[go: up one dir, main page]

EP4577557A1 - Protéines rsv-f - Google Patents

Protéines rsv-f

Info

Publication number
EP4577557A1
EP4577557A1 EP23733931.2A EP23733931A EP4577557A1 EP 4577557 A1 EP4577557 A1 EP 4577557A1 EP 23733931 A EP23733931 A EP 23733931A EP 4577557 A1 EP4577557 A1 EP 4577557A1
Authority
EP
European Patent Office
Prior art keywords
seq
rsv
substitution
protein
present disclosure
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23733931.2A
Other languages
German (de)
English (en)
Inventor
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
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
GlaxoSmithKline Biologicals SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by GlaxoSmithKline Biologicals SA filed Critical GlaxoSmithKline Biologicals SA
Priority claimed from PCT/EP2023/066332 external-priority patent/WO2024041773A1/fr
Publication of EP4577557A1 publication Critical patent/EP4577557A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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 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.
  • the RSV-F proteins generated by the inventors involve the stabilisation of multiple domains and folds of RSV-F.
  • 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).
  • 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.
  • 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 and comprises (a), (b) and (c): (a) (ai) at least one mutation relative to the wild-type in a region corresponding to positions 38- 60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (aii) at least one mutation relative to the wild-type in a region corresponding to positions 296- 318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation relative to the wild-type in a region corresponding to positions 208-216 of SEQ ID NO:1, where
  • 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 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).
  • Biolayer interferometry (BLI) of the histidine-tagged sequences indicates that designs F21 and F28 (referred to as 21 and 28) express in mammalian cells, when compared to spent media (confirmed by subsequent experiments, data not shown).
  • Figure 3. Binding affinity (K D ) of pre-fusion RSV-F-specific antibodies for “Round 1” RSV-F mutants. The prefusion conformation of the two designs F21 and F28 were tested with biolayer interferometry (BLI) against antibodies AM14 (quaternary epitope), D25 (site ⁇ ), RSB1 (site V) motavizumab (site II).
  • K D binding affinity
  • 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.
  • 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 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.
  • 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 protein in the pre-fusion conformation which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1 and comprises (a), (b) and (c): (a) (ai) at least one mutation relative to the wild-type in a region corresponding to positions 38- 60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (aii) at least one mutation relative to the wild-type in a region corresponding to positions 296- 318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation relative to the wild-
  • RSV-F protein in the pre- fusion conformation which is mutated relative to wild-type RSV-F according to SEQ ID NO: 1, and comprises (a) and (b) as defined above, and: (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.
  • the present disclosure also provides, in a sixth 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, and comprises (d) as defined above.
  • the present disclosure also provides, in a seventh independent aspect, 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 the at least one mutation introduces neither a disulphide bond nor a P residue into said wild-type protein.
  • a multimer comprising protomers, wherein at least one protomer comprises or consists of an RSV-F protein of the present disclosure (i.e.
  • SEQ ID NO: 1 which contains the two substitutions
  • wild-type in accordance with e.g. [12]
  • References to “wild-type RSV-F according to SEQ ID NO: 1” and “SEQ ID NO: 1” are herein used interchangeably.
  • 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 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 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).
  • cryo-EM is performed as in the Examples (see subsection Cryo-electron microscopy of RSV-F designs F21, F216, and F224.
  • RSV-F proteins of the present disclosure comprise (according to said first, second and third independent aspects) or may comprise (according to said fourth, fifth, sixth, seventh and eighth independent aspects): (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 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 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) may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation 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).
  • 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).
  • 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.
  • RSV-F proteins of the present disclosure comprise (according to said second and sixth independent aspects) or may comprise (according to said first, third, fourth, seventh and eighth independent aspects): 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.
  • 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 may comprise at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 (e.g.
  • 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. 1, 2, 3, 4, 5, 6, 7 or 8) 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 228 (N) for K, R, Q, N or A; preferably K, R, Q or N; preferably K, R or Q; preferably K or R; preferably K; 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; optionally a substitution at position 445
  • substitutions at positions 228, 315, 455 and 459 includes substitutions found in design F225 (F225 also has S55T, S215A 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.
  • 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%, 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 NOs: 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.
  • 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%, 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: 32 or 89 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 and S348N (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 32 and 89.
  • 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.
  • 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.
  • 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 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 portion preferably includes the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M, which are present in SEQ ID NO: 20.
  • 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 may encode an RSV-F protein comprising or consisting of an amino acid sequence according to any of SEQ ID NO: 40-49, 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: 40-49 (where applicable) relative to SEQ ID NO: 1.
  • 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 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%, 80%, 81% 82%, 83%,
  • 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).
  • 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.
  • 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).
  • 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. 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. comprising a wild-type cytoplasmic tail, e.g.
  • 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.
  • 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.
  • 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, 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) may encode an RSV-F protein of the present disclosure only (i.e. the nucleic acid encodes a single protein). Alternatively, the nucleic acid may encode multiple proteins, of which one is the RSV-F protein of the present disclosure. In some embodiments, 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. a ferritin 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. 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.
  • 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.
  • 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 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 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 overall result of this sequence of transcriptions is substantial amplification in the number of the introduced RNAs, and so the encoded RSV-F protein of the present disclosure (potentially in addition to further proteins as detailed above) becomes a major polypeptide product of the cells.
  • the total number of A ribonucleotides (“As”) in at least two non- contiguous stretches may be, for example, 10-700, such as 10-600, 10-500, 20-500, 50-500, 70-500, 100-500, 20-400, 30-300, 40-200, 50-150, 70-120, 100-120, or, in particular, 100-120.
  • the total number of As in a (in particular, only one) contiguous stretch may be, for example, 10-700; such as 10-600, 20-600 or in particular 40-600 (such as 50-600, 80-600, 80-550, 100-500; or 40-70, 50-65 or 55-65). Wherein at least two non-contiguous stretches of As are used, these may be of differing length.
  • 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'-O-dimethyladenosine; 1- methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladeno
  • 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.
  • 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).
  • 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).
  • “[may] comprise[s]... and neither [X]...nor [Y]” may be used interchangeably with the wording “[may] comprise[s]... and does not comprise... [X] and/or [Y] ”.
  • the RNA is codon-optimised.
  • 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; (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 1m ⁇ , and neither standard U ribonu
  • 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 (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).
  • 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.
  • 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
  • compositions typically further comprise a pharmaceutically acceptable excipient.
  • Pharmaceutically acceptable excipients are well-known in the art, see, e.g. [27].
  • Such compositions are generally for immunising subjects against disease, preferably against RSV.
  • pharmaceutical compositions of the present disclosure are generally considered vaccine compositions.
  • Pharmaceutical compositions of the present disclosure may comprise the RSV-F protein, nucleic acid (preferably RNA) and/or carrier (preferably lipid nanoparticle) in plain water (e.g. “w.f.i.”) or in a buffer e.g. a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer.
  • Buffer salts will typically be included in the 5-20mM range.
  • Pharmaceutical compositions of the present disclosure may have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0.
  • Pharmaceutical compositions of the present disclosure compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10 ⁇ 2 mg/mL NaCl is typical, e.g. about 9 mg/mL (or 9 mg/mL).
  • Pharmaceutical compositions of the present disclosure may include metal ion chelators (in particular, in embodiments wherein such compositions comprise RNA). These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis.
  • 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 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.
  • 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 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. Medical uses and methods of treatment
  • the present disclosure also provides, in a further independent aspect, an RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, for use in medicine.
  • 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 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.
  • 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
  • the first administration (“prime”) may induce proliferation and maturation of B and/or T cell precursors specific to one or more immunogenic epitopes present on the delivered antigen (induction phase).
  • the second (and in some cases subsequent) administration (“boost”) may further stimulate and potentially select an anamnestic response of cells elicited by the prior administration(s).
  • the different administrations may be given by the same or different routes e.g.
  • prime administration 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.
  • two prime administrations may be administered 3-9 weeks apart (e.g. 4-9, 5-9, 6-9, 7-9 or 7-8 weeks apart, or about two months apart), followed by one or more boost administrations 4-14 months after the second prime administration (e.g. 5-13, 6-13, 7-13, 8-13, 9-13, 10-13 or 11-13 months, or about one year).
  • prime administration is to a na ⁇ ve subject.
  • RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure will generally be administered directly to the subject.
  • Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or to the interstitial space of a tissue).
  • Alternative delivery routes include rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • the RSV-F proteins, nucleic acids, carriers, or pharmaceutical composition of the present disclosure will be administered intramuscularly or intradermally (in particular via a needle such as a hypodermic needle), more preferably intramuscularly.
  • the RSV-F proteins, nucleic acids, lipid carriers, or pharmaceutical compositions of the present disclosure may be used to elicit systemic and/or mucosal immunity.
  • the subject of a method of vaccination according to the present disclosure may be a child (preferably an infant) or adult (preferably an older adult or pregnant female). Immunocompromised individuals may also be the subject of such vaccination (whether children or adults).
  • 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.
  • infants preferably human infants
  • the immune systems of infants are immature (see, e.g. [30]), hence this population is susceptible to RSV infection and resulting disease.
  • Infant vaccination may prevent lower respiratory tract infection (in particular, bronchiolitis and (broncho-)pneumonia).
  • the infant may be less than one year old, such as less than: 11, 10, 9, 8, 7, 6, 5, 4 or less than 3 months old.
  • the infant may be ⁇ one month old, such as ⁇ : 2, 3, 4, 5 or ⁇ 6 months old.
  • the infant is 2-6 months old (i.e.
  • 39. The RSV-F protein of embodiment 31, wherein 16-20, such as 17-20, 18-20 or 19-20 residues are deleted from the C-terminal end of the cytoplasmic domain of the RSV-F protein. 40.
  • compositions 93-95 wherein the subject is 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 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, carrier of any of embodiments 81-89, pharmaceutical composition of embodiment 90, or vaccine composition of embodiment 91.
  • kits comprising the RSV-F protein of any of embodiments 1-64, trimer of embodiment 65, nucleic acid of any of embodiments 66-80, carrier of any of embodiments 81-89, pharmaceutical composition of embodiment 90, or vaccine of embodiment 91, and instructions for use. 104.
  • the RSV-F protein of embodiment 104 wherein: (a) the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 comprise ⁇ sheets; (b) the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 comprises a ⁇ sheet and a loop. 106.
  • the RSV-F protein of any of embodiments 104 or 105 wherein: (a) the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 38-60 of SEQ ID NO:1; and/or, optionally and, the region corresponding to positions 296-318 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 296-318 of SEQ ID NO:1; (b) the region corresponding to positions 38-60 of SEQ ID NO:1 has least 75% or 85% sequence identity to positions 208-216 of SEQ ID NO:1; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 has at least 75% or 85% sequence identity to positions 345-352 of SEQ ID NO: 1.
  • the RSV-F protein of any one of embodiments 104-106 comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: for T or N. 108.
  • the RSV-F protein of embodiment 107 comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; (b) a substitution at position 215 of SEQ ID NO: 1 for A; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: 1 for N. 109.
  • the RSV-F protein of embodiment 107 or 108 comprising a glycan linked to position 348; optionally comprising N-acetyl glucosamine.
  • the RSV-F protein of any one of embodiments 104-110 comprising an F2 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 26-108 of SEQ ID NO: 1. 112.
  • the RSV-F protein of any one of embodiments 104-111 comprising an F1 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1. 113.
  • a homotrimer comprising three RSV-F proteins of any one of embodiments 104-112 having the same amino acid sequence.
  • 114. A nucleic acid encoding the RSV-F protein of any one of embodiments 104-112. 115. The nucleic acid of embodiment 114, wherein the nucleic acid is RNA.
  • 116. A lipid nanoparticle comprising nucleic acid of embodiment 114 or 115.
  • 117. A pharmaceutical composition comprising the RSV-F protein of any of embodiments 104- 112, trimer of embodiment 113, nucleic acid of embodiment 114 or 115, or lipid nanoparticle of embodiment 116; optionally for use in medicine. 118.
  • composition for use of embodiment 117 for use in a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; a human older adult, optionally ⁇ 60 years old; or a pregnant human female, optionally ⁇ 28 weeks pregnant.
  • a method of inducing an immune response against RSV in a subject comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of embodiments 104-112, trimer of embodiment 113, nucleic acid of embodiment 114 or 115, or lipid nanoparticle of embodiment 116, or pharmaceutical composition of embodiment 118. 120.
  • Respiratory syncytial virus fusion (RSV-F) protein in the pre-fusion conformation which is mutated relative to SEQ ID NO: 1 and comprises (a), (b) and (c): (a) (i) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 38- 60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (ii) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation relative to SEQ ID NO: 1 in a region corresponding to positions 208- 216 of SEQ ID NO:1, wherein the at least one mutation increases the
  • the RSV-F protein of embodiment 120 wherein: (a) the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 comprise ⁇ sheets; (b) the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 comprises a ⁇ sheet and a loop. 122.
  • the RSV-F protein of any one of embodiments 120 or 121 wherein: (a) the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 38-60 of SEQ ID NO:1; and/or, optionally and, the region corresponding to positions 296-318 of SEQ ID NO:1 has at least 90% or 95% sequence identity to positions 296-318 of SEQ ID NO:1; (b) the region corresponding to positions 208-216 of SEQ ID NO:1 has least 75% or 85% sequence identity to positions 208-216 of SEQ ID NO:1; and/or, optionally and, (c) the region corresponding to positions 345-352 of SEQ ID NO:1 has at least 75% or85% sequence identity to positions 345-352 of SEQ ID NO: 1.
  • RSV-F protein of any one of embodiments 120-122 comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO: 1 for A, P, V, I, or F; optionally A or P; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: for T or N. 124.
  • the RSV-F protein of embodiment 123 comprising: (a) a substitution at position 55 of SEQ ID NO: 1 for T; (b) a substitution at position 215 of SEQ ID NO: 1 for A; and/or, optionally and, (c) a substitution of position 348 of SEQ ID NO: 1 for N. 125.
  • the RSV-F protein of embodiment 123 or 124 comprising a glycan linked to position 348; optionally comprising N-acetyl glucosamine.
  • the RSV-F protein of any one of embodiments 120-126 comprising an F2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to positions 26-108 or 26-109 of SEQ ID NO: 1. 128.
  • the RSV-F protein of any one of embodiments 120-127 comprising an F1 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% sequence identity to positions 137-513 of SEQ ID NO: 1. 129.
  • the RSV-F protein of any one of embodiments 120-128, comprising a heterologous trimerisation domain at the C-terminus thereof, and/or C-terminal to the F1 domain; optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain.
  • the RSV-F protein of any one of embodiments 120-128, comprising a transmembrane domain at the C-terminus thereof, and/or C-terminal to the F1 domain; and optionally a cytoplasmic domain C-terminal to said transmembrane domain.
  • the RSV-F protein of any of embodiments 120-134 wherein the RSV-F protein comprises an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1.
  • 136. A homotrimer comprising three RSV-F proteins of any one of embodiments 120-135 having the same amino acid sequence.
  • 137. A nucleic acid encoding the RSV-F protein of any one of embodiments 120-135.
  • 138. The nucleic acid of embodiment 137, wherein the nucleic acid is RNA.
  • a lipid nanoparticle comprising nucleic acid of embodiment 137 or 138. 140.
  • a pharmaceutical composition comprising the RSV-F protein of any of embodiments 120- 135, trimer of embodiment 136, nucleic acid of embodiment 137 or 138, or lipid nanoparticle of embodiment 139; optionally for use in medicine.
  • the pharmaceutical composition for use of embodiment 140 for use in a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; a human older adult, optionally ⁇ 60 years old; or a pregnant human female, optionally ⁇ 28 weeks pregnant. 142.
  • a method of inducing an immune response against RSV in a subject comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of embodiments 120-135, trimer of embodiment 136, nucleic acid of embodiment 137 or 138, or lipid nanoparticle of embodiment 139, or pharmaceutical composition of embodiment 140.
  • RSV-F protein any of embodiments 120-135, trimer of embodiment 136, nucleic acid of embodiment 137 or 138, or lipid nanoparticle of embodiment 139, or pharmaceutical composition of embodiment 140.
  • RSV-F mutants Materials & Methods Expression and Purification of RSV-F mutants, DS-Cav1 and RSV-F mAbs (Examples 3, 4, 6, 7 and 9) RSV-F mutants were synthesized (GENEWIZ/AZENTA) and cloned into a CMV-based vector with a C-terminal thrombin-cleavable double Strep tag II tag followed by a 6x His-tag.
  • DS-Cav1 and RSV-F mutants were transiently expressed in Expi293 F cells (THERMO FISHER SCIENTIFIC). Media was harvested after 4 days, and purified using affinity chromatography, either nickel affinity or strep-tag affinity.
  • cell harvest medium was passed over a HisTrap Excel column (CYTIVA) and eluted with a step gradient of imidazole.
  • CYTIVA HisTrap Excel column
  • 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.
  • RSV antibodies, AM14, D25, Motavizumab, and RSB1 were transiently transfected in EXPI293F cells (THERMO FISHER SCIENTIFIC) according to manufacturer's instructions and media was harvested 6-7 days post transfection.
  • the cell harvest media was passed over a MABSELECT SURE COLUMN (CYTIVA) and eluted with 0.1 M citrate pH 3 into 1 M Tris pH 9; buffer exchanged into 20 mM HEPES pH 7, 150 mM NaCl; followed by a final size exclusion chromatography step on a HILOAD 16/600 Superdex 30 pg column (CYTIVA) in 20 mM Hepes pH 7, 150 mM NaCl.
  • 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.
  • the microspheres 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. Following the wash, 50 ⁇ L/well of r-Phycoerythrin (r-PE) conjugated anti- mouse IgG (JACKSON IMMUNORESEARCH) was added, and plates are incubated, covered, on an orbital shaker at RT for 60 minutes.
  • r-PE r-Phycoerythrin conjugated anti- mouse IgG
  • RNA immunisation In vivo RNA immunisation (Example 11) All recombinant RNA molecules were produced by in vitro transcription using N1-methyl pseudouridine to replace all uridines. All recombinant RNA molecules comprised a cap-1 5' cap (TRILINK CLEANCAP) and a 3' poly(A) tail. The mRNAs were purified and evaluated for mRNA integrity (by capillary and glyoxal denaturing gel electrophoresis).
  • the RV39 LNP mRNA constructs were then formulated in LNPs comprising 40 mol% cationic lipid RV39; 2 mol% PEG-conjugated lipid; 48 mol% cholesterol; and 10 mol% 1,2-diastearoyl-sn- glycero-3-phosphocholine (DSPC).
  • 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 ⁇ 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
  • Cytoplasmic tail deletions (Example 12) Cloning and expression of RSV-F monoclonal antibodies: Plasmids encoding RSV-F antibodies, AM14, D25 and Motavizumab were transiently transfected in Expi293F cells (THERMO FISHER SCIENTIFIC) according to manufacturer's instructions and media was harvested 6-7 days post transfection.
  • the cell harvest media was passed over a MABSELECT SURE COLUMN (CYTIVA) and eluted with 0.1 M citrate pH 3 into 1 M Tris pH 9; buffer exchanged into 20 mM HEPES pH 7, 150 mM NaCl; followed by a final size exclusion chromatography step on a HILOAD 16/600 Superdex 30 pg column (CYTIVA) in 20 mM Hepes pH 7, 150 mM NaCl.
  • RSV F wildtype protein sequence SEQ ID NO: 107 protein was back-translated to a nucleic acid sequence using specific metrics for codon optimality.
  • the DNA gBLOCKS (INTEGRATED DNA TECHNOLOGIES) were amplified by PCR, and ligation into a vector with a polyA tail. Amino acid substitutions N67I and S215P (also known as design F(ii)) were incorporated DNA constructs and encoded in the eventual mRNA and protein. The additional variations (also known as DS-Cav1, F(iii), F(i), F318 and F319) and their amino acid substitutions are shown in the Table 7. Table 7 – substitutions in parent mRNAs designs tested in cell-based assay
  • 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.
  • RNAs were produced by in vitro transcription with capping analogue (TRILINK CLEANCAP A/G) and 100% uridine replacement (with 1m ⁇ ), followed with DNase I, phosphatase treatments (NEW ENGLAND BIOLABS) and silica column purification (QIAGEN). Newly synthesized mRNAs were validated by Tapestation (Agilent) and denaturing RNA gels.
  • 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% CO 2 .
  • DMEM LONZA 12-614F
  • FBS CORNING 35-016-CV
  • antibiotic GIBCO 15140-122
  • glutamine GBCO 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).
  • 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.
  • TRANSIT mRNA transfection reagent MIRUS mir2250
  • OPTIMEM OPTIMEM
  • Nonspecific antibody- binding for fixed cells was blocked using 1% Normal Horse Serum (GIBCO 16050-130) in PBS (1%NHS-PBS).
  • RSV F protein was labelled by incubating cell monolayers with the respective human anti-RSV F monoclonal antibodies: AM14, D25, motavizumab. Each well was incubated with 331ng of the respective antibody in blocking media overnight at 4C. Cell monolayers are rinsed 3 times with 1%NHS-PBS. Indirect immunofluorescent detection of RSV F expression was completed by incubating cell monolayers with goat anti-human antibody with ALEXA647 (THERMOFISHER A-21445) diluted 1:2000 in 1%NHS-PBS.
  • cell nuclei were co-labelled with DYECYCLE Violet (THERMOFISHER V35003) following manufacturer's recommendations.
  • Cell monolayers are rinsed 3 times with 1% NHS-PBS then cells are stored in PBS for imaging.
  • 9 fields per well were imaged in the DYECYCLE Violet and Alexa647 fluorescent channels using the 10x objective on the THERMOSCIENTIFIC Cell Insight CX7 automated imaging system.
  • Image analysis is completed using the Target Activation protocol associated with the CELLOMICS (HCS NAVIGATOR Ver 6.6.2 Build 8533) image analysis system.
  • Data analysis was completed using MICROSOFT EXCEL and PRISM GRAPHPAD.
  • 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.
  • 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.
  • Example 2 Evolutionary Sequence and Structure-Based Design of RSV-F (“Round 1”)
  • a consensus design method was used to select non-redundant evolutionary homologs for clustering, searching against the Uniclust database and concatenated to produce a combined position specific scoring matrix (PSSM) with the HHblits module.
  • PSSM position specific scoring matrix
  • the ROSETTA Protein Suite was used for structural design, constraining the trimeric chains with cyclic symmetry to produce homo-oligomers.
  • An evolutionary workflow was then used to perform a single point mutation scan to ascertain residues that surpassed certain energy thresholds and for the combinatorial design of in silico mutants ( Figure 1).
  • Example 3 Evolutionary Sequence and Structure-Based Design of RSV-F (“Round 1”)
  • the consensus sequence designs were transfected into human embryonic kidney 293 cells and tested for expression and antigenicity in supernatant.
  • Biolayer interferometry (BLI) was used to test expression via affinity-based histidine tags, resulting in the identification of two constructs (F21 and F28), see Figure 2.
  • Design F21 had a subset of 31 substitutions relative to RSV-F WT (see Figure 5), while Design F28 had 9 substitutions relative to RSV-F WT.
  • Example 8 Toluene nitrosulphonic acid (TNS) fluorescence assay for determining pKa Steps (1) – (14): (1) admixing 400 ⁇ L of 2 mM of the cationic lipid that is in 100 volume % ethanol and 800 ⁇ L of 0.3 mM of fluorescent probe TNS, which is in 90 volume % ethanol and 10 volume % methanol, thereby obtaining a lipid/TNS mixture; (2) admixing 7.5 ⁇ L of the lipid/TNS mixture and 242.5 ⁇ L of a first buffer comprising a sodium salt buffer comprising 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, wherein the first buffer has a first pH from 4.44 to 4.52, thereby obtaining a first mixture, and dispensing 100 ⁇ L of the first mixture in a first well of a 96-well
  • 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.
  • 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.
  • the GMT at a 95% confidence interval are shown.
  • mice 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 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 (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.
  • 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).
  • Example 13 In vivo mRNA immunisation (“Round 3” designs) RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319 and F319 ⁇ CT20 was administered to mice as set out in the Materials and Methods section.
  • 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.
  • SEQ ID NO: 1 Amino acid (AA) sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094 (no foldon, transmembrane domain or cytoplasmic tail). SEQ ID NO: 1 is referred to herein as wild-type.
  • SEQ ID NO: 2 AM14 light chain AA sequence
  • SEQ ID NO: 3 AM14 heavy chain AA sequence
  • SEQ ID NO: 4 D25 light chain AA sequence
  • SEQ ID NO: 5 D25 heavy chain AA sequence
  • SEQ ID NO: 6 Motavizumab light chain AA sequence
  • SEQ ID NO: 7 Motavizumab heavy chain AA sequence
  • SEQ ID NO: 8 RSB1 light chain AA sequence
  • SEQ ID NO: 9 RSB1 heavy chain AA sequence
  • SEQ ID NO: 10 AA sequence of exemplary F2-F1 linker sequence
  • SEQ ID NO: 11 AA sequence of exemplary F2-F1 linker sequence
  • SEQ ID NO: 12 AA sequence of exemplary F2-F1 linker sequence
  • SEQ ID NO: 13 AA sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094 – furin processed, without signal sequence (no foldon
  • SEQ ID NO: 72 RNA sequence of construct KM112 (encoding F217) – all U ribonucleotides are 1m ⁇ – GC content 49.90% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 73 RNA sequence of construct KM113 (encoding F224) – all U ribonucleotides are 1m ⁇ – GC content 48.90% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 74 RNA sequence of construct KM114 (encoding F225) – all U ribonucleotides are 1m ⁇ – GC content 49.00% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 75 RNA sequence of construct KM116 (encoding F315) – all U ribonucleotides are 1m ⁇ – GC content 49.80% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 79 RNA sequence of construct KM120 (encoding F319) – all U ribonucleotides are 1m ⁇ – GC content 49.80% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 80 RNA sequence of construct KM121 (encoding F320) – all U ribonucleotides are 1m ⁇ – GC content 49.90% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 143 RNA sequence of construct KM112C10 (encoding F217) – all U ribonucleotides are 1m ⁇ – GC content 49.70% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 144 RNA sequence of construct KM118C2 (encoding F317) – all U ribonucleotides are 1m ⁇ – GC content 48.40% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 145 RNA sequence of construct KM120C3 (encoding F319) – all U ribonucleotides are 1m ⁇ – GC content 49.60% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 146 RNA sequence of construct XW03C37 (encoding F(ii)) – all U ribonucleotides are 1m ⁇ – GC content 49.00% – 5' and 3' UTRs are “UTR4”
  • SEQ ID NO: 147 C-terminus (position 514 onwards) of recombinant protein constructs used in inter alia, Example 6 (linker sequences, T4 fibritin foldon trimerisation domain, thrombin cleavage site, strep tag and His-tag)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

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 et qui comprend (a), (b) et (c) : (ai) au moins une mutation par rapport au type sauvage dans une région correspondant aux positions 38-60 de SEQ ID NO : 1, ladite mutation augmentant l'hydrophobicité de la région par rapport aux positions 38-60 de SEQ ID NO : 1 ; et/ou (aii) au moins une mutation par rapport au type sauvage dans une région correspondant aux positions 296-318 de SEQ ID NO : 1, ladite mutation augmentant l'hydrophobicité de la région par rapport aux positions 296-318 de SEQ ID NO : 1, et/ou introduisant, par substitution ou insertion, un résidu choisi parmi M, F, I et V dans la région ; (b) au moins une mutation par rapport au type sauvage dans une région correspondant aux positions 208-216 de SEQ ID NO : 1, ladite mutation augmentant l'hydrophobicité de la région par rapport aux positions 208-216 de SEQ ID NO : 1, et/ou introduisant, par substitution ou insertion, un résidu P dans la région ; et (c) au moins une mutation par rapport au type sauvage dans une région correspondant aux positions 345 à 352 de SEQ ID NO : 1, ladite mutation introduisant, par substitution ou insertion, un site de glycosylation dans la région.
EP23733931.2A 2022-08-22 2023-06-16 Protéines rsv-f Pending EP4577557A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US202263399811P 2022-08-22 2022-08-22
US202263399804P 2022-08-22 2022-08-22
US202263476694P 2022-12-22 2022-12-22
US202263476696P 2022-12-22 2022-12-22
EP23155959 2023-02-10
EP23155964 2023-02-10
US202363452760P 2023-03-17 2023-03-17
US202363452793P 2023-03-17 2023-03-17
US202363452728P 2023-03-17 2023-03-17
PCT/EP2023/066332 WO2024041773A1 (fr) 2022-08-22 2023-06-16 Protéines rsv-f

Publications (1)

Publication Number Publication Date
EP4577557A1 true EP4577557A1 (fr) 2025-07-02

Family

ID=95937328

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23733931.2A Pending EP4577557A1 (fr) 2022-08-22 2023-06-16 Protéines rsv-f

Country Status (2)

Country Link
EP (1) EP4577557A1 (fr)
CN (1) CN120303288A (fr)

Also Published As

Publication number Publication date
CN120303288A (zh) 2025-07-11

Similar Documents

Publication Publication Date Title
US11964011B2 (en) Coronavirus vaccine
US11241493B2 (en) Coronavirus vaccine
JP7613685B2 (ja) 新規ｒｓｖ ｒｎａ分子及びワクチン接種用組成物
EP4147717A1 (fr) Vaccin contre le coronavirus
JP2023513544A (ja) ＳＡＲＳ－ＣＯＶ－２ ｍＲＮＡドメインワクチン
KR20230164648A (ko) SARS-CoV-2 변이체에 대한 RNA 백신
WO2024256637A1 (fr) Protéines vrs-f à substitution de cystéine
US20240181038A1 (en) Immunogenic compositions
KR20250075664A (ko) 인플루엔자 바이러스 백신
WO2024041773A1 (fr) Protéines rsv-f
EP4577557A1 (fr) Protéines rsv-f
EP4577556A1 (fr) Protéines rsv-f
WO2024193965A1 (fr) Acides nucléiques codant pour rsv-f
WO2024194153A1 (fr) Acides nucléiques codant pour rsv-f
HK40090415A (en) Coronavirus vaccine
WO2024223724A1 (fr) Vaccins contre le virus de la grippe
CN117440824A (zh) 抗SARS-CoV-2变体的RNA疫苗

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250319

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)