of CD4+ (FIG. 25A) and CD8+ (FIG. 25B) TRM cells in the BAL of control and RV-A16 VP0 mRNA immunized mice at various time points after RV-A01 infection. Bars represent the mean cell number or mean cell frequency ± SEM from 8 animals. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA. [Figure 26] A set of histograms showing the numbers of CD4+ (FIG. 26A) and CD8+ (FIG. 26B) TRM cells in the lungs of control and RV-A16 VP0 mRNA immunized mice at various time points after RV-A1 infection. Bars represent the mean cell number or mean cell frequency ± SEM from 8 animals. *p<0.05, **p<0.01, ***p<0.001, ANOVA. [Figure 27]A set of histograms showing the frequency of CD4+ (FIG. 27A) and CD8+ (FIG. 27B) TRM cells in the lungs of control and RV-A16 VP0 mRNA immunized mice at various time points after RV-A1 infection. Bars represent the mean cell number or mean cell frequency±SEM from eight animals. ****p<0.0001, ANOVA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0164] Materials and Methods Bioinformatic analysis to identify centroid RV VP0 immunogens Sequence of RV VP0 Protein sequences of RV VP0 were manually extracted from the NCBI protein database using a mixture of blast and annotation searches. Results were refined by comparison with strains annotated in the Taxonomy Browser (NCBI). VP0 protein sequences of 150 RV strains were used for analysis of 78 RV-A, 25 RV-B, and 47 RV-C sequences.

[0165] Alignment, sequence identity and similarity scores A multiple alignment of 150 RV VP0 sequences was performed using the MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithm. Identity and similarity matrices were extracted from the alignment. The identity matrix was used to calculate the pairwise percentage of identical amino acids per position between all sequences. The similarity matrix was used to calculate the pairwise percentage of amino acids with similar physicochemical properties per position between all sequences.

[0166] Hierarchical Clustering The VP0 sequences of 150 RV strains were clustered using unsupervised hierarchical clustering based on the calculated identity scores (converted to distance if necessary by the method). The tree was then divided into three clusters (Figure 1). From each cluster, a centroid RV VP0 strain sequence (i.e., central / typical, strain or medoid) was identified. Each centroid VP0 sequence was defined as the VP0 sequence in the cluster that was predicted to generate an immune response to the greatest percentage of other strains in that cluster. Centroid identification was performed using the range of identity thresholds defined below.

[0167] Fragment analysis Conservation among the VP0 protein sequences of RV strains was also analyzed at the epitope level by dividing the VP0 sequence of each strain into 12-mers with one amino acid overlap. Fragment identity was estimated by calculating the percentage of conserved 12-mer fragments between pairs of VP0 sequences (i.e., "conserved" is defined as 100% identity between two fragments). Fragment similarity was estimated by calculating the percentage of similar 12-mer fragments. Similarity was defined as 80% of the amino acids in a fragment being conserved or evolutionarily "interchangeable" by BLOSUM62 scoring.

[0168] Production and purification of RV VP0 protein [Table 1]

[0169] The same cloning strategy was applied for all recombinant proteins. Briefly, the respective nucleotide sequences were optimized for E. coli expression and the synthetic genes were cloned in frame with the SUMO sequence into the T / A cloning site of the pET-SUMO vector and then expressed using the pET-SUMO Expression System (Invitrogen). Genes were synthesized and plasmids were constructed at Crelux GmbH or Charles River Laboratories.

[0170] Escherichia coli (T7 Express, NEB) was transformed with any of the plasmids listed above. Expression was performed in 1-2 liter scale shake flasks (Ultra Yield, Thomson) using 2YT medium. Cultures were grown at 37°C to an OD600nm of 0.8 and protein production was induced by lowering the temperature to 18°C ​​and adding 1 mM IPTG prior to induction. Cells were harvested 16 hours after induction by centrifugation at 4,500g for 10 minutes and pellets were either stored at -80°C or processed immediately.

[0171] The progress of purification was monitored by SDS-PAGE after each purification step. Purification was performed at 4°C using chilled buffers (buffer components are listed in Table 2 below). The pellet was resuspended in Buffer A (10mL / 1g cells) and disrupted at 25kpsi using a cell disruptor (Constant Systems). The lysate was clarified by centrifugation at 38,000g for 30 minutes. The resulting inclusion body pellet was washed two more times with the same buffer and solubilized in Buffer B (10mL / 1g lysed cells) by agitation overnight at 4°C. The solution was then clarified by centrifugation at 38,000xg for 30 minutes and loaded at 1mL / min onto two 5mL HisTrap HP columns (Cytiva™) serially connected and equilibrated with Buffer B using the Aktaexpress system. The column was washed with Buffer B until the baseline stabilized, followed by a 20 CV wash with Buffer C and re-equilibration in Buffer B with a further 20 CV wash. A shallow gradient over 30 CV was then applied to Buffer D, after which the protein was eluted with Buffer E and immediately buffer exchanged into Buffer F using a HiPrep™ 26 / 10 desalting column (Cytiva™).

[0172] To remove the His-SUMO tag, approximately 0.4 U of Sumo protease (Lifesensors Inc.) per mg of VP0 protein was added to the eluted protein and incubated overnight at 4°C.

[0173] The cleaved protein was separated from uncleaved material and proteases by secondary nickel chromatography with gravity flow using HisSelect® affinity gel (Cytiva™) equilibrated with Buffer F.

[0174] The flow-through was collected, concentrated to approximately 2 mL, and loaded onto a Superdex S200 gel filtration column (Cytiva™) equilibrated with Buffer F and run at 1 mL / min. Fractions containing VPO protein were pooled for a final overnight dialysis into Buffer G.

[0175] The dialyzed samples were then concentrated to 0.3-0.6 mg / ml and tested for endotoxin levels using the ToxinSensor™ chromogenic LAL endotoxin assay kit (GenScript®) according to the manufacturer's instructions. Samples with levels below 5 EU / mg protein were aliquoted and stored at -80°C.

[0176] [Table 2]

[0177] Immunization of mice C57BL / 6 mice aged 8-10 weeks were immunized by the subcutaneous (SC) route on days 0 and 21.

[0178] Each mouse received 10 μg of RV VP0 protein (either RV-A16, RV-B06, RV-C01, RV-C07, RV-C19, or RV-C24) in a total volume of 100 μl together with IFA / CpG adjuvant (10 μg CPG 1826 (Invivogen, Toulouse France) + 100 μl incomplete Freund's adjuvant (IFA Sigma-Aldrich, Gillingham, United Kingdom).

[0179] Protein buffer (Tris 20 mM, NaCl 150 mM, Arginine 0.5 M, pH 8.0) with or without IFA / CpG adjuvant was used as a negative control and administered to a control group of mice following the same procedure.

[0180] Sample processing Blood and spleens were collected on day 42. Blood was collected into vacutainer tubes (BD Vacutainer SST II Nus plastic serum tubes (BD Biosciences, Le Pont-De-Claix, France)), stored overnight at 4°C, and centrifuged at 1660g for 20 min to separate serum from cells. Serum was stored at -20°C.

[0181] Spleens were rapidly harvested under sterile conditions after sacrifice.

[0182] Cytokine ELISPOT Spleens were manually homogenized using a syringe plunger through a cell strainer (BD Biosciences, San Jose, Calif.) and treated with red blood cell lysis buffer HybriMax (Sigma-Aldrich, Gillingham, United Kingdom) to lyse red blood cells. Cells were washed twice with RPMI 1640 medium containing HEPES (Gibco, Paisley, UK) supplemented with 2% decomplemented fetal calf serum (FCS) (HYCLONE, Hyclone, Logan, Utah), 50 μM 2-mercaptoethanol (Gibco), 2 mM L-glutamine (Gibco), and 100 units / mL penicillin-streptomycin (Gibco). Cells were counted using a Multisizer and resuspended in complete medium containing RPMI 1640 medium (Gibco) supplemented with 10% decomplemented FCS (HYCLONE), 50 μM 2-mercaptoethanol (Gibco), 2 mM L-glutamine (Gibco), and 100 units / mL penicillin-streptomycin (Gibco). 2 × 10 cells were cultured per well. 5Splenocytes were seeded in 96-well plates (source) and stimulated with pools of peptides corresponding to the full-length VP0 antigens of various RV-B06, RV-C01, RV-C07, RV-C19, or RV-C24 and 20 U / ml of mouse IL-2. In addition, to assess cross-reactivity, splenocytes were stimulated with peptide pools corresponding to full-length VP0 from various RV strains of each RV species, as indicated in each figure. Each peptide pool consisted of 80 15-mer peptides covering the entire VP0 protein and overlapping by 11 amino acids. Peptide pools were synthesized by Mimotopes and used at a final concentration of 1 μg / ml. Phorbol 12-myristate 13-acetate (PMA; Invivogen, Toulouse France) and ionomycin (Stemcell, Cambridge United Kingdom) were used as positive stimulation controls. An irrelevant peptide pool (PepMix™ human (HLA class I Ig-like C1 domain), obtained from JPT, Berlin Germany, 15-mer with an overlap of 11 amino acids) was used as a negative stimulation control. Plates were pre-coated with 1 μg per well of rat anti-mouse IFN-γ antibody (BD Pharmingen, San Diego, Calif.) in sterile PBS overnight at 4° C. and blocked with complete RPMI 1640 medium (Gibco) containing 10% FCS for 1 h at 37° C. Stimulation of splenocytes was performed in 5% C02 at 37° C. for 18 h.

[0183] After stimulating the splenocytes, the plates were washed three times with PBS, then three times with PBS-Tween 0.05%. Biotinylated anti-IFN-g (BD Biosciences, Le Pont-De-Claix, France) at 5 mg / ml in PBS-Tween 0.05% was added at 100 ml per well, and the plates were incubated in the dark at 20°C. The plates were then washed three times with PBS-Tween 0.05% and incubated with streptavidin-horseradish peroxidase (Southern Biotech) in PBS-Tween 0.05% for 1 h at 20°C in the dark.

[0184] Plates were then washed three times with PBS-Tween 0.05% followed by three times with PBS. 100 μl of substrate solution (3-amino-9-ethylcarbazole, AE, Sigma-Aldrich) was added per well and plates were incubated for 15 min at 20°C in the dark. The reaction was stopped by adding approximately 150 μl of distilled water per well. Substrate solution was prepared by mixing 9 ml of distilled water, 1 ml of acetate buffer, 0.250 ml of AE, and 5 μI of H2O2. The solution was filtered through a 0.22 μm filter (Sigma-Aldrich). Individual spots corresponding to IFN-γ-secreting T cells were counted by an automated ELISPOT reader (AID, Strassberg Germany). Results were compared to 10 6 It was expressed as the number of IFN-γ spots per splenocyte.

[0185] ELISPOT data for each immunogen were analyzed separately on the log10 scale using a linear mixed model with fixed effects for test and treatment groups and random effects adjusted for individual mice. Results for each treatment group were reported on the inverse transformed (antilog) scale using least squares geometric means, ratios of least squares geometric means, 95% confidence intervals, and p-values ​​for individual comparisons. Statistical significance was reported at the 5% significance level (i.e., p<0.05). No multiple comparison adjustments were performed as all comparisons were defined a priori and were against the control group (irrelevant peptide).

[0186] ELISA Anti-RV VP0 IgG2a (or IgG2c in 57Bl / 6 mice) responses were measured by ELISA. Greiner 96-well microplates (Greiner, Gloucestershire, United Kingdom) were coated overnight at 4°C with 100 ng per well of VP0 of RV-B06, C19, or C24 in PBS buffer pH 7.4 (Sigma-Aldrich). Non-specific sites were blocked for 1 h at 37°C with 150 μl per well of PBS-Tween (PBS pH 7.1, 0.05% Tween 20) and 1% skim milk. Sera were serially diluted 2-fold in VP0-coated plates from a starting dilution of 1:100 or 1:1000 in PBS-Tween 0.05% and milk 1%, incubated for 90 min, and washed 3 times with PBS-Tween.

[0187] RV VP0-specific IgG2a was detected by adding HRP-conjugated goat anti-mouse IgG2a (Southern Biotech, Birmingham, Ala.) diluted 1:4000 in PBS-Tween and 1% skim milk to each well and incubating for 90 min at 37°C.

[0188] Plates were then washed three times with PBS-Tween and tetramethylbenzidine (TMB, Tebu-bio laboratories, Le Perray-en-Yvelines, France) substrate solution was added to each well at 100 mL per well. Plates were incubated at room temperature in the dark for 10–30 min. The color reaction was stopped by adding 100 μl / well of 1 M HCl (VWR Prolabo, Fontenay-sou-Bois, France). Plates were immediately read at 450 and 650 nm in a Versamax plate reader (Molecular Devices).

[0189] In vitro mRNA studies Transfection of HEK cells with VP0 mRNA and detection of expressed RV VP0 protein HEK293 cells were cultured in DMEM medium supplemented with 10% FCS and 1% penicillin / streptomycin. For transfection, cells were seeded in 24-well plates and transfected in duplicate at approximately 80% confluency. Cells were transfected with RV VP0 mRNA along with mRNA-Fect (RJH Biosciences) according to a previously optimized protocol consisting of 4 μL of mRNA (Tri-Link), 60 μL of serum-free DMEM medium, and 4 μL of mRNA-fect per well. A non-transfected control was included, consisting of mRNA-fect without any mRNA. Prior to transfection, each mRNA was diluted to 0.1 mg / mL in RNase-free water (Promega), and mRNA-fect was used as a neat solution at 1 mg / mL according to the manufacturer's recommended protocol. Diluted mRNA (4 μL) was added to 60 μL of DMEM medium and vortexed for 3 seconds. Undiluted mRNA-fect (4 μL per transfection) was then added, and the RNA mixture was vortexed for 10 seconds and then left at room temperature for 30 minutes to allow complex formation. After incubation, the complexes (68 μL total) were added dropwise directly to each well of a 24-well plate in duplicate in a medium volume of 0.5 mL. The cells were then incubated for a set time (24-96 hours) prior to collection. For sample collection, the medium was removed, the monolayer was washed with 0.5 mL of PBS pH 7.4, and 100 μL of 2x SDS sample buffer (Invitrogen) was added to two duplicate wells. Cells were lysed by pipetting and scraping, then the duplicates were pooled to produce one sample, and the samples were stored at -80°C. Protein expression was visualized using SDS polyacrylamide gel electrophoresis (SDS-PAGE, Invitrogen) followed by protein transfer to nitrocellulose (Invitrogen) and Western blotting.Western blots consisted of probing each membrane overnight at 4°C with pooled VP0-specific mouse antisera (antisera against VP0 of A16, B06, or C24) diluted 1 / 200 in blocking buffer (5% skim milk in PBS). In one particular experiment, the membrane was probed overnight with anti-RV-A16 monoclonal antibody (Antibodies Online, Cat No. ABIN1000236) diluted 1 / 500 in blocking buffer. After incubation, the membrane was washed three times for 5 min with shaking in 30-50 mL of wash buffer (PBS pH 7.4 with 0.1% Tween 20). After washing, anti-mouse H+L chain IgG-HRP secondary antibody (Invitrogen) was added at a dilution of 1 / 5,000 in blocking buffer. The secondary antibody was incubated for 1 h with shaking, the secondary antibody was removed, and 30–50 mL of wash buffer was added for 30 min, followed by two washes of 30–50 mL of wash buffer for 5 min each. After washing, 2 mL of ECL Plus substrate (Thermo Scientific) was added for 2 min, and the membrane was subjected to imaging using a Peqlab imager following the manufacturer's recommended protocol for chemiluminescence.

[0190] Cynomolgus monkey mRNA vaccine research All studies were conducted in accordance with local laws and regulations and were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).

[0191] Preparation of mRNA vaccines On the day of use, in vivo-jetRNA® transfection reagent and mRNA buffer (both from Polyplus, France) were equilibrated to room temperature. The mRNA was diluted to 50 μg / mL in the mRNA buffer. A volume of in vivo-jetRNA® transfection reagent equal to the volume of mRNA added to the mRNA buffer was added, and the resulting solution was incubated at room temperature for 15 min before use. As a control solution, the mRNA was replaced with an equal volume of sterile water (Polyplus).

[0192] Immunization of Cynomolgus Monkeys Each animal received 100 μg of mRNA or 2 mL of transfection control reagent in a total volume of 2 mL administered intramuscularly on days 0 and 21 of the study.

[0193] Blood sampling and preparation of plasma and peripheral blood mononuclear cells Blood was collected from the saphenous or radial vein on study days 0, 14, 21, 28, 35, and 42. Blood was incubated at room temperature for 30 minutes before centrifugation, and plasma and peripheral blood mononuclear cells (PBMCs) were prepared by standard methods.

[0194] plasmaELISA 96-well plates were coated with goat anti-rhesus IgG(H+L)-UNLB (Southern Biotech, US) diluted in PBS for standard curves or with 25 μg / well RV VP0 protein diluted in PBS for detection of antigen-specific IgG in monkey plasma samples. Plates were incubated overnight at 4°C. Plates were washed three times with PBS-Tween (PBS containing 0.05% Tween 20) (Sigma-Aldrich). Nonspecific binding was blocked by incubation with PBS 5% milk (Sigma-Aldrich) for 2 h at room temperature. Plates were washed three times with PBS-Tween. 100 μL of standards or plasma samples were added per well, diluted in PBS-1% BSA, and incubated for 2 h at room temperature. An 11-point standard curve was generated using two-fold serial dilutions starting at 400 ng / mL of rhesus IgG-UNLB (Southern Biotech, US). Plates were washed three times with PBS-Tween. Bound antibodies were detected by incubation with Fcγ fragment-specific peroxidase AffiniPure goat anti-human IgG (Jackson Immunoresearch, US) diluted 1:5000 in PBS 1% BSA (Sigma-Aldrich) for 1 h at room temperature. Plates were washed three times with PBS-Tween and then developed by incubation with 100 μL TMB substrate per well for approximately 10 min, followed by stopping the reaction by adding 100 μL 0.18 M H2SO4 per well. Plates were immediately read at 450 nm using a SpectraMax plate reader (Molecular Devices).

[0195] PBMC ELISpot PBMCs were cultured with peptide pools as described above, at 2 × 10 per peptide pool stimulation. 5Cells were stimulated with 2 × 10 IgG. Each stimulation was performed in triplicate. IFN-γ or IL-4 ELISpot was performed using monkey IFN-γ ELISpot PRO kit (ALP), strips (Mabtech-3421M-2AST-10) and human IL-4 ELISpot PRO kit (ALP) (Mabtech-3410-2APW-10) according to the manufacturer's instructions. 5 The activity per PBMC was determined using an AID ELISpot machine (Model ELR088IFL).

[0196] Mouse mRNA vaccine research Preparation of mRNA vaccines On the day of use, in vivo-jetRNA® transfection reagent and mRNA buffer (both from Polyplus, France) were equilibrated to room temperature. mRNA was diluted to 200 μg / mL in mRNA buffer. An equal volume of in vivo-jetRNA® transfection reagent was added to the diluted mRNA, and the resulting solution was incubated for 15 min at room temperature before use. As a control solution, mRNA was replaced with an equal volume of sterile water (Polyplus).

[0197] Immunization of mice Each mouse received 5 μg of RV mRNA vaccine or 50 μL of transfection control reagent in a total volume of 50 μL administered intramuscularly on days 0 and 21 of the study.

[0198] Rhinovirus infection of mice Each mouse received an intranasal (in) dose of 50 μL of rhinovirus preparation (grown as described in Bartlett et al., 2008, Nat. Med.) or 50 μL of PBS (ThermoFisher Scientific, UK) administered dropwise into both nostrils under isofluorane anesthesia.

[0199] Collection and processing of mouse samples Blood samples were obtained via tail vein puncture prior to sacrifice and collected using sodium heparinized capillary tubes (Hirschmann-Laborgerate, Germany). The final blood sample was collected from the jugular vein using sodium heparinized capillary tubes. Whole blood samples were centrifuged to isolate serum from cells, and serum was subsequently stored at -80°C.

[0200] Following euthanasia, the trachea was intubated and bronchoalveolar lavage (BAL) was performed using three 0.5 mL sterile PBS washes per mouse, pooled to give a total volume of 1.5 mL BAL. Samples were centrifuged at 1500 RPM for 10 minutes at 4°C and the supernatants were stored at -80°C. Cells were resuspended in 0.2% w / v NaCl to lyse red blood cells and subsequently resuspended in an equal volume of 1.6% w / v NaCl.

[0201] Lymph nodes and spleens were collected in R10 medium (RPMI 1640 medium containing 10% fetal calf serum (FCS) and 100 U / mL penicillin / streptomycin (P / S), all from ThermoFisher Scientific) and then manually homogenized through a 100 μm filter (Greiner Bio-One, UK). Red blood cells were lysed using ACK Lysing Buffer (Fisher Scientific, UK) and the cells were then washed twice with R10 medium.

[0202] The right upper and subcaval lobes from each mouse were collected in RNAprotect (Qiagen, UK) and stored at -80°C. The remaining lung lobes were collected in R10 medium, then minced and incubated at 37°C for 30 min in R10 medium supplemented with 0.14 Wunsch units / mL Liberase (Roche, UK) and 50 μg / mL DNase1 (Roche). Lung tissue was then manually homogenized through a 100 μm filter (Greiner Bio-One). Red blood cells were lysed using ACK Lysing Buffer (Fisher Scientific, UK) and the cells were then washed twice with R10 medium.

[0203] Viability of single cell preparations was assessed using trypan blue (ThermoFisher Scientific) exclusion staining, and cells were counted using a hemocytometer (Neubauer, Germany).

[0204] Neutralization assay using mouse serum Mouse sera were serially diluted 1:2 or 1:3.14 and titrated with RV-A1 (5 × 10 4 The virus-serum complex (50 mL / well) was then added to 150 mL of HeLa cells (3 × 10 5 Cells were incubated with 1000 ng / mL of RV-A1 guinea pig serum in eight replicates in flat-bottom 96-well plates. Virus-containing cells and cells alone were used as controls. In some experiments, anti-RV-A1 guinea pig serum (ATCC Cat No V-113-501-558) was used as a positive control for neutralization in 1:3.14 serial dilutions to generate a 12-point titration curve. Plates were incubated for 3 days, then the supernatant was discarded and the cell monolayers were stained with 150 ml of crystal violet solution (0.1% in PBS) per well for 10–15 min, followed by gentle water washing to remove the stain. The stained monolayers were then incubated with 150 mL of 1% SDS solution in PBS per well for a 2-h incubation time at room temperature on an orbital shaker to solubilize the stain. The resulting optical density was measured at 560 nm using a SpectraMax plate reader (Molecular Devices, US).

[0205] Pulmonary viral load RNA was extracted from whole lung tissue using RNeasy® Mini Kit columns according to the manufacturer's instructions and converted to cDNA using the Omniscript® Reverse Transcription Kit (both Qiagen, UK). RV was quantified for 18S rRNA expression using 50 nM forward primer (5'-tgagtcctccggcccctgaatg-3'), 300 nM forward primer (5'-gtgaagagccscrtgtgct-3') and 5 μM QuantiTect Probe (Qiagen) and interpolated using an in-house generated RV plasmid standard. Quantitative rtPCR was performed using a QuantStudio 5 real-time qPCR machine (ThermoFisher Scientific).

[0206] Ex vivo cell stimulation for flow cytometry Single cell suspensions of lung or BAL cells were stimulated in R10 medium containing 40 ng / mL PMA (Invivogen, US) and 3 μg / mL ionomycin (Stemcell Technologies, Canada) in the presence of 10 μg / mL brefeldin A (Enzo Life Sciences, US), or 16 μg / mL RV peptide pool for 4 h at 37° C. Unstimulated control samples were incubated in R10 medium containing brefeldin A alone.

[0207] Flow cytometry Single cell suspensions of lung, BAL, or lymph node cells were first stained with Fixable Violet Dead Cell Stain Kit (Invitrogen, US) and then stained with anti-mouse surface marker antibodies and TruStain FcX antibodies diluted in FACS buffer (PBS containing 2% FCS and 2 mM EDTA) (Fisher Scientific) for 30–60 min. Cells were washed twice with FACS buffer and fixed with 1% formaldehyde solution (Sigma-Aldrich, UK) in FACS buffer. For intranuclear staining, cells were fixed with Transcription Factor Staining Buffer Set (eBioscience, US) and stained with anti-mouse transcription factor and cytokine antibodies diluted in permeabilization buffer (eBioscience) for 1 h. Samples were acquired using an LSRFortessa (BD, US) and analyzed using FlowJo™ (BD).

[0208] ELISA Nunc Maxisorp 96-well plates (Fisher Scientific) were coated with 50 ng / well mouse anti-IgG antibody (R&D Systems, US) diluted in PBS for standard curves and with 25 μg / well RV-A16, RV-B06, or RV-C24 protein diluted in PBS for detection of antigen-specific IgG in mouse serum samples. Plates were incubated overnight at 4°C. Plates were washed three times with PBS-Tween (PBS containing 0.05% Tween 20 (Sigma-Aldrich)). Nonspecific binding was blocked by incubation with PBS 5% milk (Sigma-Aldrich) for 1 h at room temperature. Plates were washed three times with PBS-Tween. 50 μL of standards or serum samples were added per well, diluted in PBS-5% milk, and incubated for 2 h at room temperature. A seven-point standard curve was generated using 1:3 serial dilutions of total IgG mouse ELISA standard (Invitrogen) starting at 100 μg / mL. Plates were washed three times with PBS-Tween. Bound antibody was detected by incubation with 25 ng / well mouse IgG biotinylated antibody (R&D Systems) diluted in PBS 1% BSA (Sigma-Aldrich) for 1 h at room temperature. Plates were washed three times with PBS-Tween and then incubated with 50 ng / well streptavidin-HRP conjugate (Millipore, US) for 20 min at room temperature in the dark. Plates were washed three times with PBS-Tween and then developed by incubation with 50 μL per well of TMB substrate (Fisher Scientific) for approximately 5 min, followed by addition of 50 μL per well of 0.18 M H2SO4 to stop the reaction. Plates were immediately read at 450 nm using a Petromax plate reader (Molecular Devices).

[0209] result Identification of centroid RV sequences for use as cross-protective immunogens Defining the threshold for centroid identification A study by Glanville et al. (2013) [6] demonstrated that immunization of mice with the VP0 protein from RV-A16 resulted in cellular immunity that cross-reacted with RV-B14, RV-A1, and RV-A29. Identity scores were calculated for the VP0 sequences of RV-B14, RV-A1, and RV-A29 in pairwise comparison with the RV-A16 VP0 sequence (Table 3).

[0210] [Table 3]

[0211] Glanville et al. (2013) [6] demonstrated that immunization of mice with RV-A16 VP0 protein elicited cellular immunity that cross-reacted with RV-A14, RV-A1, and RV-A29. Minimum identity and similarity scores were used as thresholds. Any VP0 proteins with sequence identity scores greater than 59.9% or similarity scores greater than 84.0% (Table 3) were predicted to elicit cross-reactive cellular immunity against each other. Any VP0 proteins with identity scores below these thresholds were not predicted to elicit cross-reactive immunity against each other.

[0212] This threshold was used to select a centroid VP0 protein from each cluster.

[0213] Centroid Identification Centroid identification was first performed using only sequence identity scores since this is the most stringent approach.

[0214] Figure 1 shows a heatmap of sequence identity scores calculated by MUSCLE between the VP0 sequences of 150 RV strains. Sequences with identity scores greater than 59.9% are shown in blue. RV-A16 VP0 has previously been demonstrated to be a potential centroid VP0 sequence for RV-A species. This data confirmed that the RV-A16 VP0 sequence has an identity score greater than 59.9% to all other RV-A species strains and is therefore predicted to elicit cellular immunity against all other A strains, leading to its selection as the centroid immunogen for RV-A species.

[0215] However, the heatmap shows that RV-A16 VP0 is not predicted to cross-immunize against many strains of RV-B and C species. Therefore, to identify additional RV VP0 protein centroids predicted to elicit cellular immunity against all other strains, hierarchical clustering based on average linkage was performed. Figure 2 shows a circular tree created by hierarchical clustering of VP0 sequences of 140 RV strains based on identity scores and divided into three clusters. Cluster 1 (red) contains all RV-A species sequences, Cluster 2 (black) contains all RV-B species strains, and Cluster 3 (green) contains all RV-C strains included in the analysis. Based on this analysis, RV-B06 VP0 was selected as the centroid for RV-B species because it has an identity score above the threshold of 59.9% to all other RV-B species VP0 protein sequences (Table 4). The centroid of the RV-C species was identified as RV-C07 because the VP0 sequence of RV-C07 was calculated to cover the VP0 protein sequences of all other RV-C species above a threshold of 59.9% (Table 4).

[0216] [Table 4]

[0217] Identification of further RV-C species centroids We next examined the number of IFN-γ producing splenocytes from mice immunized with RV-A16 VP0 or RV-B06 VP0 proteins. The data presented in Figures 3 and 4 demonstrate that RV-A16 VP0 and RV-B06 VP0 proteins, respectively, when used as immunogens in mice, elicited cellular cross-immunity against all other RV-A and RV-B species, respectively (Figures 3B and 4B). However, immunization with either RV-A16 or RV-B06 VP0 did not elicit cellular immunity against RV-C species (Figures 3C and 4C). When RV-C07 VP0 was used as the immunogen, splenocytes from mice immunized with RV-C07 responded weakly to stimulation with peptide pools from other RV-C species strains (Figure 5B) compared to the response of splenocytes from mice immunized with RV-A16 and RV-B06 to stimulation with peptides from the respective species (Figures 3B and 4B). Furthermore, there was very little cross-reactivity with peptide pools from RV-A and B species strains (Figure 5C). Therefore, further analysis was performed to select new RV-C species VP0 immunogens that would provide good cross-immunity to other RV-C species but are not similar to RV-C07.

[0218] A deeper level of analysis was required to identify RV-C centroids that are dissimilar to RV-C07 but are still predicted to cross-react with all other RV-C species strains. Therefore, a new analysis was performed using all calculated scores: sequence identity, sequence similarity, fragment identity, and fragment similarity.

[0219] Identity and similarity scores were combined as the geometric mean of the logarithmic measurements (+1 was added before taking the logarithm to avoid negative values). This value was referred to as the identity / similarity score, with higher scores indicating higher similarity and identity. Two identity / similarity scores were reported, one for the entire VP0 sequence analysis and one for the fragment analysis. The sequence identity / similarity scores were used to compare against RV-C07 VP0, and the fragment identity / similarity scores were used to predict the potential for cross-immunity based on previously calculated thresholds.

[0220] In Figure 6, the median fragment identity / similarity scores for the VP0 sequence of each RV-C strain across all other RV-C VP0 strains were plotted against the median sequence identity / similarity score for each strain relative to RV-C07 alone. When all RV-C species strains were analyzed as a single cluster (Figure 6A), VP0 of RV-C24, C25, and C49 had the highest median fragment identity / similarity scores across all other RV-C species strains tested, while having different sequence identity / similarity scores than RV-C07. When RV-C species strains were divided into two clusters (Figure 6B), RV-C19 VP0 was identified from the first cluster and RV-C01 VP0 from the second cluster as possible centroids.

[0221] Comparing the identified VP0 sequences using uncombined sequence identity and similarity to RV-C07 VP0 and uncombined identity and sequence to VP0 of all RV-C strains (Table 5), RV-C24 VP0, which has the best overall balance between predicted interstrain coverage and dissimilarity to RV-C07 VP0, was selected as the centroid RV-C species VP0. From the 2-cluster analysis, VP0 of RV-C01 and RV-C19 were selected for further study.

[0222] [Table 5]

[0223] RV-A16 VP0 immunization Figure 3 is a set of histograms showing the results of ELISPOT experiments to analyze the cellular immunity induced by immunization with RV-A16 VP0 protein against VP0 from other RV species strains. Mice were immunized twice with RV-A16 VP0 protein with adjuvant as described above, followed by collection of spleens. To quantify the level of cellular immunity induced by immunization with RV-A16 VP0, splenocytes were isolated and incubated with various stimuli as shown in Figure 3. Stimuli-activated splenocytes secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0224] The results show that splenocytes from mice immunized with RV-A16 VP0 secrete IFN-γ in response to stimulation with a pool of VP0 peptides derived from a diverse set of RVs from species A (Fig. 3B). This is in addition to the secretion of IFN-γ in response to restimulation with the same recombinant RV-A16 VP0 protein used for immunization (Fig. 3A).

[0225] When splenocytes from mice immunized with RV-A16 VP0 were stimulated with nothing (medium alone) or with an irrelevant peptide, the production of IFN-γ by the splenocytes was negligible (Figures 3A and 3B). When the extent of IFN-γ secretion by splenocytes in response to the RV-A species peptide pool was statistically compared to that produced in response to stimulation with an irrelevant peptide, the results for all peptide pools tested were highly significant (Figure 3B). These results demonstrate that immunization of mice with RV-A16 VP0 induces cellular immunity that cross-reacts with other members of the RV species A. However, when splenocytes from mice immunized with RV-A16 were stimulated with a peptide pool corresponding to the VP0 protein from strains of the RV species B and C, no significant secretion of IFN-γ was observed (Figure 3C). This indicates that immunization with RV-A16 does not induce cross-reactive cellular immunity to strains other than the RV species A.

[0226] RV-B06 VP0 immunization Figure 4 is a set of histograms showing the results of an ELISPOT experiment to analyze the cellular immunity induced by immunization with RV-B06 VP0 protein. Mice were immunized twice with RV-B06 VP0 protein with adjuvant as described above, followed by harvesting of spleens. To quantify the level of cellular immunity induced by immunization with RV-B06 VP0, splenocytes were isolated and incubated with various stimuli as shown in Figure 4. Splenocytes activated by the stimuli secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0227] The results show that splenocytes from mice immunized with RV-B06 VP0 secrete IFN-γ in response to stimulation with a VP0 peptide pool derived from a diverse set of RVs of species B (Figure 4B). This is in addition to the secretion of IFN-γ in response to the immunizing RV-B06 VP0 protein (Figure 4A) and the RV-B06 peptide pool (Figure 4B).

[0228] When splenocytes from mice immunized with RV-B06 VP0 were stimulated unstimulated (medium alone) or with an irrelevant peptide, there was negligible production of IFN-γ by the splenocytes (Figures 4A and 4B). When the extent of IFN-γ secretion by splenocytes in response to RV-B species peptide pools was statistically compared to that produced in response to stimulation with an irrelevant peptide, the results for all peptide pools tested were highly significant (Figure 4B).

[0229] These results demonstrate that immunization of mice with RV-B06 VP0 induces cellular immunity that cross-reacts with other members of the RV B species. However, when splenocytes from mice immunized with RV-B06 were stimulated with a peptide pool corresponding to VP0 proteins from strains of the RV-A and C species, significant secretion of IFN-γ was observed only in response to peptides from several RV-A species strains (Figure 4C). This indicates that immunization with RV-B06 VP0 induces limited cross-reactive cellular immunity to strains of the RV-A species, but not against strains of the RV-C species.

[0230] To further demonstrate that immunization of mice with RV-B06 VP0 elicits immunity, humoral responses to the immunizing antigen were evaluated. Mice were immunized twice with RV-B06 VP0 protein with adjuvant as described above, followed by serum collection. The amount of RV-B06 VP0-specific antibodies in mouse serum was determined by ELISA. Figure 10 is a set of line graphs showing the levels of anti-RV-B06 VP0 antibodies in serum from mice after immunization quantified by ELISA.

[0231] All mice immunized with RV-B06 VP0 had high levels of antibodies that specifically recognized RV-B06 VP0, whereas control immunized mice had no discernible levels of antibodies. These results demonstrate that in addition to the induction of cross-reactive cellular immunity by immunization with RV-B06 VP0, there was also a strong humoral response against the immunizing protein.

[0232] RV-C07 VP0 immunization Figure 5 is a set of histograms showing the results of ELISPOT experiments to analyze the cellular immunity against VP0 peptides from other strains of RV induced by immunization with RV-C07 VP0 protein. Mice were immunized twice with RV-C07 VP0 protein with adjuvant as described above, followed by harvesting of spleens. To quantify the level of cellular immunity induced by immunization with RV-C07 VP0, splenocytes were isolated and incubated with various stimuli as shown in Figure 5. Stimuli-activated splenocytes secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0233] The results show that splenocytes from mice immunized with RV-C07 VP0 secrete IFN-γ when restimulated with the RV-C07 VP0 immunogen (Figure 5A), whereas the secretion of IFN-γ in response to stimulation with peptide pools corresponding to strains of other RV-C species was less significant (Figure 5B). Moreover, there was little cross-reactivity to VP0 peptides from other RV species (Figure 5C). As a result, a deeper bioinformatic analysis was performed to identify other VP0 sequences that could act as centroid immunogens covering strains of the RV-C species.

[0234] RV-C01 VP0 immunization Figure 7 is a set of histograms showing the results of ELISPOT experiments to analyze the cellular immunity against other strains of RV induced by immunization with RV-C01 VP0 protein. Mice were immunized twice with RV-C01 VP0 protein with adjuvant as described above, followed by harvesting of spleens. To quantify the level of cellular immunity induced by immunization with RV-C01 VP0, splenocytes were isolated and incubated with various stimuli as shown in Figure 7. Stimuli-activated splenocytes secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0235] The results show that splenocytes from mice immunized with RV-C01 VP0 secrete IFN-γ in response to stimulation with VP0 peptide pools derived from a diverse set of RVs from C species (Figure 7B). This is in addition to the secretion of IFN-γ in response to the immunizing RV-C01 VP0 protein (Figure 7A) and peptide pools (Figure 7B).

[0236] When splenocytes from mice immunized with RV-C01 VP0 were stimulated with nothing (medium alone, FIG. 7A) or with an irrelevant peptide (FIG. 7B), the production of IFN-γ by the splenocytes was negligible. When the extent of IFN-γ secretion by splenocytes in response to RV-C species peptide pools was statistically compared to that produced in response to stimulation with an irrelevant peptide, the results for all peptide pools tested were highly significant, except for the RV-C19 VP0 peptide (FIG. 7B).

[0237] These results demonstrate that immunization of mice with RV-C01 VP0 induces cellular immunity that cross-reacts with other members of RV species C. Furthermore, when splenocytes from mice immunized with RV-C01 were stimulated with a pool of VP0 peptides derived from other RV species strains, there was significant secretion of IFN-γ in response to peptides from both RV species A and B (Figure 7C).

[0238] To further demonstrate that immunization of mice with RV-C01 VP0 induces immunity, humoral responses to the immunizing antigen were evaluated. Mice were immunized twice with RV-C01 VP0 protein with adjuvant as described above, followed by collection of serum. The amount of RV-C01 VP0-specific antibodies in mouse serum was determined by ELISA. Figure 10 is a set of line graphs showing the levels of anti-RV-C01 VP0 antibodies in serum from mice after immunization quantified by ELISA.

[0239] All mice immunized with RV-C01 VP0 had high levels of antibodies that specifically recognized RV-C01 VP0, whereas control-immunized mice had no discernible levels of antibodies. These results demonstrate that in addition to the induction of cross-reactive cellular immunity by immunization with RV-C01 VP0, there was also a strong humoral response against the immunizing protein.

[0240] RV-C19 VP0 immunization Figure 8 is a set of histograms showing the results of ELISPOT experiments to analyze the cellular immunity against other strains of RV induced by immunization with RV-C19 VP0 protein. Mice were immunized twice with RV-C19 VP0 protein with adjuvant as described above, followed by harvesting of spleens. To quantify the level of cellular immunity induced by immunization with RV-C19 VP0, splenocytes were isolated and incubated with various stimuli as shown in Figure 8. Stimuli-activated splenocytes secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0241] The results show that splenocytes from mice immunized with RV-C19 VP0 secrete IFN-γ in response to stimulation with VP0 peptide pools derived from a diverse set of RVs from C species (Figure 8B). This is in addition to the secretion of IFN-γ in response to the immunizing RV-C19 VP0 protein (Figure 8A) and peptide pools (Figure 8B).

[0242] When splenocytes from mice immunized with RV-C19 VP0 were stimulated unstimulated (medium alone, FIG. 8A) or with an irrelevant peptide (FIG. 8B), there was negligible production of IFN-γ by the splenocytes. When the extent of IFN-γ secretion by splenocytes in response to RV-C species peptide pools was statistically compared to that produced in response to stimulation with an irrelevant peptide, the results for all peptide pools tested were highly significant (FIG. 8B).

[0243] These results demonstrate that immunization of mice with RV-C19 VP0 elicits cellular immunity that cross-reacts with other members of the RV species C. Furthermore, when splenocytes from mice immunized with RV-C19 were stimulated with a pool of VP0 peptides derived from other RV species strains, there was significant secretion of IFN-γ in response to peptides from both RV-A and B species (Figure 8C).

[0244] To further demonstrate that immunization of mice with RV-C19 VP0 induces immunity, humoral responses to the immunizing antigen were evaluated. Mice were immunized twice with RV-C19 VP0 protein with adjuvant as described above, followed by collection of serum. The amount of RV-C19 VP0-specific antibodies in mouse serum was determined by ELISA. Figure 10 is a set of line graphs showing the levels of anti-RV-C19 VP0 antibodies in serum from mice after immunization quantified by ELISA.

[0245] All mice immunized with RV-C19 VP0 had high levels of antibodies that specifically recognized RV-C19 VP0, whereas control-immunized mice had no discernible levels of antibodies. These results demonstrate that in addition to the induction of cross-reactive cellular immunity by immunization with RV-C19 VP0, there was also a strong humoral response against the immunizing protein.

[0246] RV-C24 VP0 immunization Figure 9 is a set of histograms showing the results of ELISPOT experiments to analyze the cellular immunity against other strains of RV induced by immunization with RV-C24 VP0 protein. Mice were immunized twice with RV-C24 VP0 protein with adjuvant as described above, followed by harvesting of spleens. To quantify the level of cellular immunity induced by immunization with RV-C24 VP0, splenocytes were isolated and incubated with various stimuli as shown in Figure 9. Stimuli-activated splenocytes secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0247] The results show that splenocytes from mice immunized with RV-C24 VP0 secrete IFN-γ in response to stimulation with a VP0 peptide pool derived from a diverse set of RVs from C species (Figure 9B). This is in addition to the secretion of IFN-γ in response to the immunizing RV-C24 VP0 protein (Figure 9A) and the RV-C24 peptide pool (Figure 9B).

[0248] When splenocytes from mice immunized with RV-C24 VP0 were stimulated unstimulated (medium alone, FIG. 9A) or with an irrelevant peptide (FIG. 9B), there was negligible production of IFN-γ by the splenocytes. When the extent of IFN-γ secretion by splenocytes in response to RV-C species peptide pools was statistically compared to that produced in response to stimulation with an irrelevant peptide, the results for all peptide pools tested were highly significant (FIG. 9B).

[0249] These results demonstrate that immunization of mice with RV-C24 VP0 induces cellular immunity that cross-reacts with other members of the RV C species. Furthermore, when splenocytes from mice immunized with RV-C24 were stimulated with a pool of VP0 peptides derived from other RV species strains, there was a significant secretion of IFN-γ in response to all peptides from both RV-A and B species (Figure 9C), suggesting that RV-C24 VP0 has the potential to induce cross-reactive cellular immunity against all species of RV.

[0250] To further demonstrate that immunization of mice with RV-C24 VP0 induces immunity, humoral responses to the immunizing antigen were evaluated. Mice were immunized twice with RV-C24 VP0 protein with adjuvant as described above, followed by collection of serum. The amount of RV-C24 VP0-specific antibodies in mouse serum was determined by ELISA. Figure 10 is a set of line graphs showing the levels of anti-RV-C24 VP0 antibodies in serum from mice after immunization quantified by ELISA.

[0251] All mice immunized with RV-C24 VP0 had high levels of antibodies that specifically recognized RV-C24 VP0, whereas control-immunized mice had no discernible levels of antibodies. These results demonstrate that in addition to the induction of cross-reactive cellular immunity by immunization with RV-C24 VP0, there was also a strong humoral response against the immunizing protein.

[0252] Recombinant RV-C24 VP0 induced immunogenicity in cynomolgus monkeys. Cynomolgus monkeys were immunized twice with recombinant RV-C24 VP0 (rRV-C24 VP0) protein with or without adjuvant (control) as described, and blood was collected 28 days after the first immunization to prepare PBMCs. To quantify the level of cellular immunity induced by immunization with rRV-C24 VP0 protein, PBMCs were incubated with stimuli or controls of different peptide pools corresponding to full-length VP0 from different strains of RV, as shown on the x-axis of Figure 11. PBMCs activated by the stimuli secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0253] The results show that PBMC from a representative cynomolgus macaque immunized with rRV-C24 VP0 secreted IFN-γ in response to stimulation with VP0 peptide pools derived from a diverse set of RVs of species A, B, and C (Figure 11B). PBMC from animals immunized with adjuvant alone (control) produced negligible IFN-γ in response to VP0 peptide pool stimulation (Figure 11A).

[0254] When PBMCs from cynomolgus monkeys immunized with rRV-C24 VP0 were stimulated with an irrelevant peptide control pool (Figure 11B), IFN-γ production was negligible. When PBMCs from control cynomolgus monkeys were stimulated with an irrelevant peptide control pool or with the VP0 peptide pool (Figure 11A), IFN-γ production was again negligible.

[0255] These results demonstrate that immunization of cynomolgus macaques with rRV-C24 VP0 induces cellular immunity that cross-reacts with RV species A, B, and C strains.

[0256] To demonstrate the Th phenotype of the cellular immune response, the IFN-γ response of PBMCs from two cynomolgus monkeys immunized with adjuvant alone (control) or rRV-C24 VP0 protein was compared to the IL-4 response by ELISPOT after stimulation with peptide pools. IFN-γ is a canonical Th1 cytokine and IL-4 is a canonical Th2 cytokine. Figure 12A demonstrates that PBMCs from two cynomolgus monkeys immunized with rRV-C24 VP0 protein secrete more IFN-γ in response to stimulation with a peptide pool derived from RV C species, but not with adjuvant alone (control), than was produced in response to an irrelevant peptide control pool, but secreted negligible IL-4 (Figure 12B).

[0257] To further demonstrate that immunization with rRV-C24 VP0 induces immunity in cynomolgus monkeys, humoral responses to the immunizing antigen were evaluated. Figure 13 shows a histogram of RV-C24 VP0-binding IgG quantified by ELISA in the plasma of cynomolgus monkeys at various time points after immunization with rRV-C24 VP0 protein. The results show that immunization with rRV-C24 VP0 induced the production of RV-C24 VP0-specific IgG antibodies, which increased over time, whereas animals immunized with adjuvant alone (control) did not produce detectable RV-C24 VP0-specific IgG.

[0258] Transfection of HEK293 cells with RV VP0 mRNA To demonstrate that RV VP0 mRNA is translated into full-length VP0 protein in cells, HEK293 cells were transfected with various RV VP0 mRNAs as described above.

[0259] Figure 14A is a Western blot of lysates from HEK293 cells transfected with RV-A16 VP0 mRNA performed 24, 48, 72, or 96 hours after transfection. A positive control of recombinant RV-A16 VP0 protein was loaded on the gel (labeled rRV-A16 VP0). The results demonstrate that transfection of HEK293 cells with RV-A16 VP0 mRNA results in expression of RV-A16 VP0 protein, which peaks at 48 hours after transfection.

[0260] FIG. 14B is a Western blot of lysates from HEK293 cells transfected with RV-B06 or RV-C24 VP0 mRNA at 24, 48, 72, or 96 hours after transfection. The results demonstrate that HEK293 cells transfected with RV-B06 VP0 or C24 VP0 mRNA express RV-B06 and RV-C24 VP0 proteins, respectively. Expression of both proteins peaks at 24 and 48 hours after transfection. By comparing the VP0 band detected using monoclonal antibodies or antisera against the markers, the VP0 band is observed to have the correct approximate molecular weight of 36 kDa.

[0261] RV VP0 mRNA induced immunogenicity in mice. Mice were immunized twice with RV-C24 VP0 mRNA as described above, and spleens were harvested and splenocytes prepared 42 days after the first immunization. To quantify the level of cellular immunity induced by immunization with RV-C24 VP0 mRNA, splenocytes were incubated with peptide pools corresponding to full-length VP0 proteins from various strains of RV or a control pool, as shown in Figure 15. Stimulated splenocytes secreted the cytokine IFN-γ, and the extent of IFN-γ secretion was quantified by ELISPOT analysis.

[0262] The results show that splenocytes from mice immunized with RV-C24 VP0 mRNA secrete IFN-γ in response to stimulation with a VP0 peptide pool derived from a diverse set of C-species RVs. When splenocytes from mice immunized with RV-C24 VP0 mRNA were stimulated with an irrelevant peptide pool (Figure 15), IFN-γ production was negligible. When the response to stimulation with the VP0 peptide pool was statistically compared to the response to the irrelevant peptide pool, the results were significant. These results demonstrate that immunization of mice with RV-C24 VP0 mRNA induces cellular immunity that cross-reacts with other members of the C-species RVs.

[0263] To further demonstrate that immunization with RV-C24 VP0 mRNA induces immunity in mice, humoral responses to the immunizing antigen were evaluated. Figure 16 is a histogram showing the concentration of RV-C24 VP0-binding IgG quantified by ELISA in the serum of mice at various time points after immunization with RV-C24 VP0 mRNA. The results show that immunization with RV-C24 VP0 mRNA induced the production of RV-C24 VP0-specific IgG antibodies, which increased over time, while control animals did not produce detectable RV-C24 VP0-specific IgG.

[0264] RV VP0 mRNA induced immunogenicity in cynomolgus monkeys. Figure 17 is a histogram showing the results of an ELISPOT experiment analyzing the cellular immunity induced by immunization with mRNA concatemers consisting of RV-C24 / A16 / B06 VP0 mRNA in sequences separated by the 2A peptide coding sequence. Immunization of animals with RV VP0 mRNA concatemers allows the determination of the immune response against the respective antigen.

[0265] Cynomolgus monkeys were immunized twice with RV-C24 / A16 / B06 VP0 mRNA concatemers or controls as described above, and blood was collected 28 days after the first immunization to prepare PBMCs. To quantify the level of cellular immunity against each antigen induced by immunization with RV-C24 / A16 / B06 VP0 mRNA concatemers, PBMCs were incubated with peptide pools corresponding to full-length VP0 of RV-A16, B06, C24 or a control irrelevant peptide pool, and IFN-γ (Figure 17A) or IL-4 (Figure 17B) secretion was quantified by ELISPOT.

[0266] The results show that PBMCs from cynomolgus monkeys immunized with VP0 mRNA of RV-C24 / A16 / B06 concatemers secrete IFN-γ in response to stimulation with a VP0 peptide pool corresponding to the VP0 of full-length RV-A16, B06, and C24. When PBMCs from monkeys immunized with VP0 mRNA of RV-C24 / A16 / B06 concatemers were stimulated with an irrelevant peptide pool, IFN-γ production was negligible. PBMCs from control immunized animals produced negligible IFN-γ in response to stimulation with the RV VP0 peptide pool or an irrelevant peptide control pool. The results in Figure 17A demonstrate that immunization of cynomolgus monkeys with VP0 mRNA of RV-C24 / A16 / B06 concatemers induces cellular immunity against each of the three antigens produced by the VP0 mRNA concatemers.

[0267] To demonstrate the Th phenotype of the cellular immune response, the IFN-γ response of PBMCs from cynomolgus monkeys immunized with RV-C24 / A16 / B06 VP0 mRNA concatemers was compared to the IL-4 response by ELISPOT. IFN-γ is a canonical Th1 cytokine, and IL-4 is a canonical Th2 cytokine. Figure 17B demonstrates that PBMCs from cynomolgus monkeys immunized with RV-C24 / A16 / B06 concatemers VP0 mRNA produced negligible IL-4 compared to IFN-γ (Figure 17A) in response to stimulation with RV VP0 or a control peptide pool. This result demonstrates that the cellular immune response primed with RV-C24 / A16 / B06 concatemers VP0 mRNA is biased toward Th1.

[0268] To further demonstrate that immunization of cynomolgus macaques with RV VP0 mRNA elicits immunity, humoral responses to each antigen produced by the RV-C24 / A16 / B06 VP0 mRNA concatemer were determined.

[0269] Figure 18 shows histograms showing the concentration of RV-A16 (Figure 18A), RV-B06 (Figure 18B), and RV-C24 (Figure 18C) VP0-binding IgG quantified by ELISA in the plasma of cynomolgus monkeys at various time points after immunization. These results show that immunization with RV-C24 / A16 / B06 VP0 mRNA concatemers induces the production of RV-VP0-specific IgG antibodies against each of the three RV antigens in the concatemer in cynomolgus monkeys.

[0270] Efficacy of RV VP0 mRNA immunization in mice To demonstrate that immunization with RV VP0 mRNA is protective against subsequent heterotypic RV infection, mice were immunized with RV-A16 VP0 mRNA and then infected intranasally with RV-A1, a species A strain of RV that is heterotypic to the immunizing strain, as described above.

[0271] Figure 19 is a histogram showing the concentration of RV-A1 RNA in the lungs of control and RV-A16 VP0 mRNA immunized mice as determined by RT-qPCR. The results demonstrate that while control and immunized mice have similar levels of viral RNA in the lungs one day after infection, immunized mice have significantly less viral RNA than control mice by day 6. By day 14, the concentration of viral RNA becomes negligible in both groups of animals. The results demonstrate that immunization with RV VP0 mRNA accelerates the clearance of heterotypic RV infection from the lungs of mice.

[0272] RV VP0 mRNA induced cell-mediated immunity. To demonstrate how immunization with RV VP0 mRNA protects mice from RV infection, we harvested spleens from mice immunized with RV-A16 VP0 mRNA or control and then infected with heterotypic RV-A1 on day 14 post-infection to generate splenocytes. Splenocytes were stimulated with peptide pools corresponding to full-length VP0 from a diverse set of strains covering each species of RV or with a control irrelevant peptide pool. IFN-γ secretion in response to peptide stimulation was measured by ELISPOT.

[0273] The results shown in Figure 20 are surprising and demonstrate that splenocytes from mice immunized with RV-A16 VP0 mRNA and subsequently infected with RV-A1 secrete IFN-γ in response to stimulation with a VP0 peptide pool derived from a broad range of RV strains covering species A, B, and C. When the results obtained with the VP0 peptide pool were statistically compared to those obtained with an irrelevant peptide control pool, the results were highly significant.

[0274] In contrast, in control mice, negligible splenocytes secreted IFN-γ after stimulation with RV VP0 or a control peptide pool, as shown in Figure 21. The results in Figures 20 and 21 demonstrate that immunization with VP0 mRNA derived from a single strain of RV results in a surprising cross-strain cellular immunity.

[0275] To further substantiate the mechanism by which immunization with RV VP0 mRNA protects mice from infection with RV-A1, we performed immunophenotyping studies in the lungs of RV-A16 VP0 mRNA-immunized and non-immunized mice following infection with RV-A1. Methods are described above.

[0276] Figure 22 shows histograms showing the mean numbers of CD62- CD44+ effector CD4+ (Figure 22A) and CD8+ (Figure 22B) T cells in the lungs of mice immunized with RV-A16 VP0 mRNA and control mice, respectively, after infection with RV-A1. The results demonstrate that at 6 and 14 days post-infection, there were significantly more effector CD4+ and CD8+ cells in the lungs of immunized mice than in control mice.

[0277] Immunization with RV VP0 mRNA primes Th1-biased T cell immunity. To determine the Th phenotype primed by RV VP0 mRNA immunization, lungs were harvested from mice infected with RV-A1 after immunization with RV-A16 VP0 mRNA or control. Single cell populations were prepared from the lungs and cells were incubated with a peptide pool corresponding to full-length VP0 from RV-A1. Intracellular cytokine staining flow cytometry analysis was performed to measure the frequency of Th1 and Th2 cells as described.

[0278] Th1 cells were identified as T-bet+ IFN-γ+ CD4+ cells and Th2 cells were identified as Gata3+ IL-4+ CD4+ cells. Figure 23 is a set of correlation plots plotting the frequency of Th1 cells against the frequency of Th2 cells for each animal following stimulation of lung cells with the RV-A1 VP0 peptide pool.

[0279] The results demonstrate that the frequency of RV-A1 VP0-reactive Th1 cells is greater than that of RV-A1 VP0-reactive Th2 cells in the lungs of immunized mice (Figure 23B). The opposite was observed in control mice (Figure 23A), where the proportion of Th2 cells was greater than that of Th1 cells. The results demonstrate that RV VP0 mRNA primes a Th1-biased T cell response, resulting in a greater proportion of RV-reactive CD4+ T cells of the Th1 phenotype than the Th2 phenotype in the lungs of immunized mice.

[0280] RV VP0 primes tissue-resident T cell memory. An important component of protective cellular immunity induced by natural infection with respiratory viruses is tissue-resident memory T cells (T RM ) is the formation of these T RM The cells proliferate rapidly to eliminate the subsequent infection.

[0281] T in the airways and lungs of mice immunized with intramuscularly delivered RV VP0 mRNA vaccine RM To determine whether RV-A16 VP0 mRNA or control could prime the formation of CD4+ and CD8+ TRM cells, bronchoalveolar lavage fluid (BAL) and lungs were collected from mice infected with RV-A01 after immunization with RV-A16 VP0 mRNA or control. Single cell populations were prepared from lungs at various time points post-infection and immunophenotyping was performed to identify and enumerate CD4+ and CD8+ TRM cells. CD4+ and CD8+ TRM cells were identified as CD103+ CD69+ CD4+ or CD103+ CD69+ CD8+ cells, respectively.

[0282] FIG. 24 shows CD4+ (FIG. 24A) and CD8+ (FIG. 24B) T cells in the BAL of control and RV-A16 VP0 mRNA-immunized mice at various time points after RV-A1 infection. RM The results show that 6 days after infection, there were significantly more CD4+ and CD8+ T cells in the BAL of immunized mice than in control mice. RM This indicates that cells are present.

[0283] FIG. 25 shows CD4+ (FIG. 25A) and CD8+ (FIG. 25B) T cells in the BAL of control and RV-A16 VP0 mRNA-immunized mice at various times after RV-A1 infection. RM The results are a set of histograms showing the frequency of CD4+ and CD8+ T cells in the BAL of immunized mice 6 and 14 days after infection. RM 4A and 4B show that these cells are present at a significantly higher percentage of total CD4+ or CD8+ cells, respectively, compared to control mice.

[0284] FIG. 26 shows CD4+ (FIG. 26A) and CD8+ (FIG. 26B) T cells in the lungs of control and RV-A16 VP0 mRNA-immunized mice at various time points after RV-A1 infection. RM The results show that immunized mice had significantly more CD4+ T cells in their lungs compared to control mice 6 days after infection. RM cells, and significantly more CD8+ T cells 6 and 14 days after infection. RM This indicates that cells are present.

[0285] Figure 27 is a set of histograms showing the frequency of CD4+ (Figure 27A) and CD8+ (Figure 27B) TRM cells in the lungs of control and RV-A16 VP0 mRNA immunized mice at various times after RV-A1 infection. The results show that CD4+ and CD8+ TRM cells were present at significantly higher percentages among total CD4+ or CD8+ cells, respectively, in the lungs of immunized mice 6 and 14 days after infection compared to control mice.

[0286] These results indicate that intramuscular administration of RV VP0 mRNA vaccine inhibits T cell proliferation in the airways and lungs of mice. RM Upon subsequent infection with a heterotypic strain of RV, the vaccine primes T RM The cells were primed for rapid proliferation.

[0287] conclusion The inventors have identified RV VP0 proteins from a single representative strain that can elicit a broad cellular immune response that cross-reacts with other RV strains from the same species. In particular, as described in the Examples (Figures 3, 4, 5, 7, 8, and 9), splenocytes from mice immunized with RV-A16 cross-react with VP0 peptides from strains of other RV-A species, mice immunized with RV-B06 VP0 cross-react with VP0 peptides from members of other RV-B species and peptides from strains of several RV-A species, and splenocytes from mice immunized with VP0 of RV-C01, RV-C19, or RV-C24 cross-react with peptides from members of other RV-C species. Furthermore, immunization with VP0 proteins from RV-C species confers cellular immunity that recognizes VP0 peptides from both RV-A and RV-B species. Thus, the immunogenic compositions according to the claimed invention comprising VP0 peptides selected from RV-A16, RV-B06, RV-C01, RV-C19, and RV-C24 in any combination can be used to immunize humans against infection with all RV strains.

[0288] Furthermore, we have demonstrated that immunization with mRNA encoding the RV VP0 peptide induces a broad cellular immune response. For example, as shown in Figure 15, we have demonstrated that immunization of mice with RV-C24 VP0 mRNA induces cellular immunity that cross-reacts with other members of the RV C species.

[0289] Advantageously, therefore, the immunogenic composition according to the invention overcomes the challenge of antigenic heterogeneity across RV strains that has hindered the development of RV vaccines to date. Moreover, the immunogenic composition is particularly effective because vaccination with RV VP0 antigens provides protection to patients with chronic pulmonary pathologies and therefore at risk of RV-induced exacerbations of their pathologies, thereby reducing the associated morbidity, mortality and health care burden. (References) TIFF2025510530000034.tif161153

Claims

1. 1. An immunogenic composition comprising at least one isolated human rhinovirus (RV) VP0 peptide or an isolated polynucleotide encoding said peptide, wherein said peptide is selected from the group consisting of RV-C VP0 peptide, RV-B VP0 peptide, RV-A28 VP0, and RV-A89 VP0, or a variant or fragment thereof.

2. 2. The immunogenic composition of claim 1, comprising at least two isolated human rhinovirus (RV) VP0 peptides or isolated polynucleotides encoding said peptides, wherein said peptides are selected from the group consisting of RV-C VP0 peptide, RV-B VP0 peptide, RV-A28 VP0, and RV-A89 VP0.

3. 3. The immunogenic composition of claim 1 or 2, comprising at least three isolated human rhinovirus (RV) VP0 peptides, or isolated polynucleotides encoding said peptides, wherein said peptides are selected from the group consisting of RV-C VP0 peptide, RV-B VP0 peptide, RV-A28 VP0, and RV-A89 VP0.

4. The immunogenic composition of any one of claims 1 to 3, wherein the RV-C VP0 peptide is selected from the group consisting of RV-C07 VP0, RV-C19 VP0, RV-C24 VP0, and RV-C01 VP0, or variants or fragments thereof.

5. 5. The immunogenic composition of claim 1, wherein the RV-B VP0 peptide is RV-B06 VP0.

6. 6. The immunogenic composition of any one of claims 1 to 5, further comprising an isolated RV-A16 VP0 peptide and / or an isolated RV-A29 VP0 peptide, or an isolated polynucleotide encoding said peptide, or a variant or fragment thereof.

7. The immunogenic composition of any one of claims 1 to 6, wherein the fragment of at least one RV VP0 peptide comprises or consists of the corresponding VP2 and / or VP4 peptide.

8. The immunogenic composition of any one of claims 1 to 7, wherein at least two or more of the RV VP0 peptides form a fusion peptide.

9. 9. The immunogenic composition according to any one of claims 1 to 8, wherein said polynucleotide comprises a nucleic acid sequence encoding said peptide, preferably said nucleic acid sequence being under the control of elements necessary for its expression in a mammalian cell.

10. The immunogenic composition of claim 9, wherein the nucleic acid is DNA or RNA, optionally wherein the RNA is messenger RNA (mRNA) or self-amplifying RNA (saRNA).

11. The immunogenic composition of any one of claims 1 to 10, wherein the isolated polynucleotide encodes at least two or more of the RV VP0 peptides or wherein at least two or more of the RV VP0 peptides are encoded by multiple separate polynucleotides.

12. The immunogenic composition of any one of claims 1 to 11, comprising an isolated polynucleotide encoding the RV-C24 VP0 peptide, an isolated polynucleotide encoding the RV-A16 VP0 peptide, and an isolated polynucleotide encoding the RV-B06 VP0 peptide.

13. The immunogenic composition of any one of claims 1 to 12, wherein the at least one RV VP0 peptide comprises an amino acid sequence substantially as set forth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, and / or 25, or a variant or fragment thereof.

14. 14. The immunogenic composition of any one of claims 1 to 13, wherein the isolated polynucleotide comprises a nucleotide sequence substantially as set out in any of SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, and / or 27, or a variant or fragment thereof.

15. 15. The immunogenic composition of claim 1, further comprising an adjuvant.

16. 16. The immunogenic composition of any one of claims 1 to 15, which is a vaccine.

17. 17. An immunogenic composition according to any one of claims 1 to 16 for use in therapy or prophylaxis.

18. 17. The immunogenic composition of any one of claims 1 to 16 for use in eliciting an immune response, preferably against rhinovirus infection.

19. The immunogenic composition for use according to claim 18, which induces an immune response against at least one, at least two, or at least three species of rhinovirus, more particularly against RV-A, RV-B, and / or RV-C.

20. 20. The immunogenic composition for use according to claim 18 or 19, which induces an immune response against at least one strain of RV-A, at least one strain of RV-B, and / or at least one strain of RV-C.

21. 21. The immunogenic composition for use according to any one of claims 18 to 20, which induces an immune response against the common cold, viral wheezing diseases, asthma exacerbations, bronchiectasis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and / or chronic fibrosing lung diseases.

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