BIANCUCCI MARCO (US)
CHESTERMAN CHELSY CARYN (US)
HARSHBARGER WAYNE DANIEL (US)
MOUSAVI KAMBIZ (US)
WAHOME NEWTON MUCHUGU (US)
WANG XIAOFENG (US)
WILLIAMS JAMES ALAN (US)
MALLETT COREY (US)
PHOGAT SANJAY (US)
HOLZAPFEL GENEVIEVE (US)
PHUNG EMILY (US)
WO2019195291A1 | 2019-10-10 | |||
WO2017174564A1 | 2017-10-12 |
US20170182151A1 | 2017-06-29 | |||
US20180319846A1 | 2018-11-08 |
"GenBank", Database accession no. KT992094
"Uniprot", Database accession no. P13843
RHA ET AL., PEDIATRICS, vol. 146, no. 1, July 2020 (2020-07-01), pages e20193611
FALSEY ET AL., N ENGL J MED., vol. 352, no. 17, 28 April 2005 (2005-04-28), pages 1749 - 59
FALSEYWALSH, CLIN MICROBIOL REV., vol. 13, 2000, pages 371 - 84
GROOTHUIS ET AL., ADV THER, vol. 28, 2011, pages 110 - 25
FELTES ET AL., PEDIATR RES, vol. 70, 2011, pages 186 - 91
CARBONELL-ESTRANY ET AL., PEDIATRICS, vol. 125, 2010, pages e35 - 51
O'BRIEN ET AL., LANCET INFECT DIS, vol. 15, 2015, pages 1398 - 408
PEDIATRICS, vol. 102, 1998, pages 531 - 537
KRARUP ET AL., NAT COMMUN., vol. 6, 3 September 2015 (2015-09-03), pages 8143
MCLELLAN ET AL., SCIENCE, vol. 340, no. 6136, 2013, pages 1113 - 598
WHITEHEAD ET AL., J VIROL., vol. 72, no. 5, 1998, pages 4467 - 4471
WHITEHEAD ET AL., J VIROL., vol. 73, no. 4, 1999, pages 3438 - 3442
KYTEDOOLITTLE, J MOL BIOL., vol. 157, 1982, pages 105 - 132
SIEVERS ET AL., METHODS MOL BIOL., vol. 1079, 2014, pages 105 - 16
GILMAN ET AL., PLOSPATHOGENS., vol. 11, no. 7, 2015, pages el005035
CORTI ET AL., NATURE, vol. 501, no. 7467, 2013, pages 439 - 43
CINGOZ, MABS, vol. l, no. 5, September 2009 (2009-09-01), pages 439 - 42
LUCAS ET AL., CHEM SCI., vol. 7, 2016, pages 1038 - 1050
CHEN ET AL., MOL CELL, vol. 76, no. 1, 2019, pages 96 - 109
EDGAR, BMC BIOINFORMATICS, vol. 5, 2004, pages 113
EDGAR ET AL., BIORXIV. 2021. 06.20.449169
GENNARO: "Remington: The Science and Practice of Pharmacy", 2000
VAN DRUNNEN LITTLEVAN DEN HURK ET AL., REV MED VIROL., vol. 17, no. 1, 2007, pages 5 - 34
AW ET AL., IMMUNOLOGY, vol. 120, no. 4, 2007, pages 435 - 46
CLAIMS 1. Respiratory syncytial virus fusion (RSV-F) protein in the pre-fusion conformation, which is mutated relative to SEQ ID NO: 1, and comprises (a) and (b): (a) (ai) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; and/or (aii) a substitution at position 301 of SEQ ID NO: 1 for M, F or I, and/or at position 303 of SEQ ID NO: 1 for V, M, F or I; and (b) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 208- 216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO: 1, and/or introduces, through substitution or insertion, a P residue into the region. 2. The RSV-F protein of any one of the preceding claims, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; and (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P. 3. The RSV-F protein of claim 2, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; and (b) a substitution at position 215 of SEQ ID NO: 1 for A. 4. The RSV-F protein of any one of the preceding claims, comprising a substitution of position 228 of SEQ ID NO: 1 for K, R or Q; optionally K or R; optionally K. 5. The RSV-F protein of any one of the preceding claims, comprising at least 2 or at least 3 substitutions selected from, or comprising the substitutions: a substitution at position 228 of SEQ ID NO: 1 for K, R or Q; a substitution at position 315 of SEQ ID NO: 1 for I or V; a substitution at position 455 of SEQ ID NO: 1 for V or I; and a substitution at position 459 of SEQ ID NO: 1 for M. 6. The RSV-F protein of claim 5, comprising at least 2 or at least 3 substitutions selected from, or comprising the substitutions: a substitution at position 228 of SEQ ID NO: 1 for K; a substitution at position 315 of SEQ ID NO: 1 for I; a substitution at position 455 of SEQ ID NO: 1 for V; and a substitution at position 459 of SEQ ID NO: 1 for M. 7. The RSV-F protein of claim 6, comprising the substitutions: a substitution at position 55 of SEQ ID NO: 1 for T; a substitution at position 215 of SEQ ID NO: 1 for A; optionally a substitution at position 228 of SEQ ID NO: 1 for K; a substitution at position 315 of SEQ ID NO: 1 for I; a substitution at position 455 of SEQ ID NO: 1 for V; and a substitution at position 459 of SEQ ID NO: 1 for M. 8. The RSV-F protein of any one of the preceding claims, comprising at least 4 or at least 5 substitutions selected from, or comprising the substitutions: a substitution at position 152 of SEQ ID NO: 1 for R, L or W; optionally a substitution at position 211 of SEQ ID NO: 1 for N; a substitution at position 228 of SEQ ID NO: 1 for K, R or Q; a substitution at position 315 of SEQ ID NO: 1 for I or V; a substitution at position 346 of SEQ ID NO: 1 for Q, D, H, K, N, R, S or W; optionally a substitution at position 445 of SEQ ID NO: 1 for D; a substitution at position 455 of SEQ ID NO: 1 for V or I, and a substitution at position 459 of SEQ ID NO: 1 for M. 9. The RSV-F protein of claim 8, comprising at least 4 or at least 5 substitutions selected from, or comprising the substitutions: a substitution at position 152 of SEQ ID NO: 1 for R; optionally a substitution at position 211 of SEQ ID NO: 1 for N; a substitution at position 228 of SEQ ID NO: 1 for K; a substitution at position 315 of SEQ ID NO: 1 for I; a substitution at position 346 of SEQ ID NO: 1 for Q; optionally a substitution at position 445 of SEQ ID NO: 1 for D; a substitution at position 455 of SEQ ID NO: 1 for V; and a substitution at position 459 of SEQ ID NO: 1 for M. 10. The RSV-F protein of claim 9, comprising the substitutions: a substitution at position 55 of SEQ ID NO: 1 for T; a substitution at position 152 of SEQ ID NO: 1 for R; optionally a substitution at position 211 of SEQ ID NO: 1 for N; a substitution at position 215 of SEQ ID NO: 1 for A; optionally a substitution at position 228 of SEQ ID NO: 1 for K; a substitution at position 315 of SEQ ID NO: 1 for I; a substitution at position 346 of SEQ ID NO: 1 for Q; optionally a substitution at position 445 of SEQ ID NO: 1 for D; a substitution at position 455 of SEQ ID NO: 1 for V; and a substitution at position 459 of SEQ ID NO: 1 for M. 11. The RSV-F protein of any one of the preceding claims, comprising an F2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to positions 26-108 or 26-109 of SEQ ID NO: 1. 12. The RSV-F protein of any one of the preceding claims, comprising an F1 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1. 13. The RSV-F protein of any one of the preceding claims, comprising a heterologous trimerisation domain at the C-terminus thereof, and/or C-terminal to the F1 domain; optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain. 14. The RSV-F protein of any one of claims 1-12, comprising a transmembrane domain at the C- terminus thereof, and/or C-terminal to the F1 domain; and optionally a cytoplasmic domain C- terminal to said transmembrane domain. 15. The RSV-F protein of any one of claims 1-12 or 14, wherein the RSV-F protein comprises a cytoplasmic domain; wherein, relative to a cytoplasmic domain according to SEQ ID NO: 109 or 110, 2-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 16. The RSV-F protein of claim 15, wherein 16-20, 17-20, 18-20, 19-20 or 20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 17. The RSV-F protein of any one of the preceding claims, wherein a signal peptide is not present in the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO: 1. 18. The RSV-F protein of any one of the preceding claims, wherein a p27 peptide is not present in the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO: 1. 19. The RSV-F protein of any of the preceding claims, wherein the RSV-F protein comprises an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1. 20. A nucleic acid encoding the RSV-F protein of any one of claims 1-19. 21. The nucleic acid of claim 20, wherein the nucleic acid is RNA. 22. A lipid nanoparticle comprising nucleic acid of claim 20 or 21. 23. A pharmaceutical composition comprising the RSV-F protein of any of claims 1-19, nucleic acid of claim 20 or 21, or lipid nanoparticle of claim 22; optionally for use in medicine. 24. The pharmaceutical composition for use of claim 23, for use in a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; a human older adult, optionally ≥50 or ≥60 years old; or a pregnant human female, optionally ≥28 weeks pregnant. 25. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of claims 1-19, the nucleic acid of claim 20 or 21, the lipid nanoparticle of claim 22, or the pharmaceutical composition of claim 23. |
Said reagent is sold as CLEANCAP Reagent AG (3'OMe) (TRILINK BIOTECHNOLOGIES) Generally, the RNA comprises a 3' poly-adenosine (“poly-A”) tail, e.g. comprising 10-700 A ribonucleotides. The poly-A tail may comprise at least two non-contiguous stretches of A ribonucleotides (also referred to as a “split poly-A tail”), or a (in particular, only one) contiguous stretch of A ribonucleotides. The total number of A ribonucleotides (“As”) in at least two non- contiguous stretches may be, for example, 10-700, such as 10-600, 10-500, 20-500, 50-500, 70-500, 100-500, 20-400, 30-300, 40-200, 50-150, 70-120, 100-120, or, in particular, 100-120. The total number of As in a (in particular, only one) contiguous stretch may be, for example, 10-700; such as 10-600, 20-600 or in particular 40-600 (such as 50-600, 80-600, 80-550, 100-500; or 40-70, 50-65 or 55-65). Wherein at least two non-contiguous stretches of As are used, these may be of differing length. For example, a first stretch may be 10-150 As in length, such as 10-100, 10-50, 15-50, 20-50, 20-40, 25-40, or, in particular 25-35 As in length. For example, a second stretch may be 10-150 As in length, such as 10-150, 20-120, 30-100, 40-90, 50-90, 60-90, 65-90, 70-90, or, in particular, 80-90 As in length. The first stretch may be located 5' or 3' relative to the second stretch. However, in a particular embodiment, the first stretch is located 5' relative to the second stretch. In a further particular embodiment, the polyA tail comprises, in the 5' to 3' direction, a first and a second non- contiguous stretch of As, that are 25-35 and 80-90 As in length respectively. In a further particular embodiment, the polyA tail comprises, in the 5'-3' direction, a first and a second non-contiguous stretch of As, that are 25-35 and 65-90 As in length respectively. In some embodiments, the at least two non-contiguous stretches of As is from, or is part of, the 3' untranslated region (UTR), e.g. as detailed below. The RNA preferably comprises (in addition to any 5' cap structure) one or more modified ribonucleotides, i.e. ribonucleotides that are modified in structure relative to standard A, C, G or U ribonucleotides. In other embodiments, the RNA does not comprise modified ribonucleotides, i.e. the RNA contains standard A, C, G or U ribonucleotides only (except for any 5' cap structure, if present, e.g. as detailed above). In preferred embodiments wherein one or more modified ribonucleotides are used, said one or more modified ribonucleotides may be, or may comprise, N1- methylpseudouridine (“1mΨ”); pseudouridine (“Ψ”); N1-ethylpseudouridine; 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6- methyladenosine (m 6 A); N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1- methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2- methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-O- methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis- hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'- O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6- hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2- methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; .alpha.-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2- (propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2'-Deoxy-2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6- (methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8- (halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7- methyladenine; 1-Deazaadenosine TP; 2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP; 2-aminoadenine; 2- Aminoadenosine TP; 2-Amino-ATP; 2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b- Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'-difluoroadenosine TP; 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2'-a- thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'- Deoxy-2'-b-bromoadenosine TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine TP; 2'-Deoxy-2'-b- thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2- methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3- bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3- iodoadenosine TP; 3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'- Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8- Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7- deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5- hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 2'-O- methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O- dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'-OMe- Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; .alpha.-thio- cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxy-CTP; 2'-Deoxy-2'-a- aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3- (alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5- (alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5- (trifluoromethyl)cytosine: 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-1-deaza- pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine: 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4- thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio- pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP; 2'O-methyl-N4-Bz- cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'- b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy-2'-a-mercaptocytidine TP; 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b- azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b- fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP; 2'-Deoxy-2'- b-thiomethoxycytidine TP; 2'-O-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'- Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl- CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5'-Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl- cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2- methylguanosine; Wyosine; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7- aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7- dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2- dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo- guanosine; N1-methyl-guanosine; .alpha.-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'- Amino-2'-deoxy-GTP; 2'-Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a- azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7- (methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8- (alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8- (hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7- deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza- guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio- guanosine; 1-Me-GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP; 2'-b-Ethynylguanosine TP; 2'-b- Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a- mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP; 2'-Deoxy-2'-b- chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy- 2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'- Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy- thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5- hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; Dihydrouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5- carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl-pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3-(3-amino-3- carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine; 5- (carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O- dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O- methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5- carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine; 5- carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- caboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycaeoonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5- methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2- selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5- Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; N1- methyl-pseudo-uridine; N1-ethyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil; .alpha.-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)- pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouridine ; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)- pseudouridine; 1 (aminocazbonylethylenyl)-2(thio)-pseudouridine; 1 (aminocarbonylethylenyl)-2,4- (dithio)pseudouridine; 1 (aminocarbonylethylenyl)-4 (thio)pseudouridine; 1 (aminocarbonylethylenyl)-pseudouridine; 1 substituted 2(thio)-pseudouridine; 1 substituted 2,4- (dithio)pseudouridine; 1 substituted 4 (thio)pseudouridine; 1 substituted pseudouridine; 1- (aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouridine; 1-Methyl-3-(3-amino-3- carboxypropyl) pseudouridine TP; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl- pseudo-UTP; 2 (thio)pseudouridine; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4- (dithio)psuedouracil; 2' methyl, 2'amino, 2'azido, 2'fluoro-guanosine; 2'-Amino-2'-deoxy-UTP; 2'- Azido-2'-deoxy-UTP; 2'-Azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP; 2- methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouridine; 4- (thio)pseudouridine; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5 (2- aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouridine; 5-(alkyl)-2,4 (dithio)pseudouridine; 5-(alkyl)-4 (thio)pseudouridine; 5-(alkyl)pseudouridine; 5- (alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5- (dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5- (methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2-(thio)pseudouridine; 5-(methyl)-2,4 (dithio)pseudouridine; 5-(methyl)-4 (thio)pseudouridine; 5-(methyl)pseudouridine; 5-(methylaminomethyl)-2 (thio)uracil; 5- (methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5- (trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-1-2-ethanoic acid; Pseudouridine; 4-Thio-pseudo-UTP; 1- carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1- taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2- methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl- pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio- pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl- pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (.+-.)1-(2- Hydroxypropyl)pseudouridine TP; (2R)-1-(2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2- Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo- vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; 1-(2,2,2- Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2- Diethoxyethyl)pseudouridine TP; 1-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1-(2,4,6-Trimethyl- benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo- UTP; 1-(2-Amino-ethyl)pseudo-UTP; 1-(2-Hydroxyethyl)pseudouridine TP; 1-(2- Methoxyethyl)pseudouridine TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; 1-(3,4- Dimethoxybenzyl)pseudouridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino- propyl)pseudo-UTP; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino-4- carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1-(4-Amino-butyl)pseudo-UTP; 1-(4- Amino-phenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4- Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4- Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy- phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP; 1-(4- Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; 1-(4-Nitro-phenyl)pseudo-UTP; 1- (4-Thiomethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4- Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino- hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}- ethoxy)-propionyl]pseudouri- dine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-Acetylpseudouridine TP; I-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)- pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP; 1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl- pseudo-UTP; 1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1- Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1- Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1- Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1- Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1- Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1- Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me- 4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP; 1- Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4- morpholino)-pseudo-UTP; 1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; 1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6- bromo-pseudo-UTP; 1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP; 1-Methyl-6- cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP; 1-Methyl-6-ethoxy-pseudo-UTP; 1- Methyl-6-ethylcarboxylate-pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo- UTP; 1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP; 1-Methyl-6- hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP; 1-Methyl-6-iso-propyl-pseudo-UTP; 1- Methyl-6-methoxy-pseudo-UTP; 1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo- UTP; 1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6- trifluoromethoxy-pseudo-UTP; 1-Methyl-6-trifluoromethyl-pseudo-UTP; 1- Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1- Pivaloylpseudouridine TP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP; 1-propynyl- pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1- Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1- Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'- anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy-2'-a- mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'- Deoxy-2'-b-azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'- Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2'- Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O-Methyl-5-(1- propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'- Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5- Dimethylaminouridine TP; 5'-Homo-uridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5- Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4- Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6- Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano- pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo- UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino- pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy- pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6- Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo- UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4- methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1- [3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)- ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy)-ethoxy}-ethoxy]- ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1- methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1- 3-propionic acid; Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid; Pseudo-UTP- N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid; Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3- (aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido, 2'fluoro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'-azido-2'- deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified bases; 2'-O-methyl-ribose; 2-oxo-7- aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)- 7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4- (methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6- (methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7- (aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-y l; 7-(aminoalkylhydroxy)-1-(aza)-2- (thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7- (aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2- (oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenoxazinl-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthi azin-1-yl; 7- (guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazi n-1-yl; 7-(guanidiniumalkylhydroxy)- 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)- phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1 -yl; 7- (propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin- 1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2- substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; O6- substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin- 2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para- (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo- pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7- deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; or N6-(19-Amino- pentaoxanonadecyl)adenosine TP. In some embodiments, the percentage of standard As substituted with A-substitutable modified nucleotide (e.g. those above) is at least: 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In some embodiments, the percentage of standard As substituted with m 6 A may be 0.1-5%, in particular 0.5-2%, in particular 0.8-1.2%, such as about 1% (or 1%); in these embodiments the RNA may be circular RNA. Low substitution levels with m 6 A (e.g.1%) have been shown in inhibit innate immune activation [21]. In some embodiments, the percentage of standard Cs substituted with cytosine- substitutable modified nucleotide (e.g. those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In some embodiments, the percentage of standard Gs substituted with G-substitutable modified nucleotide (e.g. those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In preferred embodiments, the percentage of standard Us substituted with U-substitutable modified nucleotide (e.g. those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or preferably 100%; more preferably with 1mΨ and/or Ψ (even more preferably 1mΨ) . In a preferred embodiment, the one or more modified ribonucleotides detailed above is, or comprise, 1mΨ and/or Ψ, more preferably 1mΨ. In such embodiments, the RNA may comprise 1mΨ and/or Ψ, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. there are no standard U nucleotides, nor modified U ribonucleotides other than 1mΨ and/or Ψ, in the RNA; i.e. 100% U substitution). In particular, the RNA may comprise 1mΨ and/or Ψ, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e. there are no standard U nucleotides, nor modified ribonucleotides of any type - A, C, G or U substitutable - other than 1mΨ and/or Ψ, in the RNA; i.e. 100% U substitution with no other modified nucleotides being allowed). The RNA may comprise Ψ, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. 100% U substitution with Ψ). In particular, the RNA may comprise Ψ, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e. 100% U substitution with Ψ with no other modified nucleotides being allowed). More preferably, the RNA comprises 1mΨ, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. 100% U substitution with 1mΨ). In an even more preferred embodiment, the RNA comprises 1mΨ, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e.100% U substitution with 1mΨ with no other modified nucleotides being allowed). In the embodiments in this paragraph, “[may] comprise[s]... and neither [X]...nor [Y]” may be used interchangeably with the wording “[may] comprise[s]... and does not comprise... [X] and/or [Y] ”. Preferably, the RNA is codon-optimised. Codon optimisation may provide an elevated GC content, relative to non-codon optimised RNA encoding the same protein(s). The GC content (the percentage of all ribonucleotides (or, defined alternatively, all “nitrogenous bases”) in the RNA which are G or C) of the RNA may be at least 10%, such as at least 20%, 30%, 35% or at least 40%, preferably at least 45%, 46%, 47%, 48%, 49%, or at least 50%. The GC content of the RNA may be 10-70%, such as 20-65%, 30-65% or 35-65%, preferably 40-60%, 45-55%, 46-53%, 47-51%, or 48-50%. The GC content of the RNA may be 30-70%, such as 40-70%, 45-70%, 50-70%, or 55-70%. Codon optimisation may provide an elevated C content relative to non-codon optimised RNA encoding the same protein(s). The percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be least 30%, such as at least 40%, 50%, 55% or at least 60%, preferably at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% or at least 72%; The percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be 30-80%, such as 40-90%, 45-90%, 50-80%, 55-80% or 60-80%, preferably 65-75%, 66-75%, 67-75%, 68-75%, 69-75%, 70-74%, 71-74% or 72- 74%. Generally, the RNA comprises a 5' and/or a 3' untranslated region (UTR), preferably both a 5' and 3' UTR; e.g. selected from the 5'and 3' UTRs of RNA transcripts of the following genes (preferably the following human genes): beta-actin, albumin, ATP synthase beta subunit, fibroblast activation protein (“FAP”), H4 clustered histone 15 (“HIST2H4A”), glyceraldehyde-3-phosphate dehydrogenase, heat shock protein family A (Hsp70) member 8 gene,, interleukin-2 gene (“IL-2”), and transferrin. In some preferred embodiments, the RNA comprises a 5' and a 3' UTR selected from: - SEQ ID NO: 61 and 62, respectively, - SEQ ID NO: 63 and 64, respectively, - SEQ ID NO: 65 and 66, respectively, - SEQ ID NO: 67 and 68, respectively, - SEQ ID NO: 69 and 70, respectively, and - RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 61, 63, 65, 67 or 69 (for the 5' UTR) and RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 62, 64, 66, 68 or 70 (for the 3' UTR) (in particular, the pairing of 5' and 3' UTRs having such identity to SEQ ID NO: 61 and 62, SEQ ID NO: 63 and 64, SEQ ID NO: 65 and 66, SEQ ID NO: 67 and 68, and SEQ ID NO: 69 and 70, respectively); with RNA sequences according to SEQ ID NO: 61 and 62, SEQ ID NO: 67 and 68, SEQ ID NO: 69 and 70 (and RNA sequences having such identity thereto, preferably at least 95% or greater) being more preferred; and RNA sequences according to SEQ ID NO: 61 and 62 (and RNA sequences having such identity thereto, preferably at least 95% or greater) being even more preferred. Both the 3' and 5' UTR may influence expression of the RSV-F protein of the present disclosure through a variety of mechanisms. Without wishing to be found by this theory, the 5' UTR may affect the expression of at least the RSV-F protein of the present disclosure e.g. via pre-initiation complex regulation, closed-loop regulation, upstream open reading frame regulations (i.e. reinitiation), provision of internal ribosome entry sites, and provision of microRNA binding sites. Without wishing to be found by this theory, the 3' UTR may affect the expression of at least the RSV-F protein of the present disclosure e.g. via providing regulation regions that post-transcriptionally influence expression; e.g. influencing translation efficiency, localisation of the RNA, stability of the RNA, polyadenylation, and circularization of the RNA. In one specific embodiment, the RNA is circular RNA. In a preferred embodiment, the RNA fulfils any 2, 3, 4 or 5 of the following criteria (for example, (a) (b), (d) and (f); (a), (b), (c), (d) and (f); or (a), (b), (d), (e) and (f): (a) is non-self-replicating; (b) is single stranded; (c) comprises a 5' cap, which is a 7'-methylguanosine linked 5'-to-5' to the 5' first ribonucleotide by a triphosphate bridge, and wherein the first 5' ribonucleotide comprises a 2'-methylated ribose (2'-O-Me); (d) comprises a 3'poly-A tail; (e) comprises 1mΨ, and neither standard U ribonucleotides nor other modified ribonucleotides (f) comprises a 5' and a 3' UTR. More preferably, the RNA fulfils all of criteria (a) – (f), above. Generally, the RNA will comprise, in the 5' to 3' direction: 5' Cap, 5' UTR, open reading frame encoding at least an RSV-F protein of the present disclosure, 3'UTR, and 3' poly-A tail (in particular, the 5' Caps; 5' UTRs, 3'UTRs and 3' poly-A tails as detailed above throughout this subsection). In preferred embodiments, the RNA comprises or consists of the sequence: SEQ ID NO: 71; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 142; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 72; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 143; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 73; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 74; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 75; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 76; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 77; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 144; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 78; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 115; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 116; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 117; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 79; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 145; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 118; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 119; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; SEQ ID NO: 120; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; or SEQ ID NO: 80; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1. The present disclosure also provides, in a further independent aspect, a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising or consisting of: any of SEQ ID NO: 71-80, or any of the foregoing sequences having identity to any of SEQ ID NO: 71-80. In preferred embodiments, the RNA comprises an open reading frame (ORF) comprising or consisting of the sequence of: positions 32-1753 of SEQ ID NO: 71; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 142; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 72; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 143; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 73; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 74; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 75; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 76; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 77; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 144; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 78; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1744 of SEQ ID NO: 115; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1693 of SEQ ID NO: 116; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1678 of SEQ ID NO: 117; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 79; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1753 of SEQ ID NO: 145; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1744 of SEQ ID NO: 118; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1693 of SEQ ID NO: 119; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; positions 32-1678 of SEQ ID NO: 120; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1; or positions 32-1753 of SEQ ID NO: 80; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to (and numbered according to) SEQ ID NO: 1. The present disclosure also provides, in a further independent aspect, a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising an ORF; said ORF comprising or consisting of the sequence of: positions 32-1753 of any of SEQ ID NO: 71-80, or any of the foregoing sequences having identity to positions 32-1753 of any of SEQ ID NO: 71-80. Nucleic acid (e.g. RNA) alignments may be performed, for example, visually, or by any well-known algorithm; e.g. using an NCBI BLAST algorithm such as “megablast”, e.g. on default settings (available at e.g. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn& BLAST_SPEC=GeoBlast&PAGE_TY PE=BlastSearch); or e.g. using the “Muscle” algorithm (see, e.g. [22], [23]), e.g. on default settings; with the Muscle algorithm being preferred. Corresponding nucleotide or ribonucleotide positions are easily identifiable to the skilled person, and can be identified by aligning the nucleotide or ribonucleotide sequences using any well-known method (such as visual or algorithm, e.g. as detailed above). The RNA can conveniently be prepared by in vitro transcription (IVT). IVT can use a (DNA) template created and propagated in plasmid form in bacteria, or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA- dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the replicating RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the poly-A tail is usually encoded within the DNA template). Carriers comprising a nucleic acid encoding an RSV-F protein in the prefusion conformation Nucleic acid (especially RNA) by themselves and unprotected, may be degraded by the subject's nucleases and may require a carrier to facilitate target cell entry. Accordingly, the present disclosure also provides a carrier comprising a nucleic acid (preferably RNA) encoding an RSV-F protein of the present disclosure. The carrier may be lipid-based (e.g. a lipid nanoparticle or cationic nanoemulsion), polymer-based (e.g. comprising polyamines, dendrimers and/or copolymers), peptide or protein-based (e.g. comprising protamine, a cationic cell-penetrating peptide, and/or an anionic peptide conjugated to a positively charged polymer), cell-based (e.g. antigen presenting cells, such as dendritic cells loaded with the nucleic acid), or virus-based (e.g. viral replicon particles). In particular embodiments, the carrier is non-virion, i.e. free or substantially free of viral capsid. In particular, lipid-based carriers provide a means to protect the nucleic acid (preferably RNA), e.g. through encapsulation, and deliver it to target cells for protein expression. In certain embodiments, the lipid-based carrier is, or comprises, a cationic nano-emulsion (“CNE”). CNEs and methods for their preparation are described in, for example, [24]. With a CNE, the nucleic acid (preferably RNA) which encodes the RSV-F protein of the present disclosure is complexed with a CNE particle, in particular comprising an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule, thereby anchoring the molecule to the emulsion particles. In a particular embodiment, a lipid-based carrier is a lipid inorganic nanoparticle (“LION”). LNPs In a preferred embodiment, nucleic acids (preferably RNA) are encapsulated in a lipid nanoparticle (LNP). Thus, in a preferred embodiment, the present disclosure also provides an LNP encapsulating a nucleic acid (preferably RNA) which encodes an RSV-F protein of the present disclosure. A plurality of such LNPs will be part of a composition (e.g. a pharmaceutical composition as detailed in the section entitled Pharmaceutical compositions below) comprising free and/or encapsulated nucleic acid (preferably RNA), and in some embodiments the LNPs encapsulate at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least 100% of the total number of nucleic acid (preferably RNA) molecules in the composition. At least 80% of the LNPs in the composition may be 20-200 nm, 40-190 nm, 60-180 nm or, in particular, 80-160 nm in diameter. In a particular embodiment, substantially all, or all, LNPs in the composition are 20-200 nm, 40-190 nm, 60-180 nm or, in particular, 80-160 nm in diameter. The LNP can comprise multilamellar vesicles (MLV), small uniflagellar vesicles (SUV), or large unilamellar vesicles (LUV). The amount of nucleic acid (preferably RNA) per LNP can vary, and the number of individual nucleic acid molecules per LNP can depend on the characteristics of the particle being used. For RNA molecules, in general, an LNP may include 1-500 RNA molecules, e.g. <200, <100, <50, <20, <10, <5, or 1-4. Generally, an LNP includes fewer than 10 different species of RNA e.g. fewer than 5, 4, 3, or 2 different species. Preferably the LNP includes a single RNA species (i.e. all RNA molecules in the particle have the same sequence). LNPs according to the present disclosure may be formed from a single lipid (e.g. a cationic lipid) or, in particular, from a mixture of lipids. In particular, the mixture comprises various classes of lipids, such as: (a) a mixture of cationic lipids and sterols, (b) a mixture of cationic lipids and neutral lipids, (c) a mixture of cationic lipids and polymer-conjugated lipids, (d) a mixture of cationic lipids, sterols and polymer-conjugated lipids, or (e) a mixture of cationic lipids, neutral lipids and polymer-conjugated lipids; or preferably: (f) a mixture of cationic lipids, sterols and neutral lipids; or more preferably: (g) a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. Further classes of lipids, such as anionic lipids, may also be present in a mixture of lipids. The cationic lipid may have a pKa of 5.0-10.0, 5.0-9.0, 5.0-8.5, preferably 5.0-8.0, 5.0-7.9, or 5.0- 7.8, 5.0-7.7, or more preferably 5.0-7.6. The pKa of the cationic lipid is distinct to the pKa of the LNP as a whole (sometimes called “apparent pKa”). pKa may be determined via any well-known method, such as via a toluene nitrosulphonic acid (TNS) fluorescence assay or acid base titration; preferably a TNS fluorescence assay; more preferably performed according to Example 8. The cationic lipid preferably comprises a tertiary or quaternary amine group, more preferably a tertiary amine group. Exemplary cationic lipids comprising tertiary amine groups include: 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2- DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl- 3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP), 3- (N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]- dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2- DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); or MC3 (see, e.g. [25]). In some embodiments, the cationic lipid has the structure of lipid RV28, RV31, RV33, RV37, RV39 RV42, RV44, RV73, RV75, RV81, RV84, RV85, RV86, RV88, RV91, RV92, RV93, RV94, RV95, RV96, RV97, RV99 or RV101, as disclosed in [26]. In a further embodiment, the cationic lipid has the structure: In a preferred embodiment, the cationic lipid has the structure: (also referred to as lipid RV39). In another preferred embodiment, the cationic lipid has the structure:
In another preferred embodiment, the cationic lipid has the structure: The lipids in the LNP may comprise (in mole %) 20-80, 25-75, 30-70, or 35-65%, preferably 30-60, 40-55 or 40-50% cationic lipid; such as about 40% (or 40%), about 42% (or 42%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) cationic lipid. The lipids in the LNP may comprise (in mole %) at least 20, 25 or at least 35%, or preferably at least 40% cationic lipid. The lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60% or preferably no more than 50% cationic lipid. The molar ratio of protonatable nitrogen atoms in the LNP's cationic lipids to phosphates in the nucleic acid, preferably RNA (a.k.a “N:P” ratio), may be in the range of (including the endpoints) 1:1-20:1, 2:1-10:1, 3:1-9:1, or 4:1-8:1; preferably 4.5:1-7.5:1, 4.5:1-6.5:1 or 5.0:1-6.5:1. The polymer-conjugated lipid is preferably a PEGylated lipid. In an LNP, the PEGs of such PEGylated lipids may have average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8- 7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6- 2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0 (or 2.0 kDa). The average molecular weight of such PEGs may be expressed as the median molecular weight. In an LNP, the PEGs of such PEGylated lipids may have a weight average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2- 2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0 (or 2.0 kDa). Alternatively, in an LNP, the PEGs of such PEGylated lipids may have a number average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0 (or 2.0 kDa). Alternatively, in an LNP, at least 80% of the PEGs of such PEGylated lipids may have molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0, or 2.0 kDa. The PEGylated lipid may have the structure: Exemplary PEGylated lipids include 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, 1,2-dimyristoyl-sn-glycero-2- phosphoethanolamine-N-[methoxy(polyethylene glycol)] and 1,2-dimyristoyl-rac-glycerol-3- methoxypolyethylene glycol. Preferably, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. The lipids in the LNP may comprise (in mole %) 0.1-8.0, 0.4-7.0, 0.6-6.0, 0.8-4.0 or 0.8-3.5%, preferably 1.0-3.0% polymer-conjugated lipid (preferably PEGylated lipid); such as about 1.0 (or 1.0%), about 1.5% (or 1.5%), about 2.0% (or 2.0%) or about 2.5% (or 2.5%) polymer-conjugated lipid (preferably PEGylated lipid). The lipids in the LNP may comprise (in mole %) at least 0.1, 0.5 or at least 0.8%, or preferably at least 1% polymer-conjugated lipid (preferably PEGylated lipid). The lipids in the LNP may comprise (in mole %) no more than 8.0, 6.0 or 4.0% or preferably no more than 3.0% polymer-conjugated lipid (preferably PEGylated lipid). Preferably, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), although other neutral lipids available to the skilled person may also be used. The lipids in the LNP may comprise (in mole %) 0-15.0, 0.1-15.0, 2.0-14.0, 5.0-13.0, 6.0-12.0 or 7.0- 11.0%, preferably 8.0-11.0% or 9.0-11.0% neutral lipid; such as about 9.4% (or 9.4%), about 9.6% (or 9.6%), about 9.8% (or 9.8%) or about 10.0% (or 10%) neutral lipid. The lipids in the LNP may comprise (in mole %) at least 0.1, 5.0 or at least 7.0%, or preferably at least 8.0% or at least 9.0% neutral lipid. The lipids in the LNP may comprise (in mole %) no more than 15.0, 13.0 or no more than 12.0%, or preferably no more than 11.0% neutral lipid. Exemplary sterols include cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7- dehydrocholesterol, dihydrolanosterol, symosterol, lathosteriol, 14-demethyl-lanosterol, 8(9)- dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydrolanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9), 14-dien-3β-ol (dihydro-FF-MAS-d6), 4,4- dimethyl(d6)-cholest-8(9)-en-3β-ol (dihydro T-MAS-d6), zymostenol, sitostanol, campestanol, camperstanol, 7-dehydrodesmosterol, pregnenolone, 4,4-dimethyl-cholest-8(9)-en-3β-ol (dihyrdro T- MAS), Δ5-avensterol, brassicasterol, dihydro FF-MAS, 24-methylene cholesterol, oxysterols, deuterated sterols, fluorinated sterols, sulfonated sterols, phosphorylated sterols, A-ring substituted sterols, cholest-5-ene-3ß,4ß-diol, 5α-cholestan-3ß-ol, 4-cholesten-3-one, cholesta-8(9),24-dien-3- one, cholesta-8(9),24-dien-3-one, 2,2,3,4,4-pentadeuterio-5a-cholestan-3ß-ol, cholesteryl phosphocholine, cholesteryl-d7 pentadecanoate, cholesteryl-d7 palmitate, B-ring substituted sterols, cholestanol, 5ß,6ß-epoxy-d7, 3ß-hydroxy-5-cholestene-7-one, 6α-hydroxy-5α-cholestane, cholestanol, 5α,6α-epoxy, cholest-5-en-3ß,7α-diol, cholest-5-en-3ß,7ß-diol, cholestanol, 5α,6α- epoxy-d7, Δ5,7-cholesterol, cholesta-5,8(9)-dien-3ß-ol, cholesta-5,8(14)-dien-3ß-ol, 7α-hydroxy-4- cholesten-3-one, zymostenol-d7, zymostenol, 7-dehydrodesmosterol, 3b,5a-dihydroxy-cholestan-6- one, D-ring substituted sterols, 3ß-hydroxy-5α-cholest-8(14)-en-15-one, 3ß-hydroxy-5α-cholestane- 15-one, 5α-cholest-8(14)-ene-3ß,15α-diol, 5α-cholest-8(14)-ene-3ß,15ß,-diol, lanosterol-95, 5α-7,24- cholestadiene, 14-dehydro zymostenol, ergosta-5,7,9(11),22-tetraen-3ß-ol, cholest-5-ene-3ß,25-diol, cholest-(25R)-5-ene-3ß,27-diol, 24(R/S),25-epoxycholesterol, 24(S),25-epoxycholesterol, 24(R/S),25-epoxycholesterol-d6, cholest-5-ene-3ß,22(S)-diol, cholest-5-ene-3ß,22(R)-diol, cholest- 5-ene-3ß,24(S)-diol, cholest-5-ene-3ß,24(R)-diol, 27-hydroxy-4-cholesten-3-one, campestanol, N,N- dimethyl-3ß-hydroxycholenamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3ß- hydroxycholenamide, 25,27-dihydroxycholesterol, 5-cholestene-3β,20α-diol, 24S,25-epoxy-5α- cholest-8(9)-en-3β-ol, 24(S/R),25-epoxylanost-8(9)-en-3β-ol, 7-keto-27-hydroxycholesterol, 7α,27- dihydroxy-4-cholesten-3-one, 7α,27-dihydroxycholesterol, 7ß,27-dihydroxycholesterol, 5α,6ß- dihydroxycholestanol, 7α,25-dihydroxycholesterol, 7β,25-dihydroxycholesterol, 7α,24(S)- dihydroxycholesterol, 7α,24(S)-dihydroxy-4-cholesten-3-one, 7-keto-25-hydroxycholesterol, 7α,24S,27-trihydroxycholesterol, dihydrotestosterone, testosterone, estrone, estrogen, estradiol, corticosterone, cortisol, or 24S,27-dihydroxycholesterol. Preferably, the sterol is cholesterol or a cholesterol-based lipid (e.g. any of those provided in the foregoing paragraph). The lipids in the LNP may comprise (in mole %) 20-80, 25-80, 30-70, 30-60, 35-60 or 40-60%, preferably 40-50% or 41-49% sterol; such as about 42% (or 42%), about 43% (or 43%), about 44% (or 44%), about 46% (or 46%), or about 48% (or 48%) sterol. The lipids in the LNP may comprise (in mole %) at least 20, 30 or at least 35%, or preferably at least 40% or at least 41% sterol. The lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60%, or preferably no more than 50% sterol. The lipids in the LNP may have the following mole % in combination: 30-60% cationic lipid (such as 35-55%, or preferably 40-50%), 35-70% sterol (such as 40-55%, or preferably 41-49%), 0.8-4.0% polymer-conjugated lipid (such as 0.8-3.5%, or preferably 1.0-3.0%), and 0-15% neutral lipid (such as 6.0-12.0% or preferably 8.0-11.0%). Such LNPs encapsulating nucleic acids (preferably RNA) may be formed by admixing a first solution comprising the nucleic acids with a second solution comprising lipids which form the LNP. The admixing may be performed by any suitable means available to the skilled person, e.g. a T- mixer, microfluidics, or an impinging jet mixer. Admixing may be followed by filtration to obtain a desirable LNP size distribution (e.g. those as detailed above in this subsection). The filtration may be performed by any suitable means available to the skilled person, e.g. tangential-flow filtration or cross-flow filtration. According, in a further independent aspect, the present disclosure provides a method of preparing an LNP encapsulating a nucleic acid (preferably RNA) encoding a RSV-F protein of the present disclosure, comprising admixing a first solution comprising the nucleic acid and a second solution comprising lipids which form the LNP (e.g using the means as set out in the foregoing paragraph); and optionally filtering the obtained admixture (e.g using the means as set out in the foregoing paragraph). Pharmaceutical compositions In a further independent aspect, the present disclosure also provides a pharmaceutical composition comprising an RSV-F protein, nucleic acid (preferably RNA) and/or carrier (preferably lipid nanoparticle) of the present disclosure. Such compositions typically further comprise a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well-known in the art, see, e.g. [27]. Such compositions are generally for immunising subjects against disease, preferably against RSV. Accordingly, pharmaceutical compositions of the present disclosure are generally considered vaccine compositions. Pharmaceutical compositions of the present disclosure may comprise the RSV-F protein, nucleic acid (preferably RNA) and/or carrier (preferably lipid nanoparticle) in plain water (e.g. “w.f.i.”) or in a buffer e.g. a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will typically be included in the 5-20mM range. Pharmaceutical compositions of the present disclosure may have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0. Pharmaceutical compositions of the present disclosure compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/mL NaCl is typical, e.g. about 9 mg/mL (or 9 mg/mL).. Pharmaceutical compositions of the present disclosure may include metal ion chelators (in particular, in embodiments wherein such compositions comprise RNA). These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis. Thus, such compositions may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc.. Such chelators are typically present at between 10-500 μΜ e.g. 0.1 mM. A citrate salt, such as sodium citrate, can also act as a chelator, while advantageously also providing buffering activity. Pharmaceutical compositions of the present disclosure may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between 290-310 mOsm/kg. Pharmaceutical compositions of the present disclosure may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free compositions are preferred, and preservative-free vaccines can be prepared. Pharmaceutical compositions of the present disclosure may be aseptic or sterile. Pharmaceutical compositions of the present disclosure may be non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. Pharmaceutical compositions of the present disclosure may be gluten free. Pharmaceutical compositions of the present disclosure may be prepared in unit dose form. In some embodiments a unit dose may have a volume of between 0.1 -1.0 mL e.g. about 0.5mL (or 0.5mL). Pharmaceutical compositions of the present disclosure may be prepared as injectables, either as solutions or suspensions. The composition may be prepared for pulmonary administration e.g. by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops. Injectables for intramuscular administration are typical. Pharmaceutical compositions of the present disclosure comprise an immunologically effective amount of RSV-F protein. nucleic acid (preferably RNA) and/or carrier (preferably lipid nanoparticle), as well as any other components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention, preferably prevention of RSV. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other rel- evant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. In embodiments wherein pharmaceutical compositions of the present disclosure comprise RNA, the RNA content will generally be expressed in terms of the amount of RNA per dose. A preferred dose has ≤120µg RNA e.g. ≤100µg (e.g. 10-120µg or 10-100 µg, such as 10µg, 25µg, 50µg, 75µg or 100µg, or about 10µg, 25µg, 50µg, 75µg or 100µg), but expression can be seen at much lower levels e.g. ≤1µg/dose, ≤100ng/dose, ≤10ng/dose, ≤1ng/dose, etc. Pharmaceutical compositions of the present disclosure may further comprise an adjuvant (i.e. an agent that enhances an immune response in a non-specific manner), in particular, but not exclusively, when comprising an RSV-F protein of the present disclosure. Common adjuvants include suspensions of minerals (e.g. alum, aluminum hydroxide, aluminum phosphate) onto which RSV-F proteins may be adsorbed; emulsions, including water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists), and various combinations of such components. In some embodiments, the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod. In some embodiments, the adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulphate. The adjuvants described herein can be used singularly or in any combination, such as alum/TLR7 (also called AS37). Pharmaceutical compositions of the present disclosure may comprise a saponin as an adjuvant, e.g. saponin fraction QS21 (see, e.g. [28]). QS21 may be used in substantially pure form, e.g. at least 80% pure, such as at least 85, 90%, 95% or at least 98% pure. A suitable QS-21 fraction is as described in [29]. Pharmaceutical compositions of the present disclosure (preferably when comprising a lipid nanoparticle comprising a nucleic acid of the present disclosure, preferably RNA) may be lyophilised. In some embodiments, pharmaceutical compositions of the present disclosure comprise (i) a nucleic acid (preferably RNA) encoding an RSV-F protein of the present disclosure, and (ii) a further nucleic acid (preferably RNA) encoding at least one further protein. The nucleic acids of (i) and (ii) may be comprised within the same carrier (preferably lipid nanoparticle), or within separate carriers (preferably lipid nanoparticles). In preferred embodiments, the at least one further protein is an antigen; and as such may comprise, or may be, a viral, bacterial, fungal, parasitic, tumour, or allergenic (i.e. from, or derived from, an allergen) antigen. The at least one further protein will typically be a pathogen antigen. The at least one further protein will typically be an antigen that is a surface polypeptide e.g. a spike glycoprotein, a haemagglutinin, an adhesin or an envelope glycoprotein. In a particular embodiment, the at least one further protein is an antigen from, or derived from, a virus, in particular a virus causing respiratory disease, in particular a seasonal virus causing respiratory disease. In embodiments wherein the at least one further protein is an antigen from, or derived from, a virus, examples of such viruses include: Coronavirus, Orthomyxovirus, Pneumoviridae, Paramyxoviridae, Poxviridae, Picornavirus, Bunyavirus, Heparnavirus, Filovirus, Togavirus, Flavivirus, Pestivirus, Hepadnavirus, Rhabdovirus, Caliciviridae, Retrovirus, Reovirus, Parvovirus, Herpesvirus, Papovaviruses and Adenovirus. In a preferred embodiment, the at least one further protein encoded by the nucleic acid of (ii) is a further Pneumoviridae protein (in particular a Pneumoviridae antigen). Useful further Pneumoviridae proteins (in particular, antigens) can be from an Orthopneumovirus or Metapneumovirus, in particular human RSV or human Metapneumovirus (hMPV). Useful further hMPV antigens include e.g. the F, N, P, M, M2-1, and M2 antigens (in particular, the F antigen). Such hMPV proteins (in particular, antigens) may be from, or derived from, the A or B subtype. In a preferred embodiment, the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding an hMPV antigen (in particular, the F antigen). In such RNA embodiments, a preferred patient group (in which the pharmaceutical composition may be used in therapy, in particular vaccination) is infants (see section entitled Medical uses and methods of treatment, below). Useful further human RSV antigens encoded by the nucleic acid of (ii) include e.g. the G, M1, M2-1, M2-2, P, L, N, NS1, NS2 and SH antigens, in addition to further RSV-F antigens, i.e. of distinct amino acid sequence to the RSV-F protein of the present disclosure encoded by the nucleic acid. Such further human RSV proteins (in particular, antigens, in particular F antigens) may be from, or derived from, the A or B subtype, in particular the B subtype. In a preferred embodiment, the at least one further protein encoded by the nucleic acid of (ii) is a Coronavirus antigen. Useful Coronavirus antigens can be from a SARS coronavirus, in particular SARS-CoV2. Useful Coronavirus antigens (preferably SARS-CoV2 antigens) include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein. Preferably, the Coronavirus antigen is a SARS-CoV2 spike protein. Said SARS-CoV2 spike protein may be from any variant, e.g. Omicron (such as Omicron BA.1, BA.2, BA3, BA.4 or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma, or Delta. Preferably, said SARS-CoV2 spike protein includes one or more mutations relative to the wild-type protein, in particular one or more (e.g. two) mutations to proline resides. Said one or more mutations may be introduced to stabilise said SARS-CoV2 spike protein in its pre-fusion conformation. In a preferred embodiment, the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding a Coronavirus antigen, e.g. as detailed above. In such RNA embodiments, a preferred patient group (in which the pharmaceutical composition may be used in therapy, in particular vaccination) is older adults (see section entitled Medical uses and methods of treatment, below). In another preferred embodiment, the at least one further protein encoded by the nucleic acid of (ii) is an Orthomyxovirus antigen. Useful Orthomyxovirus antigens can be from an influenza A, B or C virus. Useful Orthomyxovirus antigens (in particular influenza A, B or C virus antigens) include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin. Preferably, the Orthomyxovirus antigen is an influenza A virus haemagglutinin. Said influenza A virus hemagglutinin may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. In a preferred embodiment, the nucleic acid of (i) is RNA encoding an RSV- F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding an Orthomyxovirus antigen, e.g. as detailed above. In such RNA embodiments, a preferred patient group (in which the pharmaceutical composition may be used in therapy, in particular vaccination) is older adults (see section entitled Medical uses and methods of treatment, below). In such RNA embodiments, the nucleic acid of (i) may encode an RSV-F protein of the present disclosure, the nucleic acid of (ii) may encode an Orthomyxovirus antigen, e.g. as detailed above, and (iii) a third nucleic acid may be present in the pharmaceutical composition which may encode a Coronavirus antigen, e.g. as detailed above in the preceding paragraph. In a further independent aspect, the present disclosure also provides a delivery device (e.g. syringe, nebuliser, sprayer, inhaler, dermal patch, etc.) comprising a pharmaceutical composition of the present disclosure. This device can be used to administer the composition to a vertebrate subject. In a further independent aspect, the present disclosure also provides a method of preparing a pharmaceutical composition, comprising formulating an RSV-F protein, nucleic acid (preferably RNA) or carrier (preferably lipid nanoparticle) of the present disclosure with a pharmaceutically acceptable excipient, to produce said composition. In particular, said pharmaceutical composition has the features as detailed above throughout this section. In a further independent aspect, the present disclosure also provides a kit comprising an RSV-F protein, nucleic acid, carrier, pharmaceutical composition or delivery device of the present disclosure, and instructions for use. Medical uses and methods of treatment The present disclosure also provides, in a further independent aspect, an RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, for use in medicine. Said use will generally be in a method for raising an immune response in a subject. The present disclosure also provides, in a further independent aspect, the use of an RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, in the manufacture of a medicament. Said medicament will generally be for raising an immune response in a subject. The present disclosure also provides, in a further independent aspect, a therapeutic method comprising the step of administering an effective amount of an RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure to a subject (preferably a subject in need of such administration). Said method will generally be for raising an immune response in the subject. The immune response is preferably protective and, preferably involves antibodies and/or cell- mediated immunity. Generally, the subject is a vertebrate, preferably a mammal, more preferably a human or large veterinary mammal (e.g. horses, cattle, deer, goats, pigs), even more preferably a human. The RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure may be for use in the prevention, reduction or treatment of infection or disease. In addition, or alternatively, the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure may be for use in the prevention, reduction or treatment of symptoms associated with infection or disease. The infection is generally one by, and said disease is generally one associated with, a Pneumoviridae virus. In preferred embodiments, the Pneumoviridae virus is an Orthopneumovirus, which is more preferably RSV, and even more preferable human RSV (including both the A and B subtypes thereof). Accordingly, the present disclosure also provides an RSV-F protein, nucleic acid, carrier or pharmaceutical composition of the present disclosure; for use in treating of preventing RSV (preferably a method of vaccination against RSV). The present disclosure also provides the use of an RSV-F protein, nucleic acid, carrier or pharmaceutical composition of the present disclosure, in the manufacture of a medicament for treating or preventing RSV (preferably wherein the medicament is a vaccine). The present disclosure also provides a method of inducing an immune response against RSV in a subject (preferably a method of vaccinating a subject against RSV), comprising administering to the subject an immunologically effective amount of the RSV-F protein, nucleic acid, carrier or pharmaceutical composition of the present disclosure to the subject. Vaccination according to the present disclosure may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Such methods of vaccination may comprise administration of a single dose. Alternatively, such methods of vaccination may comprise a vaccination regimen (i.e. administration of multiple doses). Such regimens may involve the repeated administration of an immunologically identical protein antigen (in the form of, or delivered via, an RSV-F protein, nucleic acid, carrier, or pharmaceutical composition of the present disclosure), in particular in a prime-boost regimen. In a prime-boost regimen, the first administration (“prime”) may induce proliferation and maturation of B and/or T cell precursors specific to one or more immunogenic epitopes present on the delivered antigen (induction phase). The second (and in some cases subsequent) administration (“boost”), may further stimulate and potentially select an anamnestic response of cells elicited by the prior administration(s). The different administrations may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. The prime administration(s) and boost administration(s) will be temporally separated, e.g. by at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more months. In some embodiments, two prime administrations may be administered 3-9 weeks apart (e.g. 4-9, 5-9, 6-9, 7-9 or 7-8 weeks apart, or about two months apart), followed by one or more boost administrations 4-14 months after the second prime administration (e.g. 5-13, 6-13, 7-13, 8-13, 9-13, 10-13 or 11-13 months, or about one year). In some embodiments, prime administration is to a naïve subject. In some embodiments, the protein antigen may be delivered in the prime and boost administrations as, or via, different formats. For example, the protein antigen may be delivered as a protein for the prime administration(s), and via a nucleic acid (in particular RNA, in particular via a carrier comprising RNA) for the boost administration(s), or vice versa. Alternatively, different nucleic acid formats may be used, e.g. the protein antigen may be delivered via RNA (in particular via a carrier comprising RNA) for the prime administration(s), and a via a viral vector (e.g. an adenoviral vector) for the boost administration(s), or vice versa. The RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure will generally be administered directly to the subject. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or to the interstitial space of a tissue). Alternative delivery routes include rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Preferably, the RSV-F proteins, nucleic acids, carriers, or pharmaceutical composition of the present disclosure will be administered intramuscularly or intradermally (in particular via a needle such as a hypodermic needle), more preferably intramuscularly. The RSV-F proteins, nucleic acids, lipid carriers, or pharmaceutical compositions of the present disclosure may be used to elicit systemic and/or mucosal immunity. The subject of a method of vaccination according to the present disclosure may be a child (preferably an infant) or adult (preferably an older adult or pregnant female). Immunocompromised individuals may also be the subject of such vaccination (whether children or adults). Infant vaccination In a preferred embodiment, the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure are administered to infants (preferably human infants), as the subject of vaccination. The immune systems of infants are immature (see, e.g. [30]), hence this population is susceptible to RSV infection and resulting disease. Infant vaccination may prevent lower respiratory tract infection (in particular, bronchiolitis and (broncho-)pneumonia). The infant may be less than one year old, such as less than: 11, 10, 9, 8, 7, 6, 5, 4 or less than 3 months old. The infant may be ≥one month old, such as ≥: 2, 3, 4, 5 or ≥6 months old. Preferably the infant is 2-6 months old (i.e. within and including the ages of 2 and 6 months), more preferably 2-4 months old. In a preferred embodiment, the infant was born from a female to whom an RSV vaccine (such as an RSV-F protein, nucleic acid, carrier, or pharmaceutical composition of the present disclosure) was administered, preferably while pregnant with said infant. The combination of maternal and infant vaccination may advantageously provide passive transfer of maternal antibodies (i.e. via the placenta and/or breast milk) to, in addition to active immunity generated by, the infant. Older adult vaccination In another preferred embodiment, the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure are administered to older adults (preferably human older adults), as the subject of vaccination. Older adults may suffer from age-related immunosenescence (reviewed in, e.g. [31]), hence this population is also susceptible to RSV infection and resulting disease. Older adult vaccination may prevent lower respiratory tract infection (in particular, pneumonia). The older adult may be ≥50 years old, such as ≥: 55, 60, 65, 70, 75, 80, 85, 90, 95 or ≥100 years old. Preferably, the older adult is ≥60 or ≥65 years old (such as 60-120 or 65-120 years old). Pregnant female vaccination In another preferred embodiment, the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure are administered to pregnant females (preferably pregnant human females), as the subject of vaccination. The primary object of maternal vaccination is to protect the infant from RSV infection when born, e.g. through passive transfer of antibodies via the placenta and/or breast milk. The pregnant female may be in her first, second or third trimester of pregnancy, preferably third trimester. The pregnant female may be ≥20 weeks pregnant, such as ≥: 22, 24, 26, 28, 30, 32, 34, 36 or ≥38 weeks pregnant. Preferably, the pregnant female is ≥28 , ≥29 or ≥30 weeks pregnant (such as 28-43, 29-43 or 30-43 weeks pregnant). General The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. The term “at least one” refers to one or more. Unless specified otherwise, where a numerical range is provided, it is inclusive, i.e., the endpoints are included. The terms “at least”, “no more than” and other such terms preceding a list of values are applicable to all members of said list (not merely the first member thereof), unless otherwise stated. The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y. The term “about” in relation to a numerical value x is optional and means, for example, x+10%. The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the present disclosure. References to charge, to cations, to anions, etc., are taken at pH 7. Embodiments The present disclosure also provides the following numbered embodiments. Combinations of features of the present disclosure presented below are exemplary, and not to be construed as exhaustive. 1. A RSV-F protein in the pre-fusion conformation, which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1 and comprises (a): (ai) at least one mutation relative to the wild-type in a region corresponding to positions 38-60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (aii) at least one mutation relative to the wild-type in a region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region. 2. The RSV-F protein of embodiment 1, comprising (b) at least one mutation relative to the wild- type in a region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a P residue into the region. 3. The RSV-F protein of embodiment 1 or 2, comprising (c) at least one mutation relative to the wild-type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, a glycosylation site into the region; or (d) at least one mutation relative to the wild-type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, at least one residue selected from N, D, F, H, K, L, Q, R, T, W and Y into the region. 4. A RSV-F protein in the pre-fusion conformation, which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1 and comprises (b) at least one mutation relative to the wild-type in a region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a P residue into the region. 5. The RSV-F protein of embodiment 4, comprising (c) at least one mutation relative to the wild- type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, a glycosylation site into the region; or (d) at least one mutation relative to the wild-type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, at least one residue selected from N, D, F, H, K, L, Q, R, T, W and Y into the region. 6. A RSV-F protein in the pre-fusion conformation, which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1 and comprises (c) at least one mutation relative to the wild-type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, a glycosylation site into the region; or (d) at least one mutation relative to the wild-type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, at least one residue selected from N, D, F, H, K, L, Q, R, T, W and Y into the region. 7. The RSV-F protein of any of embodiments 1-3, wherein the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 each comprise a β sheet. 8. The RSV-F protein of any of embodiments 1-3 or 7, wherein the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to positions 38-60 of SEQ ID NO:1; and/or the region corresponding to positions 296- 318 of SEQ ID NO:1 has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity to positions 296-318 of SEQ ID NO:1. 9. The RSV-F protein of any of embodiments 1-3, 7 or 8, comprising (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or C. 10. The RSV-F protein of any of embodiments 1-3, 7, 8 or 9, comprising (a) a substitution at position 55 of SEQ ID NO: 1 for T. 11. The RSV-F protein of any of embodiments 2-5 or 7-10, wherein the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop. 12. The RSV-F protein of any of embodiments 2-5 or 7-11, wherein the region corresponding to positions 38-60 of SEQ ID NO:1 has least 50%, 60%, 75% or 85% sequence identity to positions 208-216 of SEQ ID NO:1. 13. The RSV-F protein of any of embodiments 2-5, 7-11 or 12, comprising (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P. 14. The RSV-F protein of any of embodiments 2-5 or 7-13, comprising (b) a substitution at position 215 of SEQ ID NO: 1 for A. 15. The RSV-F protein of any of embodiments 3, 5, 6 or 7-14 wherein the region corresponding to positions 345-352 of SEQ ID NO:1 comprises a β sheet and a loop. 16. The RSV-F protein of any of embodiments 3, 5, 6 or 7-15, wherein the region corresponding to positions 345-352 of SEQ ID NO:1 has at least 50%, 60% ,75% or 85% sequence identity to positions 345-352 of SEQ ID NO:1. 17. The RSV-F protein of any of embodiments 3, 5, 6, 7-15 or 16, comprising (c) a substitution of position 348 of SEQ ID NO: 1 for T or N. 18. The RSV-F protein of any of embodiments 3, 5, 6, 7-15, 16 or 17, comprising (c) a substitution of position 348 of SEQ ID NO: 1 for N. 19. The RSV-F protein of embodiment 17 or 18, comprising a glycan linked to position 348 of SEQ ID NO: 1; optionally wherein the glycan comprises N-acetyl glucosamine. 20. The RSV-F protein of any of embodiments 3, 5, 6 or 7-16, comprising (d) a substitution of position 348 of SEQ ID NO: 1 for N, D, F, H, K, L, N, Q, R, T, W or Y; optionally N, F, H, K, N, Q, R, T, W or Y; optionally N, F, R, W or Y. 21. The RSV-F protein of embodiment 20, comprising (d) a substitution of position 348 of SEQ ID NO: 1 for N. 22. The RSV-F protein of any of embodiments 1-19, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P; and (c) a substitution of position 348 of SEQ ID NO: 1 for T or N. 23. The RSV-F protein of embodiment 22, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; (b) a substitution at position 215 of SEQ ID NO: 1 for A; and (c) a substitution of position 348 of SEQ ID NO: 1 for N; wherein the N at position 348 is linked to a glycan; which optionally comprises N-acetyl glucosamine. 24. The RSV-F protein of any of embodiments 1-16, 20 or 21, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P; and (d) a substitution of position 348 of SEQ ID NO: 1 for N, D, F, H, K, L, N, Q, R, T, W or Y; optionally N, F, H, K, N, Q, R, T, W or Y; optionally N, F, R, W or Y. 25. The RSV-F protein of embodiment 24, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; (b) a substitution at position 215 of SEQ ID NO: 1 for A; and (d) a substitution of position 348 of SEQ ID NO: 1 for N. 26. The RSV-F protein of any preceding embodiment, comprising an F2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 26-108 or 26-109 of SEQ ID NO: 1. 27. The RSV-F protein of any preceding embodiment, comprising an F1 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1. 28. The RSV-F protein of any preceding embodiment, comprising a heterologous trimerisation domain on the C-terminus thereof, and/or C-terminal to the F1 domain; optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain. 29. The RSV-F protein of embodiments 1-27, comprising a transmembrane domain on the C- terminus thereof, and/or C-terminal to the F1 domain; and optionally a cytoplasmic domain C- terminal to said transmembrane domain. 30. The RSV-F protein of any of embodiments 1-27 or 29, comprising a cytoplasmic domain; wherein, relative to a cytoplasmic domain according to SEQ ID NO: 109 or 110, 2-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 31. The RSV-F protein of embodiment 30, wherein 3-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 32. The RSV-F protein of embodiment 30 or 31, wherein 2-5, such as 2-4, 2-3, 3-4 or 3 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 33. The RSV-F protein of any of embodiments 30-32, wherein the cytoplasmic domain comprises or consists of (i) an amino acid sequence according to positions 10-31 of SEQ ID NO: 134 or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic domain does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). 34. The RSV-F protein of any of embodiments 30-32, wherein the cytoplasmic domain comprises or consists of (i) an amino acid sequence according to positions 10-29 of SEQ ID NO: 135, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic domain does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). 35. The RSV-F protein of embodiment 31, wherein 6-13, such as 7-13, 8-12, 9-11, 9-10, 10-11 or 10 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 36. The RSV-F protein of any of embodiments 30, 31 or 35, wherein the cytoplasmic domain comprises or consists of (i) an amino acid sequence according to positions 10-24 of SEQ ID NO: 136 or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic domain does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). 37. The RSV-F protein of embodiment 31, wherein 14-16, such as 14-15 or 15-16, or 15 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 38. The RSV-F protein of any of embodiments 30, 31 or 37, wherein the cytoplasmic domain comprises or consists of (i) an amino acid sequence according to positions 10-19 of SEQ ID NO: 137, or (ii) an amino acid sequence at least 60%, 70%, 80% or 90% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic domain does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). 39. The RSV-F protein of embodiment 31, wherein 16-20, such as 17-20, 18-20 or 19-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 40. The RSV-F protein of embodiment 39, wherein 20 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F domain. 41. The RSV-F protein of any of embodiments 30, 31, 39 or 40, wherein the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-14 of SEQ ID NO: 138, or (ii) an amino acid sequence at least 60% or 80% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). 42. The RSV-F protein of any of embodiments 30-41, wherein the deletions increase the cell-surface expression, optionally in human fibroblasts, optionally in human foreskin fibroblasts, optionally in human primary BJ cells, optionally the ATCC CRL-2522 cell line, of the RSV-F protein in trimeric, pre-fusion form from a nucleic acid, relative to expression in such form of an RSV-F protein having the same amino acid but absent such deletions, such as an RSV-F protein comprising a cytoplasmic domain according to SEQ ID NO: 109 or 110. 43. The RSV-F protein of embodiment 42, wherein the increased cell surface expression is for a period of at least 24, 48, 72 or 96 hours. 44. The RSV-F protein of any preceding embodiment, wherein a signal peptide is not present in the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO: 1. 45. The RSV-F protein of any preceding embodiment, wherein a p27 peptide is not present in the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO: 1. 46. The RSV-F protein of any preceding embodiment, wherein the RSV-F protein comprises an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1. 47. The RSV-F protein of any preceding embodiment, further comprising, relative to SEQ ID NO: 1: a substitution at position 152 for R, L or W; optionally R or W; optionally R; a substitution at position 210 for H, A, F, K, N, W or Y; optionally H, F, K, N, W or Y; optionally, H, F or Y; optionally H; optionally a substitution at position 211 for N; a substitution at position 228 for K, R, Q, N or A; optionally K, R, Q or N; optionally K, R, or Q; optionally K or R; optionally K; a substitution at position 241 for N a substitution at position 315 for I or V; optionally I; a substitution at position 346 for Q, D, H, K, N, R, S or W; optionally Q, D, H, K, N, R or S; optionally Q; a substitution at position 419 for D, N, S, or T; optionally D or T; optionally D; optionally a substitution at position 445 for D; a substitution at position 455 for V or I; optionally V; and/or, optionally and, a substitution at position 459 for M. 48. The RSV-F protein of any preceding embodiment, further comprising, relative to SEQ ID NO: 1: a substitution at position 152 for R, L or W; optionally R or W; optionally R; optionally a substitution at position 211 for N; a substitution at position 228 for K, R, Q, N or A; optionally K, R, Q or N; optionally K, R, or Q; optionally K or R; optionally K; a substitution at position 315 for I or V; optionally I; a substitution at position 346 for Q, D, H, K, N, R, S or W; optionally Q, D, H, K, N, R or S; optionally Q; optionally a substitution at position 445 for D; a substitution at position 455 for V or I; optionally V; and/or, optionally and; a substitution at position 459 for M. 49. The RSV-F protein of any preceding embodiment, further comprising, relative to SEQ ID NO: 1: a substitution at position 228 for K, R, Q, N or A; optionally K, R, Q or N; optionally K, R, or Q; optionally K or R; optionally K; a substitution at position 315 for I or V; optionally I; a substitution at position 241 for N a substitution at position 455 for V or I; optionally V; and/or, optionally and; a substitution at position 459 for M. 50. The RSV-F protein of any preceding embodiment, further comprising, relative to SEQ ID NO: 1: a substitution at position 228 for K, R, Q, N or A; optionally K, R, Q or N; optionally K, R, or Q; optionally K or R; optionally K; a substitution at position 315 for I or V; optionally I; a substitution at position 455 for V or I; optionally V; and/or, optionally and; a substitution at position 459 for M. 51. The RSV-F protein of any preceding embodiment, comprising a substitution of position 228 of SEQ ID NO: 1 for K, R, Q, N or A; optionally K, R, Q or N; optionally K, R, or Q; optionally K or R. 52. The RSV-F protein of embodiment 51, comprising a substitution of position 228 of SEQ ID NO: 1 for K. 53. The RSV-F protein of any preceding embodiment, comprising a substitution at position 152 of SEQ ID NO: 1 for R, and/or a substitution at position 346 of SEQ ID NO: 1 for Q. 54. The RSV-F protein of any preceding embodiment, comprising a substitution at position 211 of SEQ ID NO: 1 for N, and/or a substitution at position 445 of SEQ ID NO: 1 for D, optionally both substitutions. 55. The RSV-F protein of any preceding embodiment, wherein the RSV-F protein is of the A subtype. 56. The RSV-F protein of any of embodiments 1-54, wherein the RSV-F protein is of the B subtype. 57. The RSV-F protein of any preceding embodiment, which is specifically bound by a pre-fusion mAb with a K D , as measured by SPR, of less than 10 nM; optionally 1 pM – 10 nM. 58. The RSV-F protein of any preceding embodiment, which is specifically bound by a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 2 and 3 respectively with a K D , as measured via SPR, of less than 1000, 900, 800700, 650, 600, 550, 100, 90, 80, 70, 60, 50 or 35 pM; wherein the RSV-F protein is in the form of a trimer. 59. The RSV-F protein of any preceding embodiment, which is specifically bound by a pre-fusion mAb comprising a LC and HC according SEQ ID NO: 4 and 5 respectively with a K D , as measured via SPR, of less than 200, 180, 160, 140, 130, 100, 95, 90, 85, 80 or 70 pM. 60. The RSV-F protein of any preceding embodiment, which is specifically bound by a pre-fusion mAb comprising a LC and HC according SEQ ID NO: 8 and 9 respectively with a K D , as measured via SPR, of less than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM. 61. The RSV-F protein of any preceding embodiment, which is specifically bound by a pre-fusion mAb comprising a LC and HC according SEQ ID NO: 8 and 9 respectively with a K D , as measured via SPR, of less than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM. 62. The RSV-F protein of any preceding embodiment, which is specifically bound by a mAb comprising a LC and HC according SEQ ID NO: 6 and 7 respectively with a K D , as measured via SPR, of less than 200, 180, 160, 140, 120, 110, 100, 95, 80, 70, 60 , 55, 50, 45, or 40 pM. 63. A recombinant RSV-F protein in the pre-fusion conformation, comprising at least one mutation relative to wild-type RSV-F according to SEQ ID NO: 1, wherein at least one mutation introduces neither an artificial disulphide bond nor a P residue into said wild-type protein. 64. The RSV-F protein according to embodiment 63, comprising the features of any of embodiments 1-62, with the proviso that P residues are not introduced into the protein by the at least one mutation. 65. A trimer comprising three RSV-F proteins according to any preceding embodiment. 66. A nucleic acid encoding the RSV-F protein of any of embodiments 1-64. 67. The nucleic acid of embodiment 66, wherein the nucleic acid is, or is comprised within, a viral vector; optionally wherein the viral vector is an adenovirus vector. 68. The nucleic acid of embodiment 66, wherein the nucleic acid is DNA; optionally wherein the DNA is a DNA plasmid. 69. The nucleic acid of embodiment 66, wherein the nucleic acid is RNA. 70. The RNA of embodiment 69, which is non-self-replicating RNA. 71. The RNA of embodiment 69, which is self-replicating RNA. 72. The RNA of any of embodiments 69-71, comprising, in the 5' to 3' direction: a 5' Cap, a 5' UTR, an open reading frame encoding at least an RSV-F protein according to any of embodiments 1-64, a 3'UTR, and a 3' poly-A tail. 73. The RNA of embodiment 72, wherein the 5' cap comprises a 7'-methylguanosine linked 5'-to-5' to the 5' first ribonucleoside by a triphosphate bridge, and wherein the first 5' ribonucleoside comprises a 2'-methylated ribose (2'-O-Me). 74. The RNA of embodiment 72 or 73, wherein the 3' poly-A tail comprises a contiguous stretch of 100-500 A ribonucleotides. 75. The RNA of embodiment 72 or 73, wherein the 3' poly-A tail comprises at least two non- contiguous stretches of A ribonucleotides; optionally 25-35 and 65-90 ribonucleotides in length respectively; optionally orientated in the 5' to 3' direction. 76. The RNA of any of embodiments 69-75, comprising a modified ribonucleotide. 77. The RNA of embodiment 76, wherein the modified ribonucleotide is 1mΨ 78. The RNA of embodiment 77, wherein the RNA comprises 1mΨ and neither standard U ribonucleotides nor other modified U ribonucleotides; optionally wherein the RNA comprises 1mΨ and neither standard U ribonucleotides nor other modified ribonucleotides. 79. The RNA of any of embodiments 69-78, having a GC content of 55-70%. 80. The RNA of any of embodiments 69-78, having a GC content of 40-60%. 81. A carrier comprising nucleic acid of any of embodiments 66, 68 or 69-80. 82. The carrier of embodiment 81, which is a lipid nanoparticle. 83. The lipid nanoparticle of embodiment 82, comprising a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. 84. The lipid nanoparticle of embodiment 83, wherein the cationic lipid has a pKa of 5.0-8.0; optionally 5.0-7.6. 85. The lipid nanoparticle of embodiment 83 or 84, wherein the cationic lipid comprises a tertiary amine group. 86. The lipid nanoparticle of any of embodiments 83-85, wherein the polymer-conjugated lipid is a PEGylated lipid; optionally wherein the PEG has an average molecular weight of 1-3 kDa. 87. The lipid nanoparticle of embodiment 86, wherein the PEG has a weight average molecular weight of 1-3 kDa. 88. The lipid nanoparticle of any of embodiments 83-87, wherein the sterol is cholesterol or a cholesterol-based lipid. 89. The lipid nanoparticle of any of embodiments 83-88 comprising (in mole %) 30-60% cationic lipid, 35-70% sterol, 0.8-4.0% polymer-conjugated lipid, and 0-15% neutral lipid; optionally 40- 50% cationic lipid, 41-49% sterol, 1.0-3.0% polymer-conjugated lipid and 8.0-11.0% neutral lipid. 90. A pharmaceutical composition comprising the RSV-F protein of any of embodiments 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, or carrier of any of embodiments 81-89; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant. 91. A vaccine composition comprising the RSV-F protein of any of embodiments 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, or carrier of any of embodiments 81- 89; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant 92. The pharmaceutical composition of embodiment 90, for use in medicine. 93. The pharmaceutical composition for use of embodiment 92, for use in a method of raising an immune response in a subject; optionally a protective immune response in a subject. 94. The pharmaceutical composition for use of embodiment 92 or 93, for use in the treatment or prevention of RSV. 95. The pharmaceutical composition for use of embodiment 94, for use in a method of vaccinating a subject against RSV; optionally wherein the vaccination is prophylactic. 96. The pharmaceutical composition for use of any of embodiments 93- 95, wherein the subject is a human infant; optionally 2-6 months old. 97. The pharmaceutical composition for use of any of embodiments 93- 95, wherein the subject is a human older adult; optionally ≥60 years old. 98. The pharmaceutical composition for use of any of embodiments 93-95, wherein the subject is a pregnant human female; optionally ≥28 weeks pregnant. 99. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of embodiments 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, carrier of any of embodiments 81-89, pharmaceutical composition of embodiment 90, or vaccine composition of embodiment 91. 100. Use of the RSV-F protein of any of any of embodiments 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, or carrier of any of embodiments 81-89, in the manufacture of a medicament. 101. Use according to embodiment 84, wherein the medicament is for treating of preventing RSV. 102. Use according to embodiment 85, wherein the medicament is a vaccine; optionally a prophylactic vaccine. 103. A kit comprising the RSV-F protein of any of embodiments 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, carrier of any of embodiments 81-89, pharmaceutical composition of embodiment 90, or vaccine of embodiment 91, and instructions for use. 104. A Respiratory syncytial virus fusion (RSV-F) protein in the pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO: 1 and comprises (a), (b) and (c): (a) (ai) at least one mutation relative to the wild-type in a region corresponding to positions 38- 60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (aii) at least one mutation relative to the wild-type in a region corresponding to positions 296- 318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation relative to the wild-type in a region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a P residue into the region; and (c) at least one mutation relative to the wild-type in a region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, a glycosylation site into the region. 105. The RSV-F protein of embodiment 104, wherein: (a) the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 comprise β sheets; (b) the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 comprises a β sheet and a loop. 106. The RSV-F protein of any of embodiments 104 or 105, wherein: (a) the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 38-60 of SEQ ID NO:1; and/or, optionally and, the region corresponding to positions 296-318 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 296-318 of SEQ ID NO:1; (b) the region corresponding to positions 38-60 of SEQ ID NO:1 has least 75% or 85% sequence identity to positions 208-216 of SEQ ID NO:1; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 has at least 75% or 85% sequence identity to positions 345-352 of SEQ ID NO: 1. 107. The RSV-F protein of any one of embodiments 104-106, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: for T or N. 108. The RSV-F protein of embodiment 107, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; (b) a substitution at position 215 of SEQ ID NO: 1 for A; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: 1 for N. 109. The RSV-F protein of embodiment 107 or 108, comprising a glycan linked to position 348; optionally comprising N-acetyl glucosamine. 110. The RSV-F protein of any one of embodiments 104-109, comprising a substitution of position 228 of SEQ ID NO: 1 for K, R, Q or N; optionally K or R; optionally K. 111. The RSV-F protein of any one of embodiments 104-110, comprising an F2 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 26-108 of SEQ ID NO: 1. 112. The RSV-F protein of any one of embodiments 104-111, comprising an F1 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1. 113. A homotrimer comprising three RSV-F proteins of any one of embodiments 104-112 having the same amino acid sequence. 114. A nucleic acid encoding the RSV-F protein of any one of embodiments 104-112. 115. The nucleic acid of embodiment 114, wherein the nucleic acid is RNA. 116. A lipid nanoparticle comprising nucleic acid of embodiment 114 or 115. 117. A pharmaceutical composition comprising the RSV-F protein of any of embodiments 104- 112, trimer of embodiment 113, nucleic acid of embodiment 114 or 115, or lipid nanoparticle of embodiment 116; optionally for use in medicine. 118. The pharmaceutical composition for use of embodiment 117, for use in a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; a human older adult, optionally ≥60 years old; or a pregnant human female, optionally ≥28 weeks pregnant. 119. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of embodiments 104-112, trimer of embodiment 113, nucleic acid of embodiment 114 or 115, or lipid nanoparticle of embodiment 116, or pharmaceutical composition of embodiment 118. 120. Respiratory syncytial virus fusion (RSV-F) protein in the pre-fusion conformation, which is mutated relative to SEQ ID NO: 1 and comprises (a), (b) and (c): (a) (i) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 38- 60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (ii) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 208- 216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a P residue into the region; and (c) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 345- 352 of SEQ ID NO:1, wherein the at least one mutation introduces, through substitution or insertion, a glycosylation site into the region. 121. The RSV-F protein of embodiment 120, wherein: (a) the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 comprise β sheets; (b) the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 comprises a β sheet and a loop. 122. The RSV-F protein of any one of embodiments 120 or 121 wherein: (a) the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 38-60 of SEQ ID NO:1; and/or, optionally and, the region corresponding to positions 296-318 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 296-318 of SEQ ID NO:1; (b) the region corresponding to positions 208-216 of SEQ ID NO:1 has least 75% or 85% sequence identity to positions 208-216 of SEQ ID NO:1; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 has at least 75% or85% sequence identity to positions 345-352 of SEQ ID NO: 1. 123. The RSV-F protein of any one of embodiments 120-122, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: for T or N. 124. The RSV-F protein of embodiment 123, comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; (b) a substitution at position 215 of SEQ ID NO: 1 for A; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: 1 for N. 125. The RSV-F protein of embodiment 123 or 124 comprising a glycan linked to position 348; optionally comprising N-acetyl glucosamine. 126. The RSV-F protein of any one of embodiments 120-125, comprising a substitution of position 228 of SEQ ID NO: 1 for K, R, Q or N; optionally K or R; optionally K. 127. The RSV-F protein of any one of embodiments 120-126, comprising an F2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to positions 26-108 or 26-109 of SEQ ID NO: 1. 128. The RSV-F protein of any one of embodiments 120-127, comprising an F1 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1. 129. The RSV-F protein of any one of embodiments 120-128, comprising a heterologous trimerisation domain at the C-terminus thereof, and/or C-terminal to the F1 domain; optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain. 130. The RSV-F protein of any one of embodiments 120-128, comprising a transmembrane domain at the C-terminus thereof, and/or C-terminal to the F1 domain; and optionally a cytoplasmic domain C-terminal to said transmembrane domain. 131. The RSV-F protein of any one of embodiments 120-128, wherein the RSV-F protein comprises a cytoplasmic domain; wherein, relative to a cytoplasmic domain according to SEQ ID NO: 109 or 110, 2-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 132. The RSV-F protein of embodiment 131, wherein 17-20, 18-20, 19-20 or 20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 133. The RSV-F protein of any one of embodiments 120-132, wherein a signal peptide is not present in the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO: 1. 134. The RSV-F protein of any one of embodiments 120-133, wherein a p27 peptide is not present in the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO: 1. 135. The RSV-F protein of any of embodiments 120-134, wherein the RSV-F protein comprises an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1. 136. A homotrimer comprising three RSV-F proteins of any one of embodiments 120-135 having the same amino acid sequence. 137. A nucleic acid encoding the RSV-F protein of any one of embodiments 120-135. 138. The nucleic acid of embodiment 137, wherein the nucleic acid is RNA. 139. A lipid nanoparticle comprising nucleic acid of embodiment 137 or 138. 140. A pharmaceutical composition comprising the RSV-F protein of any of embodiments 120- 135, trimer of embodiment 136, nucleic acid of embodiment 137 or 138, or lipid nanoparticle of embodiment 139; optionally for use in medicine. 141. The pharmaceutical composition for use of embodiment 140, for use in a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; a human older adult, optionally ≥60 years old; or a pregnant human female, optionally ≥28 weeks pregnant. 142. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of embodiments 120-135, trimer of embodiment 136, nucleic acid of embodiment 137 or 138, or lipid nanoparticle of embodiment 139, or pharmaceutical composition of embodiment 140. EXAMPLES Many modifications and variations of the present disclosure are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, a skilled person in the art would recognise that the invention may be practiced otherwise than as specifically described. The illustrative embodiments and examples should not be construed as limiting the invention. Materials & Methods Expression and Purification of RSV-F mutants, DS-Cav1 and RSV-F mAbs (Examples 3, 4, 6, 7 and 9) RSV-F mutants were synthesized (GENEWIZ/AZENTA) and cloned into a CMV-based vector with a C-terminal thrombin-cleavable double Strep tag II tag followed by a 6x His-tag. DS-Cav1 and RSV-F mutants were transiently expressed in Expi293 F cells (THERMO FISHER SCIENTIFIC). Media was harvested after 4 days, and purified using affinity chromatography, either nickel affinity or strep-tag affinity. Briefly, for nickel affinity chromatography, cell harvest medium was passed over a HisTrap Excel column (CYTIVA) and eluted with a step gradient of imidazole. For strep-tag affinity, the harvest medium was buffer exchanged into 50 mM Tris pH 8, 300 mM NaCl, passed over a StrepTrap HP column (CYTIVA) and eluted with elution buffer (100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA and 2.5 mM desthiobiotin). This was followed by a final size exclusion chromatography polishing step. The oligomeric state and protein integrity of DS-Cav1 and RSV-F mutants were confirmed by High Performance Liquid Chromatography (HPLC) on a WATERS ALLIANCE HPLC system. 20 µg of purified DS-Cav1 or RSV-F mutant were loaded onto a SUPEROSE 6 Increase 3.2/300 column (CYTIVA) and an isocratic gradient of a mobile phase composed of 20 mM Tris pH 7.5, 150 mM NaCl at a flow rate of 0.05 mL/min for a total run time of 60 min was used. RSV antibodies, AM14, D25, Motavizumab, and RSB1, were transiently transfected in EXPI293F cells (THERMO FISHER SCIENTIFIC) according to manufacturer's instructions and media was harvested 6-7 days post transfection. The cell harvest media was passed over a MABSELECT SURE COLUMN (CYTIVA) and eluted with 0.1 M citrate pH 3 into 1 M Tris pH 9; buffer exchanged into 20 mM HEPES pH 7, 150 mM NaCl; followed by a final size exclusion chromatography step on a HILOAD 16/600 Superdex 30 pg column (CYTIVA) in 20 mM Hepes pH 7, 150 mM NaCl. Initial Quantitation and Antigenicity using Biolayer Interferometry (Examples 3, 4, 6 and 7) Quantitation experiments were performed on the unpurified cell harvest media of 6x His-tagged DS- Cav1 and RSV-F mutants using the Octet Red 384 instrument (SARTORIUS). Purified DS-Cav1 diluted in EXPI293 expression media with 0.1% BSA, 0.05% Tween-20 was used to make a standard curve. BSA and Tween-20 were added to DS-Cav1 and RSV-F mutants unpurified cell harvest media to a final concentration of 0.1% and 0.05%, respectively. 6x His-tagged purified DS-Cav1 and RSV- F mutant unpurified cell harvest media was captured on HIS2 biosensors for 2 min and the capture level was recorded. The concentrations were determined using unweighted 4 parameter logistics curve fitting in the manufacturer's analysis software (Data Analysis HT 12.0.1.55). Initial antigenicity experiment was performed on the unpurified cell harvest media of DS-Cav1 and RSV-F mutants to determine binding to AM14, D25, Motavizumab, and RSB1 mAbs using the OCTET Red 384 (SARTORIUS). mAbs were diluted to 10 µg/mL in 1xPBS with 0.1% BSA and 0.05% Tween-20. BSA and Tween-20 was added to DS-Cav1 and RSV-F mutant unpurified cell harvest media to a final concentration of 0.1% and 0.05%, respectively. AHC biosensors were regenerated in 10 mM Glycine pH 1.5 before and between experiments. AHC biosensors were washed in 1x PBS with 0.1% BSA and 0.05% Tween-20 for 30 sec, mAbs were loaded for 60 sec, and washed for 30 sec before capturing DS-Cav1 or RSV-F mutants from the unpurified cell harvest media. Binding and dissociation of DS-Cav1 and RSV-F mutants was measured for 180 sec each. The response of DS-Cav1 binding to each mAb was compared to the RSV-F mutants' response to each mAb to determine yes or no binding. Thermostability using nanoDSF (Examples 4, 6 and 7) Thermostability experiments were performed in duplicate on a PROMETHEUS NT.48 instrument (NANOTEMPER TECHNOLOGIES). Capillaries were filled with 10 µL of DS-Cav1 and RSV-F mutants, placed in the sample holder, and the temperature was increased from 25 to 95°C at a ramp rate of 1°C/min. The reported ratio of the recorded emission intensities (Em350 nm/Em330 nm) and its first derivative was calculated with the manufacturer's software (PR.ThermControl v2.1.1) to determine melting temperatures. Binding Kinetics using BIACORE (Example 4) Single cycle kinetics experiments were performed in duplicate on a BIACORE 8K+ (CYTIVA) using a ligand capture method at 25°C. HBS-EP+ was used as both a running buffer and sample diluent. A blank run of buffer as the ligand was followed by runs with IgGs captured to 100-200 RUs in flow cell 2 on a Protein A chip, leaving flow cell 1 as a reference. DS-Cav1 and RSV-F mutants were injected in both flow cells at 30 µL/min for 120 sec followed by 2400 sec dissociation. Antigen concentrations ranged from 0-10 nM. Reference- and blank-subtracted sensograms were fitted using a 1:1 binding model to calculate k on , k off , and K D . Cryo-electron microscopy of RSV F designs F21, F216, F224 and F310 (Example 5 and 6) Purified RSV-F mutants were complexed with molar excess AM14 Fab domain for a minimum of 1 hour at 4° Celsius prior to isolation of the F21:AM14 Fab and F216:AM14 Fab complexes by gel filtration using a SUPEROSE 6 increase 3.2/300 GL column in 20mM Tris + 150mM NaCl, pH 7.5 buffer. Gel filtration was not used to isolate the F224:AM14 Fab complex or the F310:AM14 Fab complex. Samples were concentrated to ~0.5 mg/mL prior to depositing 3.0 µL onto QUANTIFOIL 1.2/1.3300 mesh grids that had been glow discharged using a PELCO EASIGLOW Glow Discharge Cleaning System. EM grids were blotted for 3.5s prior to plunge freezing into liquid ethane using a Mark IV Vitrobot vitrification apparatus set to 100% humidity and 4° Celsius (THERMOFISHER). Vitrified grids were clipped into autogrids and loaded into either a Titan Krios equipped with a BIOQUANTUM K3 direct electron detector (F21:AM14 Fab complex) or GLACIOS TEM equipped with a FALCON 3 direct electron detector. (F216:AM14 Fab, F224:AM14 Fab and F310:AM14 Fab complexes). Single particle analysis was carried out using the RELION 3.1 image processing suite. Processing was carried out similarly for all specimens with relevant data and imaging parameter listed in Figure 16. Expression of RSV-F mutants from mRNA (Example 7) RSV-F mutant protein sequences were back-translated with specified metrics to DNA sequences. The T7 promotor region and UTRs (“UTR4”) were appended to 5' and 3' of the coding regions. The DNA GBLOCKS (INTEGRATED DNA TECHNOLOGIES) were used in two methods to generate mRNAs: Method 1, GBLOCKS were amplified by a series of Polymerase Chain Reactions (PCR) with the reverse primer contains polyA tail at the 3' end. Purified PCR products were used as the templates for in vitro RNA transcription. Method 2, GBLOCKS were amplified by PCR, followed with introduction of restriction sites, then ligated (NEW ENGLAND BIOLABS) into vector with a polyA tail. The plasmids were linearized with the BspQ1 restriction enzyme (NEW ENGLAND BIOLABS) to produce the DNA templates for in vitro transcription. mRNAs were produced by in vitro transcription with capping analogue (TRILINK CLEANCAP) and 100% uridine replacement (with 1mΨ), followed with DNase I and phosphatase treatments (NEW ENGLAND BIOLABS) and silica column purification (QIAGEN). Two micrograms of mRNAs were electroporated with a GENE PULSER X-CELL (BIO-RAD) with 1 million either HEK293 cells (ATCC) or Human Skeletal Muscle cells (LONZA). After 18 hours of post transfection incubation, cells were fixed and permeabilized with CYTOFIX/CYTOPERM buffer (BD BIOSCIENCES). Cells were treated with 10ug/ml primary antibodies (D25, RSB1, AM14, Motavizumab), followed with fluorophore-conjugated secondary antibody (THERMO FISHER) treatment. IQUE3 Flow Cytometer (SARTORIUS) was run to measure the protein expression in the cells. The gating threshold was set based on single cell size and fluorescent intensity of negative and positive cells. The positive cell percentage, positive cell Geomean and positive cell number were quantified. Rapid Stability Assay (BIACORE) (Example 7) The rapid stability assay was performed in duplicate on a BIACORE 8K+ (CYTIVA) using a ligand capture method at 25°C. HBS-EP+ was used as both a running buffer and sample diluent. A blank run of buffer as the ligand was followed by runs with 10 µg/mL IgGs captured in flow cell 2 on a Protein A chip, leaving flow cell 1 as a reference. 5 µg/mL DS-Cav1 and PreF mutants, incubated at 50 or 60°C for 0, 30, 60, or 120 min, were injected in both flow cells at 10 µL/min for 120 sec followed by 60 sec dissociation. The relative analyte stability early response from blank subtracted sensograms was normalized to the time 0 response and plotted in EXCEL. Biacore Potency Assay for long term stability studies (Example 9) DS-Cav1, F216, F217, F224, and F225 were diluted to 240 µg/mL or 480 µg/mL in 20 mM Hepes pH 7, 150 mM Sodium chloride. Samples were incubated at 4 or 25°C for 0, 3, 7, 10, 15, or 21 days and stored at -80°C until the experiment was performed. The potency assay was performed in duplicate on a BIACORE 8K+ (CYTIVA) using a ligand capture method at 25°C. HBS-EP+ was used as both a running buffer and sample diluent. A blank run of buffer as the ligand was followed by runs with 10 µg/mL IgGs captured in flow cell 2 on a Protein A chip, leaving flow cell 1 as a reference. DS-Cav1 and PreF mutant standard curves (80, 40, 20, 10, 5, 2.5, 1.25, 0.625 µg/mL) and long-term stability samples, diluted 1:20, were injected in both flow cells at 10 µL/min for 120 sec followed by 60 sec dissociation. A 4-parameter logistic fit of the standard curves in SOFTMAX PRO (MOLECULAR DEVICES) was used to back calculate standard curve and sample concentrations. The following were used to determine system suitability, % Relative error for the back calculated standard curve concentrations must be ≤20%, %CV ≤10%, and %Recovery for Day 0 samples must be between 80-120%. Thermostability for long term stability studies (Example 9) DS-Cav1, F216, F217, F224, and F225 were diluted to 2 mg/mL in 20 mM Hepes pH 7, 150 mM Sodium chloride. Samples were incubated at 4 or 25°C for 0, 3, 7, 10, 15, or 21 days and stored at - 80°C until the experiment was performed. Thermostability experiments were performed in duplicate on a PROMETHEUS NT.48 instrument (NANOTEMPER TECHNOLOGIES). Capillaries were filled with 10 µL of DS-Cav1 and PreF mutants, placed in the sample holder, and the temperature was increased from 25 to 95°C at a ramp rate of 1°C/min. The reported ratio of the recorded emission intensities (Em350 nm/Em330 nm) and its first derivative was calculated with the manufacturer's software (PR.THERMCONTROL v2.1.1) to determine melting temperatures. For the melting temperatures for which a clear peak was present, but no melting temperature was reported by the software, melting temperatures were manually determined by examining the Ratio (First Derivative) in EXCEL analysis file from PR.THERMOCONTROL. In vivo protein immunisation study (Example 10) Study Design: Purified protein was diluted 1:1 with ASO3 adjuvant to arrive at a final dose of 3 µg or 0.3ug RSVF antigen. Formulation was performed at room temperature within 1 hour of dosing. BALB/c female mice were immunized on day 0 and day 21 with either the 3 µg or 0.3ug dose of RSVF antigen plus AS03 adjuvant at each injection. Following immunization, samples were collected on day 21 and day 35. Blood sat at room temperature for a minimum of 30 minutes then was centrifuged prior to transfer and storage at -80°C. Samples were analyzed for total PreF IgG and for neutralizing antibody response. IgG Binding Assay: The LUMINEX assay was designed to measure the levels of RSV Pre-Fusion protein specific IgG binding antibodies from immunized mice. LUMINEX microspheres (MAGPLEX microspheres, LUMINEX CORP from Austin, TX) were coupled with RSV preF antigen using sulfo-NHS and EDC, according to manufacturer's instructions. In a 96-well plate, 2,000 microspheres/well are added in a volume of 50 µl PBS with 1% BSA + 0.05% Na Azide (assay buffer) to 100 µl of mouse serum serial diluted. After incubation of the microspheres and serum on an orbital shaker, covered, at RT for 60 minutes, the microspheres are washed 2 times with 200 µl/well of PBS, 0.05% Tween-20 (wash buffer) on a plate washer using a magnet to allow settling of beads between washes. Following the wash, 50 µL/well of r-Phycoerythrin (r-PE) conjugated anti- mouse IgG (JACKSON IMMUNORESEARCH) was added, and plates are incubated, covered, on an orbital shaker at RT for 60 minutes. After a final plate wash (same as described above), the samples were resuspended in PBS, covered, and incubated at RT on an orbital shaker for 20 minutes. Fluorescent intensity is measured using a LUMINEX FLEXMAP 3D. The raw data was analyzed using a SOFTMAX PRO template, where the serum sample binding potency was interpolated based on a four-parameter logistic fit of the standard curve. Serum anti-RSV preF IgG binding was calculated in terms of Assay Units (AU) using a reference standard assigned to a concentration of 100 AU. Neutralizing Antibody Response: The neutralization assay was performed at SIGMOVIR (SOP 7.2.1). HEp-2 cells were seeded and incubated to achieve confluency. Serum samples were heat inactivated and stored in -20°C. Serum was diluted. The RSV virus stock was diluted to approximately 25-50 plaque forming units (PFU) per 25 μL inoculum in EMEM. Virus was added to serum and the plates were incubated for 1 hour at 25-30°C. The serum-virus mix was added to plates and incubated for 1 hour at 37°C, 5% CO2. After incubation, 1 mL of methyl cellulose overlay was added to each well. Plates were incubated at 37°C, 5% CO2 for 4 days. After 4-day incubation, the cells were fixed and stained with 0.5 mL of crystal violet and incubated for 2-4 hours at 25-30°C. After incubation, plates were rinsed and left to air dry. The number of PFU was counted from each well and the 60% reduction end-point was calculated by multiplying the average virus control (PFU/well) by 40% (1:40). Neutralizing antibody titres were determined at the 60% reduction end- point of the virus control using the statistics program “plqrd.manual.entry”. Statistics: Post 1 and post 2 IgG binding titres data were analyzed by dose on the log10 scale using analysis of variance (ANOVA) models for repeated measurements with group, day and the interaction group*day as fixed effects. Observations below LOD were set to 0.0005. Homogeneity of variances between groups was considered in the model with 3 µg dose groups. Heterogeneity of variances between groups was considered in the model with 0.3 µg dose groups. Post 2 RSV neutralization titres data were analyzed on the log10 scale using an ANOVA model with group as fixed effect. Homogeneity of variances between groups was considered. For both IgG binding and RSV neutralizing titres, geometric means of titres with 95% CI were computed and comparisons with reference groups (gr 2 or 3, DsCav1) were assessed through geometric mean ratios with 90% CI (2-sided test, alpha=0.05). Multiplicity of comparisons was not taken into account and data from the saline group were not included. In vivo RNA immunisation (Example 11) All recombinant RNA molecules were produced by in vitro transcription using N1-methyl pseudouridine to replace all uridines. All recombinant RNA molecules comprised a cap-1 5' cap (TRILINK CLEANCAP) and a 3' poly(A) tail. The mRNAs were purified and evaluated for mRNA integrity (by capillary and glyoxal denaturing gel electrophoresis). The RV39 LNP mRNA constructs were then formulated in LNPs comprising 40 mol% cationic lipid RV39; 2 mol% PEG-conjugated lipid; 48 mol% cholesterol; and 10 mol% 1,2-diastearoyl-sn- glycero-3-phosphocholine (DSPC). Female BALB/c mice were 7 - 8 weeks old at day 0 of the study. A 28 - gauge needle was used to administer 50 µL (25 µL in each hindleg thigh muscle) of either saline or a high (2 µg) or low (0.2 µg) dose of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319 (mRNA constructs XW03C37 (SEQ ID NO: 146), XW02C23 (SEQ ID NO: 141), KM111C2 (SEQ ID NO: 142), KM112C10 (SEQ ID NO: 143), KM118C2 (SEQ ID NO: 144) and KM120C3 (SEQ ID NO: 145), respectively) into each mouse on day 0 and day 21. The groups of animals, formulation lot numbers, stock concentrations, number of vials, and storage temperatures were as follows (Table 6): Table 6 – first in vivo RNA immunisation study design On day 21, mice were anesthetized under isoflurane to collect 100 μL of whole blood (40 μL of serum) by submandibular collection method. On day 35, mice were anesthetized under isoflurane and terminally exsanguinated by cardiac stick to obtain an estimated 200 μL to 500 μL of whole blood, (100 μL of serum). RSV pre-F IgG binding antibody titres and RSV A neutralizing antibody titres were measured on day 21 and day 35. Pre-F IgG binding antibody titres were determined by LUMINEX binding assay as per Example 10. RSV A neutralizing antibody titre assay: Heat-inactivated sera (incubated for 30 min at 56°C) were diluted 3-fold starting at 1/8 (for a final dilution of 1/16). A control serum (WYETH Human Reference Sera from WHO/NIBSC) was included at a starting dilution of 1/64 (1/128 final). For the serial dilutions, 30μL of diluted serum was added on top of 60μL of RSV media (Biorich DMEM supplemented with 3%-fetal bovine serum (FBS; MOREGATE, FBSAE1000), 2 mM L-Glutamine, and 50 μg/mL Gentamicin). RSV lab-adapted A-Long virus was diluted to approximately 50-150 foci-forming units per 25μL. 60μL of virus was added into the wells with the same volume of serum dilutions and incubated for 2 hours at 35°C 5% CO2. After incubation, 50μL of the serum-virus mixture was added on top of the vero cells (seeded the day before the test at a density of 15000 cells/well, to reach a minimum of 80% confluency) and incubated for 2 hours at 35°C 5% CO2. After incubation, serum-virus supernatant was removed and 200μL of 0.5% carboxymethyl cellulose + RSV media was added on top of the cells. Plates were incubated for 2 days (max of 42 hours) at 35°C 5% CO2. Plates were then washed 2 times with 100μL of PBS and 50μL of 1% paraformaldehyde was added per well. Plates were covered in aluminium and incubated overnight at 4°C. The next day, plates were rinsed 3 times with 150μL of PBS. 100μL of blocking solution (2% milk + PBS) was added on top of the wells and incubated for 1 hour at 37°C. After incubation, plates were rinsed 3 times with 200μL of PBS. 50μL of primary goat anti-RSV polyclonal Ab (BIODESIGN, B65860G) diluted 1:400 in blocking solution was added per well and plates were incubated for 1 hour at 37°C. 50μL of secondary Ab rabbit anti-goat HRP (AGRISERA, AS10659) diluted 1:1500 in blocking solution was added per well and plates were incubated for 1 hour at 37°C. After 1 hour, plates were rinsed 3 times with 200μL of PBS and 50μL of TRUEBLUE Peroxidase substrate (KPL, 5510-0049) was added on top. After an incubation for 5-15 minutes, plaques were then washed extensively with DI water and let to dry. Imaging of the plaques was done using an AXIOVISION microscope. Effective dilution 60 (ED60) values, corresponding to the reciprocal serum dilution associated with 60% reduction in FFU counts, were determined using a linear model. Statistical Analysis: RSVA neutralization and anti-Pre-F IgG titres were analysed separately. Only high dose groups (gr 2, 4, 6, 8, 10, 12) were included in the statistical analyses. Post-1 and post-2 IgG data were analysed on the log10 scale using analysis of variance (ANOVA) models for repeated measurements with group, day, and the interaction group*day as fixed effects. Heterogeneity of variances between groups was considered. Post-2 RSV neutralization titres were analysed on the log10 scale using an ANOVA model with group as fixed effect. Homogeneity of variances between groups was considered. For both responses, geometric means of titres with 95% CI were computed. Non-inferiority to reference groups (gr 2 or 4) was assessed through geometric mean ratios with 90% CI. Multiplicity of comparisons was not considered. Cytoplasmic tail deletions (Example 12) Cloning and expression of RSV-F monoclonal antibodies: Plasmids encoding RSV-F antibodies, AM14, D25 and Motavizumab were transiently transfected in Expi293F cells (THERMO FISHER SCIENTIFIC) according to manufacturer's instructions and media was harvested 6-7 days post transfection. The cell harvest media was passed over a MABSELECT SURE COLUMN (CYTIVA) and eluted with 0.1 M citrate pH 3 into 1 M Tris pH 9; buffer exchanged into 20 mM HEPES pH 7, 150 mM NaCl; followed by a final size exclusion chromatography step on a HILOAD 16/600 Superdex 30 pg column (CYTIVA) in 20 mM Hepes pH 7, 150 mM NaCl. Cloning of specific RSV-F mutants from mRNA: RSV F wildtype protein sequence (SEQ ID NO: 107) protein was back-translated to a nucleic acid sequence using specific metrics for codon optimality. The DNA gBLOCKS (INTEGRATED DNA TECHNOLOGIES) were amplified by PCR, and ligation into a vector with a polyA tail. Amino acid substitutions N67I and S215P (also known as design F(ii)) were incorporated DNA constructs and encoded in the eventual mRNA and protein. The additional variations (also known as DS-Cav1, F(iii), F(i), F318 and F319) and their amino acid substitutions are shown in the Table 7. Table 7 – substitutions in parent mRNAs designs tested in cell-based assay
For CT truncating experiments, the CT was selectively removed in part or in whole from these constructs to create deletion (ΔCT) mutants. For this, NEB Q5 Site-Directed Mutagenesis Kit (NEB # E0554) was used to generate 7 CT deletion constructs: FL (“full length”, or “reference CT”), ΔCT3, ΔCT5, ΔCT10, ΔCT15, ΔCT20 and ΔCT25 Table 8 – C-terminal, cytoplasmic tail (CT) variations Briefly, the full-length constructs were combined with a forward primer combined with a unique reverse primer to create each ΔCT construct. The reverse primers were designed in which the 3' end PCR annealing starting points are at 7 different positions: position d0, end of coding region; position d3, 3 amino acid residues upstream of the end of coding region; position Δ5, 5 residues to the end; position Δ10, 10 residues to the end; position Δ15, 15 residues to the end; position Δ20, 20 residues to the end; position Δ25, 25 residues to the end, which is the entire CT region. The PCR reaction was heated to 98 °C for 30 seconds, followed by 16 cycles at 98 °C for 10 seconds, 69 °C for 30 seconds, 72 °C for 30 seconds. and a final extension of 72 °C for 2 min. The 7 PCR products were treated with KLD enzyme (NEB E0554) at room temperature for 5 minutes. Transformation with competent cells (NEB C3040H) was carried out by following manufacture instructions. 24 hours after, colonies were screened to identify correct sequences. In the DNA sequences of CT deletion constructs, the T7 promotor region and the UTRs were appended to 5' and 3' of the coding regions (5' and 3' “UTR4”) and a polyA tail is after 3' UTR region. The final plasmids were validated by Sanger sequencing and purified for mRNA production. In vitro transcription to generate mRNA for RSV-F variations: The plasmids were linearized with the BspQI restriction enzyme (NEW ENGLAND BIOLABS) to produce the DNA templates for in vitro transcription. mRNAs were produced by in vitro transcription with capping analogue (TRILINK CLEANCAP A/G) and 100% uridine replacement (with 1mΨ), followed with DNase I, phosphatase treatments (NEW ENGLAND BIOLABS) and silica column purification (QIAGEN). Newly synthesized mRNAs were validated by Tapestation (Agilent) and denaturing RNA gels. Cell culture conditions: Primary BJ cells (ATCC, CRL-2522) were maintained by routine passaging in growth media (DMEM (LONZA 12-614F) supplemented with 10% FBS (CORNING 35-016-CV), antibiotic (GIBCO 15140-122) and glutamine (GIBCO 25030-081) and grown at 37°C, 5% CO 2 . Forward transfection of candidate mRNAs: BJ cells were seeded in growth media at 1.5x10 5 cells/mL onto 96-well, clear-bottom, black-walled imaging microwell plates (PERKIN ELMER 6055302). The following day, target mRNAs were complexed with TRANSIT mRNA transfection reagent (MIRUS mir2250) in OPTIMEM (GIBCO 31985-070). Each target mRNA was forward transfected into BJ cell monolayers using 0.35% transfection reagent (final concentration) with mRNAs diluted to 0.454ng/uL (final concentration), or water-only negative control. The transfected BJ cells were incubated according to the time-course assay. Indirect immunofluorescent labelling and detection of surface-expressed RSV F: At the appropriate hours post-transfection (hpt), (1, 8, 24, 48, 72, 96 hpt), the cell media was removed from cells in 96- well format and cell monolayers were rinsed once with PBS with calcium and magnesium (THERMOFISHER 14080055). The cell monolayers were fixed in 4% paraformaldehyde (THERMOFISHERSCIENTIFIC J19943-K2) for 15min. Fixed cells were stored in PBS at 4C until cells can be immunolabeled as a batch. The fixed cell monolayers were rinsed twice with PBS (VWR 02-0119-1000). Nonspecific antibody- binding for fixed cells was blocked using 1% Normal Horse Serum (GIBCO 16050-130) in PBS (1%NHS-PBS). RSV F protein was labelled by incubating cell monolayers with the respective human anti-RSV F monoclonal antibodies: AM14, D25, motavizumab. Each well was incubated with 331ng of the respective antibody in blocking media overnight at 4C. Cell monolayers are rinsed 3 times with 1%NHS-PBS. Indirect immunofluorescent detection of RSV F expression was completed by incubating cell monolayers with goat anti-human antibody with ALEXA647 (THERMOFISHER A-21445) diluted 1:2000 in 1%NHS-PBS. Additionally, cell nuclei were co- labelled with DYECYCLE Violet (THERMOFISHER V35003) following manufacturer's recommendations. Cell monolayers are rinsed 3 times with 1% NHS-PBS then cells are stored in PBS for imaging. 9 fields per well were imaged in the DYECYCLE Violet and Alexa647 fluorescent channels using the 10x objective on the THERMOSCIENTIFIC Cell Insight CX7 automated imaging system. Image analysis is completed using the Target Activation protocol associated with the CELLOMICS (HCS NAVIGATOR Ver 6.6.2 Build 8533) image analysis system. Data analysis was completed using MICROSOFT EXCEL and PRISM GRAPHPAD. In vivo RNA immunisation (Example 13) RNAs were produced and formulated into LNPs as per Example 11 (though encoding different RSV- F proteins, as detailed below). Female BALB/c mice were 7 - 8 weeks old at day 0 of the study. An insulin syringe with a permanently attached needle was used to administer 50 µL (25 µL in each hindleg thigh muscle) of either saline or a high (2 µg) or low (0.2 µg) dose of F(iii) which includes a full cytoplasmic tail deletion (dCT), F(i), F(i) ΔCT20, F(ii), F(ii) ΔCT20 (low dose only), DS-Cav1 (high dose only), F318, F318 ΔCT20, F319, or F319 ΔCT20 (mRNA constructs detailed in Table 10, below) into each mouse on day 0 and day 21. The groups of animals, formulation lot numbers, stock concentrations, number of vials, and storage temperatures were as follows (Table 9): Table 9 – second in vivo RNA immunisation study design
Table 10 – second in vivo RNA immunisation study – construct details On day 21, mice were anesthetized under isoflurane to collect 100 μL of whole blood (40 μL of serum) by submandibular collection method. On day 35, mice were anesthetized under isoflurane and terminally exsanguinated by cardiac stick to obtain an estimated 200 μL to 500 μL of whole blood, (100 μL of serum). RSV pre-F IgG binding antibody titres and RSV A neutralizing antibody titres were measured on day 21 and day 35, as per Example 11. Statistical analysis: with N=8/vaccinated group, the study was powered detect non-inferiority by a 4- fold margin between vaccinated groups, assuming a max standard deviation (log) of 0.45. Both Pre-F IgG and RSVA neutralization analyses were run on log10 transformed data of all vaccinated groups (saline group excluded from analyses). For each endpoint, an ANOVA model for repeated measures with antigen and dose and all interactions (vaccine*dose, vaccine*day, dose*day, vaccine*dose*day) as fixed effects was fitted on data, assuming heterogeneity of variances between groups. No correction for multiplicity of comparisons was applied. Geometric mean of titres (GMT) with 95%CI by group were computed from those models as well as geometric mean ratio (GMR) of interest. Comparisons of primary interest were tested through GMR with 90% CI, assessing the non- inferiority of new designs with respect to benchmarks, by dose. Additional comparisons consist of within-antigen comparisons (ΔCT20 vs. unmodified) by dose. Example 1 - Computational Modelling and Structure Preparation for Design No apo-structure of native pre-fusion RSV-F antigen exists in structural databases. While more than 30 crystal structures of pre-fusion RSV-F have been deposited in the Protein Data Bank, those structures were either solved with an antibody or small molecule bound or consist of multiple stabilizing mutations. To best utilize the available structural information and to generate a suitable model for consensus sequence design, a systematic approach was implemented to select an appropriate template on which to thread the native sequence. Homology models of 15 RSV-F pre-fusion crystal structures were generated with symmetry-based comparative modeling (ROSETTACM), in the presence of density map constraints. These ROSETTACM runs produced 100 decoys for each template, with the final models being selected based on energetics and C-alpha backbone similarity to the crystal structure template. Based on ROSETTA free energy (kcal/mol) and root mean square deviation (RMSD), models from PDB: 5EA4 (see https://www.rcsb.org/structure/5ea4) were selected as the initial decoys for evolutionary consensus design. Example 2 - Evolutionary Sequence and Structure-Based Design of RSV-F (“Round 1”) A consensus design method was used to select non-redundant evolutionary homologs for clustering, searching against the Uniclust database and concatenated to produce a combined position specific scoring matrix (PSSM) with the HHblits module. The ROSETTA Protein Suite was used for structural design, constraining the trimeric chains with cyclic symmetry to produce homo-oligomers. An evolutionary workflow was then used to perform a single point mutation scan to ascertain residues that surpassed certain energy thresholds and for the combinatorial design of in silico mutants (Figure 1). Example 3 - Evolutionary Sequence and Structure-Based Design of RSV-F (“Round 1”) The consensus sequence designs were transfected into human embryonic kidney 293 cells and tested for expression and antigenicity in supernatant. Biolayer interferometry (BLI) was used to test expression via affinity-based histidine tags, resulting in the identification of two constructs (F21 and F28), see Figure 2. The results indicated that design F21 has slightly lower expression relative to DS- Cav1 (of reference [10]), while the expression of construct F28 was low. The two designs were then tested for the presence of pre-fusion specific epitopes using four known antibodies: AM14 (quaternary epitope), D25 (site 0), RSB1 (site V) Motavizumab (site II) (see Figure 4 for models of Fab binding). Both designs were able to bind to the four antibodies, with F21 having lower affinity than DS-Cav1 for binding to D25, AM14, and Motavizumab (Figure 3). Therefore, “Round 1” identified designs with near-equivalent expression and antigenicity profiles to DS-Cav1,and produced a mutational landscape of 33 substitutions that played a role in the stabilisation of the pre-fusion conformation (see Table 1). Within those 33 substitutions, Design F21 had a subset of 31 substitutions relative to RSV-F WT (see Figure 5), while Design F28 had 9 substitutions relative to RSV-F WT. Table 1 – substitutions relative to wild-type in “Round 1” designs F21 and F28
* Indicates substitution found in design F28. F21 substitutions are presented in Figure 5. Example 4 - Expression and Binding Studies of RSV-F Consensus Designs (“Round 2”) To optimise the expression and antigenicity of the “Round 1” designs, a second round of consensus sequence design was performed. In this iteration, the reduced landscape of 33 mutants (Table 1) was used to generate the protein specific scoring matrix (PSSM) for design in ROSETTA. For “Round 2”, the human RSV-F A2 subtype wild-type sequence (SEQ ID NO: 1) was chosen as a structural template design, resulting in 15 constructs for testing. These 15 constructs were tested in mammalian cells for expression and antibody binding in a similar manner as “Round 1” (see Figure 7 for antibody binding). Four constructs were identified for further characterization based on these assays, indicating equivalent or higher expression relative to DS-Cav1, with similar binding to the four pre-fusion antibodies (antibody binding in Figure 7, see designs F216, F217, F224, F225; protein expression in Figure 8). These four designs exhibited a range of optimal biophysical properties: expression, antigenicity, and thermostability as measured by nano-DSF (see Figure 10 for summary), In addition, the four designs had a relatively low the number of substitutions relative to wild-type (see Figure 9 – additional construct F214 also presented)). The three-dimensional protomer structure and substitutions of F224 and F216 are shown in Figures 11 and 12, respectively. Example 5 - Structural Analysis of RSV-F Consensus Designs (“Round 1” & “Round 2” designs) To verify the pre-fusion structure of the designed constructs, cryo-EM studies were performed on designs F21, F216 and F224 in complex with the quaternary antibody, AM14. The structural studies showed a majority of the 2D & 3D populations had a trimeric pre-fusion conformation and maintained complete binding to AM14 Fab fragments (Figure 13A shows 2D populations of F21). Electron density maps for F21, F216 and F224 are shown in Figures 13B, 14 and 15, respectively. The substitutions were further analysed on the model for design F224 (Table 2, Figure 14), indicating that the consensus approach was introducing substitutions that rigidified the pre-fusion trimeric structure, while limiting any hinge motions or structural rearrangements in the F1 domain and the heptad repeat A and B regions (HRA and HRB) that would allow for the transition from pre-fusion to post-fusion conformations. This shows the strength of using evolutionary sequences and rational structure-based design to find combinatorial sets of substitutions that might otherwise not work single point substitutions Table 2 – substitutions relative to wild-type in “Round 2” design F224 *Not present in design F225 (A241, as per wild-type) Example 6 – Minimal substitution screen (“Round 3” designs) Substitutions present in design F225 were (i) individually removed to generate sequences F301 – F307 with 6 substitutions each (Figure 17, dashed boxes; Table 3), or (ii) individually added to the WT sequence to generate sequences F308 – F313 and F226, with 1 mutation each (Figure 17, black boxes except for F226; Table 3). C-terminal sequences (positions 514 onwards) of all recombinant protein constructs were according to SEQ ID NO: 147. Table 3 – substitutions in “Round 3” designs tested in minimal substitution screen *Generated in “Round 2” Mutants were produced and screened as described for Rounds 1 and 2. All mutants in the group F301 – F307 expressed and had binding to mAbs AM14, D25, and RSB1 equivalent to DS-Cav1 (Figure 19), confirming a pre-fusion confirmation. This means a pre-fusion confirmation can be obtained with different combinations of the disclosed substitutions. Sequences F308, F309, and F311 which contained only the S55T, S215A, or S348N substitutions respectively, resulted in protein production and had some binding to mAbs AM14, D25, and RSB1 (Figures 18 & 19 respectively), though less binding than DS-Cav1. Thus, these substitutions are likely important in the stabilization of the pre- fusion confirmation. Finally, sequence F310, containing substitution N228K had both protein expression and binding to AM14, D25, and RSB1 that was equivalent to DS-Cav1 (Figures 18 & 19 respectively), indicating that this substitution has a significant contribution to the stabilization of pre- fusion RSV F, and is able to stabilize the pre-fusion conformation independently. Cryo EM analysis of F310 complexed with AM14 Fab confirmed its pre-fusion conformation (Figure 26). F301-F307 were further characterized and showed optimal biophysical properties including thermostability similar to F225 by nano-DSF (See Table 3B, below). Long term stability of F310 was tested and is shown in Figure 27. Table 3B – Thermostability measured by nano-DSF Example 7 – mRNA expression and epitope recovery screen (“Round 2” & “Round 3” designs) Given the lower affinity of antibodies AM14 and D25 for designs F216 and F217 compared to F224 and F225 (see Figure 10), an epitope recovery screen was performed to assess whether reverting substitutions in the quaternary and site Ø epitopes back to wild-type would recover antibody binding. The following RSV-F mutants were expressed from mRNA, which lacked either the K445D substitution (to recover AM14 binding), the S211N substitution (to recover D25 binding), or both (see Figure 20 for study design). Table 4 – substitutions in “Round 3” designs tested in epitope recovery screen Designs F224 (KM113) and F225 (KM114) were also expressed from mRNA, alongside controls DS-Cav1 and a further positive control RSV-F protein. The designs were shown to be expressed by mRNA (results shown in Figure 21). RSV-F mutations F216, F315, F317, F319, F217, F316, F318, and F320 were also expressed as protein and showed binding to AM14, D25, and RSB1 equivalent to DS-Cav1 (Figures 28 & 29), confirming their prefusion confirmation. This indicates S211N and K445D are not necessary for the prefusion confirmation in the F216 or F217 background. However, both mutations appear to improve protein stability after heat stress (Figure 30). The usefulness of these mutations in improving stability is also reflected in the changes in thermostability data of the epitope recovery mutants compared to F216 and F217 (Table 4B, below). Table 4B – Thermostability measured by nano-DSF Example 8 – Toluene nitrosulphonic acid (TNS) fluorescence assay for determining pKa Steps (1) – (14): (1) admixing 400 μL of 2 mM of the cationic lipid that is in 100 volume % ethanol and 800 μL of 0.3 mM of fluorescent probe TNS, which is in 90 volume % ethanol and 10 volume % methanol, thereby obtaining a lipid/TNS mixture; (2) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a first buffer comprising a sodium salt buffer comprising 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, wherein the first buffer has a first pH from 4.44 to 4.52, thereby obtaining a first mixture, and dispensing 100 μL of the first mixture in a first well of a 96-well plate, which has a clear bottom; (3) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a second buffer comprising the sodium salt buffer, wherein the second buffer has a second pH of 5.27, thereby obtaining a second mixture, and dispensing 100 μL of the second mixture in a second well of the 96-well plate; (4) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a third buffer comprising the sodium salt buffer, wherein the third buffer has a third pH of from 6.15 to 6.21, thereby obtaining a third mixture, and dispensing 100 μL of the third mixture in a third well of the 96-well plate; (5) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a fourth buffer comprising the sodium salt buffer, wherein the fourth buffer has a fourth pH of 6.57, thereby obtaining a fourth mixture, and dispensing 100 μL of the fourth mixture in a fourth well of the 96-well plate; (6) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a fifth buffer comprising the sodium salt buffer, wherein the fifth buffer has a fifth pH of from 7.10 to 7.20, thereby obtaining a fifth mixture, and dispensing 100 μL of the fifth mixture in a fifth well of the 96-well plate; (7) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a sixth comprising the sodium salt buffer, wherein the sixth buffer has a sixth pH of from 7.72 to 7.80, thereby obtaining a sixth mixture, and dispensing 100 μL of the sixth mixture in a sixth well of the 96-well plate; (8) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a seventh buffer comprising the sodium salt buffer, wherein the seventh buffer has a seventh pH of from 8.27 to 8.33, thereby obtaining a seventh mixture, and dispensing 100 μL of the seventh mixture in a seventh well of the 96-well plate; (9) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of an eighth buffer comprising the sodium salt buffer, wherein the eighth buffer has an eighth pH of from 10.47 to 11.12, thereby obtaining an eighth mixture, and dispensing 100 μL of the eighth mixture in an eighth well of the 96- well plate; (10) measuring the absolute fluorescence at a wavelength of 431 nm with an excitation wavelength of 322 nm and a cut-off below 420 nm of each of the first through eighth wells and an empty well of the 96-well plate; (11) subtracting the absolute fluorescence of the empty well from each of the absolute fluorescence values of the first through the eighth wells, thereby obtaining a blank-subtracted fluorescence for each of the first through eighth mixtures; (12) normalising each of the blank-subtracted fluorescence values of the first through eighth mixtures to the blank-subtracted fluorescence of the first mixture, thereby obtaining a relative fluorescence for each of the first through eighth mixtures, the relative fluorescence of the first mixture being 1; (13) regressing by the Henderson-Hasselbalch equation, the first through eighth pH values versus the respective relative fluorescence values of the first through eighth mixtures thereby obtaining a line of best fit; and (14) determining the pKa as the pH at which a relative fluorescence of 0.5 is obtained on the line of best fit. Example 9 – Long term stability studies (“Round 2” designs) The thermostability of F216, F217, F224 and DS-Cav1 (as measured by nano-DSF) remained stable after incubation of the protein at 4 or 25°C for up to 21 days (see Figure 31). Binding to RSV-F-specific antibodies (AM14, D25 and RSB1) via BIACORE potency assay was also tested after the same incubations as above (see Figure 32). Incubation at 4°C or 25°C for up to 21 days did not substantively affect F216 and F217 binding to any RSV-F-specific antibodies. F224 binding to the RSV-F-specific antibodies decreased after incubation at 25°C, but not 4°C, indicating F224 is more stable at 4°C. In contrast, incubation of DS-Cav1 at both 4°C and 25°C decreased binding to the RSV-F prefusion antibodies, indicating it is not stable at either temperature. Hence, in this, assay Round 2 designs F216, F217 and F224 exhibited greater long-term stability than DS- Cav1. Example 10 – In vivo protein immunisation study (“Round 2” designs) Designs F216, F217, F224, F225 (referred to as “PreF Design 16”, “PreF Design 17”, “PreF Design 24” and “PreF Design 25” respectively in the relevant Figures) and DS-Cav1 were administered to mice as set out in the Materials and Methods section. PreF IgG response was measured at 2 weeks post second injection. Data for the 3μg dose shows that F216 and F224 induce a statistically similar PreF IgG response when compared to DS-Cav1 (Figure 33A and B; Table 5A). The neutralizing antibody response was also measured. Data for the high dose, 3μg, shows that F217 induces a statistically similar neutralizing response as compared to DS-Cav1. At the lower dose, 0.3μg, F216, F217, F224 F225 all induce a statistically similar RSV neutralizing response when compared to DS- Cav1 (Figure 35A and B; Table 5C and D). Table 5A - Total level of RSV pre-fusion protein specific IgG binding antibodies from immunized mice with a 3µg dose at day 21 and day 35. Table 5A legend: The total level of RSV pre-fusion protein specific IgG binding antibodies from immunized mice with a 3µg dose was measured at day 21 and day 35 by a Luminex assay. The geometric mean titres (GMT) at a 95% confidence interval are shown. The number of responding mice (N resp) above the limit of detection (LOD) out of 8 total mice at each time point is also shown. Two mice were not above the LOD at day 21. All mice were above the LOD on day 35. The saline group was not included in the statistical analysis. Table 5B - Total level of RSV pre-fusion protein specific IgG binding antibodies from immunized mice with a 0.3µg dose at day 21 and day 35. Table 5B legend: The total level of RSV pre-fusion protein specific IgG binding antibodies from immunized mice with a 0.3µg dose was measured at day 21 and day 35 by a Luminex assay. The GMT at a 95% confidence interval are shown. The number of responding mice (N resp) above the LOD out of 8 total mice at each time point is also shown, except for F225 day 35 where the total number of mice was 7 ( ^). All mice were above the LOD on day 35. The saline group was not included in the statistical analysis. Table 5C - RSV neutralizing antibody titres measured with a neutralization assay for mice immunized with a 3.0 or 0.3 µg dose at day 35.
Table 5C legend: The RSV neutralizing antibody titres were measured with a neutralization assay for mice immunized with a 3.0 or 0.3 µg dose at day 35. The GMT at a 95% confidence interval was calculated. The number of responding mice (N resp) above the LOD out of 8 total mice at each time point was reported. The saline group was not included in the statistical analysis. Table 5D - RSV neutralizing antibody titres for mice immunized with a 3.0 or 0.3 µg dose at day 35 Table 5D legend: The RSV neutralizing antibody titres were measured with a neutralization assay for mice immunized with a 3.0 or 0.3 µg dose at day 35. The GMR at a 90% confidence interval comparing constructs to DS-Cav1 was calculated. Example 11 – In vivo mRNA immunisation (“Round 2” designs) RNA encoding F216, F217, F317, F319, DS-Cav1 and F(ii) was administered to mice as set out in the Materials and Methods section. Figures 36A and B display the RSV pre-F IgG binding antibody geometric mean titres on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either 2 μg (Figure 36A) or 0.2 μg (Figure 36B) of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319 (where each point represents an individual animal). There were no binding antibody responses in the saline control group (data not shown). On day 21, a 2 μg dose of F317 (geometric mean ratio GMR=1.75, lower confidence interval LCI=1.14, upper confidence interval UCI=2.68), 217 (GMR=3.05, LCI=2.25, UCI=4.13), or 216 (GMR=2.01, LCI=1.53, UCI=2.63) elicited statistically significantly higher pre-F IgG titres compared to DS-Cav1 (Figures 36A and C). On day 35, pre-F IgG titres elicited from a 2 μg dose of F317, F217, or F216 performed similarly when compared to F(ii) and DS-Cav1. The trends in RSV pre-F IgG binding titres remained the same even at the lower dose (0.2 μg). Most notably, F216, F217, F317, and F319 elicited higher pre-F IgG binding titres compared to DS-Cav1 at a 0.2 μg dose (Figure 36B). Due to response levels, statistical analysis was not conducted on the 0.2 μg dose group. Figures 37A and B display the RSV A neutralizing antibody titres (ED60) on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either 2 μg (Figure 37A) or 0.2 μg (Figure 37B) of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319 (where each point represents an individual animal). The saline group did not generate a measurable neutralization response to RSV A (data not shown). Out of all the exploratory constructs tested (F216, F217, F317, and F319), F217 generated the highest neutralization to RSV A. At a lower dose (0.2 μg), neutralizing antibodies to RSV A were low, but the trends remained the same. RSV A neutralization elicited by a 0.2 μg dose of F217 trended higher than DS-Cav1 (Figure 37B). Due to response levels, statistical analysis was not conducted on the 0.2 μg dose group. Example 12 – Cytoplasmic tail deletions (“Round 3” designs) Select truncation of the RSV-F CT was used to produce a mRNA vaccine designs with unique features. 12A Human primary BJ cells are permissive for the cell-surface expression of RSV F protein encoded by exogenous mRNAs. The steady-state, total cell-surface RSV F protein expression of the design, F318 CT Δ20, is observed to increase from 8 hours post transfection (Figure 38A”) to 24 hours post transfection (Figure 38B”) in BJ cells and decay in the subsequent 3 days (Figure 38, C”-E”). Quantification of RSV F levels using High Content imaging and image analysis in individual BJ cells in the transfected cell monolayer is shown (Figure 38, F-J) and exhibits a corresponding shift in the population distribution indicates increasing RSV F levels over the first day and decay in the subsequent days. 12B The RSV-F variant design F(ii) (Figure 39A) expresses AM14-(+) RSV F protein, as does designs, F318 and F319 (Figure 39B and 39C, respectively), and design F(i) (Figure 39D). As shown by area under the curve (AUC), the four constructs perform similarly (Figure 39E). Design F(ii), with CT deletions (in whole or in part), expresses AM14(+) RSV F to a greater degree than F(ii) parental molecule (i.e. absent CT deletions) (Figure 39A). Using F318, F319 or F(i), (Figures 39B, C and D, respectively), deletions within the CT improves RSV F AM14+ signal by at least 2x, at 24 hours post transfection. The maximal effect from CT deletion is consistently observed with 20AA deletion while 3AA deletion supersedes complete deletion of the CT (Figure 39E). 12C The surface expression of immunogenic RSV F may include, but is not limited to, monomeric to multimeric F states and any abundant RSV F conformations. Total expression of RSV F protein at the cell surface can be captured using the monoclonal antibody motavizumab. As shown, Motavizumab(+) RSV F variant F(ii) is readily detected 24 hours post transfection, while 3 amino acid, 20 amino acid and complete CT deletion, respectively, engineered into F protein unambiguously increases expression (Figure 40A). Similarly, the RSV F variants F318, F319 and F(i), each demonstrate substantial increases for RSV F expression when carrying CT deletions (Figure 40B, 4C & 4D, respectively). Deletions in the CT universally increase RSV F expression (Figure 40E) 12D The RSV F protein variants F(ii) and DS-Cav1 (Table 7), were each modelled as their respective mRNA doppelgangers for an in vivo study (Example 13). Designs F(i) and F(iii) (control) were additionally synthesized (Table 7). mRNA-based models of F(ii), F(i), F318 and F319 were created to include the CT deletion of 20 amino acids (ΔCT20) (Table 8). The total RSV F expression (Figure 41A & C) is improved by ΔCT20. As a measure of the total, pre-fusion state, RSV F expression is increased by the ΔCT20 (Figure 41E and G). In congruence, the ΔCT20 universally improves cell surface expression of the trimeric, pre-fusion RSV F conformation (Figure 41I & K). While the level of RSV F measured varied across a broad range (Figure 41A, C, E, G, I & K), transfection of diverse mRNAs and the expressed cognate proteins did not meaningfully impact the integrity of the cell monolayers at either 24 (Figure 41B, F & J) or 67 hours post transfection (Figure 41D, H & L) and were not acutely toxic in primary cells. 12E Protein expression at the cell surface varied as a function of RSV F CT amino acid length. BJ cells were transfected with mRNAs encoding F(ii) RSV F protein that varied in the CT length across a range from no CT to the full length, 25AA CT (See Table 8). BJ cell monolayers were fixed at time points either 20 or 47 hours post transfection. Surface exposed, trimeric RSV F (Figure 42A) or whole-cell, prefusion RSV F (Figure 42B) was quantified by High Content imaging following immunolabeling of the fixed, BJ cells. Expression level varied according to the length of CT. For example, when assessed by AM14 binding (Figure 42A), trimeric, prefusion RSV-F expression was highest with the ΔCT20 variant (CT length of 5 residues). Example 13 – In vivo mRNA immunisation (“Round 3” designs) RNA encoding F(iii), F(i), F(i) ΔCT20, F(ii), F(ii) ΔCT20, DS-Cav1, F318, F318 ΔCT20, F319 and F319 ΔCT20 was administered to mice as set out in the Materials and Methods section. Figure 43 displays the RSV pre-F IgG binding antibody geometric mean titres on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either 2 μg (Figure 43A) or 0.2 μg (Figure 43B) of RNA encoding F(iii), F(i), F(i) ΔCT20, F(ii), F(ii) ΔCT20, DS-Cav1, F318, F318 ΔCT20, F319, or F319 ΔCT20 (where each point represents an individual animal). There were no binding antibody responses in the saline control group (data not shown). On day 21, all constructs elicited measurable pre-F-specific IgG binding antibodies with a 2μg dose. A single dose of DS-Cav1 elicited the lowest pre-F-specific IgG binding antibodies compared to the other constructs. By day 35, all pre-F-specific IgG antibodies were boosted, and elicited similar antibody titres. The two immunizations with a 2μg dose of F318, F318 ΔCT20, F319, and F319 ΔCT20 boosted pre-F specific IgG antibodies to levels that were noninferior to the benchmark controls F(i), F(ii), F(iii) and DS-Cav1 (Figures 43 A and C- F). At the low dose, F318 ΔCT20 achieved noninferiority when compared to F(ii) and F(iii) at day 35 (2wp2) (Figures 43B, D and F). One 0.2μg dose of F318 ΔCT20 or F319 ΔCT20 elicited significantly higher pre-F IgG titres compared to the non- ΔCT20 counterparts (Figure 43B and G). Figure 44 displays the RSV A neutralizing antibody titres (ED60) on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either (Figure 44A) 2 μg or (Figure 44B) 0.2 μg of RNA encoding F(iii), F(i), F(i) ΔCT20, F(ii), F(ii) ΔCT20, DS-Cav1, F318, F318 ΔCT20, F319, or F319 ΔCT20 (where each point represents an individual animal). The saline group did not generate a measurable neutralization response to RSV A (data not shown). All neutralization titres were boosted with a second vaccination. At the high (2μg) dose, one vaccination generated measurable neutralization to RSV A (Figure 44A). At the high (2μg dose), F318 ΔCT20, F319, and F319 ΔCT20 reached noninferiority compared to F(iii) at day 35 (2wp2) (Figure 44A and C). At the high (2μg) dose, F318 ΔCT20, F319, and F319 ΔCT20 generated significantly higher neutralization titres compared to DS-Cav1 on day 21 (3wp1). By day 35, F318 ΔCT20, F319, and F319 ΔCT20 were noninferior to DS-Cav1 (Figure 44A and E). DS-Cav1 vaccination generated the lowest neutralization titres with one 0.2μg dose (Figure 44B). F318 ΔCT20 reached noninferiority when compared to F(iii) (Figure 44B and C) at day 35 (2wp2). The effect of ΔCT20 was most prominent in the low dose (Figure 44B). At the low (0.2μg) dose, F(ii) and F318 and F319 containing the ΔCT20 elicited higher neutralization titres to RSV A compared to their non - ΔCT20 counterparts (Figure 44B) , which was statistically significant for F318 and F319 (Figure 44E). At the high (2μg) dose (Figure 44A, E), the impact of the ΔCT20 may have been masked due to possible saturation effects. Example 14 – Cytoplasmic tail deletions (further designs – incremental deletions) The RSV F protein expression of F(ii) with varying CT lengths was characterized in primary human fibroblasts for the cell-surface trimeric, pre-fusion (Figure 45A, B, C) or cell-surface pre-fusion (Figure 46A, B, C) RSV F protein. See Table 11, below, for CT lengths tested. Table 11 – CT variations (incremental deletions)
In agreement with prior results (see Example 12), the deletion of the full-length CT (Figure 45A & 46A, see F(ii) ΔCTD) increases expression of RSV F at the cell surface compared to the parent (absent any deletions from the CT) (Figure 45A & 46A, F(ii)). The mRNA encoding CT deletion variants (Figure 45A, see F(ii) CTD Δ15, F(ii) CTD Δ15 F(ii) CTD Δ17, and F(ii) CTD Δ20) offered the best cell-surface, trimeric pre-fusion RSV F protein expression, while F(ii) CTD Δ16 offered a substantial improvement out to at least 72 hours post transfection. In contrast, both F(ii) CTD Δ21 and F(ii) ΔCTD each exhibit weaker expression (Figure 45A) for the duration of the assay. The data is summarized using area under the curve (AUC) and shown in Figures 45B & C and 46B & C. Consistent with the time-course shown in Figure 45A, the peak cell-surface, trimeric, prefusion RSV F expression is specific to variants using the CTD length at least 5 amino acids long, and in contrast, CTD lengths less than 5 amino acids are associated with reduced F protein expression (Figure 45B).
SEQUENCES SEQ ID NO: 1: Amino acid (AA) sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094 (no foldon, transmembrane domain or cytoplasmic tail). SEQ ID NO: 1 is referred to herein as wild-type. SEQ ID NO: 2: AM14 light chain AA sequence SEQ ID NO: 3: AM14 heavy chain AA sequence SEQ ID NO: 4: D25 light chain AA sequence SEQ ID NO: 5: D25 heavy chain AA sequence SEQ ID NO: 6: Motavizumab light chain AA sequence SEQ ID NO: 7: Motavizumab heavy chain AA sequence SEQ ID NO: 8: RSB1 light chain AA sequence SEQ ID NO: 9: RSB1 heavy chain AA sequence SEQ ID NO: 10: AA sequence of exemplary F2-F1 linker sequence SEQ ID NO: 11: AA sequence of exemplary F2-F1 linker sequence SEQ ID NO: 12: AA sequence of exemplary F2-F1 linker sequence SEQ ID NO: 13: AA sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail). SEQ ID NO: 13 is referred to herein as wild- type. SEQ ID NO: 14: AA sequence of T4 fibritin foldon domain SEQ ID NO: 15: AA sequence of RSV-F WT transmembrane domain SEQ ID NO: 16: AA sequence of RSV-F WT cytoplasmic domain Q SEQ ID NO 17: AA sequence of design F216 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO 18: AA sequence of design F217 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 19: AA sequence of design F224 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 20: AA sequence of design F225 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 21: AA sequence of WT with S55T, S215A and S348N substitutions only (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 22: AA sequence of design F315 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 23: AA sequence of design F317 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 24: AA sequence of design F319 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 25: AA sequence of design F316 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 26: AA sequence of design F318 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 27: AA sequence of design F320 (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 28: AA sequence of design F216 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 29: AA sequence of design F217 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 30: AA sequence of design F224 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 31: AA sequence of design F225 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 32: AA sequence of WT with S55T, S215A and S348N substitutions only – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 33: AA sequence of design F315 (no foldon, transmembrane domain or cytoplasmic tail) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 34: AA sequence of design F317 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 35: AA sequence of design F319 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 36: AA sequence of design F316 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 37: AA sequence of design F318– furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 38: AA sequence of design F320 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 39: AA sequence of isoleucine substituted GCN4 leucine zipper SEQ ID NO: 40: AA sequence of design F301 (F225 substitutions minus V459M) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 41: AA sequence of design F302 (F225 substitutions minus T455V) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 42: AA sequence of design F303 (F225 substitutions minus S348N) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 43: AA sequence of design F304 (F225 substitutions minus K315I) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 44: AA sequence of design F305 (F225 substitutions minus N228K) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 45: AA sequence of design F306 (F225 substitutions minus S215A) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 46: AA sequence of design F307 (F225 substitutions minus S55T) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 47: AA sequence of design F308 (S55T substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 48: AA sequence of design F309 (S215A substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 49: AA sequence of design F311 (S348N substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 50: AA sequence of design F301 (F225 substitutions minus V459M) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 51: AA sequence of design F302 (F225 substitutions minus T455V) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 52: AA sequence of design F303 (F225 substitutions minus S348N) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 53: AA sequence of design F304 (F225 substitutions minus K315I) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 54: AA sequence of design F305 (F225 substitutions minus N228K) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 55: AA sequence of design F306 (F225 substitutions minus S215A) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 56: AA sequence of design F307 (F225 substitutions minus S55T) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 57: AA sequence of design F308 (S55T substitution only) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 58: AA sequence of design F309 (S215A substitution only) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 59: AA sequence of design F311 (S348N substitution only) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 60: AA sequence of exemplary linker sequence (between F1 domain and trimerisation domain) SEQ ID NO: 61: RNA sequence of 5' UTR of “UTR4” (HIST2H4A 5' UTR) – n.b., spacing to be ignored (sequence to be read as one continuous sequence) SEQ ID NO: 62: RNA sequence of 3' UTR of “UTR4” (HIST2H4A 3' UTR) – n.b., spacing to be ignored (sequence to be read as one continuous sequence) SEQ ID NO: 63: RNA sequence of 5' UTR of “UTR3” (FAP 5' UTR) – n.b., spacing to be ignored (sequence to be read as one continuous sequence) SEQ ID NO: 64: RNA sequence of 3' UTR of “UTR3” (FAP 3' UTR) – n.b., spacing to be ignored (sequence to be read as one continuous sequence) SEQ ID NO: 65: RNA sequence of 5' UTR of “UTR7” (IL-25' UTR) – n.b., spacing to be ignored (sequence to be read as one continuous sequence) SEQ ID NO: 66: RNA sequence of 3' UTR of “UTR7” (IL-23' UTR) – n.b., spacing to be ignored (sequence to be read as one continuous sequence) SEQ ID NO: 67: RNA sequence of possible 5' UTR SEQ ID NO: 68: RNA sequence of possible 3' UTR SEQ ID NO: 69: RNA sequence of possible 5' UTR SEQ ID NO: 70: RNA sequence of possible 3' UTR SEQ ID NO: 71: RNA sequence of construct KM111 (encoding F216) – all U ribonucleotides are 1mΨ – GC content 49.90% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 72: RNA sequence of construct KM112 (encoding F217) – all U ribonucleotides are 1mΨ – GC content 49.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 73: RNA sequence of construct KM113 (encoding F224) – all U ribonucleotides are 1mΨ – GC content 48.90% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 74: RNA sequence of construct KM114 (encoding F225) – all U ribonucleotides are 1mΨ – GC content 49.00% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 75: RNA sequence of construct KM116 (encoding F315) – all U ribonucleotides are 1mΨ – GC content 49.80% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 76: RNA sequence of construct KM117 (encoding F316) – all U ribonucleotides are 1mΨ – GC content 49.80% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 77: RNA sequence of construct KM118 (encoding F317) – all U ribonucleotides are 1mΨ– GC content 49.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 78: RNA sequence of construct KM119 (encoding F318) – all U ribonucleotides are 1mΨ – GC content 50.00% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 79: RNA sequence of construct KM120 (encoding F319) – all U ribonucleotides are 1mΨ – GC content 49.80% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 80: RNA sequence of construct KM121 (encoding F320) – all U ribonucleotides are 1mΨ – GC content 49.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 81: AA sequence of design F310 (N228K substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 82: AA sequence of design F310 (N228K substitution only) – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 83: RNA sequence of construct KM123 (encoding F310) – all U ribonucleotides are 1mΨ – GC content 49.50% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 84: AA sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail). SEQ ID NO: 84 is referred to herein as wild-type. SEQ ID NO 85: AA sequence of design F216 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO 86: AA sequence of design F217 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 87: AA sequence of design F224 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 88: AA sequence of design F225 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 89: AA sequence of WT with S55T, S215A and S348N substitutions only – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 90: AA sequence of design F315 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 91: AA sequence of design F317 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 92: AA sequence of design F319 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 93: AA sequence of design F316 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 94: AA sequence of design F318 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 95: AA sequence of design F320 – furin processed, without signal sequence (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 96: AA sequence of design F301 – furin processed, without signal sequence (F225 substitutions minus V459M) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 97: AA sequence of design F302 – furin processed, without signal sequence (F225 substitutions minus T455V) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 98: AA sequence of design F303 – furin processed, without signal sequence (F225 substitutions minus S348N) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 99: AA sequence of design F304 – furin processed, without signal sequence (F225 substitutions minus K315I) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 100: AA sequence of design F305 – furin processed, without signal sequence(F225 substitutions minus N228K) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 101: AA sequence of design F306 – furin processed, without signal sequence (F225 substitutions minus S215A) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 102: AA sequence of design F307 – furin processed, without signal sequence (F225 substitutions minus S55T) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 103: AA sequence of design F308 – furin processed, without signal sequence (S55T substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 104: AA sequence of design F309 – furin processed, without signal sequence (S215A substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 105: AA sequence of design F311 – furin processed, without signal sequence (S348N substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 106: AA sequence of design F310 – furin processed, without signal sequence (N228K substitution only) (no foldon, transmembrane domain or cytoplasmic tail) SEQ ID NO: 107: Amino acid (AA) sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094 with full cytoplasmic tail. SEQ ID NO: 107 is referred to herein as wild-type. SEQ ID NO: 108: AA sequence of wild-type RSV-F B subtype strain 18537 (Uniprot ID: P13843) with full cytoplasmic tail SEQ ID NO: 109: Cytoplasmic tail of SEQ ID NO: 107 (AA sequence) SEQ ID NO: 110: Cytoplasmic tail of SEQ ID NO: 108 (AA sequence) SEQ ID NO: 111: AA sequence of DS-CAV1 (without CT deletions) SEQ ID NO: 112: AA sequence of F(ii) construct (without CT deletions) SEQ ID NO: 113: AA sequence of F(i) construct (without CT deletions) SEQ ID NO: 114: AA sequence of F(iii) construct (including full deletion of CT) SEQ ID NO: 115: RNA sequence of construct KM119d3 (encoding F318d3) – all U ribonucleotides are 1mΨ – GC content 49.80% – 5' and 3' UTRs are “UTR4” (reference to d3, d20 and so forth, when referencing an encoded protein in the sequences, means ΔCT3, ΔCT20, and so forth). SEQ ID NO: 116: RNA sequence of construct KM119d20 (encoding F318d20) – all U ribonucleotides are 1mΨ – GC content 49.70% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 117: RNA sequence of construct KM119dCT (encoding F318dCT) – all U ribonucleotides are 1mΨ – GC content 49.60% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 118: RNA sequence of construct KM120d3 (encoding F319d3) – all U ribonucleotides are 1mΨ – GC content 49.70% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 119: RNA sequence of construct KM120d20 (encoding F319d20) – all U ribonucleotides are 1mΨ – GC content 49.60% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 120: RNA sequence of construct KM120dCT (encoding F319dCT) – all U ribonucleotides are 1mΨ – GC content 49.60% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 121: RNA sequence of construct KM173 (encoding F(i)) – all U ribonucleotides are 1mΨ – GC content 48.00% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 122: RNA sequence of construct KM173d3 (encoding F(i) d3) – all U ribonucleotides are 1mΨ – GC content 49.30% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 123: RNA sequence of construct KM173d20 (encoding F(i) d20) – all U ribonucleotides are 1mΨ – GC content 49.40% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 124: RNA sequence of construct KM173dCT (encoding F(i) dCT) – all U ribonucleotides are 1mΨ – GC content 49.20% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 125: RNA sequence of construct KM135 (encoding F(ii)) – all U ribonucleotides are 1mΨ – GC content 48.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 126: RNA sequence of construct KM136 (encoding F(ii) d3) – all U ribonucleotides are 1mΨ – GC content 48.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 127: RNA sequence of construct KM137 (encoding F(ii) d5) – all U ribonucleotides are 1mΨ – GC content 48.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 128: RNA sequence of construct KM138 (encoding F(ii) d10) – all U ribonucleotides are 1mΨ – GC content 49.00% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 129: RNA sequence of construct KM139 (encoding F(ii) d15) – all U ribonucleotides are 1mΨ – GC content 49.00% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 130: RNA sequence of construct KM140 (encoding F(ii) d20) – all U ribonucleotides are 1mΨ – GC content 48.90% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 131: RNA sequence of construct KM141 (encoding F(ii) dCT) – all U ribonucleotides are 1mΨ – GC content 48.80% – 5' and 3' UTRs are “UTR4” SEQ ID NO:132: RNA sequence of construct KM03 (encoding DS-Cav1) – all U ribonucleotides are 1mΨ – GC content 49.50% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 133: Reference construct C-terminus, including additional transmembrane (TM) domain residues N-terminal to CT start (AA sequence) SEQ ID NO: 134: ΔCT3 construct C-terminus, including additional transmembrane (TM) domain residues N-terminal to CT start (AA sequence) SEQ ID NO: 135: ΔCT5 construct C-terminus, including additional transmembrane (TM) domain residues N-terminal to CT start (AA sequence) SEQ ID NO: 136: ΔCT10 construct C-terminus, including additional transmembrane (TM) domain residues N-terminal to CT start (AA sequence) SEQ ID NO: 137: ΔCT15 construct C-terminus, including additional transmembrane (TM) domain residues N-terminal to CT start (AA sequence) SEQ ID NO: 138: CTΔ20 construct C-terminus, including additional transmembrane (TM) domain residues N-terminal to CT start (AA sequence) SEQ ID NO: 139: ΔCT25 construct: transmembrane (TM) domain residues only (AA sequence) SEQ ID NO: 140: RNA sequence of construct KM126 (encoding F(iii) construct (including full deletion of CT)) – all U ribonucleotides are 1mΨ – GC content 56.40% SEQ ID NO: 141: RNA sequence of construct XW02C23 (encoding DS-Cav1) – all U ribonucleotides are 1mΨ – GC content 49.40% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 142: RNA sequence of construct KM111C2 (encoding F216) – all U ribonucleotides are 1mΨ – GC content 49.70% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 143: RNA sequence of construct KM112C10 (encoding F217) – all U ribonucleotides are 1mΨ – GC content 49.70% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 144: RNA sequence of construct KM118C2 (encoding F317) – all U ribonucleotides are 1mΨ – GC content 48.40% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 145: RNA sequence of construct KM120C3 (encoding F319) – all U ribonucleotides are 1mΨ – GC content 49.60% – 5' and 3' UTRs are “UTR4”
SEQ ID NO: 146: RNA sequence of construct XW03C37 (encoding F(ii)) – all U ribonucleotides are 1mΨ – GC content 49.00% – 5' and 3' UTRs are “UTR4” SEQ ID NO: 147: C-terminus (position 514 onwards) of recombinant protein constructs used in inter alia, Example 6 (linker sequences, T4 fibritin foldon trimerisation domain, thrombin cleavage site, strep tag and His-tag)
REFERENCES [1] Rha et al. Pediatrics.2020 Jul;146(1):e20193611 [2] Falsey et al. N Engl J Med.2005 Apr 28;352(17):1749-59. [3] Falsey and Walsh. Clin Microbiol Rev.2000;13:371–84. [4] Groothuis et al. Adv Ther 2011;28:110-25. [5] Feltes et al. Pediatr Res 2011; 70:186-91. [6] Carbonell-Estrany et al. Pediatrics 2010;125:e35–51. [7] O'Brien et al. Lancet Infect Dis 2015;15:1398-408. [8] The IMpact-RSV study group. Pediatrics 1998;102:531-537. [9] Krarup et al. Nat Commun.2015 Sep 3;6:8143. [10] McLellan et al. Science.2013, 342, 6158: 592-598 [11] Whitehead et al. J Virol.1998, 72, 5: 4467-4471 [12] Whitehead et al. J Virol.1999, 73, 4: 3438-3442 [13] Kyte & Doolittle. J Mol Biol.1982; 157, 105-132 [14] Sievers et al. Methods Mol Biol.2014;1079:105-16. [15] Gilman et al. PLOSPathogens.2015; 11(7), e1005035 [16] McLellan et al. Science.2013.340, 6136.1113-7 [17] Corti et al. Nature.2013;501(7467):439–43 [18] Cingoz MAbs.2009 Sep-Oct;1(5):439-42. [19] Lucas et al. Chem Sci.2016.7, 1038-1050 [20] WO 2005/113782 [21] Chen et al. Mol Cell.201976, 1: 96-109 [22] Edgar. BMC Bioinformatics.2004.5, 113 [23] Edgar et al. bioRxiv.2021.06.20.449169 [24] WO 2012/006380 [25] US20100324120 [26] WO 2021/038508 [27] Gennaro. 2000. Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [28] WO 1988/09336 [29] WO 2019/10692 [30] van Drunnen Little-van den Hurk et al. Rev Med Virol.2007.17(1): 5-34 [31] Aw et al. Immunology.2007.120(4): 435-46
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