WO2024041772A1 - Protéines rsv-f - Google Patents

Protéines rsv-f Download PDF

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WO2024041772A1
WO2024041772A1 PCT/EP2023/066330 EP2023066330W WO2024041772A1 WO 2024041772 A1 WO2024041772 A1 WO 2024041772A1 EP 2023066330 W EP2023066330 W EP 2023066330W WO 2024041772 A1 WO2024041772 A1 WO 2024041772A1
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rsv
seq
protein
present disclosure
positions
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PCT/EP2023/066330
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Nicholas John BARROWS
Marco BIANCUCCI
Chelsy Caryn CHESTERMAN
Wayne Daniel HARSHBARGER
Kambiz MOUSAVI
Newton Muchugu WAHOME
Xiaofeng Wang
James Alan WILLIAMS
Corey Mallett
Sanjay Phogat
Genevieve HOLZAPFEL
Emily PHUNG
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Glaxosmithkline Biologicals Sa
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18571—Demonstrated in vivo effect

Definitions

  • RSV-F PROTEINS FIELD The present disclosure is in the field of vaccinology, in particular structure-based protein design of vaccine antigens.
  • BACKGROUND Respiratory syncytial virus (“RSV”) is a ribonucleic acid virus of the Pneumoviridae family of which two antigenically distinct subgroups, referred to as RSV A and RSV B, exist.
  • RSV is a leading cause of infant and older adult hospitalisation and mortality. Each year in the United States, RSV leads to approximately 58,000 hospitalisations with 100-500 deaths among children under five [1], and 177,000 hospitalisations with 14,000 deaths among adults aged 65 years and above [2].
  • ribavirin is the only approved antiviral therapy for RSV treatment, but its use is restricted to severe hospitalized cases in infants and young children [3].
  • palivizumab Synagis
  • motavizumab two RSV-specific humanized monoclonal antibodies, palivizumab (Synagis) and motavizumab, are confirmed to be safe and effective in reducing RSV hospitalization rates and serious complications among high- risk children in multiple clinical settings [4, 5, 6, 7, 8].
  • Available treatment for RSV in older adults is generally supportive in nature, consisting of supplemental oxygen, intravenous fluids and bronchodilators.
  • RSV-F RSV fusion protein
  • RSV-F adopts a metastable “pre-fusion” conformation in the viral envelope as a homotrimer, and then an irreversible and distinct “post-fusion” conformation during fusion with the host cell membrane (see Figure 2 of [9]).
  • the pre-fusion conformation is more immunogenic, and is bound by most RSV-F-specific neutralising antibodies in human sera.
  • the native pre- fusion conformation is not energetically favourable. Therefore, pre-fusion RSV-F antigens, for use in vaccination, need to be stabilised to prevent irreversible folding to the post-fusion conformation. Structure-based antigen design strategies have previously been used in attempts to stabilise the pre- fusion conformation.
  • pre-fusion RSV-F protein designs which Docket No.: 70221WO01 may be used as vaccine antigens, and in particular, which are amenable to high expression yields when expressed from nucleic acids.
  • the inventors have created new RSV-F proteins in the pre-fusion conformation. Using a computational model of wild-type pre-fusion RSV-F (A2 strain), the inventors firstly identified an in silico residue substitution landscape that enhanced the expression and stability of trimeric, pre-fusion RSV-F (see e.g. Example 2). This strategy employed a combination of sequence-based evolutionary bioinformatics and structure-based thermodynamic design.
  • Example 6 design F310, which has the N228K substitution).
  • Three-dimensional structural analysis by the inventors revealed that the introduced residue (K) appears to form an intra-protomer hydrogen bond (H bond) with a proximal residue, Y250 (see Figure 25).
  • K introduced residue
  • H bond intra-protomer hydrogen bond
  • Y250 proximal residue
  • Mutations of this type may therefore be useful either alone, in order to achieve pre-fusion RSV-F per se, or in combination with further mutations (preferably substitutions) to stabilise RSV-F in the pre-fusion conformation (e.g. to provide longer term stability).
  • exemplary RSV-F proteins according to the present disclosure exhibit higher expression yields in vitro than DS-Cav1 of reference [10] (see e.g. Examples 4 and 6; Figures 8 and 18). Exemplary RSV-F proteins according to the present disclosure also exhibit greater long-term stability than DS-Cav1 (see, e.g. Example 9; Figure 32). In the in vivo context, exemplary RSV-F proteins according to the present disclosure elicit a pre- fusion RSV-F-specific antibody response, and moreover a neutralising antibody response against e.g. RSV A, when administered in a murine model (see e.g. Examples 10, 11 and 13; Figures 34-37, 43 and 44).
  • RSV-F proteins generated by the inventors may be useful as vaccine antigens, namely to be used in prophylactic vaccination against RSV. Docket No.: 70221WO01 Accordingly, in a first independent aspect, the present disclosure provides: RSV-F protein in the pre-fusion conformation, which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1; wherein the RSV-F protein comprises at least one mutation relative to the wild-type in a region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation introduces, through substitution or insertion, a residue comprising an H bond donor and/or acceptor moiety in its side chain.
  • the present disclosure provides a nucleic acid (preferably RNA) encoding an RSV-F protein of the present disclosure.
  • the present disclosure provides a host cell comprising a nucleic acid of the present disclosure.
  • the present disclosure provides an in vitro method for the production of an RSV-F protein of the present disclosure, comprising expressing a nucleic acid of the present disclosure (preferably, an expression vector) in a host cell, and optionally purifying the RSV-F protein.
  • the present disclosure provides a carrier (preferably, a lipid nanoparticle) comprising a nucleic acid of the present disclosure.
  • the present disclosure provides a pharmaceutical composition comprising an RSV-F protein, nucleic acid (preferably RNA) or carrier (preferably lipid nanoparticle) of the present disclosure.
  • the present disclosure provides an RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, for use in medicine.
  • the present disclosure provides a therapeutic method comprising the step of administering an effective amount of the 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).
  • FIGURES Figure 1 Sequence and structure-based consensus design to stabilize the prefusion conformation of RSV-F.
  • A the ROSETTA protein design suite was used to find combinatorial substitutions at different in silico energy thresholds, resulting in 12 sequences ranging from -0.5 kcal/mol to -6 kcal/mol (calculated in 0.5 kcal/mol increments), relative to wildtype;
  • B The ensuing substitution Docket No.: 70221WO01 landscape is shaded to illustrate sequence diversity among potential designs (darker shading representing greater sequence diversity), relative to known epitope positions (sites ⁇ , I, II,III, IV, V).
  • Figure 2 the substitution Docket No.: 70221WO01 landscape is shaded to illustrate sequence diversity among potential designs (darker shading representing greater sequence diversity), relative to known epitope positions (sites ⁇ , I, II,III, IV, V).
  • Octet BLI of the “Round 3” minimal substitution designs bound to RSV-F antibodies (AM14, D25, RSB1, motavizumab), relative to DS-Cav1. Negative control (EXPIFECTAMINE and cell culture supernatant), F225 and F300 (wild-type) also shown.
  • Figure 20 Round 3 (mRNA) – study design of epitope recovery experiment only.
  • Figure 21 Positive cell percentage of “Round 2” and “Round 3“ RSV-F designs and controls (DS- Cav1, positive control RSV-F construct, and negative control JW27 (NCBI:txid65840)) expressed from mRNA, with detection by RSB1, AM14, motavizumab and 4D7 antibodies.
  • the LOD is represented by a dashed line.
  • the neutralizing antibody levels are shown with circles.
  • the GMT at a 95% confidence interval are shown (bars).
  • B The RSV neutralizing antibody titres were measured with a neutralization assay for mice immunized with a 3.0 or 0.3 ⁇ g dose at day 35. The GMR at a 90% confidence interval was calculated.
  • FIG. 37 RSV A neutralizing antibody titres (ED60) on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either (A) 2 ⁇ g or (B) 0.2 ⁇ g of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319. Each point represents an individual animal.
  • C Statistical analysis: GMR, UCI and LCI of 2ug dose results. Docket No.: 70221WO01 Figure 38. Human primary BJ cells support surface expression of RSV F protein from candidate mRNAs.
  • Representative images from a 4-day time course assay are shown.
  • indirect immunofluorescence and imaging (10x objective) captures the individual cell nuclei (denoted ‘) and the cell surface RSV F (denoted “) variant F318 with 3 amino acids removed from the cytoplasmic tail (CT) in cells fixed approximately 8 (A’ & A”), 24 (B’ & B”), 48 (C’ & C”), 72 (D’ & D”) or 96 (E’ & E”) hours post transfection and labelled by using the primary antibody motavizumab.
  • the population distribution from High Content imaging (HCi) and analysis for BJ cells transfected and labelled corresponding to the representative images in panels A-E is shown at approximately 8 (F), 24 (G), 48 (H), 72 (I) and 96 (J) hours post transfection.
  • F-J The population distribution was binned and plotted by GraphPad Prism using the cell-specific RSV F average intensity values from High Content Imaging (HCi) and analysis.
  • Figure 39 Deletion of the RSV F CT increases cell-surface expression of the pre-fusion RSV F trimer.
  • RSV F trimer protein was evaluated by indirect immunofluorescent labelling using monoclonal antibody AM14 followed by quantification using high content imaging and analysis across a 4-day time course.
  • human BJ cells were forward transfected in 96-microwell format with mRNAs encoding RSV F variants F(ii) (A), F318 (B), F319 (C) or F(i) (D) (solid dot, solid line) or the respective CT deletion variations CT ⁇ 3 (solid dot, dashed line), ⁇ CT20(open circle, dashed line), or ⁇ CT (i.e. deletion of the entire CT - open circle, solid line).
  • RSV F was labelled and imaged using a 10x objective.
  • each plotted value expresses the average intensity of the Alexa647 signal for cells identified by automated image analysis from 9 imaged fields per well.
  • Each point on the line graph represents the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 biological replicates.
  • the area under the curve (AUC) for each line graph is shown (E) with 1 standard error of the mean (SEM).
  • the means, AUC and variability shown on the line and bar graphs were calculated by GraphPad Prism software.
  • Figure 40 Total expression of the RSV F protein increases for mRNA vaccine candidates with CT deletions.
  • RSV F protein was evaluated by indirect immunofluorescent labelling using the primary anti-RSV F antibody motavizumab followed by quantification using HCi and analysis across a 4-day time course.
  • Primary, human BJ cells were forward transfected in 96-microwell format with mRNAs encoding RSV F variants F(ii) (A), F318 (B), F319 (C) or F(i) (D) (solid dot, solid line) or the respective CT deletion variations CT ⁇ 3 (solid dot, dashed line), ⁇ CT20(open circle, dashed line), or ⁇ CT (open circle, solid line).
  • RSV F RNA sequence encoding RSV F
  • the cell monolayers were fixed and RSV F protein expression was evaluated by indirect immunofluorescence coupled with HCi and image analysis.
  • the mRNAs encode RSV F variants including DS-CAV1, F(ii), F(iii) and F(i) proteins or the F318 and F319 protein constructs. Results for corresponding variants lacking the CT 20 amino acids ( ⁇ CT20) are also shown.
  • RSV F surface protein expression was quantified 1 day post infection by labelling cells using the anti-RSV F antibodies Motavizumab (A), D25 (E) or AM14 (I) or 3 days post transfection (Motavizumab (C), D25 (G) or AM14 (K)).
  • the average cell count for three imaged wells is shown and corresponds to the RSV F expression values for 1 day post infection (Motavizumab (B), D25 (F) or AM14 (J) or 3 days post transfection (motavizumab (D), D25 (H) or AM14 (L)).
  • Each graph depicts the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 biological replicates as calculated by GraphPad Prism software. Figure 42.
  • a short, 5 amino acid CT (See Table 8, Row 6) for RSV F protein maximally enhanced RSV F protein expression both within the cell and at the cell surface.
  • In vitro transcribed mRNAs that encoded variations of F(ii) CT lengths (0, 5, 10, 15, 20, 22 amino acids & full length) were forward transfected into primary, BJ cell monolayers. The 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.
  • Trimeric pre-fusion RSV F (identified by AM14), or prefusion F (identified by D25), was quantified.
  • FIG. 43 RSV pre-F IgG binding antibody geometric mean titres on day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 ⁇ g or (B) 0.2 ⁇ g of RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (where each point represents an individual animal).
  • the optimal length of the RSV F CT that supports cell-surface expression of the trimeric, pre-fusion RSV F protein includes CTs of at least 5, but not longer than 10, amino acids.
  • the cell- surface expression of trimeric, pre-fusion RSV F protein was evaluated by indirect immunofluorescent labelling using monoclonal antibody AM14 followed by quantification using high content imaging and analysis across a 4-day time course.
  • Primary, human fibroblast (BJ) cells were forward transfected in 96-well format with mRNAs encoding RSV F variant F(ii). In (A), select CT variations are shown.
  • the parent (F(ii), solid line, solid box) was modified by deletion of the RNA sequence encoding the terminal 15 amino acids (F(ii) CTD ⁇ 15, solid line, solid circle), 16 amino acids ((F(ii) CTD ⁇ 16, dotted line, solid circle), 17 amino acids (F(ii) CTD ⁇ 17, dotted line, open circle), 20 amino acids (F(ii) CTD ⁇ 20, solid line, open circle), 21 amino acids (F(ii) CTD ⁇ 21, dotted line, solid box), or complete deletion of the CT domain (F(ii) CTD ⁇ 25, solid line, open box).
  • RSV-F proteins in the pre-fusion conformation which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1; wherein the RSV-F protein comprises at least one mutation relative to the wild-type in a region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation introduces, through substitution or insertion, a residue comprising an H bond donor and/or acceptor moiety in its side chain.
  • the present disclosure also provides, in a second independent aspect, RSV-F protein in the pre- fusion conformation, which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1; wherein the RSV-F protein comprises a substitution at position 228 of SEQ ID NO: 1 for K or A. Docket No.: 70221WO01
  • the present disclosure also provides, in a third independent aspect, a multimer comprising a plurality of protomers of RSV-F proteins in the pre-fusion conformation, said protomers of RSV-F proteins comprising at least one mutation relative to wild-type RSV-F according to SEQ ID NO: 1; wherein the at least one mutation introduces or stabilises, through substitution or insertion, a cross-protomer interaction.
  • the cross-protomer interaction may be a hydrogen bond, or tertiary cation-pi-anion interaction, between residues of two adjacent protomers of RSV-F proteins.
  • the tertiary cation-pi-anion interaction is between all of (i), (ii) and (iii); (i) and (ii) being positions 232 and 250 respectively on a first protomer (more preferably E232 and Y250), and (iii) being position 235 (more preferably R235) on a second protomer (numbering according to SEQ ID NO: 1).
  • the at least one mutation may introduce, through substitution or insertion, at least one residue comprising a hydrogen bond donor and/or acceptor moiety in its side chain (and preferably into a region corresponding to positions 217-239 of SEQ ID NO:1). More preferably, the at least one mutation comprises a substitution at position 228 of SEQ ID NO: 1 (N) for K, R, Q or N; or K, R or Q (preferably K or R, more preferably K).
  • the multimer is a trimer. More preferably, the multimer is a homotrimer comprising protomers of three RSV-F proteins, each comprising at least one mutation as detailed above in this paragraph.
  • the present disclosure also provides, in a fourth independent aspect, a multimer comprising a plurality of protomers, wherein at least one protomer comprises or consists of an RSV-F protein of according to the first or second independent aspect of the present disclosure.
  • RSV-F proteins according to said first or second independent aspect, and protomers of RSV-F proteins according to said second or third independent aspect are “RSV-F proteins of the present disclosure” as referred to herein.
  • the wild-type RSV-F (A2 subtype) sequences of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 84 and 107 are not “RSV-F proteins of the present disclosure” as referred to herein.
  • the wild-type RSV-F sequence of SEQ ID NO: 108 (B subtype strain 18537) is also not an “RSV-F protein of the present disclosure”, as referred to herein.
  • “Mutation” as used herein encompasses substitution, insertion and deletion of residues, although substitution and insertion are preferred according to all aspects of the present disclosure, with substitution being more preferred. Mutations which “introduce, through substitution or insertion” a given residue may be referred to interchangeably as “substitutions [for] or insertions [of]” the residue.
  • Both the RSV-F proteins of the present disclosure, and generally the mutations which they comprise relative to SEQ ID NO: 1, are “engineered”.
  • RSV-F proteins of the present disclosure do not occur in nature.
  • the mutations which they comprise are generally “engineered” in the sense that such mutations may individually occur in nature, but have been deliberately selected and introduced into the proteins, in order to stabilise the pre-fusion conformation.
  • RSV-F proteins of the present disclosure may also be considered “recombinant” Docket No.: 70221WO01 (“engineered” and “recombinant” may be used interchangeably in this context).
  • RSV proteins of the present disclosure generally comprise engineered mutations relative to SEQ ID NO: 1, as defined throughout the present disclosure.
  • SEQ ID NO: 1 is an RSV-F sequence from a strain of human RSV of the A2 subtype that contains 2 mutations (K66E and Q101P) relative to GenBank Accession number KT992094, herein referred to as “wild-type”.
  • wild-type the F protein substitutions K66E and Q101P resulted through passaging of the A2 strain deposited under GenBank Accession number KT992094 (also wild-type), see e.g. [11].
  • SEQ ID NO: 1 which contains the two substitutions
  • wild-type in accordance with e.g. [12]).
  • SEQ ID NO: 1 comprises neither a trimerisation domain, transmembrane domain, nor cytoplasmic domain at the C-terminus, as the domain(s) included at the C-terminus may vary according to the format of the RSV-F protein when used as a vaccine antigen (e.g. RSV-F protein-based vaccine, or nucleic acid-based vaccine encoding RSV-F).
  • RSV proteins of the present disclosure may also comprise mutations relative to SEQ ID NO: 1 found in RSV-F proteins from other strains and subtypes, both naturally-occurring and engineered (e.g.
  • RSV-F proteins of other A subtype strains, or B subtype strains may be of the A or the B subtype.
  • “[W]herein the at least one mutation increases the hydrophobicity of [a given sequence / region] relative to the wild-type [corresponding sequence / region]” refers to the sum total hydrophobicity of all residues in a region being increased relative to the corresponding wild-type region, as a result of the at least one mutation. For example, considering a single substitution in a given sequence / region, S may be substituted for a residue selected from I, V, L, F, C, M, A, G, T and W (all of which are more hydrophobic than S).
  • hydrophobicity may be measured using the Kyte and Doolittle scale [13], see Table 2, “Hydropathy index”, as set out below (greater values meaning greater hydrophobicity).
  • positions 55, 215 and 348 of SEQ ID NO: 1, and so forth are easily identifiable to the skilled person, and can be identified by aligning the amino acid sequences using any well-known method (visual or algorithm, e.g. as detailed above).
  • “heptad repeat A” domain (“HRA”) refers to positions 149-206 of SEQ ID NO: 1
  • HRB refers to positions 474-523 of SEQ ID NO: 1
  • “heptad repeat C” domain (“HRC”) refers to positions 53-100 of SEQ ID NO: 1.
  • Docket No.: 70221WO01 RSV-F proteins of the present disclosure are preferably antigens (or, phrased differently, are antigenic).
  • RSV-F proteins of the present disclosure preferably elicit an immune response when administered in vivo, namely against RSV.
  • the immune response may comprise an antibody response (usually including IgG) and/or a cell-mediated immune response, in particular an antibody response.
  • the immune response will typically recognise the three-dimensional structure of the corresponding wild-type pre-fusion RSV-F, in particular one or more epitopes present on the (solvent-exposed) surface of the protein when in the pre-fusion conformation.
  • RSV-F proteins of the present disclosure may also be considered antigens (or, phrased differently, are antigenic) given their ability to be bound by antibodies AM14, D25, RSB1 and motavizumab (in particular AM14, D25 and RSB1, in particular AM14), e.g. with a dissociation constant (K D ), as measured by SPR, of less than 10 nM such as 1 pM – 10 nM, e.g. as detailed below.
  • K D dissociation constant
  • the incorporation of both naturally and non-naturally occurring amino acids is envisaged in RSV-F proteins of the present disclosure, although naturally occurring amino acids are preferred.
  • RSV-F proteins of the present disclosure elicit a pre-fusion RSV-F-specific antibody response against RSV in vivo, e.g. an IgG antibody response (see, e.g. Examples 10, 11 and 13).
  • RSV-F proteins of the present disclosure elicit a neutralising antibody response against RSV in vivo, e.g. against RSV A (see, e.g. Examples 10, 11 and 13).
  • Said neutralising antibody response may inhibit replication of RSV in the respiratory system of a subject (such as in the lungs).
  • Said neutralising antibody response may yield protective immunity against RSV in a subject.
  • Pre-fusion conformation Generally, RSV-F proteins of the present disclosure may be considered as stabilised in the pre-fusion conformation.
  • pre-fusion conformation of RSV-F proteins of the present disclosure may be confirmed via binding of pre-fusion RSV-F-specific monoclonal antibodies (“pre-fusion mAbs”).
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a light chain and a heavy chain (LC and HC) selected from the group consisting of: SEQ ID NO: 2 and 3 respectively, SEQ ID NO: 4 and 5 respectively, and SEQ ID NO: 8 and 9 respectively.
  • LC and HC light chain and a heavy chain
  • the foregoing are the LC and HC sequences of prefusion mAbs AM14, D25, and RSB1, respectively; see, e.g. [15, 16, 17].
  • Specific binding of the pre-fusion mAb(s) may be determined via surface plasmon resonance (“SPR”) or biolayer interferometry (“BLI”), however SPR is preferred.
  • SPR may be performed using a BIACORE system; preferably as performed in the Examples (see subsection Binding kinetics using BIACORE).
  • RSV-F proteins of the present disclosure may be specifically bound by any of the pre-fusion mAbs above with a dissociation constant (K D ), as Docket No.: 70221WO01 measured by SPR, of less than 10 nM, such as 1 pM – 10 nM; in particular less than 1 nM (1000 pM), such as 1-1000 pM.
  • AM14 is preferred. Unlike the other pre-fusion mAbs, AM14 is specific for RSV-F in the pre-fusion conformation when in an intact trimer.
  • the antibody motavizumab (see, e.g. [18]) was also used in the examples (LC and HC of SEQ ID NO: 6 and 7 respectively), but also binds to the post-fusion conformation, and so is not preferred for confirming pre-fusion conformation.
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 2 and 3 respectively (or, defined differently, antibody AM14), with a K D , as measured via SPR, of: less than 1000, 900, 800700, 650, or 600 pM; or, in certain embodiments, less than 550 pM; or, in certain embodiments, less than 100, 90, 80, 70, 60, 50, or 40 pM; or, in certain embodiments, less than 35 pM.
  • K D as measured via SPR
  • RSV-F proteins according to present disclosure designated F216, F217, F224, and F225 are specifically bound by such a mAb with K D s, as measured via SPR, of 598, 546, 37.8 and 30.2 pM respectively (see, e.g. Example 4, Figure 10).
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 2 and 3 respectively (or, defined differently, antibody AM14) with a K D , as measured via SPR, in the range of 1-1000, 1-900, 1-8001-700, 1-6501-6001- 5501-100, 1-90, 1-80, 1-70, 1-60, 1-50, or 1-40 pM; such as 10-1000, 10-900, 10-800, 10-700, 10- 650, 10- 600, 10-550, 10- 100, 10-90, 10-80, 10-70, 10-60, 10-50, or 10-40 pM; such as 20-1000, 20- 900, 20-800, 10-700, 20-650, 20-600, 20-550, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, or 20-40 pM.
  • RSV-F proteins of the disclosure are generally assembled in trimeric form, as a homotrimer.
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a LC and HC according SEQ ID NO: 4 and 5 respectively (or, defined differently, antibody D25), with a K D , as measured via SPR, of: less than 200, 180, 160, 140, or 130 pM; or, in certain embodiments, less than 100, 95, 90, or 85 pM; or, in certain embodiments, less than 80 pM; or, in certain embodiments, less than 70 pM.
  • RSV-F proteins according to present disclosure designated F216, F217, F224, and F225 are specifically bound by such a mAb with K D s, as measured via SPR, of 119, 75.2, 67.8 and 83.6 pM respectively (see, e.g. Example 4; Figure 10).
  • RSV-F proteins of the present disclosure may be specifically bound by a pre- fusion mAb comprising a LC and HC according to SEQ ID NO: 4 and 5 respectively (or, defined differently, antibody D25) with a KD, as measured via SPR, in the range of 1-200, 1-180, 1-160, 1- 140, 1-130, 1-100, 1-95, 1-90, 1-85, 1-80 or 1-70 pM; such as 20-200, 20-180, 20-160, 20-140, 20- 130, 20-100, 20-95, 20-90, 20-85, 20-80 or 20-70 pM; such as 40-200, 40-180, 40-160, 40-140, 40- Docket No.: 70221WO01 130, 40-100, 40-95, 40-90, 40-85, 40-80 or 40-70 pM; such as 50-200, 50-180, 50-160, 50-140, 50- 130, 50-100, 50-95, 50-90, 50-85, 50-
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 8 and 9 (or, defined differently, antibody RSB1) respectively with a KD, as measured via SPR, of: less than 150, 120, 110, 100, 105, 95, or 90 pM; or, in certain embodiments, less than 80, 75, or 70 pM; or, in certain embodiments, less than 60, 55, or 50 pM; or, in certain embodiments, less than 45 pM.
  • RSV-F proteins according to present disclosure designated F216, F217, F224, and F225 are specifically bound by such a mAb with KDs, as measured via SPR, of 85.6, 67.6, 40.4 and 46.5 pM respectively (see, e.g. Example 4, Figure 10).
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 6 and 7 respectively (or, defined differently, antibody RSB1) with a K D , as measured via SPR, in the range of 1-150, 1-120, 1- 110, 1-100, 1-105, 1-95, 1-80, 1-75, 1-70, 1-60, 1-55, 1-50 or 1-45 pM; such as 10-150, 10-120, 10- 110, 10-100, 10-105, 10-95, 10-80, 10-75, 10-70, 10-60, 10-55, 10-50 or 10-45 pM; such as 20-150, 20-120, 20-110, 20-100, 20-105, 20-95, 20-80, 20-75, 20-70, 20-60, 20-55, 20-50 or 20-45 pM.
  • RSV-F proteins of the present disclosure are specifically bound by: (i) a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 2 and 3 respectively (or, defined differently, antibody AM14), with a K D of less than 1000, 900, 800, 700 or less than 650 pM (such as 1-1000, 1-900, 1-800, 1-700 or 1-650 pM); (ii) a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 4 and 5 respectively (or, defined differently, antibody D25), with a K D of less than 300, 250, 200, 150 or less than 130 pM; optionally less than 100 or 80 pM (such as 1-300, 1- 250, 1-200, 1-150 or 1-130 pM; optionally 1-100 or 1-80 pM); and/or (iii) a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 8 and 9 respectively (or, defined differently,
  • an RSV-F protein of the present disclosure meets 2, or more preferably all 3 of criteria (i), (ii) and (iii).
  • proteins F216 and F217 meet all of said criteria (see e.g. Example 4, Figure 10), with F217 meeting the optional criteria set out in (ii).
  • RSV-F proteins may be bound by an antibody comprising a LC and HC according to SEQ ID NO: 6 and 7 respectively (or, defined differently, motavizumab), with a KD as measured via SPR of less than 200, 150, 100 or less than 80 pM (such as 1-200, 1-150, 1-100 or 1-80 pM).
  • RSV-F proteins of the present disclosure are specifically bound by: (iv) a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 2 and 3 respectively (or, defined differently, antibody AM14), with a KD of less than 200, 150, 100, 80, 60 or 40 pM (such as 1-200, 1-150, 1-100, 1-80, 1-60 or 1-40 pM); (v) a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 4 and 5 respectively (or, defined differently, antibody D25), with a K D of less than 200, 150, 100, 90 or 85 pM; optionally less than 70 pM (such as 1-200, 1-150, 1-100, 1-90 or 1-85 pM; optionally 1-70 pM); and/or (vi) a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 8 and 9 respectively (or,
  • an RSV-F protein of the present disclosure 2, or more preferably all 3 of criteria (iv), (v) and (vi).
  • proteins F224 and F225 meets all of said criteria (see e.g. Example 4, Figure 10), with F224 meeting the optional criteria set out in (v).
  • RSV-F proteins may be bound by an antibody comprising a LC and HC according to SEQ ID NO: 6 and 7 respectively (or, defined differently, motavizumab), with a KD as measured via SPR of less than 40 pM (such as 1-40 pM).
  • RSV-F proteins of the present disclosure may be bound by a pre-fusion mAb (in particular, any of those defined above) over a time of, for example, at least: 24 hours, 1 week, 2 week, 3 weeks, 4 weeks, 5 weeks or 6 weeks, 7 weeks or 8 weeks; for example wherein the RSV-F protein is stored at 4° or 25°C in a buffer for said period(s) and then assayed to determine the presence or absence of specific binding of a pre-fusion mAb (in particular AM14 or D25), or an antigen binding fragment thereof (e.g. a Fab fragment thereof). Said binding over time may be determined via, for example, SPR or BLI.
  • the buffer may be HEPES buffer, e.g.
  • 20mM HEPES comprising 150mM NaCl.
  • Thermostability e.g. using Nano-DSF, e.g. as performed in the Examples
  • Aggregation of the protein may also be assessed (e.g. via high-performance liquid chromatography (“HPLC”), e.g. as performed in the Examples).
  • HPLC high-performance liquid chromatography
  • RSV-F proteins of the present disclosure may also be bound by an antibody comprising a LC and HC according to SEQ ID NO: 6 and 7 respectively (or, defined differently, motavizumab) with a K D , as measured via SPR, of: less than 200, 180, 160, 140, or 120 pM; or, in certain embodiments, less than 110, 100 or 95 pM; or, in certain embodiments, less than 80, 70, 60 or 55 pM; or, in certain embodiments, less than 50, 45, or 40 pM.
  • a K D as measured via SPR
  • RSV-F proteins according to present disclosure designated F216, F217, F224, and F225 are specifically bound by such a mAb with K D s, as measured via SPR, of 74.8, 117, 38.6 and 52.8 pM respectively (see, e.g. Example 4, Figure 10).
  • RSV-F proteins of the present disclosure may be specifically bound by a pre- Docket No.: 70221WO01 fusion mAb comprising a LC and HC according to SEQ ID NO: 6 and 7 respectively (or, defined differently, motavizumab) with a K D , as measured via SPR, in the range of 1-200, 1-180, 1-160, 1- 140, 1-120, 1-110, 1-100, 1-95, 1-80, 1-70, 1-55, 1-50, 1-45 or 1-40 pM; such as 10-200, 10-180, 10- 160, 10-140, 10-120, 10-110, 10-100, 10-95, 10-80, 10-70, 10-55, 10-50, 10-45 or 10-40 pM such as 20-200, 20-180, 20-160, 20-140, 20-120, 20-110, 20-100, 20-95, 20-80, 20-70, 20-55, 20-50, 20-45 or 20-40 pM.
  • cryo-EM cryo-electron microscopy single particle analysis
  • such cryo-EM comprises the steps: complexing the RSV-F protein of the present disclosure with an antigen binding fragment, such as a Fab fragment, of a pre-prefusion mAb (preferably of AM14, preferably a Fab fragment of AM14) to form complexes; isolating (e.g.
  • cryo-EM is performed as in the Examples (see subsection Cryo-electron microscopy of RSV-F designs F21, F216, and F224.
  • a residue comprising a hydrogen bond donor and/or acceptor moiety in its side chain RSV-F proteins of the present disclosure comprise (according to the first independent aspect) or may comprise (according to the third or fourth independent aspect) at least one mutation relative to the wild-type in a region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation introduces, through substitution or insertion, a residue comprising an H bond donor and/or acceptor moiety in its side chain.
  • the region in which the at least one mutation is located comprises or consists of an ⁇ helix.
  • the residue forms a hydrogen bond with a further residue in the RSV-F protein; preferably wherein the further residue is within a region corresponding to positions 239-254 of SEQ ID NO: 1; more preferably wherein the further residue is at position 250 of SEQ ID NO: 1; more preferably wherein the further residue is Y250 (wild-type) or D250, even more preferably Y250.
  • Docket No.: 70221WO01 The introduction of both naturally and non-naturally occurring residues comprising H bond donor and/or acceptor moieties in their side chains is within the scope of the present disclosure.
  • residues comprising such moieties in their side chains include R, K, W (comprising H bond donor moieties), D, E (comprising H bond acceptor moieties), N, Q, H, S, T and Y (comprising both H bond donor and acceptor moieties).
  • residues comprising an H bond donor and/or acceptor moiety in its side chain encompasses residues comprising, in their side chains, (i) H bond donor moieties, (ii) H bond acceptor moieties, (iii) H bond donor moieties and H bond acceptor moieties (i.e.
  • H bond donor and acceptor moieties i.e. moieties able to act as both donors and acceptors.
  • a “residue comprising an H bond donor and/or acceptor moiety...” may be used interchangeably with “residue comprising an H bond donor and/or acceptor atom...”
  • “corresponding to...” encompasses a sequence / region of the RSV-F protein of the disclosure which aligns with the corresponding wild-type sequence / region.
  • RSV-F proteins of the present disclosure may comprise: at least one mutation relative to SEQ ID NO: 1 in a region of the protein (preferably which forms an ⁇ helix) which aligns with positions 217-239 of SEQ ID NO: 1, wherein the at least one mutation introduces, through substitution or insertion, a residue comprising an H bond donor and/or acceptor moiety in its side chain.
  • RSV-F proteins of the present disclosure comprise: at least one mutation relative to SEQ ID NO: 1 within positions 217-239 of SEQ ID NO: 1, wherein the at least one mutation introduces, through substitution or insertion, a residue comprising an H bond donor and/or acceptor moiety in its side chain.
  • positions 217-239 form the ⁇ 5 helix.
  • introducing an H bond donor and/or acceptor moiety may result in an intra-protomer (i.e. between residues within a single protomer) H bond with residues in a loop corresponding to positions 239-254 of SEQ ID NO: 1.
  • the H bond may provide a stabilising interaction favouring the pre-fusion conformation.
  • the H bond and/or resulting stabilising interaction may inhibit, at least partly inhibit, or completely inhibit, the transition of RSV-F from pre-fusion to post-fusion conformation. This stabilisation mechanism is in contrast to reference [19], which introduces either F or L residues into the 217-239 region (in addition to other substitutions).
  • F and L residues may be amenable to van der Waals (VDW) contacts, but lack H bond acceptor and/or donor moieties (e.g. N or O) in their side chains.
  • RSV-F proteins of the present disclosure may comprise at least one mutation relative to SEQ ID NO: 1 in a region (preferably which forms an ⁇ helix) corresponding to positions 217-239 of SEQ ID NO: 1, wherein the at least one mutation introduces, through substitution or insertion, a residue selected from K, R, N, W, D, E, Q, H, S, T and Y into the region. Residues comprising hydrogen bond donor moieties in their side chains (i.e.
  • K, R, W, N, Q, H, S, T and Y are generally preferred; with K, R, Q Docket No.: 70221WO01 and N being preferred out of such residues (more preferably K or R, even more preferably K). Substitutions at position 228 of SEQ ID NO: 1 are generally preferred.
  • the at least one mutation may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 substitutions or insertions (preferably substitutions) relative to positions 217-239 of SEQ ID NO: 1; in particular only 1, 2, 3, 4, 5, 6, 7, or 8 such substitutions or insertions (preferably substitutions), in particular only 1, 2, 3, 4 or 5 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such substitutions or insertions (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).
  • the region (preferably which forms an ⁇ helix) corresponding to positions 217-239 of SEQ ID NO:1 may have at least 50%, 60%, 70%, 80% sequence identity, or preferably at least 85%, 90% or 95% sequence identity to positions 217-239 of SEQ ID NO:1.
  • one or more wild-type residues (in particular 1 or 2 residues, in particular only 1 residue) in the region corresponding to positions 220-235 (in particular 227-232, in particular 228-232) of SEQ ID NO: 1 may be substituted for a residue selected from K, R, N, W, D, E, Q, H, S, T and Y (e.g.
  • RSV-F proteins of the present disclosure comprise a substitution at position 228 (N) of SEQ ID NO: 1 for K, R, Q or N; or K, R or Q (preferably K or R, preferably K), and/or a substitution at position 232 (E) of SEQ ID NO: 1 for N.
  • N at position 232 also provides an H bond donor moiety, within a suitable distance for potential H bonding with Y250 (see Figure 25).
  • RSV-F proteins of the present disclosure comprise a substitution at position 228 (N) of SEQ ID NO: 1 for K, R, Q or N; or K, R or Q. In more preferred embodiments, RSV-F proteins of the present disclosure comprise a substitution at position 228 (N) of SEQ ID NO: 1 for K or R. In even more preferred embodiments, RSV-F proteins of the present disclosure comprise a substitution at position 228 (N) of SEQ ID NO: 1 for K.
  • Such preferred substitutions at positions 228 and/or 232 may be the only mutation which introduces a residue comprising an H bond donor and/or acceptor moiety within the region corresponding to positions 217-239 of SEQ ID NO: 1; and optionally the only mutation in the region corresponding to positions 217-239 of SEQ ID NO:1.
  • position 232 has either the wild-type residue of SEQ ID NO: 1 (E), or is substituted for D (also a negatively charged residue). E or D at position 232 may help to provide the tertiary cation-pi-anion interaction discussed in the paragraph below.
  • position 250 has either the wild-type residue of SEQ ID NO: 1 (Y), or is substituted for D. Docket No.: 70221WO01
  • a minimal substitution screen performed by the inventors revealed the N228K substitution alone to be able to achieve pre-fusion RSV-F (see Figure 19; design F310).
  • K in place of N at position 228 appears to result in an H bond with Y250 on the same protomer (see Figure 25, dashed line indicating hydrogen bond).
  • Said H bonding may stabilise Y250 to form a tertiary cation-pi-anion interaction between E232, Y250 and R235 (E232 and Y250 being on one protomer, with R235 being on an adjacent protomer).
  • E, Y and R are one of the dominant triads for such a tertiary cation-pi-anion interaction (see, e.g. [20]).
  • residues with other H bond donors in their side chains (such as R) at position 228 may also provide this stabilising H bond with Y250.
  • Q also provides an H bond donor moiety, with a large enough side chain for potential H bonding with Y250.
  • RSV-F proteins of the present disclosure may comprise a substitution at position 250 (Y) of SEQ ID NO: 1 for D.
  • a Y250D substitution may strengthen a cross-protomer interaction with R235 (wild-type residue) by forming a salt bridge between the two residues.
  • D comprises an H bond acceptor moiety and so the Y250D substitution would maintain the preferred hydrogen bond between positions 250 and 228 (in preferred embodiments comprising the N228K or N228R substitution, more preferably the N228K substitution).
  • the mutations detailed throughout this subsection may provide core stabilisation in the F1 domain (positions 137-513 of SEQ ID NO: 1), proximal to the heptad repeat A (“HRA”) domain and antibody binding site ⁇ (”site ⁇ ”).
  • the mutations detailed throughout this subsection may provide a H bond with Y250, e.g. which stabilises Y250 to provide a tertiary cation-pi-anion interaction between positions (i) 232 (preferably E232, as in wild-type, or D232 if substituted), (ii) Y250 and (ii) R235 across different RSV-F protomers.
  • Y250 e.g. which stabilises Y250 to provide a tertiary cation-pi-anion interaction between positions (i) 232 (preferably E232, as in wild-type, or D232 if substituted), (ii) Y250 and (ii) R235 across different RSV-F protomers.
  • Such core stabilisation, H bonds and/or tertiary anion-pi-cation interactions may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • the mutations detailed throughout this subsection may inhibit, at least partly inhibit, or completely inhibit, the transition from pre- fusion to post-fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure may further comprise (according to all independent aspects of the present disclosure): (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.
  • the region in which the at least one mutation according to (ai) is located comprises or consists of a ⁇ sheet, and the at least one mutation increases the hydrophobicity of the ⁇ sheet relative to the wild-type ⁇ sheet (i.e. positions 38-60 of SEQ ID NO: 1).
  • the region in which the at least one mutation according to (aii) is located comprises or consists of a ⁇ sheet, and the at least one mutation increases the hydrophobicity of the ⁇ sheet relative to the wild-type ⁇ sheet (i.e. positions 296-318 of SEQ ID NO: 1).
  • RSV-F proteins of the present disclosure may comprise: (ai) at least one mutation relative to SEQ ID NO: 1 in a region of the protein (preferably which forms a ⁇ sheet) which aligns with positions 38-60 of SEQ ID NO: 1, wherein the at least one mutation increases the hydrophobicity of said region relative to positions 38-60 of SEQ ID NO: 1; and /or (aii) at least one mutation relative to SEQ ID NO: 1, in region of the protein (preferably which forms a ⁇ sheet) which aligns with positions 296-318 of SEQ ID NO: 1, wherein the at least one mutation increases the hydrophobicity of said region relative to positions 296-318 of SEQ ID NO: 1 and/or introduces a residue selected from M, F, I and V into said region
  • RSV-F proteins of the present disclosure comprise: (ai) at least one mutation relative to SEQ ID NO: 1 in a region of the protein (preferably which forms a ⁇ sheet) which aligns with positions 38-60 of SEQ ID NO: 1, wherein the at least one
  • positions 38-60 and 296-318 form two ⁇ sheets, which form at least part of a largely hydrophobic pocket at the interface between the F1 domain (positions 137-513 of SEQ ID NO: 1) and the heptad repeat A (“HRA”) domain, see Figure 22.
  • HRA heptad repeat A
  • increasing the hydrophobicity (relative to wild-type) of either or both of the corresponding ⁇ sheets in RSV-F proteins of the present disclosure may provide new, energetically- favourable van der Waals (VDW) contacts within the largely hydrophobic pocket.
  • VDW van der Waals
  • VDW contacts may inhibit, at least partly inhibit, or completely inhibit, the transition of RSV-F from pre-fusion to post- fusion conformation.
  • the at least one mutation according (ai) may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 mutations (preferably substitutions) relative to positions 38-60 of SEQ ID NO: 1; in particular only 1, 2, 3, 4, 5, 6, 7, or 8 such mutations (preferably substitutions), in particular only 1, 2, 3, 4 or 5 such mutations (preferably substitutions), in particular only 1 or 2 such mutations (preferably substitutions), in preferably only 1 such mutation (preferably substitution).
  • the at least one mutation according (aii) may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 mutations (preferably substitutions) relative to positions 296-318 of SEQ ID NO: 1; in particular only 1, 2, 3, 4, 5, 6, 7, or 8 such mutations (preferably substitutions), in particular only 1, 2, 3, 4 or 5 such mutations (preferably substitutions), in particular only 1 or 2 such mutations (preferably substitutions), preferably only 1 such mutation (preferably substitution).
  • the region (preferably which forms a ⁇ sheet) corresponding to positions 38-60 of SEQ ID NO:1 may have at least 50%, 60%, 70%, 80% sequence identity, or preferably at least 85%, 90% or 95% sequence identity to positions 38-60 of SEQ ID NO:1.
  • the region (preferably which forms a ⁇ sheet) corresponding to positions 296-318 of SEQ ID NO:1 may have at least 50%, 60%, 70%, 80% sequence identity, or preferably at least 85%, 90% or 95% sequence identity to positions 296-318 of SEQ ID NO:1.
  • one or more S residues in the wild-type ⁇ sheet according to positions 38-60 of SEQ ID NO: 1 may be substituted for residues more hydrophobic than S (e.g. I, V, L, F, C, M, A, G, T or W).
  • positions 38, 41, 46, and/or 55 of SEQ ID NO: 1 may be substituted for T, C, V, I or F, in particular T, C or V, preferably T.
  • RSV-F proteins of the present disclosure may comprise a substitution at position 301 for a residue selected from M, F and I; and/or at position 303 for a residue selected from V, M, F and I; in particular, such substitutions are present at both positions 301 and 303.
  • V301 and L303 side chains point into the largely hydrophobic pocket discussed above (see Figure 22).
  • introducing relatively large and/or hydrophobic side chains by way of Docket No.: 70221WO01 substitution at these positions may, in particular, provide energetically-favourable VDW contacts within the pocket.
  • RSV-F proteins of the present disclosure comprise a substitution at position 55 of SEQ ID NO: 1 (S) for a more hydrophobic residue (e.g. I, V, L, F, C, M, A, G, T or W, which are more hydrophobic than S at the wild-type position 55).
  • RSV-F proteins of the present disclosure may comprise a substitution at position 301 for a residue selected from M, F and I; and/or at position 303 for a residue selected from V, M, F and I; in particular, such substitutions are present at both positions 301 and 303.
  • RSV-F proteins of the present disclosure comprise a substitution at position 55 of SEQ ID NO: 1 (S) for T, C, V, I or F. In more preferred embodiments, RSV-F proteins of the present disclosure comprise a substitution at position 55 of SEQ ID NO: 1 (S) for T, C or V. In more preferred embodiments, RSV-F proteins of the present disclosure comprise a substitution at position 55 of SEQ ID NO: 1 (S) for T or V. In even more preferred embodiments, RSV-F proteins of the present disclosure comprise a substitution at position 55 of SEQ ID NO: 1 (S) for T.
  • Such preferred substitutions at position 55 may be the only mutation according to (ai); and optionally the only mutation in the region corresponding to positions 38-60 of SEQ ID NO:1.
  • Such preferred substitutions at position 55 may be the only mutation according to (ai), wherein (aii) mutations are absent; and optionally the only mutation in the region corresponding to positions 38- 60 of SEQ ID NO:1.
  • a minimal substitution screen performed by the inventors revealed the S55T mutation to be a likely driver of the pre-fusion conformation (design F308).
  • T in place of S at position 55 provides a slightly larger residue which (from in silico three-dimensional structural analysis, see Figure 22) appears to be accommodated well in the hydrophobic pocket discussed above, without generating significant steric clashes.
  • the addition of the CH3 group of T appears to provide new, energetically favourable VDW contacts of the type discussed above.
  • alternative substitutions provided for position 55 by ROSETTA software include C and V (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0, -0.1 or -0.5 being used).
  • mutations according to (a) may stabilise the interface between the F1 domain (positions 137-513 of SEQ ID NO: 1) and the heptad repeat A (“HRA”) domain. Such stabilization may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (a) may provide energetically-favourable VDW contacts within a hydrophobic pocket of RSV-F, at the interface between the F1 domain and the HRA domain.
  • Such contacts may inhibit, at least partly inhibit, or completely inhibit, the transition from Docket No.: 70221WO01 pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (a) may inhibit refolding of the HRA and HRC domains. Such refolding may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (a) (preferably substitutions, preferably such substitutions at position 55 as detailed above) may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure may further comprise (according to all independent aspects of the present disclosure): 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.
  • the region in which the at least one mutation according to (b) is located comprises or consists of a loop (more preferably, a loop connecting two ⁇ helices) and the at least one mutation increases the hydrophobicity of the loop relative to the wild-type hinge (i.e. positions 208-216 of SEQ ID NO:1), and/or introduces at least one P residue into the hinge.
  • Loop as referred to herein may also be referred to as a “loop region” or “flexible loop”, or, if portions of the protein hinge about said loop during a conformational change (as is the case for inter alia, positions 208-216), a “hinge loop” “hinge” or “hinge region”.
  • RSV-F proteins of the present disclosure may comprise: (b) at least one mutation relative to SEQ ID NO: 1 in a sequence of the protein (preferably which forms a loop, more preferably a loop connecting two ⁇ helices) which aligns with 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 a P residue into the region.
  • RSV-F proteins of the present disclosure comprise: (b) at least one mutation relative to SEQ ID NO: 1 within positions 208-216 of SEQ ID NO: 1; wherein the at least one mutation results in in increased hydrophobicity of the relative to said positions, and/or introduces a P residue into said positions.
  • positions 208-216 form a loop which connects two ⁇ helices (the ⁇ 4 and ⁇ 5 helices in wild-type), see Figure 23.
  • increasing the hydrophobicity of, and/or introducing a P residue into, the loop may stabilise or Docket No.: 70221WO01 rigidify it, and/or favour packing away from the surface of RSV-F.
  • Such stabilisation, rigidification and/or packing may inhibit, at least partly inhibit, or completely inhibit, the transition of RSV-F from pre-fusion to post-fusion conformation (in particular, by inhibiting the relative motion of the two ⁇ helices adjacent to the loop, generally the ⁇ 4 and ⁇ 5 helices of RSV-F).
  • the at least one mutation according to (b) may comprise or consist of 1, 2, 3, 4, 5, 6, 7 or 8 substitutions or insertions (preferably substitutions) relative to positions 208-216 of SEQ ID NO: 1; in particular only 1, 2, 3 or 4, such substitutions or insertions (preferably substitutions), in particular only 1, 2, or 3 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such substitutions or insertions (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).
  • the region (preferably which forms a loop, more preferably a loop connecting two ⁇ helices) corresponding to positions 38-60 of SEQ ID NO:1 may have at least 50% or 60% sequence identity, or preferably at least 75% or 85% sequence identity to positions 208-216 of SEQ ID NO:1.
  • one or more S residues in the wild-type loop according to positions 208-216 of SEQ ID NO: 1 may be substituted for residues more hydrophobic than S (e.g. I, V, L, F, C, M, A, G, T or W).
  • RSV-F proteins of the present disclosure comprise a substitution at position 215 of SEQ ID NO: 1 (S) for A, P, V, I, or F.
  • RSV-F proteins of the present disclosure comprise a substitution at position 215 of SEQ ID NO: 1 (S) for A, V, I, or F.
  • RSV-F proteins of the present disclosure comprise a substitution at position 215 of SEQ ID NO: 1 (S) for A or P.
  • RSV-F proteins of the present disclosure comprise a substitution at position 215 of SEQ ID NO: 1 (S) for A.
  • Such preferred substitutions at position 215 may be the only mutation according to (b); and optionally the only mutation in the region corresponding to positions 208-216 of SEQ ID NO: 1.
  • a minimal substitution screen performed by the inventors revealed the S215A mutation to be a likely driver of the pre-fusion conformation (design F309). Without wishing to be bound by this theory, removal of the hydrophilic OH group (as S is substituted for A) is likely favourable to the packing and rigidity of the loop (see Figure 23).
  • the A residue at position 215 may provide energetically-favourable VDW contacts with positions 79206 (I residues in wild-type), L203, and/or T219.
  • Such packing, rigidification and/or VDW contacts may inhibit, at least partly inhibit, or completely inhibit the transition from pre-fusion to post-fusion conformation of RSV-F (in particular, inhibition of the relative motion of the two ⁇ helices adjacent to the Docket No.: 70221WO01 loop(generally the ⁇ 4 and ⁇ 5 helices of RSV-F), or, defined differently, inhibition of refolding of the HRC and HRA domains).
  • the side chains of P, V, I or F may also reduce conformational freedom of the loop, thus also being favourable to the packing and rigidification of the loop.
  • mutations according to (b) may stabilise or rigidify the loop corresponding to positions 208-216 of SEQ ID NO: 1.
  • Such stabilisation or rigidification may inhibit, at least partly inhibit, or completely inhibit, the transition of RSV-F from pre-fusion to post-fusion conformation (in particular, by inhibiting the relative motion of the two ⁇ helices adjacent to the loop, generally the ⁇ 4 and ⁇ 5 helices of RSV-F).
  • such mutations according to (b) may inhibit, at least partly inhibit, or completely inhibit, the transition of RSV-F from pre-fusion to post-fusion conformation (in particular, by inhibiting of the relative motion of the two ⁇ helices adjacent to the loop (generally the ⁇ 4 and ⁇ 5 helices of RSV-F), or, defined differently, by inhibiting refolding of the HRC and HRA domains).
  • RSV-F proteins of the present disclosure may further comprise (according to all independent aspects of the present disclosure): 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.
  • the region in which the at least one mutation according to (c) is located comprises a ⁇ sheet and a loop (and optionally at least part of a further ⁇ sheet), and the at least one mutation introduces a glycosylation site into the region.
  • RSV-F proteins of the present disclosure may comprise: (c) at least one mutation relative to SEQ ID NO: 1 in a region of the protein (preferably comprising a ⁇ sheet and a loop) which aligns with positions 345-352 of SEQ ID NO:1; wherein the at least one mutation introduces a glycosylation site into the region.
  • RSV-F proteins of the present disclosure comprise: (c) at least one mutation relative to SEQ ID NO: 1 within positions 345-352 of SEQ ID NO: 1, wherein the at least one mutation introduces a glycosylation site into said positions.
  • positions 348-352 of SEQ ID NO:1 form a ⁇ sheet
  • positions 346-347 of SEQ ID NO: 1 form a loop
  • position 345 is the C-terminal residue of a further ⁇ sheet.
  • the at least one mutation according to (c) results in glycosylation at a residue within the region (preferably which comprises a ⁇ sheet and a loop); or, defined differently, the at least one mutation according to (c) results in the introduction of a glycan linked to a residue within the region (preferably which comprises a ⁇ sheet and a loop).
  • glycosylation at a position within the above region may provide a stabilising cross-protomer interaction with a charged patch at approximately positions 416- 422 of SEQ ID NO: 1 (in particular K419; see Figure 24), thereby inhibiting, at least partly inhibiting, or completely inhibiting the transition of RSV-F from pre-fusion to post-fusion conformation.
  • Such interaction may be maintained or enhanced by the introduction of further mutations in positions 416-422 of SEQ ID NO: 1, as discussed below.
  • the at least one mutation according (c) may comprise or consist of one or a plurality of substitutions or insertions (preferably substitutions) relative to positions 345-352 of SEQ ID NO:1; in particular only 1, 2 or 3 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such mutations (preferably substitutions), in particular only 1 such substitution or insertion (preferably substitution).
  • the region (preferably which comprises a ⁇ sheet and a loop) corresponding to positions 345-352 of SEQ ID NO:1 may have at least 50% or 60% sequence identity, or preferably at least 75% or 85% sequence identity to positions 345-352 of SEQ ID NO:1.
  • the glycosylation site / glycosylation may be introduced into, or the glycan may be linked to, the region (preferably which comprises a ⁇ sheet and a loop) through the introduction, by mutation, of at least one N residue (resulting in N-linked glycosylation), or at least one S and/or T residue (resulting in O-linked glycosylation).
  • at least one N residue is introduced by substitution, resulting in an NXT or NXS motif (as required for N-linked glycosylation), wherein X is any amino acid other than P (as required for N-linked glycosylation.
  • the glycosylation / glycan will comprise a core structure comprising or consisting of N- acetyl glucosamine (GlcNAc).
  • the glycosylation / glycan will comprise or consist of GlcNAc.
  • the glycosylation site / glycosylation is introduced into, or the glycan is linked to a residue in, a ⁇ sheet corresponding to positions 348-352 of SEQ ID NO: 1, by mutation.
  • the glycosylation site / glycosylation is introduced into, or the glycan is linked to, said ⁇ sheet by substitution of position 348 of SEQ ID NO: 1 (S) for N.
  • Said glycosylation site may be conserved by maintaining the wild-type residue at position 350 (S), or substituting S350 for T.
  • Such preferred substitutions at position 348 may be the only mutation Docket No.: 70221WO01 according to (c); and optionally the only mutation in the region corresponding to positions 345-352 of SEQ ID NO: 1.
  • a minimal substitution screen performed by the inventors revealed the S348N mutation to be a likely driver of the pre-fusion conformation (design F311).
  • RSV-F proteins of the present disclosure further comprise at least one mutation (preferably substitution) in a region (preferably which forms a loop) corresponding to positions 416-422 of SEQ ID NO: 1, wherein the at least one mutation increases the negative charge of the region (e.g.
  • the at least one mutation is a substitution at position 419 of SEQ ID NO: 1 for D or E.
  • the at least one mutation is a substitution at position 419 of SEQ ID NO: 1 for D (as in design F216, see Figure 24A).
  • Such mutations in a region (preferably which forms a loop) corresponding to positions 416-422 of SEQ ID NO: 1 may enhance cross-protomer interactions, thereby helping to inhibit the transition of RSV-F from pre-fusion to post-fusion conformation.
  • mutations according to (c) may stabilise or rigidify the loop region in the F1 domain corresponding to positions 346-347 of SEQ ID NO: 1. Such stabilisation or rigidification may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (c) may provide a cross-protomer interaction (such as a hydrogen bond). Such cross-protomer interaction may be with one or more charged residues, including e.g.
  • Such cross-protomer interaction may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (c) preferably such substitutions, preferably such substitutions at position 348 as detailed above
  • RSV-F proteins of the present disclosure may further comprise (according to all independent aspects of the present disclosure): Docket No.: 70221WO01 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.
  • the region in which the at least one mutation according to (d) is located comprises a ⁇ sheet and a loop (and optionally at least part of a further ⁇ sheet).
  • RSV-F proteins of the present disclosure may comprise: (d) at least one mutation relative to SEQ ID NO: 1 in a region of the protein (preferably comprising a ⁇ sheet and a loop) which aligns with positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from D, F, H, K, L, N, Q, R, T, W and Y into the region.
  • RSV-F proteins of the present disclosure comprise: (d) at least one mutation relative to SEQ ID NO: 1 within positions 345-352 of SEQ ID NO: 1, wherein the at least one mutation introduces at least one residue selected from D, F, H, K, L, N, Q, R, T, W and Y into said positions.
  • the at least one mutation according (d) may comprise or consist of 1, 2, 3, 4, 5, 6, 7 or 8 substitutions or insertions (preferably substitutions) relative to positions 345-352 of SEQ ID NO: 1; in particular only 1, 2, 3, 4, 5, such substitutions or insertions (preferably substitutions), in particular only 1, 2 or 3 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such mutations (preferably substitutions), in preferably only 1 such substitution or insertion (preferably substitution).
  • the region corresponding to positions 345-352 of SEQ ID NO:1 may have at least 50% or 60% sequence identity, or preferably at least 75% or 85% sequence identity to positions 345-352 of SEQ ID NO:1.
  • one or more S residues in the wild-type ⁇ sheet according to positions 348-352 of SEQ ID NO: 1 may be substituted for N, D, F, H, K, L, Q, R, T, W or Y.
  • positions 348 and/or 350 of SEQ ID NO: 1 may be substituted for N, F, H, K, N, Q, R, T, W or Y, in particular N, F, R, W or Y, preferably N.
  • RSV-F proteins of the present disclosure comprise a substitution at position 348 of SEQ ID NO: 1 (S) for N, D, F, H, K, L, Q, R, T, W or Y.
  • RSV-F proteins of the present disclosure comprise a substitution at position 348 of SEQ ID NO: 1 (S) for N, F, H, K, N, Q, R, T, W or Y.
  • RSV-F proteins of the present disclosure comprise a substitution at position 348 of SEQ ID NO: 1 (S) for N, Docket No.: 70221WO01 F, R, W or Y.
  • RSV-F proteins of the present disclosure comprise a substitution at position 348 of SEQ ID NO: 1 (S) for N.
  • Such preferred substitutions at position 348 may be the only mutation according to (d); and optionally the only mutation in the region corresponding to positions 345-352 of SEQ ID NO: 1.
  • a minimal substitution screen performed by the inventors revealed the S348N mutation to be a likely driver of the pre-fusion conformation (design F311).
  • alternative substitutions provided for position 348 by ROSETTA software include: D, F, H, K, L, Q, R, T, W and Y (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0 or -0.1 being used), F, H, K, N, Q, R, T, W and Y (same parameters except for an energy threshold of -0.5 being used), and F, R, W or Y (same parameters except for an energy threshold of -2 being used).
  • Mutations according to (d) which introduce N or T residues may introduce a glycosylation site into the above region (preferably comprising a ⁇ sheet and a loop); preferably resulting in glycosylation at a residue within the region, or, defined differently, resulting in the introduction of a glycan linked to a residue within region.
  • the glycosylation site may be conserved by maintaining the wild-type residue at position 350 (S), or substituting S350 for T.
  • RSV-F proteins of the present disclosure further comprise at least one mutation (preferably substitution) in a region (preferably which forms a loop) corresponding to positions 416-422 of SEQ ID NO: 1, wherein the at least one mutation increases the negative charge of the region (e.g.
  • the mutation is a substitution at position 419 of SEQ ID NO: 1 for D or E.
  • the mutation is a substitution at position 419 of SEQ ID NO: 1 for D.
  • Such mutations in a region preferably which forms a loop) corresponding to positions 416-422 of SEQ ID NO: 1 may enhance (in the presence of absence of glycosylation) cross-protomer interactions, thereby helping to inhibit the transition of RSV-F from pre-fusion to post-fusion conformation.
  • mutations according to (d) may stabilise or rigidify the loop region in the F1 domain corresponding positions 346-347 of SEQ ID NO: 1. Such stabilisation or rigidification may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (d) may provide a cross-protomer interaction. Such cross- protomer interaction may be with one or more charged residues, including e.g. at position 419 of SEQ ID NO: 1 (e.g., a K, E or D residue at position 419).
  • Such cross-protomer interaction may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • mutations according to (d) preferably such substitutions, Docket No.: 70221WO01 preferably such substitutions at position 348 as detailed above
  • RSV-F proteins of the present disclosure comprise, relative to SEQ ID NO: 1: (a) a substitution at position 55 of SEQ ID NO: 1 (S) for T, C, V, I, preferably T, C or V, preferably T or V, more preferably T; (b) a substitution at position 215 of SEQ ID NO: 1 (S) for A, P, V, I, or F, preferably A, V, I, or F, preferably A or P, more preferably A; and (c) a substitution at position 348 of SEQ ID NO: 1 (S) for N or T, more preferably N; optionally wherein a glycan is linked to said N or T at position 348.
  • RSV-F proteins of the present disclosure comprise, relative to SEQ ID NO: 1: (a) a substitution at position 55 of SEQ ID NO: 1 (S) for T, C, V, I, preferably T, C or V, preferably T or V, more preferably T; (b) a substitution at position 215 of SEQ ID NO: 1 (S) for A, P, V, I, or F, preferably A, V, I, or F, preferably A or P, more preferably A; and (d) a substitution at position 348 of SEQ ID NO: 1 (S) for N, D, F, H, K, L, Q, R, T, W or Y, preferably N, F, H, K, N, Q, R, T, W or Y, preferably N, F, R, W or Y, more preferably N.
  • RSV-F proteins of the present disclosure comprise, relative to SEQ ID NO: 1, the substitutions S55T, S215A and S348N; optionally wherein a glycan is linked to said N at position 348; optionally in addition to further mutations (preferably substitutions) as detailed below.
  • the foregoing are preferably the only mutations according to (a), (b) and ((c) or (d)) relative to SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure Docket No.: 70221WO01 may comprise at least one further mutation, preferably at least one further substitution, relative to SEQ ID NO: 1.
  • said at least one further substitution is selected from (numbering and original residues according to SEQ ID NO: 1): A74R; a substitution at position 152 (V) for R, L or W; S169E; S180E; S190I; a substitution at position 210 (Q) for H, A, F, K, N, W or Y; S211N; E218; K226L; a substitution at position 228 (N) for K, R or A; A241N; M251L; S275L; M289L; V296I; L305I; a substitution at position 315 (K) for I or V; T326D, a substitution at position 346 (A) for Q, D, H, K, N, R, S or W; S350I, K359I; V384K; a substitution at position 419 (K) for D, N, S, or T; K445D; a substitution at position 455 (T) for V or I; V459M; F
  • RSV-F proteins of the present disclosure may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or at least 50 of the foregoing substitutions; such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 of the foregoing substitutions.
  • RSV-F proteins of the present disclosure comprise, in addition to (a), (b), and/or ((c) or (d)), no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 of the foregoing substitutions, such as no more than 14 such as no more than 11, such as no more than 8, such as no more than 5, such as no more than 4 of the foregoing substitutions.
  • the substitutions V152R and/or A346Q may be present in RSV-F proteins of the present disclosure. Such substitutions may enhance expression of the RSV-F protein.
  • V152R and A346Q are surface-exposed substitutions present in both designs F216 and F217, which shows higher in vitro expression levels from mRNA than F224 and F225 (buried substitutions only), see e.g. Example 7; Figure 21.
  • the substitutions S211N and/or K445D may be present in RSV-F proteins of the present disclosure.
  • the presence of the S211N and K445D appears to improve stability of the protein following heat stress (see Figure 30, comparing F217 (having both substitutions) to F318 (lacking S211N), and comparing F216 (having both substitutions) to F319 (lacking both substitutions)).
  • RSV-F proteins of the present disclosure may comprise at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 (e.g.
  • substitutions selected from (numbering and original residues according to SEQ ID NO: 1): a substitution at position 152 (V) for R, L or W, preferably R or W, preferably R; Docket No.: 70221WO01 a substitution at position 210 (Q) for H, A, F, K, N, W or Y, preferably H, F, K, N, W or Y, preferably, H, F or Y; preferably H; optionally a substitution at position 211 (S) for N; a substitution at position 241 (A) for N a substitution at position 315 (K) for I or V, preferably I; a substitution at position 346 (A) for Q, D, H, K, N, R, S or W, preferably Q, D, H, K, N, R or S, preferably Q; a substitution at position 419 (K) for D, N, S, or T, preferably D or T, preferably D; optionally a substitution at position 210 (V) for R, L or
  • substitutions at positions 152, 210, 211, 241, 315, 346, 419, 445, 455 and 459 includes substitutions found in design F216 (F216 also has S55T, S215A, N228K and S348N), in addition to alternative residues provided by ROSETTA software (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0 being used) and/or visual analysis of three- dimensional structure; more stringent ROSETTA energy thresholds and/or visual analysis used to generate subsets.
  • RSV-F proteins of the disclosure comprise, relative to SEQ ID NO: 1, the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M, and optionally S211N and/or K445D (all of which are found in in design F216 – see e.g. Example 4), optionally wherein a glycan is linked to said N at position 348; optionally with no further mutations present relative to SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure may comprise at least 1, such as at least 2, 3, 4, 5, 6 or at least 7 (e.g.
  • substitutions selected from (numbering and original residues according to SEQ ID NO: 1): a substitution at position 152 (V) for R, L or W, preferably R or W, preferably R; optionally a substitution at position 211 (S) for N; a substitution at position 315 (K) for I or V, preferably I; a substitution at position 346 (A) for Q, D, H, K, N, R, S or W, preferably Q, D, H, K, N, R or S, preferably Q; Docket No.: 70221WO01 optionally a substitution at position 445 (K) for D; a substitution at position 455 (T) for V or I, preferably V; and a substitution at position 459 (V) for M.
  • substitutions at positions 152, 211, 315, 346, 445, 455 and 459 includes substitutions found in design F217 (F217 also has S55T, S215A, N228K and S348N), in addition to alternative residues suggested by ROSETTA software (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0 being used) and/or visual analysis of three-dimensional structure; more stringent ROSETTA energy thresholds and/or visual analysis used to generate subsets .
  • RSV-F proteins of the disclosure comprise, relative to SEQ ID NO: 1, the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M, and optionally S211N and/or K445D (e.g as found in design F217 – see e.g. Example 4); optionally wherein a glycan is linked to said N at position 348; optionally with no further mutations present relative to SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure may comprise at least 1, such as at least 2, 3 or at least 4 (e.g. 1, 2, 3, 4 or 5) substitutions selected from (numbering and original residues according to SEQ ID NO: 1): a substitution at position 315 (K) for I or V, preferably I; a substitution at position 241 (A) for N a substitution at position 455 (T) for V or I, preferably V; and a substitution at position 459 (V) for M.
  • substitutions at positions 241, 315, 455 and 459 includes substitutions found in design F224 (F224 also has S55T, S215A, N228K and S348N), in addition to alternative residues provided by ROSETTA software (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0 being used) and/or visual analysis of three-dimensional structure; more stringent ROSETTA energy thresholds and/or visual analysis used to generate subsets.
  • RSV-F proteins of the disclosure comprise, relative to SEQ ID NO: 1, the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M (e.g as found in design F224 – see e.g. Example 4); optionally wherein a glycan is linked to said N at position 348; optionally with no further mutations present relative to SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure may comprise at least 1, such as at least 2 or at least Docket No.: 70221WO01 3 (e.g.1, 2, 3 or 4) substitutions selected from (numbering and original residues according to SEQ ID NO: 1): a substitution at position 315 (K) for I or V, preferably I; a substitution at position 455 (T) for V or I, preferably V; and a substitution at position 459 (V) for M.
  • at least 1 such as at least 2 or at least Docket No.: 70221WO01 3 (e.g.1, 2, 3 or 4) substitutions selected from (numbering and original residues according to SEQ ID NO: 1): a substitution at position 315 (K) for I or V, preferably I; a substitution at position 455 (T) for V or I, preferably V; and a substitution at position 459 (V) for M.
  • substitutions at positions 315, 455 and 459 includes substitutions found in design F225 (F225 also has S55T, S215A, N228K and S348N), in addition to alternative residues provided by ROSETTA software (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0 being used) and/or visual analysis of three-dimensional structure; more stringent ROSETTA energy thresholds and/or visual analysis used to generate subsets.
  • RSV-F proteins of the disclosure comprise, relative to SEQ ID NO: 1, the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M (as found in design F225 – see e.g. Example 4); optionally wherein a glycan is linked to said N at position 348; optionally with no further mutations present relative to SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure generally have two domains (in the N-terminal to C-terminal direction, an “F2” domain and an “F1” domain), which may or may not be linked via peptide bonds (although in the wild-type protein they are not so linked; linkage typically occurring through disulphide bonds).
  • the F2 domain may have at least 70% sequence identity to positions 26-108 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 26-108; and the F1 domain may have at least 70% sequence identity to positions 137-513 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 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.
  • the F2 domain may have at least 70% sequence identity to positions 26-109 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity to positions 26-109 of SEQ ID NO: 1; and the F1 domain may have at least 70% sequence identity to positions 137-513 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Docket No.: 70221WO01 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1.
  • a signal peptide is not present in the RSV-F protein of the present disclosure, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is positions 1-25 of SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure comprise an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1.
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to SEQ ID NO: 13, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 13.
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to SEQ ID NO: 84, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 84.
  • SEQ ID NO: 13 and 84 are mature, furin-processed sequences of wild-type RSV-F from the A2 subtype (that is, SEQ ID NO: 1 without signal sequence and p27).
  • positions 84 (R) and 85 (F) of SEQ ID NO: 13, 28-38, 50-59 and 84-106 are typically non-contiguous, and may or may not be (preferably are not) linked by an intervening amino acid sequence, such as a linker sequence.
  • positions 1-84 of SEQ ID NO: 13, 28-38, 50-59 and 84-106 form (in whole or in part) the F2 domain
  • positions 85-461 of said sequences form (in whole or in part) the F1 domain
  • said F2 and F1 domains may or may not be (preferably are not) linked by an intervening amino acid sequence, such as a linker sequence, between positions 84 and 85 of said sequences.
  • p27 peptide may still be present as a result of furin cleavage at only one site, e.g. the p27 peptide is linked via a peptide bond to one of the F2 or F1 domains.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 28 or 85 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitution S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 28 and 85.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 29 or 86 or a portion thereof, such as a portion at least 70%, Docket No.: 70221WO01 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 29 and 86.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 30 or 87 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 30 and 87.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 31 or 88 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains, in whole or in part (that is, at least parts of said domains compared to their full- length sequences).
  • Said portion preferably includes the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 31 and 88.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 33 or 90 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 33 and 90.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 34 or 91 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 34 and 91.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 35 or 92 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 Docket No.: 70221WO01 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 35 and 92.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 36 or 93 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 36 and 93.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 37 or 94 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 37 and 94.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 38 or 95 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion preferably includes the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 38 and 95.
  • RSV-F proteins of the present disclosure comprise or consist of an amino acid sequence according to SEQ ID NO: 82 or 106 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences).
  • Said portion includes the substitution N228K (numbering according to SEQ ID NO: 1), which is present in SEQ ID NO: 82 and 106.
  • RSV-F proteins of the present disclosure may comprise or consist of an amino acid sequence according to any of SEQ ID NO: 50-53, 55 or 56, or a portion of any of the foregoing, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains Docket No.: 70221WO01 compared to their full-length sequences).
  • Said portion preferably includes all substitutions present in the amino acid sequence according to any of SEQ ID NO: 50-53, 55, 56, (where applicable) relative to SEQ ID NO: 13.
  • RSV-F proteins of the present disclosure may comprise or consist of an amino acid sequence according to any of SEQ ID NO 96-99, 101 or 102, or a portion of any of the foregoing, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably encompasses the F2 and F1 domains (that is, at least parts of said domains compared to their full-length sequences). Said portion preferably includes all substitutions present in the amino acid sequence according to any of SEQ ID NO: 96- 99, 101 or 102 (where applicable) relative to SEQ ID NO: 84.
  • the F2 and F1 domains are linked via peptide bonds (e.g. those of an intervening amino acid sequence) they may be linked by a linker sequence.
  • the linker sequence will join the C and N terminal regions / residues of said F2 and F1 domains.
  • the linker sequence may be glycine-serine rich or consist of G and S residues, for example GSGSG (SEQ ID NO: 10), GSGSGRS (SEQ ID NO: 11), or GS (SEQ ID NO: 12).
  • the “F2” and “F1” domains may be linked by linker comprising or consisting of SEQ ID NO: 11 (or a linker having at least 55%, 75% or 85% identity thereto).
  • the “F2” and “F1” domains may be linked by linker comprising or consisting of SEQ ID NO: 12 (or either a G or an S residue).
  • the “F2” and “F1” domains are not linked via peptide bonds, they may be linked by at least one disulphide bond (typically two such bonds, which are typically naturally-occurring, e.g. as in the wild-type protein).
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13; in particular at least 75% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 80% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 85% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 90% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 95% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 99% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 99.4% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 99.5% sequence identity to SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 75% sequence identity to SEQ ID NO: 13 over at
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to SEQ ID NO: 13, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 13 over 100% of SEQ ID NO 13.
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84; in particular at least 75% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 80% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 85% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 90% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 95% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 99% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 99.4% sequence identity to SEQ ID NO: 84 over at least 80% of SEQ ID NO: 84, at least 99.5% sequence identity to SEQ ID NO: 84 over at least
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to SEQ ID NO: 84, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 84 over 100% of SEQ ID NO 84.
  • RSV-F proteins of the present disclosure comprise a heterologous trimerisation domain on the C-terminus thereof (“heterologous” meaning not being native to the viral protein).
  • the trimerisation domain may be positioned C-terminal to the F1 domain.
  • a trimerisation domain is a sequence which promotes assembly of RSV-F proteins of the present disclosure (i.e. individual promoters) into trimers, namely in particular via associations with other trimerisation domains (i.e. those on other protomers). Trimerisation domains may, in some embodiments, fold into a coiled-coil.
  • trimerisation domains include: a T4 fibritin foldon domain; a yeast GCN4 isoleucine zipper, e.g. according to SEQ ID NO: 39 (or an amino acid sequence, in particular having a trimerisation function, at least 50%, 60%, 70%, 80%, 90% or 95% identical thereto); TRAF2 (GENBANK Accession No. Q12933 [gi:23503103]; amino acids 299-348); Thrombospondin 1 (Accession No. PO7996 [gi:135717]; amino acids 291-314); Matrilin-4 (Accession No. 095460 [gi:14548117]; amino acids 594-618; CMP (matrilin-1) (Accession No.
  • NP_002370 [gi:4505111]; amino acids 463- 496; HSF1 (Accession No. AAX42211 [gi:61362386]; amino acids 165-191; Cubilin (Accession No. NP_001072 [gi:4557503]; amino acids 104-138); a trimerisation domain from an influenza hemagglutinin; a trimerisation domain from a SARS spike protein, a trimerisation domain from HIV gp41; NadA; and ATCase
  • the trimerisation domain is a T4 fibritin foldon domain, more preferably comprising or consisting of an amino acid sequence according to SEQ ID NO: 14 (or an amino acid sequence, preferably having a trimerisation function, at least 50%, 60%, 70%, 80%, 90% or 95% identical thereto).
  • the trimerisation domain is preferably linked to the C-terminus of RSV-F proteins of the present disclosure (i.e. the F1 domain) via a linker sequence.
  • Said linker sequence preferably comprises or consists of an amino acid sequence according to SEQ ID NO: 60 (or an amino acid sequence at least 50% or 75% identical thereto).
  • a fourth independent aspect of the present disclosure is a multimer comprising protomers, wherein at least one protomer is an RSV-F protein of the present disclosure.
  • the multimer is a trimer of RSV-F protein of the present disclosures.
  • the trimer is a homotrimer (that is, comprising three RSV-F proteins of the present disclosure comprising or consisting of the same primary amino acid sequence).
  • RSV-F proteins in the prefusion conformation can be prepared by routine methods, such as by expression in a recombinant host system using a nucleic acid expression vector (e.g. an expression vector as detailed in the section entitled Nucleic acids encoding RSV-F proteins, below).
  • a nucleic acid expression vector e.g. an expression vector as detailed in the section entitled Nucleic acids encoding RSV-F proteins, below.
  • Docket No.: 70221WO01 Suitable recombinant host cells include, for example, insect cells (e.g. Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells); mammalian cells (e.g. Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g.
  • HEK293 cells are preferred, more preferably Expi 293 cells (as were used in the examples). Accordingly, the present disclosure also provides, in one independent aspect, a host cell (in particular, those detailed above) comprising a nucleic acid (in particular, an expression vector as detailed below) encoding an RSV-F protein of the present disclosure.
  • the present disclosure also provides, in a further independent aspect, a host cell (in particular, those detailed above) comprising and/or expressing an RSV-F protein of the present disclosure.
  • a host cell in particular, those detailed above
  • a composition comprising a host cell (in particular, those detailed above) and (i) a nucleic acid (in particular, an expression vector as detailed below) encoding an RSV-F protein of the present disclosure, and/or (ii) an RSV-F protein of the present disclosure.
  • the present disclosure also provides, in a further independent aspect, an in vitro method for the production of an RSV-F protein of the present disclosure, comprising expressing a nucleic acid (in particular, an expression vector as detailed below) encoding the RSV-F protein in a host cell (in particular, those detailed above), and optionally purifying the RSV-F protein.
  • RSV-F proteins of the present disclosure can be purified, following expression from a host cell, by routine methods, such as precipitation and chromatographic methods (e.g. hydrophobic interaction, ion exchange, affinity, chelating or size exclusion chromatography).
  • the RSV-F proteins of the present disclosure can include a tag that facilitates purification, such as an epitope tag or a histidine (HIS) tag, to facilitate purification e.g. by affinity chromatography.
  • Nucleic acids encoding RSV-F proteins in the pre-fusion conformation The present disclosure also provides, in a further independent aspect, a nucleic acid encoding an RSV-F protein of the present disclosure.
  • General sequence features of RSV-F proteins in the pre-fusion conformation, when encoded by nucleic acids e.g.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 1.
  • SEQ ID NO: 1 is the sequence of wild-type RSV-F from the A2 subtype which includes the signal sequence (positions 1-25 of SEQ ID NO: 1), and the p27 peptide (positions 109-136 or 110-136 of SEQ ID NO: 1) which is, in the mature protein, cleaved out by furin processing.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure comprising an F2 domain having at least 70% sequence identity to positions 26-109 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity to positions 26-109 of SEQ ID NO: 1; and an F1 domain having at least 70% sequence identity to positions 137-513 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO:
  • RSV-F proteins of the present disclosure comprise an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1.
  • the signal peptide (positions 1-25 of SEQ ID NO: 1) is not considered in the above sequence identity assessment.
  • nucleic acids of the present disclosure encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 26-513 SEQ ID NO: 1.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 17; or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M , which are present in SEQ ID NO: 17.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 18; or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M, which are present in SEQ ID NO: 18.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 19; or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M, which are present in SEQ ID NO: 19.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 20; or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said Docket No.: 70221WO01 portion preferably includes the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M, which are present in SEQ ID NO: 20.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 22 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M , which are present in SEQ ID NO: 22.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 23 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M , which are present in SEQ ID NO: 23.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 24 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M , which are present in SEQ ID NO: 24.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 25 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M, which are present in SEQ ID NO: 25.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 26 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M, which are present in SEQ ID NO: 26.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 27 or a portion thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M, which are present in SEQ ID NO: 27.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of an amino acid sequence according to SEQ ID NO: 81 or a portion Docket No.: 70221WO01 thereof, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion includes the substitution N228K, which is present in SEQ ID NO: 81.
  • nucleic acids of the present disclosure may encode an RSV-F protein comprising or consisting of an amino acid sequence according to any of SEQ ID NO: 40-43, 45 or 46, or a portion of any of the foregoing, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes all substitutions present in the amino acid sequence according to any of SEQ ID NO: 40-43, 45 or 46 (where applicable) relative to SEQ ID NO: 1.
  • Two furin cleavage sites exist between positions 108 and 137 of SEQ ID NO: 1 (positions 109-136 or 110-136 of SEQ ID NO: 1 defining the “p27” peptide).
  • nucleic acids of the present disclosure encode an RSV-F protein of the present disclosure in which the p27 peptide is artificially absent (i.e. there is an artificial deletion of the p27 peptide, e.g. through recombinant means, at the level of the encoding nucleic acid).
  • the fusion peptide (positions 137-157 of SEQ ID NO: 1) may also be artificially absent.
  • the p27 peptide (and, optionally, also the fusion peptide) may be replaced by a linker sequence encoded by the nucleic acid.
  • the linker sequence may be glycine-serine rich (or consist of G and S residues), for example GSGSG (SEQ ID NO: 10), GSGSGRS (SEQ ID NO: 11), or GS (SEQ ID NO: 12).
  • the p27 peptide (or at least 80%, 85%, 90% or 95% of the residues thereof) is artificially absent and is replaced by a linker comprising or consisting of SEQ ID NO: 11 (or a linker having at least 55%, 75% or 85% identity thereto).
  • both the p27 and fusion peptides are artificially absent and are replaced by a linker comprising or consisting of SEQ ID NO: 12 (or either a G or an S residue).
  • nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure comprising two domains (in the N-terminal to C-terminal direction, the “F2” and “F1” domains); the F2 domain having at least 70% sequence identity to positions 1-108 or 1-109 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 1-108 or 1-109 of SEQ ID NO: 1; and the F1 domain having at least 70% sequence identity to positions 137-513 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%,
  • nucleic acids of the present disclosure may encode an RSV-F Docket No.: 70221WO01 protein of the present disclosure comprising two domains (in the N-terminal to C-terminal direction, the “F2” and “F1” domains); the F2 domain having at least 70% sequence identity to positions 26- 108 or 26-109 of SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 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; and the F1 domain having at least 70% sequence identity to positions 137-513 of SEQ ID NO: 1, such as at least 75%
  • Nucleic acids of the present disclosure may also encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1; in particular at least 75% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 80% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 85% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 90% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 95% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 99% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 99.4% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 99.5% sequence identity to SEQ ID NO: 1 over at least 80% of SEQ ID NO: 1, at least 75%
  • Nucleic acids of the present disclosure preferably encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 1 over 100% of SEQ ID NO 1.
  • Nucleic acids of the present disclosure preferably encode an RSV-F protein comprising a transmembrane domain, and, optionally, C-terminal to said transmembrane domain, a cytoplasmic domain, linked (directly or indirectly) to the C-terminus thereof (i.e. C-terminal to position 513 of SEQ ID NO: 1, or defined differently, C-terminal to the F1 domain).
  • a cytoplasmic domain is absent in whole.
  • a transmembrane domain comprises or consists of an amino acid sequence according to SEQ ID NO: 15 (or a sequence at least 80%, 85%, 90%, or 95% identical thereto).
  • a cytoplasmic domain if present, comprises or consists of an amino acid sequence according to SEQ ID NO: 16, 109 or 110 (or a sequence at least 80%, 85%, 90%, 95% or 95% identical thereto).
  • Nucleic acids encoding RSV-F proteins comprising cytoplasmic tail deletions The terms “cytoplasmic domain” and “cytoplasmic tail” are used interchangeably herein (including in the appended numbered embodiments and claims).
  • the cell-surface expression of trimeric, pre-fusion RSV-F protein when expressed from nucleic acids in vitro, has been enhanced through the deletion of residues from the C-terminal cytoplasmic tail (see e.g. Example 12).
  • deletion of 15, 16, 17 and 20 C-terminal residues resulted in higher trimeric pre-fusion RSV-F expression at 72 and 96 hours post-transfection, compared to the deletion of 21 C-terminal residues (see e.g. Example 14; Figure 45A).
  • RSV-F constructs comprising a cytoplasmic tail deletion generally elicited higher neutralising antibody titres against e.g. RSV of the A subtype (see e.g. Example 13; Figure 44B), in comparison to their counterparts with a fully intact cytoplasmic tail.
  • cytoplasmic tail deletions may allow for protective efficacy against RSV to be achieved at lower doses of a nucleic acid-based vaccine, leading to further possible benefits, e.g. reduced reactogenicity.
  • RSV-F proteins comprise cytoplasmic tail deletions (as defined in this subsection, and in the appended numbered embodiments and claims)
  • an RSV-F protein having a “cytoplasmic tail” refers to the presence of residues (e.g.
  • an RSV-F protein having a “cytoplasmic tail” refers to the presence of residues (e.g. 5 residues) that are C-terminal to position 549 of the RSV-F protein.
  • the RSV-F construct referred to as ⁇ CT25 used in the examples see e.g.
  • Table 8 does not comprise any residues C- terminal to the Y at position 549, and hence does not comprise a cytoplasmic tail.
  • Reference to e.g. deletion of 2-20 residues (and the like) from the C-terminal end of the CT refers to deletion of at least the two, and no more than the 20, most C-terminal residues from the CT.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising a cytoplasmic tail; wherein, relative to a cytoplasmic tail according to SEQ ID NO: 109 or 110, 2-20 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein. In some embodiments, 3-20 residues are deleted from said C-terminal end.
  • At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or at least 19 residues are deleted from said C- terminal end. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues are deleted from said C-terminal end. In some embodiments, 2-5, 3-5, 6-20, 7-20, 8-20, 9- 20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20 residues are deleted from said C-terminal end.
  • 2-5 such as 2-4, 2-3 or 3-4, and preferably 3, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110).
  • deletion of the 3 C-terminal residues (“ ⁇ CT3”) enhanced cell-surface trimeric, pre-fusion RSV-F expression from nucleic acids (as measured by AM14 antibody binding) over a period of 96 hours post-transfection, relative to expression of the parental molecule with either an intact or a fully deleted CD.
  • the cytoplasmic tail 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 tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail 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 tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii)
  • 6-13 such as 7-13, 8-12, 9-11, 9-10 or 10-11, and preferably 10 residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 109 or 110).
  • deletion of the 10 C-terminal residues (“ ⁇ CT10”) enhanced cell-surface trimeric, pre- fusion RSV-F expression from nucleic acids (as measured by AM14 antibody binding) over a period of 47 hours post-transfection, relative to expression of the parental molecule with either an intact or a Docket No.: 70221WO01 fully deleted CT.
  • the cytoplasmic tail 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 tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • 14-16 such as 14-15 or 15-16, and preferably 15, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 109 or 110).
  • the cytoplasmic tail 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 tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • 16-20 such as 17-20, 18-20 or 19-20, and preferably 20, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 109 or 110).
  • deletion of the 20 C-terminal residues (“ ⁇ CT20”) enhanced cell-surface trimeric, pre- fusion RSV-F expression from nucleic acids (as measured by AM14 antibody binding) over a period of 96 hours post-transfection, relative to expression of the parental molecule with either an intact or a fully deleted CT.
  • 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).
  • the deletions outlined above increase the cell-surface expression of RSV-F protein from RNA, relative to an RSV-F protein having the same amino acid sequence absent deletions, e.g. Docket No.: 70221WO01 comprising a wild-type cytoplasmic tail, e.g. according to SEQ ID NO: 109 or 110 (e.g. over at least 24, 48, 72 or 96 hours; or e.g. over 24, 48, 72 or 96 hours).
  • the deletions outlined above increase the cell-surface expression of RSV-F protein in trimeric, pre-fusion form from RNA, relative to expression in such form of an RSV-F protein having the same amino acid sequence absent such deletions, e.g.
  • trimeric, pre-fusion RSV-F expression is typically assessed using AM14 antibody binding (or defined differently, using binding of an antibody comprising a light chain (LC) according to SEQ ID NO: 2 and a heavy chain (HC) according SEQ ID NO: 3).
  • AM14 antibody binding may be assayed using indirect immunofluorescent labelling, e.g. using the protocol in the examples (see subsection “Indirect immunofluorescent labelling and detection of surface-expressed RSV F”).
  • the nucleic acid of the present disclosure may be DNA or RNA (including hybrids thereof), preferably RNA.
  • DNA and RNA analogues such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases, are within the scope of the present disclosure.
  • the nucleic acid may be linear, circular and/or branched, but will generally be linear.
  • the nucleic acid will be in recombinant form, i.e. a form which does not occur in nature.
  • the nucleic acid may be for the expression of an RSV-F protein of the present disclosure in vitro from a host cell (i.e. the nucleic acid is, or is part of, an expression vector).
  • Suitable nucleic acid expression vectors can comprise, for example, (1) an origin of replication; (2) a selectable marker gene; (3) one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, or a terminator), and/or one or more translation signals; and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g. those as detailed in the section entitled Preparing RSV-F proteins, above).
  • the nucleic acid is for the expression of an RSV-F protein of the present disclosure in vivo in a subject (i.e.
  • the nucleic acid is, or is part of, a nucleic acid-based vaccine).
  • the nucleic acid may comprise one or more heterologous sequences, such as a sequence encoding a further protein (e.g. as detailed below) and/or a control sequence, in particular a promoter or an internal ribosome entry site. Docket No.: 70221WO01 Nucleic acids of the present disclosure may be codon optimised. In some embodiments, nucleic acids of the present disclosure may be codon optimised for expression in human cells.
  • Codon optimisation refers to the use of specific codons, which, while not altering the sequence of the expressed protein (given genetic code redundancy), may increase translation efficacy and/or half- life of the nucleic acid.
  • codon optimised RNA are discussed in more detail in the subsection entitled RNA below.
  • nucleic acids of the present disclosure are in the form of a viral vector, such as a replicating or replication-deficient viral vector; including both DNA and RNA-based viral vectors.
  • viral vectors for encoding an RSV-F protein of the present disclosure include, for example: adenovirus vectors, such as replication-deficient or replication-competent adenovirus vectors; pox virus vectors, such as vaccinia virus vectors (e.g. modified vaccinia Ankara virus (MVA), NYVAC, avipox vectors, canarypox (e.g.
  • adenovirus vectors such as replication-deficient or replication-competent adenovirus vectors
  • pox virus vectors such as vaccinia virus vectors (e.g. modified vaccinia Ankara virus (MVA), NYVAC, avipox vectors, canarypox (e.g.
  • Alphavirus vectors such as Sindbis virus, Semlike Forest virus (SFV), Ross River virus, Venezuelan equine encephalitis (VEE) virus, and chimeras derived from Alphavirus vectors such as the foregoing; herpes virus vectors, such as cytomegalovirus (CMV)-derived vectors; arena virus vectors, such as lymphocytic choriomeningitis virus (LCMV) vectors; measles virus vectors; vesicular stomatitis virus vectors; pseudorabies virus vectors; adeno-associated virus vectors; retrovirus vectors; lentivirus vectors; and viral-like particles.
  • CMV cytomegalovirus
  • LCMV lymphocytic choriomeningitis virus
  • the nucleic acid is in the form of a DNA plasmid.
  • the viral vector is an adenovirus vector, such as a replication-incompetent adenovirus type 26 (“Ad26”) or a replication-incompetent chimpanzee-adenovirus-155 (“ChAd155”), preferably a replication- incompetent Ad26.
  • Ad26 replication-incompetent adenovirus type 26
  • ChoAd155 replication-incompetent chimpanzee-adenovirus-155
  • Ad26 replication-incompetent chimpanzee-adenovirus-155
  • Ad26 replication-incompetent chimpanzee-adenovirus-155
  • the adenovirus vector (preferably replication-incompetent Ad26) may also be co- formulated with an RSV-F protein (i.e. the protein per se) of the present disclosure, which may have the same, or a distinct, primary amino acid sequence to the RSV-F protein of the present disclosure encoded by the adenovirus.
  • the adenovirus vector (preferably replication-incompetent Ad26) may be co-formulated with a further RSV-F protein (i.e.
  • the protein per se that is not an RSV-F protein according to the present disclosure
  • an RSV-F protein with the p27 region deleted (or without the p27 region deleted) and optionally at least 2, 3, 4 or 5 mutations relative to wildtype RSV-F such as N67I and S215P; N67I, S215P and E487Q; or K66E, N67I, I76V, S215P and D486N; in particular the latter set of five mutations.
  • a particular patient group of interest in which the co-formulation may be used in therapy, in particular vaccination
  • is older adults see section entitled Medical uses and methods of treatment, below).
  • the co-formulation may be administered as, Docket No.: 70221WO01 or as part of, a prime-boost regimen, in particular involving administration of the co-formulation as both prime administration(s) and boost administration(s).
  • the nucleic acid preferably RNA
  • the nucleic acid may encode multiple proteins, of which one is the RSV-F protein of the present disclosure.
  • the nucleic acid encodes at least (i) an RSV-F protein of the present disclosure; and (ii) at least one further protein.
  • the at least one further protein may be a nanoparticle, e.g.
  • 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; typically encoded by a separate open reading frame to the RSV-F protein of the invention.
  • 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.
  • 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.
  • 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.
  • the at least one further protein detailed above is a further Pneumoviridae protein (in particular a Pneumoviridae antigen).
  • Useful further Pneumoviridae proteins 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.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to an hMPV antigen (in particular, the F antigen).
  • RNA embodiments a preferred patient group (in which the RNA 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 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
  • the nucleic acid is a viral vector (in particular, a poxvirus vector, in particular an MVA vector) encoding an RSV-F protein of the present disclosure in addition to a plurality of further RSV proteins (in particular, antigens); in particular at least 2, 3, or Docket No.: 70221WO01 4 further RSV proteins / antigens; in particular selected from G (from or derived from the A subtype: “G A ”), G (from or derived from the B subtype: “G B ”) N and either M2-1 or M2-2; in particular G A, GB, N and either of M2-1 or M2-2.
  • a viral vector in particular, a poxvirus vector, in particular an MVA vector
  • the at least one further protein detailed above is a Coronavirus antigen.
  • Useful Coronavirus antigens can be from a SARS coronavirus, in particular SARS-CoV2.
  • Useful Coronavirus antigens include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein.
  • the Coronavirus antigen is a SARS-CoV2 spike protein.
  • Said SARS-CoV2 spike protein may be from any variant, e.g.
  • 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.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to a Coronavirus antigen, e.g. as detailed above.
  • a preferred patient group in which the RNA may be used in therapy, in particular vaccination
  • the at least one further protein detailed above is an Orthomyxovirus antigen.
  • Useful Orthomyxovirus antigens can be from an influenza A, B or C virus.
  • Useful Orthomyxovirus antigens include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin.
  • 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.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to an Orthomyxovirus antigen, e.g. as detailed above.
  • a preferred patient group in which the RNA may be used in therapy, in particular vaccination
  • is older adults see section entitled Medical uses and methods of treatment, below).
  • the RNA may encode (i) an RSV-F protein of the present disclosure, (ii) a Coronavirus antigen, e.g. as detailed above, and (iii) an Orthomyxovirus antigen, e.g. as detailed above.
  • a plurality of nucleic acids of the present disclosure is, in particular, provided in purified or substantially purified form; that is, substantially free from other nucleic acids (e.g. free or substantially free from naturally-occurring nucleic acids, such as further nucleic acids expressed by a Docket No.: 70221WO01 host cell).
  • Said plurality of nucleic acids is generally at least 50% pure (by weight), such as at least 60%, 70%, 80%, 90%, or 95% pure (by weight).
  • the present disclosure also provides, in a further independent aspect, a vector comprising one or more nucleic acids of the present disclosure.
  • Nucleic acids encoding an RSV-F protein of the present disclosure may be delivered naked, or preferably in conjunction with a carrier (e.g. as detailed in the section entitled Carriers comprising a nucleic acid encoding an RSV-F protein in the prefusion conformation, below).
  • nucleic acids (preferably RNA) of the present disclosure, and the RSV-F proteins encoded thereby elicit a pre-fusion RSV-F-specific antibody response against RSV in vivo, e.g. an IgG antibody response (see, e.g. Examples 11 and 13).
  • nucleic acids (preferably RNA) of the present disclosure, and the RSV-F proteins encoded thereby elicit a neutralising antibody response against RSV in vivo, e.g. against RSV-A (see, e.g. Examples 11 and 13).
  • Said neutralising antibody response may inhibit replication of RSV in the respiratory system of a subject (such as in the lungs).
  • Said neutralising antibody response may yield protective immunity against RSV in a subject.
  • RNA refers to an artificial (or, defined differently, recombinant) ribonucleic acid encoding an RSV-F protein of the present disclosure, which may be translated in a cell (i.e. mRNA).
  • mRNA a cell
  • the RNA is neither, nor comprised within, a viral vector or virus-based vaccine (such as a live-attenuated virus vaccine).
  • RNA molecules can have various lengths but are typically 500-20,000 ribonucleotides long e.g.
  • the RNA can be non-self-replicating (also referred to as “conventional” RNA), or self-replicating; preferably non-self-replicating.
  • the RNA is self-replicating.
  • Self-replicating RNA can be produced using replication elements derived from, e.g., alphaviruses, and substituting sequences encoding the structural viral proteins with that encoding at least an RSV-F protein of the present disclosure.
  • a self-replicating RNA molecule is typically a positive-strand molecule which can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of the Docket No.: 70221WO01 encoded protein (i.e. the RSV-F protein of the present disclosure); or may be transcribed to provide further transcripts with the same sense as the delivered RNA, which are translated to provide in situ expression of the encoded protein.
  • the RNA may encode (i) an RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA and (ii) an RSV-F protein of the present disclosure.
  • the polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4.
  • Such alphavirus-based self-replicating RNA can use a replicase from, for example, a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus (EEEV), or a Venezuelan equine encephalitis virus (VEEV).
  • a self-replicating RNA encoding an RSV-F protein of the present disclosure may have two open reading frames. The first (5') open reading frame encodes a replicase, in particular an alphavirus replicase (e.g.
  • the second (3') open reading frame encodes the RSV-F protein of the present disclosure. Further open reading frames may also be present, encoding (i) one or more further proteins (preferably one or more further antigens, e.g. as detailed above); and/or (ii) accessory polypeptides.
  • the RNA comprises a 5’ cap, such as a 7’-methylguanosine (a.k.a 7-methylguanosine / m 7 G / m7G), which may be added via enzymatic means or a non-enzymatic reaction.
  • the RNA may have the following exemplary 5’ caps: - a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge (also referred to as “Cap O”); - 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) (also referred to as “Cap 1”); - a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotides by a triphosphate bridge, and wherein the first and second 5’ ribonucleotides comprise a 2’-methylated ribose (2’-O- Me) (also referred to as “Cap 2”); - or a 7’-methylguanosine linked 5’
  • the 5’ cap is 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’- Docket No.: 70221WO01 methylated ribose (2’-O-Me), e.g. the 5’ end of the RNA has the structure m7G(5')ppp(5')(2'OMeA)pG.
  • this cap is added non-enzymatically through the use of the following reagent: Said reagent is sold as CLEANCAP Reagent AG (TRILINK BIOTECHNOLOGIES).
  • a cap may be added resulting in the 5’ end of the RNA having the structure m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.
  • This cap may be added non-enzymatically through the use of the following reagent: Said reagent is sold as CLEANCAP Reagent AG (3’OMe) (TRILINK BIOTECHNOLOGIES) Docket No.: 70221WO01
  • 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).
  • at least two non-contiguous stretches of As are used, these may be of differing length.
  • 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.
  • 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.
  • the first stretch is located 5’ relative to the second stretch.
  • 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.
  • 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.
  • 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.
  • 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).
  • said one or more modified ribonucleotides may be, or may comprise, N1- methylpseudouridine (“1m ⁇ ”); pseudouridine (“ ⁇ ”); 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'-
  • the percentage of standard As substituted with A-substitutable modified nucleotide 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%.
  • 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.
  • the percentage of standard Cs substituted with cytosine- substitutable modified nucleotide 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%.
  • the percentage of standard Gs substituted with G-substitutable modified nucleotide e.g.
  • the percentage of standard Us substituted with U-substitutable modified nucleotide 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%.
  • the percentage of standard Us substituted with U-substitutable modified nucleotide 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 1m ⁇ and/or ⁇ (even more preferably 1m ⁇ ) .
  • the one or more modified ribonucleotides detailed above is, or comprise, 1m ⁇ and/or ⁇ , more preferably 1m ⁇ .
  • the RNA may comprise 1m ⁇ and/or ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. there are no standard U nucleotides, nor modified U ribonucleotides other than 1m ⁇ and/or ⁇ , in the RNA; i.e. 100% U substitution).
  • the RNA may comprise 1m ⁇ and/or ⁇ , 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 1m ⁇ and/or ⁇ , in the RNA; i.e. 100% U substitution with no other modified nucleotides being allowed).
  • the RNA may comprise ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. 100% U substitution with ⁇ ).
  • the RNA may comprise ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e. 100% U substitution with ⁇ with no other modified nucleotides being allowed). More preferably, the RNA comprises 1m ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. 100% U substitution with 1m ⁇ ). In an even more preferred embodiment, the RNA comprises 1m ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e.100% U substitution with 1m ⁇ with no other modified nucleotides being allowed). In the embodiments in this paragraph, “[may] comprise[s]...
  • 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%.
  • 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 Docket No.: 70221WO01 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.
  • UTR untranslated region
  • 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
  • Both the 3’ and 5’ UTR may influence expression of the RSV-F protein of the present disclosure through a variety of mechanisms.
  • 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.
  • 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.
  • the RNA is circular RNA.
  • 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; Docket No.: 70221WO01 (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 1
  • the RNA fulfils all of criteria (a) – (f), above.
  • 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).
  • 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
  • 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.
  • a DNA construct preferably a DNA plasmid
  • 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%, 9
  • 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 Docket No.: 70221WO01 (available at e.g.
  • 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).
  • a DNA-dependent RNA polymerase such as the bacteriophage T7, T3 or SP6 RNA polymerases
  • 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.
  • 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.
  • the carrier is non-virion, i.e. free or substantially free of viral capsid.
  • 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.
  • the lipid-based carrier is, or comprises, a cationic nano-emulsion (“CNE”).
  • RNA lipid inorganic nanoparticle
  • LNP lipid nanoparticle
  • the present disclosure also provides an LNP encapsulating a nucleic acid (preferably RNA) which encodes an RSV-F protein of the present disclosure. Docket No.: 70221WO01 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.
  • nucleic acid
  • 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.
  • 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.
  • an LNP may include 1-500 RNA molecules, e.g. ⁇ 200, ⁇ 100, ⁇ 50, ⁇ 20, ⁇ 10, ⁇ 5, or 1-4.
  • an LNP includes fewer than 10 different species of RNA e.g. fewer than 5, 4, 3, or 2 different species.
  • 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.
  • 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.
  • lipids such as: (a) a mixture of cationic lipids and sterols, (b) a mixture of cationic lipids and neutral lipids, (c)
  • 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
  • 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 [28].
  • the cationic lipid has the structure:
  • the cationic lipid has the structure: Docket No.: 70221WO01 (also referred to as lipid RV39).
  • 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 Docket No.: 70221WO01 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 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.
  • 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.
  • 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).
  • 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
  • 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).
  • 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
  • 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.
  • 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
  • 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.
  • 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 Docket No.: 70221WO01 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).
  • 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.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine
  • 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-3 ⁇ -ol (dihydro-FF-MAS-d6), 4,4- dimethyl(d6)-cholest-8(9)-en-3 ⁇ -ol (dihydro T-MAS-d6), zymostenol, sitostanol, campestanol
  • 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 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.
  • 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).
  • compositions in a further independent aspect, 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. [29].
  • 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.
  • 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).
  • 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.
  • such compositions may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc..
  • chelators are typically present at between 10-500 ⁇ 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.
  • 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. Docket No.: 70221WO01 Pharmaceutical compositions of the present disclosure may be prepared in unit dose form.
  • 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
  • carrier preferably lipid nanoparticle
  • 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.
  • 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 ⁇ 120 ⁇ g RNA e.g. ⁇ 100 ⁇ g (e.g. 10-120 ⁇ g or 10-100 ⁇ g, such as 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g, or about 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g), but expression can be seen at much lower levels e.g. ⁇ 1 ⁇ g/dose, ⁇ 100ng/dose, ⁇ 10ng/dose, ⁇ 1ng/dose, etc.
  • Pharmaceutical compositions of the present disclosure may further comprise an adjuvant (i.e.
  • 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.
  • minerals e.g. alum, aluminum hydroxide, aluminum phosphate
  • emulsions including water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions)
  • liposaccharides e.g. alum, aluminum hydroxide,
  • the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod.
  • 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. [30]). Docket No.: 70221WO01 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.
  • compositions of the present disclosure may be lyophilised.
  • 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).
  • 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.
  • 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.
  • 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.
  • the at least one further protein encoded by the nucleic acid of (ii) is a further Pneumoviridae protein (in particular a Pneumoviridae antigen).
  • a further Pneumoviridae protein in particular a Pneumoviridae antigen.
  • Useful further Pneumoviridae proteins 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.
  • 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).
  • 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.
  • 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 include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein.
  • 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.
  • 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.
  • 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.
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • 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 include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin.
  • 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.
  • 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.
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • the nucleic acid of (ii) may encode an RSV-F protein of the present disclosure
  • the nucleic acid of (ii) may encode an Orthomyxovirus antigen, e.g.
  • 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.
  • 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.
  • 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 Docket No.: 70221WO01 acceptable excipient, to produce said composition.
  • said pharmaceutical composition has the features as detailed above throughout this section.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 Docket No.: 70221WO01 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.
  • 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.
  • the first administration 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.
  • 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).
  • prime administration is to a na ⁇ ve subject.
  • the protein antigen may be delivered in the prime and boost administrations as, or via, different formats.
  • 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.
  • a nucleic acid in particular RNA, in particular via a carrier comprising RNA
  • 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.
  • RSV-F proteins, nucleic Docket No.: 70221WO01 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. [32]), 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.
  • 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.
  • an RSV vaccine such as an RSV-F protein, nucleic acid, carrier, or pharmaceutical composition of the present disclosure
  • 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. [33]), 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.
  • the older adult is ⁇ 60 or ⁇ 65 years old (such as 60-120 or 65-120 years old). Docket No.: 70221WO01
  • 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.
  • 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.
  • 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.
  • RSV-F protein in the pre-fusion conformation which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1; wherein the RSV-F protein comprises at least one mutation relative Docket No.: 70221WO01 to the wild-type in a region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation introduces, through substitution or insertion, a residue comprising an H bond donor and/or acceptor moiety in the side chain thereof.
  • the RSV-F protein of any preceding embodiment further comprising (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 Docket No.: 70221WO01 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. 11.
  • the RSV-F protein of any preceding embodiment 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. 12.
  • the RSV-F protein of any preceding embodiment 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. 13.
  • the RSV-F protein of any of embodiments 10-14 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. 16.
  • the RSV-F protein of any of embodiments 10-15 comprising (a) a substitution at position 55 of SEQ ID NO: 1 for T. 17.
  • the RSV-F protein of any of embodiments 10-16 wherein the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop. 18.
  • the RSV-F protein of any of embodiments 10-21 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.
  • the RSV-F protein of any of embodiments 10-22 comprising (c) a substitution of position 348 of SEQ ID NO: 1 for T or N.
  • the RSV-F protein of embodiment 26 comprising (d) a substitution of position 348 of SEQ ID NO: 1 for N. 28.
  • the RSV-F protein of any of embodiments 10-25 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. 29.
  • the RSV-F protein of embodiment 28, 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. 30.
  • the RSV-F protein of any of embodiments 10-22, 26 or 27, comprising: Docket No.: 70221WO01 (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. 31.
  • the RSV-F protein of embodiment 30, 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. 32.
  • 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. 33.
  • the RSV-F protein of any preceding embodiment comprising an F1 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 137-513 of SEQ ID NO: 1.
  • the RSV-F protein of any preceding embodiment comprising a heterologous trimerisation domain on the C-terminus thereof, optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain. 35.
  • 36. The RSV-F protein of any of embodiments 1-33 or 35, 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.
  • 39. The RSV-F protein of any of embodiments 35-38, 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 Docket No.: 70221WO01 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).
  • 41. The RSV-F protein of embodiment 37, 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. 42.
  • the RSV-F protein of embodiment 37, 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. 44.
  • 45. The RSV-F protein of embodiment 37, wherein 16-20, such as 17-2018-20 or 19-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 46.
  • the RSV-F protein of embodiment 45 wherein 20 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F domain.
  • the RSV-F protein of embodiment 48 wherein the increased cell surface expression is for a period of at least 24, 48, 72 or 96 hours.
  • 50 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.
  • 51 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. 52.
  • 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. 53.
  • 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 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
  • RSV-F protein of any preceding embodiment further comprising, relative to SEQ ID NO: 1: Docket No.: 70221WO01 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 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. 55.
  • the RSV-F protein of any preceding embodiment further comprising, relative to SEQ ID NO: 1: 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. 56.
  • the RSV-F protein of any preceding embodiment further comprising, relative to SEQ ID NO: 1: 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. 57.
  • 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. 58.
  • 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.
  • the RSV-F protein of any preceding embodiment which is specifically bound by a pre-fusion mAb with a KD, as measured by SPR, of less than 10 nM; optionally 1 pM – 10 nM. 62.
  • 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 KD, 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. 63.
  • 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 KD, as measured via SPR, of less than 200, 180, 160, 140, 130, 100, 95, 90, 85, 80 or 70 pM. 64.
  • 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. 65.
  • 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 KD, as measured via SPR, of less than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM. 66.
  • 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 KD, as measured via SPR, of less than 200, 180, 160, 140, 120, 110, 100, 95, 80, 70, 60 , 55, 50, 45, or 40 pM. 67.
  • 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.
  • the RSV-F protein according to embodiment 67 comprising the features of any of embodiments 1-66, with the proviso that P residues are not introduced into the protein by the at least one mutation.
  • a trimer comprising three RSV-F proteins according to any preceding embodiment.
  • 70. A nucleic acid encoding the RSV-F protein of any of embodiments 1-68. 71.
  • nucleic acid of embodiment 70 wherein the nucleic acid is, or is comprised within, a viral vector; optionally wherein the viral vector is an adenovirus vector.
  • the nucleic acid of embodiment 70, wherein the nucleic acid is DNA; optionally wherein the DNA is a DNA plasmid. Docket No.: 70221WO01 73.
  • the nucleic acid of embodiment 70, wherein the nucleic acid is RNA.
  • the RNA of embodiment 73 which is non-self-replicating RNA.
  • the RNA of embodiment 73 which is self-replicating RNA. 76.
  • RNA of any of embodiments 73-75 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 1-68, a 3’UTR, and a 3’ poly-A tail.
  • RNA of embodiment 76 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).
  • RNA of embodiment 76 or 77 or, wherein the 3’ poly-A tail comprises a contiguous stretch of 100-500 A ribonucleotides.
  • 79. The RNA of embodiment 76 or 77, 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.
  • 80. The RNA of any of embodiments 73-79, comprising a modified ribonucleotide.
  • RNA of embodiment 81 wherein the RNA comprises 1m ⁇ and neither standard U ribonucleotides nor other modified U ribonucleotides; optionally wherein the RNA comprises 1m ⁇ and neither standard U ribonucleotides nor other modified ribonucleotides.
  • 83 The RNA of any of embodiments 73-82, having a GC content of 55-70%.
  • the RNA of any of embodiments 73-82 having a GC content of 40-60%.
  • a carrier comprising nucleic acid of any of embodiments 70, 72 or 73-83.
  • the carrier of embodiment 85 which is a lipid nanoparticle. 87.
  • the lipid nanoparticle of embodiment 86 comprising a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. 88.
  • the lipid nanoparticle of any of embodiments 87-92 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.
  • a pharmaceutical composition comprising the RSV-F protein of any of embodiments 1-68, trimer of embodiment 69, nucleic acid of any of embodiments 70-83, or carrier of any of embodiments 85-93; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant. 95.
  • a vaccine composition comprising the RSV-F protein of any of embodiments 1-68, trimer of embodiment 69, nucleic acid of any of embodiments 70-83, or carrier of any of embodiments 85- 93; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant 96.
  • the pharmaceutical composition of embodiment 94 for use in medicine.
  • the pharmaceutical composition for use of embodiment 96 for use in a method of raising an immune response in a subject; optionally a protective immune response in a subject.
  • the pharmaceutical composition for use of embodiment 96 or 97 for use in the treatment or prevention of RSV. 99.
  • the pharmaceutical composition for use of any of embodiments 97-99, wherein the subject is a pregnant human female; optionally ⁇ 28 weeks pregnant. Docket No.: 70221WO01 103.
  • 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 1-68, trimer of embodiment 69, nucleic acid of any of embodiments 70-83, carrier of any of embodiments 85-93, pharmaceutical composition of embodiment 94, or vaccine composition of embodiment 95.
  • 104 Use of the RSV-F protein of any of any of embodiments 1-68, trimer of embodiment 69, nucleic acid of any of embodiments 70-83, or carrier of any of embodiments 85-93, in the manufacture of a medicament.
  • kits comprising the RSV-F protein of any of embodiments 1-68, trimer of embodiment 69, nucleic acid of any of embodiments 70-83, carrier of any of embodiments 85-93, pharmaceutical composition of embodiment 94, or vaccine of embodiment 95, and instructions for use. 108.
  • Respiratory syncytial virus fusion (RSV-F) protein in the pre-fusion conformation which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1; wherein the RSV-F protein comprises at least one mutation relative to the wild-type in a region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation introduces, through substitution or insertion, a residue comprising a hydrogen bond donor and/or acceptor moiety in its side chain.
  • the RSV-F protein of embodiment 108 or 109 wherein the region corresponding to positions 217-239 of SEQ ID NO:1 has at least 80%, 85%, 90% or 95% sequence identity to positions 217- 239 of SEQ ID NO:1.
  • the RSV-F protein of embodiment 111 comprising a substitution at position 228 of SEQ ID NO: 1 for K, R, Q or N; optionally K, R or Q; optionally K or R; optionally K; Docket No.: 70221WO01 and/or a substitution at position 232 of SEQ ID NO: 1 for N. 113.
  • the RSV-F protein of embodiment 113 comprising a substitution at position 228 (N) of SEQ ID NO: 1 for K. 115.
  • the RSV-F protein of any one of embodiments 108-114 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. 116.
  • the RSV-F protein of embodiment 115 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.
  • the RSV-F protein of embodiment 115 or 116 comprising a glycan linked to position 348; optionally comprising N-acetyl glucosamine. 118.
  • the RSV-F protein of any one of embodiments 108-117 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.
  • the RSV-F protein of any one of embodiments 108-118 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. 120.
  • a homotrimer comprising three RSV-F proteins of any one of embodiments 108-119 having the same amino acid sequence.
  • 121. A nucleic acid encoding the RSV-F protein of any one of embodiments 108-119. 122. The nucleic acid of embodiment 121, wherein the nucleic acid is RNA. Docket No.: 70221WO01 123.
  • a lipid nanoparticle comprising the nucleic acid of embodiment 121 or 122.
  • a pharmaceutical composition comprising the RSV-F protein of any of embodiments 108- 119, trimer of embodiment 120, nucleic acid of embodiment 121 or 122, or lipid nanoparticle of embodiment 123; optionally for use in medicine. 125.
  • composition for use of embodiment 124 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.
  • 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 108-119, trimer of embodiment 120, nucleic acid of embodiment 121 or 122, or lipid nanoparticle of embodiment 123, or pharmaceutical composition of embodiment 124.
  • 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.
  • 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. Docket No.: 70221WO01 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.
  • HPLC High Performance Liquid Chromatography
  • 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).
  • 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.
  • Binding Kinetics using BIACORE 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 ⁇ L/min for 120 sec followed by 2400 sec dissociation. Antigen concentrations ranged from 0-10 nM.
  • 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. Docket No.: 70221WO01 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.
  • 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. Docket No.: 70221WO
  • mRNAs were produced by in vitro transcription with capping analogue (TRILINK CLEANCAP) and 100% uridine replacement (with 1m ⁇ ), 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).
  • 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 ⁇ g/mL IgGs captured in flow cell 2 on a Protein A chip, leaving flow cell 1 as a reference.
  • 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 ⁇ g/mL or 480 ⁇ g/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.
  • % Relative error for the back calculated Docket No. 70221WO01 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.
  • 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.
  • microspheres/well are added in a volume of 50 ⁇ l PBS with 1% BSA + 0.05% Na Azide (assay buffer) to 100 ⁇ l 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 ⁇ l/well of PBS, 0.05% Tween-20 (wash buffer) on a plate washer using a magnet to allow settling of beads between washes.
  • r-PE r-Phycoerythrin conjugated anti- mouse IgG
  • JACKSON IMMUNORESEARCH r-Phycoerythrin conjugated anti- mouse IgG
  • Serum anti-RSV preF IgG binding was Docket No.: 70221WO01 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 ⁇ L inoculum in EMEM. Virus was added to serum and the plates were incubated for 1 hour at 25-30°C.
  • PFU plaque forming units
  • 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”.
  • 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 ⁇ g dose groups. Heterogeneity of variances between groups was considered in the model with 0.3 ⁇ g 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.
  • 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).
  • DSPC 1,2-diastearoyl-sn- glycero-3-phosphocholine
  • a 28 - gauge needle was used to administer 50 ⁇ L (25 ⁇ L in each hindleg thigh muscle) of either saline or a high (2 ⁇ g) or low (0.2 Docket No.: 70221WO01 ⁇ g) 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.
  • mice were anesthetized under isoflurane to collect 100 ⁇ L of whole blood (40 ⁇ L of serum) by submandibular collection method.
  • mice were anesthetized under isoflurane and terminally exsanguinated by cardiac stick to obtain an estimated 200 ⁇ L to 500 ⁇ L of whole blood, (100 ⁇ L 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).
  • RSV media Biorich DMEM supplemented with 3%-fetal bovine serum (FBS; MOREGATE, FBSAE1000), 2 mM L-Glutamine, and 50 ⁇ g/mL Gentamicin.
  • RSV lab-adapted A-Long virus was diluted to approximately 50-150 foci-forming units per 25 ⁇ L. 60 ⁇ L of virus was added into the wells with the same volume of serum dilutions and incubated for 2 hours at 35°C 5% CO2.
  • 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.
  • ANOVA analysis of variance
  • 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.
  • 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 Docket No.: 70221WO01 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.
  • 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 ⁇ 5, 5 residues to the end; position ⁇ 10, 10 residues to the end; position ⁇ 15, 15 residues to the end; position ⁇ 20, 20 residues to the end; position ⁇ 25, 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.
  • 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.
  • BJ cells 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% CO2.
  • FBS CORNING 35-016-CV
  • antibiotic GEBCO 15140-122
  • glutamine GIBCO 25030-081
  • 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.
  • THERMOFISHERSCIENTIFIC J19943-K2 4% paraformaldehyde
  • 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.
  • 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.
  • F(iii) which includes a full cytoplasmic tail deletion (dCT), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20 (low dose only), DS-Cav1 (high dose only), F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (mRNA constructs detailed in Table 10, below) into each mouse on day 0 and day 21.
  • mice were anesthetized under isoflurane to collect 100 ⁇ L of whole blood (40 ⁇ L of serum) by submandibular collection method.
  • mice were anesthetized under isoflurane and terminally exsanguinated by cardiac stick to obtain an estimated 200 ⁇ L to 500 ⁇ L of whole blood, (100 ⁇ L 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.
  • 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.
  • Design F21 Docket No.: 70221WO01 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 Docket No.: 70221WO01 * 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.
  • 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.
  • 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.
  • 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 3 ⁇ g 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.
  • Table 5A The total level of RSV pre-fusion protein specific IgG binding antibodies from immunized mice with a 3 ⁇ g 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 0.3 ⁇ g 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 0.3 ⁇ g 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 ⁇ g 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 ⁇ g 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 ⁇ g 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 ⁇ g dose at day 35.
  • 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 ⁇ g ( Figure 36A) or 0.2 ⁇ g ( Figure 36B) of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319 Docket No.: 70221WO01 (where each point represents an individual animal). There were no binding antibody responses in the saline control group (data not shown).
  • 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 ⁇ g ( Figure 37A) or 0.2 ⁇ g ( 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).
  • F217 generated the highest neutralization to RSV A.
  • the steady-state, total cell-surface RSV F protein expression of the design, F318 CT ⁇ 20 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.
  • 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). Docket No.: 70221WO01 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).
  • 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).
  • 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 were each modelled as their respective mRNA doppelgangers for an in vivo study (Example 13).
  • 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 ⁇ g ( Figure 43A) or 0.2 ⁇ g ( Figure 43B) of RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (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 2 ⁇ g 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 2 ⁇ g dose of F318, F318 ⁇ CT20, F319, and F319 ⁇ CT20 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).
  • F318 ⁇ CT20 achieved noninferiority when compared to F(ii) and F(iii) at day 35 (2wp2) ( Figures 43B, D and F).
  • One 0.2 ⁇ g dose of F318 ⁇ CT20 or F319 ⁇ CT20 elicited significantly higher pre-F IgG titres compared to the non- ⁇ CT20 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 ⁇ g or ( Figure 44B) 0.2 ⁇ g of RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (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.
  • 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: 13 is referred to herein as wild-type.
  • SEQ ID NO: 84 is referred to herein as wild-type.
  • SEQ ID NO: 107 is referred to herein as wild-type.

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Abstract

La présente divulgation concerne, entre autres, une protéine de fusion du virus respiratoire syncytial (VRS-F) dans la conformation de pré-fusion, qui est mutée par rapport au VRS-F de type sauvage selon SEQ ID NO : 1; la protéine RSV-F comprenant au moins une mutation par rapport au type sauvage dans une région correspondant aux positions 217-239 de SEQ ID NO : 1; la ou les mutations introduisant, par substitution ou insertion, un résidu comprenant une fraction donneur et/ou accepteur de liaison hydrogène dans sa chaîne latérale.
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