WO2024256637A1 - Protéines vrs-f à substitution de cystéine - Google Patents

Protéines vrs-f à substitution de cystéine Download PDF

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WO2024256637A1
WO2024256637A1 PCT/EP2024/066562 EP2024066562W WO2024256637A1 WO 2024256637 A1 WO2024256637 A1 WO 2024256637A1 EP 2024066562 W EP2024066562 W EP 2024066562W WO 2024256637 A1 WO2024256637 A1 WO 2024256637A1
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rsv
seq
protein
positions
present disclosure
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Nicholas John BARROWS
Wayne Daniel HARSHBARGER
Genevieve Anne HOLZAPFEL
Corey Mallett
Kambiz MOUSAVI
Sanjay Phogat
Emily PHUNG
James Alan WILLIAMS
Marco BIANCUCCI
Chelsy Caryn CHESTERMAN
Xiaofeng Wang
Yuejiao XIAN
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • 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

Definitions

  • 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.
  • exemplary RSV-F proteins exhibit particularly high stability of the pre-fusion conformation.
  • exemplary RSV-F proteins exhibit higher melting temperatures than a number of control designs, including DS-Cav1 (see, e.g. Example 2, Table 4).
  • RSV-F proteins may consistently retain the immunogenic pre-fusion conformation over time, either when expressed on the cell surface following delivery via nucleic acid, or when administered as a recombinant protein. Hence, the subject’s immune system may be exposed to pre-fusion RSV-F for longer, leading to more potent immune responses.
  • a nucleic acid e.g.
  • RNA-based vaccine high expression levels from nucleic acids may further potentiate the immunogenicity of a stable pre-fusion RSV-F design.
  • exemplary RSV-F proteins according to the present disclosure exhibit such high expression levels from nucleic acids in vitro (see, e.g. Example 4; Figures 16-18) – an effect which is enhanced through selective truncation of the C-terminal cytoplasmic tail of the RSV-F protein.
  • Example 6, Figures 23 and 24 where exemplary RSV-F proteins (delivered via nucleic acid) elicited higher neutralising antibody titres against RSV of the A and B subtypes, in comparison to a number of control constructs.
  • Neutralising antibody titres generally correlate with inhibition of viral replication in the lungs and other respiratory sites, and thus protective efficacy in a subject.
  • RSV-F proteins of the present disclosure may allow for protective efficacy against RSV to be achieved at lower doses of a nucleic acid-based vaccine, leading to further possible benefits, such as reduced reactogenicity. See also e.g.
  • such disulphide bonds contribute to the stability of RSV-F proteins of the present disclosure.
  • Analysis by cryo-EM also revealed further, surprising, structural effects of such disulphide bonds (see, e.g. Example 12).
  • such disulphide bonds may reposition the side chain of wild- type aromatic residue F488 to introduce a pi-pi stacking interaction with wild-type residue aromatic F137 in the fusion peptide (see, e.g. Figures 36A and D).
  • the additional interaction may result in a cation-pi-pi trio-stacking interaction between residues K339, F137 and F488, which may further restrict movement of the fusion peptide, thereby helping to stabilise the pre-fusion conformation (see, e.g. Figures 36C and D).
  • exemplary RSV-F proteins according to the present disclosure also elicit neutralising antibody titers when administered as an adjuvanted recombinant protein, see e.g. Example 14 ( Figure 49). Given all of the above, 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: An RSV-F protein, comprising at least two mutations relative to SEQ ID NO: 1 or 3 within a region of the protein corresponding to positions 474-523 of SEQ ID NO: 1 or 3; wherein the at least two mutations introduce, through substitution or insertion, a pair of C residues into the region, which form a disulphide bond.
  • 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.
  • a carrier preferably, a lipid nanoparticle
  • 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).
  • a subject preferably a subject in need of such administration.
  • Biolayer Interferometry (BLI) of the histidine- tagged sequences indicates design F528 is expressed particularly well in mammalian cells, when compared to spent media (confirmed by subsequent experiments). Error bars represent standard error.
  • Figure 2. OCTET BLI of Round 5 sequences bound to RSV-F antibodies (AM14, D25, RSB1). Responses normalized to DS-Cav1.
  • Figure 3. Binding of (A) DS-Cav1, (B) F504, and (C) F528 incubated at 50 or 60°C for 30, 60, or 120 min to RSV-F antibodies (AM14 and D25) was determined using OCTET BLI. Results are reported as response relative to control (Time 0) sample.
  • Figure 5. OCTET BLI of Round 6 designs in RSV-F A2 subtype (“F300”) background bound to RSV- F antibodies (AM14, D25, RSB1, motavizumab). Responses normalized to DS-Cav1.
  • Figure 6. OCTET BLI of Round 6 designs in F420 and F528 backgrounds quantified for expression with a histidine-tag. *Indicates concentrations were too high to determine (>300 ⁇ g/mL before Docket No.: 70348WO01 subtraction of negative control).
  • Results are reported as response relative to control (Time 0) sample.
  • Figure 9. Round 6 design final protein yield from 100 mL of EXPI293 cell harvest media. Quantification of final protein yield from 100 mL culture for 4 Round 6B designs, as compared to controls (F528 and F420), DS-Cav1, F(i), and F(ii).
  • Figure 10. Round 6 design affinity for RSV-F antibodies (AM14, D25, RSB1, motavizumab) as determined on BIACORE.
  • Figure 11. Round 6 (part 1) designs summary Figure 12.
  • E486C and A490C were predicted to form an inter-protomer disulphide bond in design F528. E486 and A490 are labeled and individual protomers are shaded differently for clarity. Image was generated using CHIMERAX molecular visualization program.
  • FIG. 15 Structural characteristics of RSV F647 determined by cryo-EM.
  • A Cryo-EM structure of the RSV F647-RSB1 Fab complex. For simplicity, one protomer is shown in ribbon while the other two protomers of the F647 trimer are shown in surface presentation. Mutations introduced in F647 design relative to wild-type are indicated by black spheres and labelled accordingly. Two of the bound RSB1 Fabs are shown in white and light grey ribbon.
  • B A zoom view of intra-protomer disulphide Docket No.: 70348WO01 bonds captured by cryo-EM.
  • the EM density map is shown in mesh. The D486C-A490C disulphide bond is emphasised with diagonal lines.
  • C The stabilized electrostatic repulsive ring in design F647.
  • D The electrostatic repulsive ring in wild-type RSV F protein. In both (C) and (D), the disulphide bond and nearby negatively charged residues are shown in sticks and the distances of the negatively charged residues at the trimer centre are labelled.
  • E The electrostatic distribution of the stabilized electrostatic repulsive ring in design F647.
  • F The electrostatic distribution repulsive ring in wild-type RSV F protein.
  • Each bar depicts the average intensity of the Alexa647 signal for cells identified by automated image analysis from 9 imaged fields per well, and as shown, represents the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 biological replicates, as calculated by GRAPHPAD PRISM software.
  • Figure 17. 26 RSV F-encoding mRNAs were screened in primary human BJ cells for their ability to express the RSV F antigen on the cell surface. RSV F pre-fusion surface expression was detected by indirect immunofluorescent labelling (using D25 antibody) followed by quantification using high content imaging and analysis. At (A) 25 hours, and (B) 72 hours post-transfection (hpt) cell monolayers are fixed, then RSV-F was labelled and imaged using a 10x objective.
  • Each bar depicts the average intensity of the Alexa647 signal, as per Figure 16.
  • Figure 18. 26 RSV F-encoding mRNAs were screened in primary human BJ cells for their ability to express the RSV F antigen on the cell surface. Total RSV F surface (pre- or post-fusion conformation) expression was detected by indirect immunofluorescent labelling (using motavizumab antibody) followed by quantification using high content imaging and analysis. At (A) 25 hours, and (B) 72 hours post-transfection (hpt) cell monolayers are fixed, then RSV-F was labelled and imaged using a 10x objective. Each bar depicts the average intensity of the Alexa647 signal, as per Figures 16 and 17. Figure 19.
  • Residues forming the hydrophobic pocket and involved in van der Waals contacts with T55 are shown as sticks (including hydrophobic pocket).
  • Figure 21 Zoomed in view of substitution 215A from cryo-EM structure of a parental design to inter alia, F217, F528 and F647, called F21 (structure as depicted here also applicable to aforementioned designs), including proximal ⁇ helices.
  • A215 is depicted as stick with transparent surface.
  • Residues forming a hydrophobic region that may be involved in van der Waals contacts with A215 are shown as sticks.
  • Figure 22 Figure 22.
  • Each bar depicts the average intensity of the Alexa647 signal for cells identified by automated image analysis from 9 imaged fields per well, and as shown, represents the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 technical replicates, as calculated by GraphPad Prism software.
  • Figure 28 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.
  • Figure 29 As for Figure 28 but with D25 antibody binding being assessed.
  • Figure 30 HPLC chromatograms assessing monodispersity of F310 or F310_v2 (2x Strep tag removed relative to F310) following purification, incubation at 4°C overnight, or one freeze/thaw cycle.
  • Figure 31 pre-F IgG binding antibody titres on (A) day 21 (3wp1) and (B) day 35 (2wp2) in animals immunised with varying doses of RNA encoding F647 ⁇ CT20, F647 ⁇ CT20 (codon optimised), F(iii), F(i) and F(i) ⁇ CT20.
  • Figure 32 The means, AUC and variability shown on the line and bar graphs were calculated by GraphPad Prism software.
  • Figure 29 As for Figure 28 but with D25 antibody binding being assessed.
  • Figure 30 HPLC chromatograms assessing monodispersity of F310 or F310_v2 (2x Strep tag removed relative to F310) following purification, incuba
  • Figure 33 Percentage of RSV A specific (A) CD4+ and (B) CD8+ T cells from mice immunised with F647 ⁇ CT20 and F647 ⁇ CT20 (codon optimised) (1.5 ⁇ g and 0.5 ⁇ g doses).
  • Figure 34 Percentage of RSV A specific (A) CD4+ and (B) CD8+ T cells from mice immunised with F647 ⁇ CT20 and F647 ⁇ CT20 (codon optimised) (1.5 ⁇ g and 0.5 ⁇ g doses).
  • FIG. 1 A Ribbon diagram overview of the fusion peptide and HRB regions in design F647 (the repositioning of F488 relative to wild-type is indicated by three black arrows).
  • FIG. 2 B Ribbon diagram overview of the fusion peptide and HRB regions in wild-type RSV- F, with the wild-type position of F488 circled by a dashed line.
  • C Cryo-EM density of the fusion peptide in F647, showing that all residues are well-resolved.
  • D Cryo-EM density of residues K339, Docket No.: 70348WO01 F137, F488 and the 486:490 disulphide in F647. In (C) and (D), cryo-EM density is shown in mesh.
  • fusion peptide residues are indicated with diagonal lines.
  • Figure 37 Structural comparison between F647, F651 and 2 nd generation DS-Cav1 (positioning of ⁇ 10 helices at the trimer base). Distances between the three ⁇ 10 helices in the trimer were measured from the C ⁇ of residue 501 of (A) F647, (B) F651 and (C) 2 nd generation DS-Cav1.
  • A Expression of “Round 6” RSV-F designs and comparators in A2 and M16 strain background (wild-type) sequences, relative to DS-Cav1 A2, measured using biolayer interferometry (BLI) of histidine-tagged sequences. Error bars represent standard error.
  • B OCTET BLI of RSV-F designs in A2 and M16 background sequences bound to RSV-F antibodies (AM14, D25, Motavizumab, and RSB1). Response normalised to DS-Cav1 A2.
  • C Binding affinity (K D ) of pre- fusion- RSV-F-specific antibodies AM14, D25, RSB1 and motavizumab for RSV-F designs, determined using BIACORE.
  • FIG 39 RSV A neutralizing antibody titer elicited by RNA encoding F647 ⁇ CT20 (codon optimised), measured from serum collected on day 21 and day 35 plus monthly for six months. Each mouse is presented as a dot. The horizontal dotted line represents the Limit of Detection (LOD). Data with observed geometric mean titer (GMT) and 95% confidence interval (CI) by dose. Observed GMT values are noted at the bottom. (A) saline group; (B) 0.497 ⁇ g dose group (two administrations, on days 0 and 21); (C) 0.497 ⁇ g dose group (one administration, on day 0). Figure 40.
  • Cytokines were gated on time/live/lymphocytes/singlets/CD3+ CD4+/CD44+ or CD8+/CD44+, and phenotypic subsets were Docket No.: 70348WO01 determined as described in methods by the Boolean Combination Gate Tool and defined in Phenotype Subset.
  • the geometric means (GM) with 95% confidence interval (CI) are displayed by phenotype. Figure 42.
  • T follicular helper (Tfh) and germinal center (GC) responses elicited by (1) saline; (2) 0.2 ⁇ g F647 A subtype RNA; (3) 0.2 ⁇ g F647 B subtype RNA; (4) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-formulated; (5) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-administered; (6) 0.4 ⁇ g F647 A subtype RNA; or (7) 0.4 ⁇ g F647 B subtype RNA.
  • GM geometric means
  • CI 95% confidence interval
  • GC B cells were gated on time/live/lymphocytes/singlets/CD3-/B220+CD19+/IgM-IgD-/CD95+GL7+/F647.
  • Figure 44 RSV A neutralizing antibody titers elicited by (1) saline; (2) 0.2 ⁇ g F647 A subtype RNA; (3) 0.2 ⁇ g F647 B subtype RNA; (4) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-formulated; (5) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-administered; (6) 0.4 ⁇ g F647 A subtype RNA; or (7) 0.4 ⁇ g F647 B subtype RNA.
  • RSV A neutralizing antibody titers elicited by (1) saline; (2) 0.2 ⁇ g F647 A subtype RNA; (3) 0.2 ⁇ g F647 B subtype RNA; (4) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-formulated; (5) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-administered; (6) 0.4 ⁇ g F647 A subtype RNA; or (7) 0.4 ⁇ g F647 B subtype RNA.
  • Neutralizing antibody titers were measured from serum collected on day 35. Each mouse is presented as a dot.
  • the horizontal dotted Docket No.: 70348WO01 line represents the Limit of Detection (LOD). Data with observed geometric mean titer (GMT) and 95% confidence interval (CI) by dose. Observed GMT values are noted at the bottom. Figure 46.
  • RSV B neutralizing antibody titers elicited by (1) saline; (2) 0.2 ⁇ g F647 A subtype RNA; (3) 0.2 ⁇ g F647 B subtype RNA; (4) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-formulated; (5) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-administered; (6) 0.4 ⁇ g F647 A subtype RNA; or (7) 0.4 ⁇ g F647 B subtype RNA.
  • Neutralizing antibody titers were measured from serum collected on day 21. Each mouse is presented as a dot.
  • the horizontal dotted line represents the Limit of Detection (LOD). Data with observed geometric mean titer (GMT) and 95% confidence interval (CI) by dose. Observed GMT values are noted at the bottom.
  • Figure 47 RSV B neutralizing antibody titers elicited by (1) saline; (2) 0.2 ⁇ g F647 A subtype RNA; (3) 0.2 ⁇ g F647 B subtype RNA; (4) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-formulated; (5) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-administered; (6) 0.4 ⁇ g F647 A subtype RNA; or (7) 0.4 ⁇ g F647 B subtype RNA.
  • RSV-F proteins The present disclosure provides, in a first independent aspect, an RSV-F protein, comprising at least two mutations relative to SEQ ID NO: 1 or 3 within a region of the protein corresponding to positions 474-523 of SEQ ID NO: 1 or 3; wherein the at least two mutations introduce, through substitution or insertion, a pair of C residues into the region, which form a disulphide bond. Docket No.: 70348WO01
  • the present disclosure also provides, in a second independent aspect, an RSV-F protein comprising a C residue at position 486 and a C residue at position 490; wherein the C residues form a disulphide bond.
  • the present disclosure also provides, in a third independent aspect, a trimer comprising three RSV-F proteins according to said first and second independent aspects.
  • the trimer is a homotrimer.
  • the homotrimer will generally comprise or consist of three RSV-F proteins, each of which comprise or consist of the same amino acid sequence.
  • RSV-F proteins according to said first and second independent aspects are “RSV-F proteins of the present disclosure”, as referred to herein.
  • Nucleic acids e.g. RNA
  • nucleic acids of the present disclosure are “nucleic acids of the present disclosure”, as referred to herein (or e.g.
  • RNA of the present disclosure in preferred embodiments, “RNA of the present disclosure”, as referred to herein).
  • the RSV-F sequences that are wild-type (in whole or in large part) according to SEQ ID NO: 1, 2, 3 and 4 are not “RSV-F proteins of the present disclosure” as referred to herein. Consequently, nucleic acids encoding SEQ ID NO: 1, 2, 3 or 4 are also not “nucleic acids of the present disclosure” as referred to herein.
  • RSV-F proteins of the present disclosure and the mutations which they comprise (relative to a wild- type RSV-F protein), are “engineered”, in the sense that such mutations have been deliberately selected and introduced into the proteins, at least in part in order to enhance pre-fusion stability and/or expression from nucleic acids.
  • RSV-F proteins of the present disclosure may also be considered “recombinant” (“engineered” and “recombinant” may be used interchangeably in this context). Accordingly, RSV-F proteins of the present disclosure are mutated relative to SEQ ID NO: 1 or 3. RSV-F proteins of the present disclosure are also mutated relative to SEQ ID NO: 2 or 4.
  • SEQ ID NO: 1 is an RSV-F sequence from a strain of human RSV of the A2 subtype that contains two mutations (K66E and Q101P) relative to GenBank Accession number KT992094 (said mutations resulting from in vitro passaging, see [10]).
  • SEQ ID NO: 3 is the RSV-F sequence from B subtype strain M16 (GenBank accession no.
  • SEQ ID NO: 2 and 4 are recombinant protein sequences comprising the wild-type sequence from SEQ ID NO: 1 and 2, respectively, up to position 513, and various domains (including a bacteriophage T4 fibritin foldon trimerisation domain) and linkers from position 514 onwards in place of inter alia, a transmembrane domain and cytoplasmic tail.
  • SEQ ID NO: 1, 2, 3 and 4 and any wild-type RSV-F sequence e.g. RSV-F proteins of other A or B subtype strains
  • SEQ ID NO: 5 and 6 may also be referred to as a “wild type cytoplasmic tail”.
  • residue positions as defined throughout the present disclosure are numbered according to SEQ ID NO: 1, 2, 3 or 4, which will generally correspond to the residue numbering of the RSV-F protein of the present disclosure.
  • residue numbering of the F1 domain will diverge from SEQ ID NO: 1, 2, 3 or 4.
  • substitutions in such RSV-F proteins relative to wild-type e.g.486C, and so forth are numbered according to SEQ ID NO: 1, 2, 3 or 4.
  • references to a sequence / region of an RSV-F protein of the present disclosure “corresponding to positions x-y” of SEQ ID NO: 1, 2, 3 or 4 encompasses sequences / regions which align with positions x-y of SEQ ID NO: 1, 2, 3 or 4 (which, for the avoidance of doubt, includes positions x and y).
  • the mutations as defined throughout the present disclosure are introduced within positions x-y of SEQ ID NO: 1, 2, 3 or 4 (again, including positions x and y). Alignments may be performed visually, or by any well-known algorithm; e.g. using an NCBI BLAST algorithm, e.g. “blastp”, e.g.
  • Heptad repeat A domain refers to positions 149-206 of SEQ ID NO: 1, 2, 3 or 4 and “heptad repeat C” (“HRC”) domain refers to positions 53-100 of SEQ ID NO: 1, 2, 3 or 4.
  • Heptad repeat B (“HRB”) domain refers to positions 474-523 of SEQ ID NO: 1 and 3, and positions 474-513 of SEQ ID NO: 2 and 4.
  • RSV-F proteins of the present disclosure are preferably antigens (or, phrased differently, are antigenic). As such, RSV-F proteins of the present disclosure preferably elicit an immune response when administered in vivo, namely against RSV.
  • the immune response may comprise an antibody response (usually including IgG) and/or a cell-mediated immune response, in particular an antibody response.
  • the immune response will typically recognise the three-dimensional structure of the corresponding wild-type pre-fusion RSV-F, in particular one or more epitopes present on the (solvent-exposed) surface of the protein when in the pre-fusion conformation.
  • RSV-F proteins of the present disclosure elicit a pre-fusion RSV-F-specific antibody response in vivo, e.g. an IgG antibody response (see, e.g. Examples 6, 11 and 14).
  • RSV-F proteins of the present disclosure elicit a neutralising antibody response against RSV (RSV A or B) in vivo, e.g. against RSV A (see, e.g. Examples 6, 11, 14, 15 and 16). Said neutralising antibody response may inhibit replication of RSV (such as RSV A or B) in the respiratory system of a subject, such as in the lungs. Said neutralising antibody response may yield protective immunity against RSV (RSV A or B) in a subject, e.g. against RSV A. Generally, RSV-F proteins of the present disclosure elicit a cross-neutralising antibody response against RSV in vivo, e.g.
  • RSV-F proteins of the present disclosure are generally in the pre-fusion conformation (e.g. following expression from nucleic acids), and may generally be considered as stabilised in the pre-fusion conformation.
  • pre-fusion conformation of RSV-F proteins of the present disclosure may be confirmed via binding of pre-fusion RSV-F-specific monoclonal antibodies (“pre-fusion mAbs”).
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a light chain and a heavy chain (LC and HC) selected from the group consisting of: SEQ ID NO: 7 and 8 respectively, SEQ ID NO: 9 and 10 respectively, and SEQ ID NO: 11 and 12 respectively.
  • LC and HC light chain and a heavy chain
  • SEQ ID NO: 7 and 8 respectively, SEQ ID NO: 9 and 10 respectively, and SEQ ID NO: 11 and 12 respectively.
  • Specific binding of the pre-fusion mAb(s) (or lack thereof) may be determined via surface plasmon resonance (“SPR”) or biolayer interferometry (“BLI”), however SPR is preferred.
  • SPR surface plasmon resonance
  • BBI biolayer interferometry
  • SPR may be performed using a BIACORE system; preferably as performed in the Examples (see subsection Binding kinetics using BIACORE).
  • RSV-F proteins of the present disclosure may be specifically bound by any of the pre-fusion mAbs above with a dissociation constant (K D ), as measured by SPR, of less than 10 nM, such as 1 pM – 10 nM; in particular less than 2 nM (2000 pM), such as 1- 2000 pM.
  • K D dissociation constant
  • AM14 is preferred. Unlike the other pre-fusion mAbs, AM14 is specific for RSV-F in the pre-fusion conformation when in an intact trimer.
  • 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: 7 and 8 respectively (or, defined differently, antibody AM14), with a K D , as measured via SPR, of: less than 1000, 900, 800, 700, 600, 500, 400, 350 or 320 pM; or, in some embodiments, less than 300, 200, 150, 100, 90, 80, 70, 60, 50, or 40 pM; or, in some embodiments, less than 35, 30, 25 or 20 pM.
  • a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 7 and 8 respectively (or, defined differently, antibody AM14), with a K D , as measured via SPR, of: less than 1000, 900, 800, 700, 600, 500, 400, 350 or 320 pM; or, in some embodiments, less than 300, 200, 150, 100, 90, 80, 70, 60, 50, or 40
  • RSV-F proteins according to present disclosure designated F528, F647, F651 are specifically bound by such a mAb with K D s, as measured via SPR, of 37.8, 309.5 and 18.9 pM respectively (see, e.g. Example 2, Table 5).
  • 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: 7 and 8 respectively (or, defined differently, antibody AM14) with a KD, as measured via SPR, in the range of: 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-350 or 1-320 pM (such as 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-350 or 10-320 pM); or, in some embodiments, 1-300, 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, or 1-40 pM (such as 10-300, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, or 10- 40 pM); or, in some embodiments, 1-35, 1-30, 1-25 or 1-20 pM (such as 10-35, 10-30, 10-25 or 10-20 pM.
  • RSV-F proteins of the disclosure are generally assembled in trimeric form, as a homotrimer.
  • RSV-F proteins of the present disclosure may be specifically bound by a pre-fusion mAb comprising a LC and HC according SEQ ID NO: 9 and 10 respectively (or, defined differently, antibody D25), with a K D , as measured via SPR, of: less than 5000, 4000, 3000, 2500, 2000, 1900 or 1850 pM; or, in some embodiments, less than 1500, 1000, 800, 600, 400, 200, 100, 90, 80, 75 or 70 pM; or, in some embodiments, less than 65 pM.
  • 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: 9 and 10 respectively (or, defined differently, antibody D25) with a KD, as measured via SPR, in the range of: 1-5000, 1-4000, 1-3000, 1-2500, 1- 2000, 1-1900 or 1-1850 pM (such as 10-5000, 10-4000, 10-3000, 10-2500, 10-2000, 10-1900 or 10- 1850 pM); or, in some embodiments, 1-1500, 1-1000, 1-800, 1-600, 1-400, 1-200, 1-100, 1-90, 1-80, 1-75 or 1-70 pM (such as 10-1500, 10-1000, 10-800, 10-600, 10-400, 10-200, 10-100, 10-90, 10-80, 10-75 or 10-70); or, in some embodiments, 1-65 pM (such as 10-65 pM).
  • a pre-fusion mAb comprising a LC and HC according to SEQ ID NO
  • RSV-F proteins according to present disclosure designated F528, F647, F651 are specifically bound by such a mAb with KDs, as measured via SPR, of 189.1, 120.6 and 72.9 pM respectively (see, e.g. Example 2, Table Docket No.: 70348WO01 5).
  • 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: 11 and 12 respectively (or, defined differently, antibody RSB1) with a K D , as measured via SPR, in the range of: 1-1000, 1-500, 1-450, 1-400, 1-350, 1-300, 1-250, 1-200 or 1-190 pM (such as 10-1000, 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-200 or 10-190 pM); or, in some embodiments, 1-180, 1-170, 1-160, 1-150, 1-140, 1-130 or 1-125 pM (such as 10-180, 10-170, 10-160, 10-150, 10-140, 10-130 or 10-125 pM); or, in some embodiments, 1-120, 1-100, 1-90, 1-80, 1-70 or 1-65 pM (such as 10-120, 10-100, 10-90, 10-80, 10- 70 or 10-65 pM).
  • a pre-fusion mAb compris
  • RSV-F protein of the present disclosure meets 2, or preferably all 3 of criteria (vii), (viii) and (ix).
  • protein F651 meets all of said criteria (see, e.g. Example 2, Table 5).
  • 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.
  • cryo-EM comprises the steps: complexing the RSV-F protein of the present disclosure with an antigen binding fragment, such as a Fab fragment, of a pre-fusion mAb (preferably of AM14 or RSB1, preferably a Fab fragment of AM14 or RSB1) to form complexes; Docket No.: 70348WO01 isolating (e.g. via gel filtration) and concentrating said complexes; depositing said complexes onto an electron microscopy grid, and vitrifying the complexes and grid (e.g.
  • an antigen binding fragment such as a Fab fragment
  • Disulphide bond RSV-F proteins of the present disclosure comprise (according to the first, second and third independent aspects) a disulphide bond formed between two, generally non-naturally occurring, C residues. According to the first independent aspect of the present disclosure, a pair of C residues is introduced into the region of the RSV-F protein corresponding to positions 474-523 of SEQ ID NO: 1 or 3 (a.k.a. the HRB domain) which form a disulphide bond.
  • this is an intra-protomer disulphide bond (i.e. linking two C residues within the same protomer).
  • the presence of an intra-protomer disulphide bond may be confirmed via e.g. (i) cryo-EM (e.g. using the method set out in the preceding subsection, in particular as performed in Example 3), or (ii) X-ray crystallography (e.g. performed in accordance with the method in [15]).
  • the pair of C residues may be within the region corresponding to positions 474-513 of SEQ ID NO: 1 or 3.
  • position 513 is the typical C-terminal residue of the HRB domain, prior to, typically, a linker sequence connecting the HRB domain to a trimerisation domain (e.g. a bacteriophage T4 fibritin foldon trimerisation domain, e.g. according to SEQ ID NO: 19) and other sequences as needed.
  • a first C residue of said pair may be within a region of the RSV-F protein corresponding to positions 478-501 of SEQ ID NO: 1 or 3, and/or (optionally and) a second C residue of said pair may be within a region corresponding to positions 482-504 of SEQ ID NO: 1 or 3.
  • C residue pairs include those at positions: 486 and 490, 485 and 494, 480 and 497, 490 and 494, 479 and 482, 484 and 498, 487 and 490, 491 and 494, 482 and 502, 478 and 483, 481 and 501, 482 and 499, 486 and 489, 486 and 488, 485 and 494, 480 and 487, or 501 and 504 of SEQ ID NO: 1 or 3.
  • C residues in these positions were (i) computationally predicted to form intra-protomer disulphide bonds, based on a distance criterion of 5 ⁇ between C ⁇ atoms in the RSV-F pre-fusion conformation (see e.g.
  • Example 5 and/or (ii) are present in designs F526, F527 and F528 (see e.g. Example 1) .
  • a first C residue of said pair may be within a region of the RSV-F protein corresponding to positions 478-491 of SEQ ID NO: 1 or 3, and/or (optionally and) a second C residue of said pair may be within a region corresponding to positions 482-502 of SEQ ID NO: 1 or 3.
  • C residue pairs include those at positions: 486 and 490, 485 and 494, 480 and 497, Docket No.: 70348WO01 490 and 494, 479 and 482, 484 and 498, 487 and 490, 491 and 494, 482 and 502, or 478 and 483 of SEQ ID NO: 1 or 3.
  • C residues in these positions were (i) computationally predicted to form intra- protomer disulphide bonds, based on a distance criterion of 5 ⁇ between C ⁇ atoms in the RSV-F pre- fusion conformation and (ii) stabilise said conformation (see e.g. Example 5); and/or are present in designs F526, F527 and F528 (see e.g.
  • the pair of C residues is at positions 486 and 490 of SEQ ID NO: 1 or 3.
  • design F528 (486:490 disulphide bond) demonstrated the highest expression level of all constructs tested (both control and non-control), and comparable or greater prefusion mAb binding than controls DS-Cav1, F(i) and F(ii).
  • the 486:490 disulphide bond was confirmed to be intra-protomer by cryo-EM in Example 3.
  • the RSV-F protein comprises a C residue at position 486 and a C residue at position 490, which form a disulphide bond. Said residue numbering will typically correspond to that of SEQ ID NO: 1 or 3.
  • this disulphide bond is an intra-protomer disulphide bond, the presence of which may be confirmed using the methods detailed above in relation to the first independent aspect of the present disclosure.
  • the substitutions for/insertions of C residues (and the resulting disulphide bond) detailed throughout this subsection may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure do not comprise a deletion of position 137.
  • Such RSV-F proteins of the present disclosure preferably comprises neither (i) a deletion of the fusion peptide (in whole), nor (ii) substitution of the fusion peptide (in whole) for a linker sequence e.g. GSGSG (SEQ ID NO: 16), GSGSGRS (SEQ ID NO: 17), or GS (SEQ ID NO: 18).
  • Such RSV-F proteins of the present disclosure preferably comprise (i) a deletion of p27 (in whole) and the fusion peptide (in whole), nor (ii) substitution of p27 (in whole) and the fusion peptide (in whole) for a linker sequence e.g.
  • RSV-F proteins of the present disclosure preferably comprise: (a) an aromatic residue at position 137 (such as F, W, Y or H; optionally F, W or Y; optionally F); (b) an aromatic residue at position 488 (such as F, W, Y or H; optionally F, W or Y; optionally F); and/or (c) a positively charged residue at position 339 (such as K, R or H, optionally K or R, optionally K); and Docket No.: 70348WO01 preferably all of (a), (b) and (c).
  • an aromatic residue at position 137 such as F, W, Y or H; optionally F, W or Y; optionally F
  • an aromatic residue at position 488 such as F, W, Y or H; optionally F, W or Y; optionally F
  • a positively charged residue at position 339 such as K, R or H, optionally K or R, optionally K
  • Docket No.: 70348WO01 preferably all of (a
  • Such RSV-F proteins of the present disclosure preferably comprise a pi-pi stacking interaction between (a) and (b), and preferably comprise a pi-pi-cation stacking interaction between (a), (b) and (c).
  • the (preferably intra-protomer) disulphide bond is preferably formed by a C residue at position 486 and a C residue at position 490 (as per preferred embodiments of the first independent aspect of the present disclosure, or as per the second independent aspect of the present disclosure).
  • the (preferably intra-protomer) disulphide bond is preferably formed by a C residue at position 486 and a C residue at position 490 (as per preferred embodiments of the first independent aspect of the present disclosure, or as per the second independent aspect of the present disclosure).
  • cryo-EM analysis of design F647 demonstrated that the 486:490 disulphide bond repositions the side chain of wild-type aromatic residue F488 to introduce a pi-pi stacking interaction with wild-type residue aromatic F137 in the fusion peptide (see, e.g. Figures 36 A and D).
  • This additional interaction resulted in a cation-pi-pi trio-stacking interaction between residues K339, F137 and F488, which further restricts movement of the fusion peptide, thereby helping to stabilise the pre-fusion conformation of RSV-F (see, e.g. Figures 36C and D).
  • RSV-F proteins of the present disclosure comprising linker sequences joining their F1 and F2 domains (e.g. F651 a.k.a R715; SEQ ID NO: 32 / 73).
  • Deletions and residue positioned identified in this paragraph are relative to and numbered according to SEQ ID NO: 1, 2, 3 or 4, which will generally correspond to the residue numbering of the RSV-F protein of the present disclosure.
  • SEQ ID NO: 1, 2, 3 or 4 will generally correspond to the residue numbering of the RSV-F protein of the present disclosure.
  • RSV-F proteins of the present disclosure may comprise an electrostatic repulsive ring comprising three negatively residues, each of which is in the HRB domain of an RSV-F protein in the trimer.
  • the negatively charged residues are typically at position 487 of each of the RVF-proteins, and may be E or D residues, typically E residues.
  • the distances between each of the negatively-charged residues in the trimer may be increased relative to such distances in a trimer comprising three RSV-F proteins comprising or consisting of SEQ ID NO: 1, 2, 3 or 4 (i.e.
  • such distances may be at least 5.0, 5.5, 6.0, 6.5, 7.0, 7.2 or 7.4 ⁇ (or e.g. 5.0-8.0 ⁇ , such as 6.0-8.0, 6.5-7.5 or 7.0-7.5 ⁇ ). Such distances may be assessed e.g. using cryo-EM, (e.g. using the method set out in the preceding subsection, in particular as performed in Example 3). In wild type, such distances are generally lower, such as 4.3 ⁇ (see e.g. Figure 15 D).
  • RSV-F proteins of the present disclosure preferably comprise one or more further mutations (relative to wild type, e.g. SEQ ID NO: 1 or 3), such as at least 2, 3, 4, 5, 6 or 7 further mutations.
  • the one or more further mutations are, or comprise, one or more Docket No.: 70348WO01 substitutions (relative to wild type, e.g.
  • the one or more mutations are, or comprise, the substitutions 103C, 148C and 190I, or 103C, 148C, 190I and 486S (optionally numbered according to SEQ ID NO: 1 or 3).
  • the one or more mutations are, or comprise, the substitutions 67I and 215P (optionally numbered according to SEQ ID NO: 1 or 3).
  • the one or more mutations are, or comprise, the substitutions 66E, 67I, 76V, 215P and 486G (optionally numbered according to SEQ ID NO: 1 or 3). In some embodiments, the one or more mutations are, or comprise, the substitutions 149C, 155C, 190F, 207L, 290C and 458C (optionally numbered according to SEQ ID NO: 1 or 3), optionally with a linker sequence joining the F2 and F1 domains of the RSV-F protein (e.g. GS / SEQ ID NO: 18).
  • the one or more mutations are, or comprise, the substitutions 102A, 149C, 155C, 190F, 207L, 290C, 373R, 379V, 447V and 458C (optionally numbered according to SEQ ID NO: 1 or 3), optionally with a linker sequence joining the F2 and F1 domains of the RSV-F protein (e.g. GS / SEQ ID NO: 18).
  • the one or more mutations are, or comprise, the substitutions 155C, 190F, 207L and 290C (optionally numbered according to SEQ ID NO: 1 or 3).
  • the one or more mutations are, or comprise, those set out in the following four subsections.
  • RSV-F proteins of the present disclosure comprise a substitution at position 228 for K, R or Q (optionally K or R), and/or a substitution at position 232 for N.
  • RSV proteins of the present disclosure comprise a substitution at position 228 for K, R or Q (optionally K or R).
  • it is more preferred that RSV proteins of the present disclosure comprise a substitution at position 228 for K (e.g. as found in designs such as F217, F528 and F647).
  • position 232 has either the wild-type residue (E),or is substituted for D (also a negatively charged residue). E or D at position 232 may help to provide a tertiary cation-pi-anion interaction discussed below.
  • position 250 has either the wild-type residue (Y), or is substituted for D.
  • substitutions at position 228 and/or 232 may be the only mutation(s) in the region of the RSV-F protein corresponding to positions 217-239 of SEQ ID NO: 1 or 3, relative to a corresponding wild- type region, e.g. positions 217-239 of SEQ ID NO: 1 or 3.
  • a minimal substitution screen revealed the 228K substitution alone to be able to achieve pre-fusion RSV-F (see Figure 26; design F310).
  • K at position 228 appears to result in an H bond with Y250 on the same protomer (see Figure 19, dashed line indicating hydrogen bond).
  • Said H bonding may stabilise Y250 to form a tertiary cation-pi-anion interaction between E232, Y250 and R235 (E232 and Y250 being on one protomer, with R235 being on an adjacent protomer).
  • E, Y and R are one of the dominant triads for such a tertiary Docket No.: 70348WO01 cation-pi-anion interaction (see, e.g. [16]).
  • residues with other H bond donors in their side chains (such as R, Q or N, in particular R) at position 228 may also provide this stabilising H bond with Y250.
  • substitution for N may also provide a stabilising hydrogen bond with Y250.
  • RSV-F proteins of the present disclosure may comprise a substitution at position 250 for D.
  • a 250D substitution may strengthen a cross- protomer interaction with R235 (wild-type residue) by forming a salt bridge between the two residues.
  • R235 wild-type residue
  • D comprises an H bond acceptor moiety and so the Y250D substitution would maintain the preferred hydrogen bond between positions 250 and 228.
  • the substitutions detailed throughout this subsection may provide core stabilisation in the F1 domain (positions 137-513 of SEQ ID NO: 1 or 3), proximal to the heptad repeat A (“HRA”) domain and antibody binding site ⁇ (”site ⁇ ”).
  • substitutions detailed throughout this subsection may provide a H bond with Y250, e.g. which stabilises Y250 to provide a tertiary cation-pi-anion interaction between positions (i) 232 (preferably E232, as in wild-type, or D232 if substituted), (ii) Y250 and (ii) R235 across different RSV-F protomers.
  • Such core stabilisation, H bonds and/or tertiary anion-pi-cation interactions may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure comprise a substitution at position 55 for T, C, V, I or F (optionally T, C or V; optionally T or C). In such embodiments, it is preferred that RSV proteins of the present disclosure comprise a substitution at position 55 for T (e.g. as found in designs such as F217, F528 and F647).
  • substitutions at position 55 may be the only mutation(s) in the region of the RSV-F protein corresponding to positions 38-60 of SEQ ID NO:1 or 3 relative to a corresponding wild-type region, e.g. positions 38-60 of SEQ ID NO: 1 or 3.
  • a minimal substitution screen revealed the S55T substitution to be a likely driver of the pre-fusion conformation (see Figure 26; design F308).
  • T in place of S (wild-type) at position 55 provides a slightly larger residue which (from in silico three-dimensional structural analysis, see Figure 20) appears to be accommodated well in the hydrophobic pocket discussed above, without generating significant steric clashes.
  • the substitutions detailed throughout this subsection may provide energetically-favourable Van der Waals (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.
  • VDW Van der Waals
  • the substitutions detailed throughout this subsection 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.
  • the substitutions detailed throughout this subsection may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure comprise a substitution at position 215 for A, P, V, I, or F (optionally A or P).
  • RSV proteins of the present disclosure comprise a substitution at position 215 for A (e.g. as found in designs such as F217, F528 and F647).
  • substitutions at position 215 may be the only mutation(s) in the region of the RSV-F protein corresponding to positions 208-216 of SEQ ID NO:1 or 3 relative to a corresponding wild-type region, e.g. positions 38-60 of SEQ ID NO: 1 or 3.
  • RSV-F proteins of the present disclosure may comprise a substitution at position 211 and/or (optionally and) position 216 for P (see e.g. designs in Example 2).
  • a minimal substitution screen revealed the S215A substitution to be a likely driver of the pre-fusion conformation (see Figure 26; design F309).
  • removal of the hydrophilic OH group, as S (wild-type) is substituted for A is likely favourable to the packing and rigidity of the loop (see Figure 21).
  • the A residue at position 215 may provide energetically-favourable VDW contacts with positions 79, 206, 203, and/or T219.
  • Such packing, rigidification and/or VDW contacts may inhibit, at least partly inhibit, or completely inhibit the transition from pre-fusion to post-fusion conformation of RSV-F (in particular, inhibition of the relative motion of the two ⁇ helices adjacent to the loop (generally the ⁇ 4 and ⁇ 5 helices of RSV-F), or, defined differently, inhibition of refolding of the HRC and HRA domains).
  • the side chains of P, V, I or F may also reduce conformational freedom of the loop, thus also being favourable to the packing and rigidification of the loop.
  • Docket No.: 70348WO01 Generally, the substitutions detailed throughout this subsection may stabilise or rigidify the loop corresponding to positions 208-216 of SEQ ID NO: 1 or 3.
  • 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 substitutions detailed throughout this subsection may inhibit, at least partly inhibit, or completely inhibit, the transition of RSV-F from pre-fusion to post-fusion conformation (in particular, by inhibiting of the relative motion of the two ⁇ helices adjacent to the loop (generally the ⁇ 4 and ⁇ 5 helices of RSV-F), or, defined differently, by inhibiting refolding of the HRC and HRA domains).
  • RSV-F proteins of the present disclosure preferably comprise combinations of substitutions as detailed in the preceding three subsections, such as: a substitution at position 228 for K, R or Q (optionally K or R; wherein substitution for K is preferred); a substitution at position 55 for T, C, V, I (optionally T, C or V; optionally T or V, wherein substitution for T is preferred); and a substitution at position 215 for A, P, V, I, or F (optionally A, V, I, or F; optionally A or P; wherein substitution for A is preferred).
  • RSV-F proteins of the present disclosure preferably comprise further substitutions, such as: a substitution at position 152 for R, L or W (optionally R or W; wherein substitution for R is preferred); a substitution at position 315 for I or V (wherein substitution for I is preferred); a substitution at position 346 for Q, D, H, K, N, R, S or W (optionally Q, D, H, K, N, R or S; wherein substitution for Q is preferred); a substitution at position 445 for D; a substitution at position 455 for V or I (wherein substitution for V is preferred); and/or a substitution at position 459 for M; in particular: a substitution at position 152 for R, L or W (optionally R or W; wherein substitution for R is preferred); Docket No.: 70348WO01 a substitution at position 315 for I or V (wherein substitution for I is preferred); a substitution at position 346 for Q, D, H, K, N, R, S or W (optionally Q, D, H, H,
  • RSV-F proteins of the present disclosure may, in preferred embodiments, comprise: a substitution at position 55 for T; a substitution at position 152 for R; a substitution at position 215 for A; a substitution at position 228 for K; a substitution at position 315 for I; a substitution at position 346 for Q; a substitution at position 445 for D; a substitution at position 455 for V; and a substitution at position 459 for M; more preferably: a substitution at position 55 for T; a substitution at position 152 for R; a substitution at position 215 for A; Docket No.: 70348WO01 a substitution at position 228 for K; a substitution at position 315 for I; a substitution at position 346 for Q; a substitution at position 445 for D; a substitution at position 455 for V; a substitution at position 459 for M; a substitution at position 486 for C; and a substitution at position 490 for C.
  • RSV-F proteins of the present disclosure further comprise a substitution at position 211 for N and/or (optionally and) a substitution at position 348 for N.
  • further substitutions as detailed throughout this subsection may inhibit, at least partly inhibit, or completely inhibit, the transition from pre-fusion to post-fusion conformation of RSV-F. Melting temperature When in the form of a homotrimer (e.g.
  • RSV-F proteins of the present disclosure may have a first melting temperature (T m 1) of at least 65.0 °C, such as at least 65.5, 66.0, 66.5, 67.0, 67.5, 68.0, 68.5, 69.0, 69.5, 70.0, 70.5, 71.0, 71.5, 72.0, 72.5, 73.0, 73.5, or 74.0 °C.
  • the T m 1 may be 65.0-80.0°C, such as 70.0-80.0, 70.0-75.0, 71.0- 75.0, 72.0-75.0, 73.0-75.0, 73.0-74.5, 73.5-74.5 or 74.0-74.5 °C.
  • RSV-F proteins of the present disclosure may have a second melting temperature (T m 2) of at least 78.0 °C, such as at least 78.5, 79.0, 79.5.5, 80.0, 80.5, 81.0, 81.5, 82.0, 82.5, 83.0, or 83.5.
  • T m 2 may be 78.0-90.0°C, such as 78.0-85.0, 79.0-85.0, 80.0-85.0, 80.0-84.0, 81.0-84.0, 82.0-84.0, 83.0-84.0, or 83.0-83.5 °C.
  • the Tm2 may be at least, or may be, 80.8 °C; and optionally the T m 1 may be at least, or may be, 65.7°C (see, e.g. Example 2, design F651).
  • the Tm2 may be at least, or may be, 79.4 °C; and optionally the Tm1 may be at least, or may be, 72.3 °C (see, e.g. Example 2, design F528).
  • the T m 2 may be at least, or may be, 80.7 °C; and optionally the Tm1 may be at least, or may be, 74.4 °C (see, e.g. Example 2, design F647).
  • the Tm1 and/or Tm2 may be determined via differential scanning fluorimetry (DSF), preferably nanoDSF (e.g. using a PROMETHEUS NT.48 instrument from NANOTEMPER TECHNOLOGIES), Docket No.: 70348WO01 preferably as determined in the Examples (see Examples 1 and 2, nanoDSF experiments, and associated materials and methods).
  • DSF measures protein unfolding events resulting from increasing temperature by monitoring changes in fluorescence, generally corresponding to the exposure of W or Y residues which were previously buried in the protein structure.
  • fluorescence may be measured as the ratio of the recorded emission intensities (Em350 nm/Em330 nm).
  • the T m 1 may be determined by exposing the RSV-F protein trimer to an increasing temperature (e.g. from 25 to 95°C, e.g. at a ramp rate of 1°C/min) and measuring the temperature at which a first peak in fluorescence occurs (see e.g. Figure 22A-C for examples).
  • the Tm2 may be determined by exposing the RSV-F protein trimer to an increasing temperature (e.g. from 25 to 95°C, e.g. at a ramp rate of 1°C/min) and measuring the temperature at which a second peak in fluorescence occurs (see e.g. Figure 22A-C for examples).
  • the RSV-F protein comprises trimerisation domain at the C-terminus thereof, and/or C-terminal to the F1 domain (e.g. a bacteriophage T4 fibritin foldon trimerisation domain, e.g. according to SEQ ID NO: 19).
  • the RSV-F protein may comprise a C- terminal domain comprising or consisting of positions 514-596 of SEQ ID NO: 2 or 4.
  • RSV-F proteins of the present disclosure generally have two domains (in the N-terminal to C-terminal direction, an “F2” domain and an “F1” domain), which may or may not be linked via peptide bonds (although in the wild-type protein they are not so linked; linkage typically occurring through disulphide bonds).
  • RSV-F proteins of the present disclosure may comprise or consist of (i) an F2 domain comprising or consisting of an amino acid sequence having at least 70% sequence identity to positions 26-109 of SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1), such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to positions 26-109 of SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1); and (ii) an F1 domain comprising or consisting of an amino acid sequence having at least 70% sequence identity to positions 137-513 of SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1), such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9
  • RSV-F proteins of the present disclosure comprising an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1 or 3.
  • 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 or 3.
  • a p27 peptide is not present in RSV-F Docket No.: 70348WO01 proteins of the present disclosure, optionally as a result of furin cleavage, optionally wherein the p27 peptide is positions 110-136 of SEQ ID NO: 1 or 3.
  • the p27 peptide may still be present as a result of furin cleavage at only one site, e.g. the p27 peptide is linked via a peptide bond to one of the F2 or F1 domains.
  • the F2 and F1 domains are linked via peptide bonds (e.g.
  • linker sequence will join the C and N terminal regions / residues of said F2 and F1 domains.
  • the linker sequence may be glycine-serine rich or consist of G and S residues, for example GSGSG (SEQ ID NO: 16), GSGSGRS (SEQ ID NO: 17), or GS (SEQ ID NO: 18).
  • the F2 and F1 domains may be linked by a linker sequence comprising or consisting of SEQ ID NO: 18.
  • F2 and F1 domains are not linked via peptide bonds, they may be linked by at least one disulphide bond (typically two such bonds, which are typically naturally-occurring, e.g. as in the wild- type protein).
  • RSV-F proteins of the present disclosure (protein per se) generally comprise a heterologous trimerisation domain on the C-terminus thereof (“heterologous” meaning not being native to the viral protein).
  • the trimerisation domain may be positioned C-terminal to the F1 domain.
  • a trimerisation domain is a sequence which promotes assembly of RSV-F proteins of the present disclosure (i.e. individual promoters) into trimers, namely in particular via associations with other trimerisation domains (i.e. those on other protomers).
  • Trimerisation domains may, in some embodiments, fold into a coiled-coil.
  • Exemplary trimerisation domains include: a T4 fibritin foldon domain; a yeast GCN4 isoleucine zipper, e.g. according to SEQ ID NO: 39 (or an amino acid sequence, in particular having a trimerisation function, at least 50%, 60%, 70%, 80%, 90% or 95% identical thereto); TRAF2 (GENBANK Accession No. Q12933 [gi:23503103]; amino acids 299-348); Thrombospondin 1 (Accession No. PO7996 [gi:135717]; amino acids 291-314); Matrilin-4 (Accession No.
  • RSV-F proteins of the present disclosure generally comprise a heterologous trimerisation domain C-terminal to the F1 domain; e.g. a T4 fibritin foldon domain e.g. according to SEQ ID NO: 19, or a trimerising amino acid sequence at least 50%, 60%, 70%, 80%, 90% or 95% identical to SEQ ID NO: 19.
  • RSV-F protein preferably comprises a linker sequence joining the F1 and F2 domains which is preferably a GS linker (SEQ ID NO: 18).
  • RSV- F proteins of the present disclosure generally comprise a heterologous trimerisation domain C-terminal to the F1 domain; e.g. a T4 fibritin foldon domain e.g. according to SEQ ID NO: 19, or a trimerising amino acid sequence at least 50%, 60%, 70%, 80%, 90% or 95% identical to SEQ ID NO: 19.
  • RSV-F proteins of the present disclosure comprise: (i) an F2 domain comprising or consisting of an amino acid sequence according to positions 26-109 of SEQ ID NO: 28 and an F1 domain comprising or consisting of an amino acid sequence according to positions 137-513 of SEQ ID NO: 28; or (ii) an F2 domain comprising or consisting of an amino acid sequence having at least 70%, 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% identity to positions 26-109 of SEQ ID NO: 28, and an F1 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 95%, 95%,
  • RSV-F proteins of the present disclosure generally comprise a heterologous trimerisation domain C-terminal to the F1 domain; e.g. a T4 fibritin foldon domain e.g. according to SEQ ID NO: 19, or a trimerising amino acid sequence at least 50%, 60%, 70%, 80%, 90% or 95% identical to SEQ ID NO: 19.
  • RSV-F proteins of the present disclosure comprise: - (i) an F2 and an F1 domain according to positions 26-109 and 137-513, respectively, of SEQ ID NO: 25; or (ii) an F2 domain and an F1 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and optionally comprising the substitutions present in positions 26-109 and 137-513 of SEQ ID NO: 25, relative to positions 26-109 and 137-513 of SEQ ID NO: 1; - (i) an F2 and an F1 domain according to positions 26-109 and 137-513, respectively, of SEQ ID NO: 27; or (ii) an F2 domain and an F1 domain comprising or consisting of an amino acid sequence having at least 70%, 75%
  • RSV-F proteins of the present disclosure may comprise or consist of an amino acid sequence having at least 70% sequence identity to positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 over at least 80% of positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 respectively, such as at least 75% sequence identity to positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 over at least 80% of positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 respectively, at least 80% sequence identity to positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 over at least 80% of positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 respectively, at least 85% sequence identity to positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 over at least 80% of positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 respectively, at least 90% sequence identity to positions 26-109 and 137-544 of SEQ ID NO: 2 or 4 over at least 80% of
  • RSV-F proteins of the present disclosure may have at least 70% sequence identity to positions 26-109 and 137-544 of SEQ ID NO: 2, 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 26-109 and 137-544 of SEQ ID NO: 2 over 100% of positions 26-109 and 137-544 of SEQ ID NO: 2.
  • RSV-F proteins of the present disclosure can be prepared by routine methods, such as by expression in a recombinant host system using a nucleic acid expression vector (e.g. an expression vector as detailed in the section entitled Nucleic acids encoding RSV-F proteins, below).
  • a nucleic acid expression vector e.g. an expression vector as detailed in the section entitled Nucleic acids encoding RSV-F proteins, below.
  • Suitable recombinant host cells include, for example, insect cells (e.g. Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells); mammalian cells (e.g. Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g.
  • the present disclosure also provides, in one independent aspect, a host cell (in particular, those detailed above) comprising a nucleic acid (in particular, an expression vector as detailed below) encoding an RSV-F protein of the present disclosure.
  • a host cell in particular, those detailed above
  • a host cell comprising and/or expressing an RSV-F protein of the present disclosure.
  • the present disclosure also provides, in a further independent aspect, a composition comprising a host cell (in particular, those detailed above) and (i) a nucleic acid (in particular, an expression vector as detailed below) encoding an RSV-F protein of the present disclosure, and/or (ii) an RSV-F protein of the present disclosure.
  • a composition comprising a host cell (in particular, those detailed above) and (i) a nucleic acid (in particular, an expression vector as detailed below) encoding an RSV-F protein of the present disclosure, and/or (ii) an RSV-F protein of the present disclosure.
  • the present disclosure also provides, in a further independent aspect, an in vitro method for the production of an RSV-F protein of the present disclosure, comprising expressing a nucleic acid (in particular, an expression vector as detailed below) encoding the RSV-F protein in a host cell (in particular, those detailed above), and optionally purifying the RSV- F protein.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure comprising or consisting of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1), such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1).
  • SEQ ID NO: 1 and 3 are full-length wild-type A and B subtype sequences respectively, Docket No.: 70348WO01 which include the signal sequence (positions 1-25 of SEQ ID NO: 1 and 3) and p27 peptide (positions 110-136 of SEQ ID NO: 1 and 3) both of which are typically cleaved out in the mature, furin processed, protein.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure comprising (i) an F2 domain comprising or consisting of an amino acid sequence having at least 70% sequence identity to positions 26-109 of SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1), such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to positions 26-109 of SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1); and (ii) an F1 domain comprising or consisting of an amino acid sequence having at least 70% sequence identity to positions 137-523 of SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1), such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%
  • nucleic acids of the present disclosure encode an RSV-F protein of the present disclosure comprising an E residue at position 66, and a P residue at position 101 of SEQ ID NO: 1 or 3.
  • the signal peptide (positions 1-25 of SEQ ID NO: 1 and 3) is not considered in the above sequence identity assessment.
  • nucleic acids of the present disclosure encode an RSV-F protein of the present disclosure comprising an amino acid sequence having at least 70% sequence identity to positions 26-574 SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1), such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 26-574 SEQ ID NO: 1 or 3 (in particular to SEQ ID NO: 1).
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 47; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 47, which preferably comprises the substitutions 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V, 459M, 486C and 490C relative to a wild-type RSV-F sequence, e.g.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 57; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 57, which preferably comprises the substitutions 55T, 152R, 211N, Docket No.: 70348WO01 215A, 228K, 315I, 346Q, 348N, 445D, 455V, 459M, 486C, 490C and ⁇ 555-574 relative to a wild- type RSV-F sequence, e.g.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 73; or (ii) an amino acid sequence having at least 70%, 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: 73, which preferably comprises the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C relative to a wild-type RSV- F sequence, e.g.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 87; or (ii) an amino acid sequence having at least 70%, 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: 87, which preferably comprises the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C, 490
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 69; or (ii) an amino acid sequence having at least 70%, 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: 69, which preferably comprises the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 4
  • nucleic acids of the present disclosure encode an RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 83; or (ii) an amino acid sequence having at least 70%, 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: 83, which preferably comprises the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C, 490C and ⁇ 555-574 relative to a wild-type RSV-F sequence, e.g.
  • nucleic acids of the present disclosure encode: - An RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 110; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, Docket No.: 70348WO01 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 110, which preferably comprises the mutations present in SEQ ID NO: 49 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 112; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 112, which preferably comprises the mutations present in SEQ ID NO: 49 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 49; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 49, which preferably comprises the mutations present in SEQ ID NO: 49 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 51; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 51, which preferably comprises the mutations present in SEQ ID NO: 51 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 53; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 53, which preferably comprises the mutations present in SEQ ID NO: 53 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 55; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 55, which preferably comprises the mutations present in SEQ ID NO: 55 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 71; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, Docket No.: 70348WO01 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 71, which preferably comprises the mutations present in SEQ ID NO: 71 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 75; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 75, which preferably comprises the mutations present in SEQ ID NO: 75 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 77; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 77, which preferably comprises the mutations present in SEQ ID NO: 77 relative to a wild-type RSV-F sequence, e.g.
  • RSV-F protein comprising or consisting of (i) an amino acid sequence according to SEQ ID NO: 79; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 79 which preferably comprises the mutations present in SEQ ID NO: 79 relative to a wild-type RSV-F sequence, e.g. according to SEQ ID NO: 1 or 3.
  • 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 and 3) 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: 16), GSGSGRS (SEQ ID NO: 17), or GS (SEQ ID NO: 18).
  • the F2 and F1 domains may be linked by a linker sequence comprising or consisting of SEQ ID NO: 18.
  • Nucleic acids of the present disclosure may also encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of SEQ ID NO: 1 or 3 respectively; in particular at least 75% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of Docket No.: 70348WO01 SEQ ID NO: 1 or 3 respectively, at least 80% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of SEQ ID NO: 1 or 3 respectively, at least 85% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of SEQ ID NO: 1 or 3 respectively, at least 90% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of SEQ ID NO: 1 or 3 respectively, at least 95% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of SEQ ID NO: 1 or 3 respectively, at least 99% sequence identity to SEQ ID NO: 1 or 3 over at least 80% of SEQ ID NO: 1 or 3 respectively, at least 99.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 1, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to SEQ ID NO: 1 over 100% of SEQ ID NO 1.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure having at least 70% sequence identity to SEQ ID NO: 3, 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: 3 over 100% of SEQ ID NO 3.
  • 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 tail. Docket No.: 70348WO01
  • a cytoplasmic tail is absent in whole.
  • a transmembrane domain comprises or consists of an amino acid sequence according to SEQ ID NO: 21 (or a sequence at least 80%, 85%, 90%, or 95% identical thereto).
  • a cytoplasmic tail if present, comprises or consists of an amino acid sequence according to SEQ ID NO: 5 or 6 (or a sequence at least 80%, 85%, 90%, 95% or 95% identical thereto).
  • Nucleic acids encoding RSV-F proteins comprising cytoplasmic tail deletions The cell-surface expression of trimeric, pre-fusion RSV-F protein, when expressed from nucleic acids in vitro, has been enhanced through the deletion of residues (e.g.20 residues) from the C-terminal end of the cytoplasmic tail (see e.g. Example 4).
  • residues e.g.20 residues
  • 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 9; Figure 28A).
  • deletion of 20 C-terminal residues from an RNA-delivered RSV-F protein of the present disclosure enhanced titres of neutralising antibodies elicited against RSV strains of both the A and B subtypes (see, e.g. Example 6; comparing constructs “647” and “647 ⁇ CT20”; Figures 23A and B).
  • Neutralising antibody titres generally correlate with inhibition of viral replication in the lungs and other respiratory sites, and thus protective efficacy in a subject.
  • the cytoplasmic tail deletions disclosed herein may allow for protective efficacy against RSV to be achieved at lower doses of a nucleic acid-based vaccine, leading to further possible benefits, e.g.
  • an RSV-F protein having a “cytoplasmic tail” refers to the presence of residues (e.g. 5 residues) that are C-terminal to the residue which aligns with position 549 of SEQ ID NO: 1 or 3 (Y), when the F1 and transmembrane domains of the RSV-F protein is aligned with positions 137-549 of SEQ ID NO: 1 or 3. Accordingly, the cytoplasmic tail is positioned C-terminal to the transmembrane domain.
  • an RSV-F protein having a “cytoplasmic tail” refers to the presence of residues (e.g.
  • references to e.g. deletion of 2-20 residues (and the like) from the C-terminal end of the CT refers to deletion of at least the two, and no more than the 20, most C-terminal residues from the CT. That is, at least the deletion of C- terminal residues SN or SK relative to SEQ ID NO: 5 or 6 respectively, and no more than the deletion of C-terminal residues TPVTLSKDQLSGINNIAFSN or TPVTLSKDQLSGINNIAFSK relative to SEQ ID NO: 5 or 6 respectively.
  • nucleic acids of the present disclosure encode an RSV-F protein comprising a cytoplasmic tail; wherein, relative to a cytoplasmic tail according to SEQ ID NO: 5 or SEQ ID NO: 6, 2-23 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein.
  • 2-22, 2-21, 2-20 or 3-20 residues are deleted from said C-terminal end.
  • at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or at least 19 residues are deleted from said C-terminal end.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Docket No.: 70348WO01 16, 17, 18, 19 or 20 residues are deleted from said C-terminal end.
  • 2-5, 3-5, 6- 20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20 or 16-20 residues are deleted from said C-terminal end.
  • 2-5, such as 2-4, 2-3, 3-4 or 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: 5 or 6).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-22 of SEQ ID NO: 5, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii) (meaning, the RSV-F protein sequence ends with the amino acid sequence of (i) or (ii)).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-22 of SEQ ID NO: 6, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-20 of SEQ ID NO: 5, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C- terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-20 of SEQ ID NO: 6, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • 6-13 such as 7-13, 8-12, 9-11, 9-10, 10-11 or 10 residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 5 or 6).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-15 of SEQ ID NO: 5, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-15 of SEQ ID NO: 6, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C- terminal to the amino acid sequence of (i) or (ii).
  • 14-16 such as 14-15, 15-16 or 15 residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 5 or 6).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence Docket No.: 70348WO01 according to positions 1-10 of SEQ ID NO: 5, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-10 of SEQ ID NO: 6, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C- terminal to the amino acid sequence of (i) or (ii).
  • 16-20 such as 17-20, 18-20 or 19-20, and preferably 20, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 5 or 6). See, e.g.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-5 of SEQ ID NO: 5, 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 cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-5 of SEQ ID NO: 6, 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 nucleic acids (e.g. 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: 5 or 6 (e.g.
  • deletions outlined above increase the cell-surface expression of RSV-F protein in trimeric, pre-fusion form from nucleic acids (e.g. 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. according to SEQ ID NO: 5 or 6 (e.g. over at least 24, 48, 72 or 96 hours; or e.g. over 24, 48, 72 or 96 hours).
  • nucleic acids e.g. RNA
  • SEQ ID NO: 5 or 6 e.g. over at least 24, 48, 72 or 96 hours; or e.g. over 24, 48, 72 or 96 hours.
  • trimeric, pre-fusion RSV-F expression is typically assessed using AM14 antibody binding (or defined differently, using binding of an antibody comprising a light chain (LC) according to SEQ ID NO: 7 and a heavy chain (HC) according SEQ ID NO: 8).
  • AM14 antibody binding may be assayed using indirect immunofluorescent labelling, e.g. using the protocol in the examples (see subsection “Indirect immunofluorescent labelling and detection of surface-expressed RSV F”).
  • fibroblasts preferably human fibroblasts, preferably human foreskin fibroblasts, preferably human primary BJ cells, preferably the CRL-2522 cell line (deposited at American Type Culture Collection (ATCC) under said accession number and publicly available). Docket No.: 70348WO01
  • 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. Typically, the nucleic acid will be in recombinant form, i.e. a form which does not occur in nature.
  • the nucleic acid may be for the expression of an RSV-F protein of the present disclosure in vitro from a host cell (i.e. the nucleic acid is, or is part of, an expression vector).
  • Suitable nucleic acid expression vectors can comprise, for example, (1) an origin of replication; (2) a selectable marker gene; (3) one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, or a terminator), and/or one or more translation signals; and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g. those as detailed in the section entitled Preparing RSV-F proteins, above).
  • the nucleic acid is for the expression of an RSV-F protein of the present disclosure in vivo in a subject (i.e.
  • the nucleic acid is, or is part of, a nucleic acid-based vaccine).
  • the nucleic acid may comprise one or more heterologous sequences, such as a sequence encoding a further protein (e.g. as detailed below) and/or a control sequence, in particular a promoter or an internal ribosome entry site.
  • 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 nucleic acid of the present disclosure is a viral vector
  • the viral vector is an adenovirus vector (e.g. Ad26).
  • 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).
  • the nucleic acid may encode multiple proteins, of which one is the RSV-F protein of the present disclosure.
  • the nucleic acid encodes at least (i) an RSV-F protein of the present disclosure; and (ii) at least one further protein.
  • the at least one further protein may be a nanoparticle, e.g. a ferritin nanoparticle (e.g. which is encoded, along with the RSV-F protein of the present disclosure, by a single open reading frame, resulting in expression of a single polypeptide).
  • the at least one further protein is an antigen; and, as such, the at least one further protein may comprise, or may be, a viral, bacterial, fungal, parasitic, tumour, or allergenic (i.e. from, or derived from, an allergen) antigen.
  • the at least one further protein will typically be 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.
  • the at least one further protein is an antigen from, or derived from, a virus
  • examples of such viruses include: Coronavirus, Orthomyxovirus, Pneumoviridae, Paramyxoviridae, Poxviridae, Picornavirus, Bunyavirus, Heparnavirus, Filovirus, Togavirus, Flavivirus, Pestivirus, Hepadnavirus, Rhabdovirus, Caliciviridae, Retrovirus, Reovirus, Parvovirus, Herpesvirus, Papovaviruses and Adenovirus.
  • the at least one further protein detailed above is a further Pneumoviridae protein (in particular a Pneumoviridae antigen).
  • Useful further Pneumoviridae proteins can be from an Orthopneumovirus or Metapneumovirus, in particular human RSV or human Metapneumovirus (hMPV).
  • Useful further hMPV antigens include e.g. the F, N, P, M, M2-1, and M2 antigens (in particular, the F antigen).
  • Such hMPV proteins (in particular, antigens) may be from, or derived from, the A or B subtype.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to an hMPV antigen (in particular, the F antigen).
  • RNA embodiments a preferred patient group (in which the RNA may be used in therapy, in particular vaccination) is infants (see section entitled Medical uses and methods of treatment, below).
  • Useful further human RSV antigens include e.g. the G, M1, M2-1, M2-2, P, L, N, NS1, NS2 and SH antigens, in addition to further RSV-F antigens, i.e. of distinct amino acid sequence to the RSV-F protein of the present disclosure encoded by the nucleic acid.
  • Such further human RSV proteins in particular, antigens; in particular F, antigens
  • the nucleic acid is a viral vector (in particular, a poxvirus vector, in particular an MVA vector) encoding an RSV-F protein of the present disclosure in addition to a plurality of further RSV proteins (in particular, antigens); in particular at least 2, 3, or 4 further RSV proteins / Docket No.: 70348WO01 antigens; in particular selected from G (from or derived from the A subtype: “G A ”), G (from or derived from the B subtype: “GB”) N and either M2-1 or M2-2; in particular GA, GB, N and either of M2-1 or M2-2.
  • a viral vector in particular, a poxvirus vector, in particular an MVA vector
  • RSV-F protein of the present disclosure in addition to a plurality of further RSV proteins (in particular, antigens); in particular at least 2, 3, or 4 further RSV proteins / Docket No.: 70348WO01 antigens; in particular selected from G (from or derived from the A subtype: “G A
  • the at least one further protein detailed above is a Coronavirus antigen.
  • Useful Coronavirus antigens can be from a SARS coronavirus, in particular SARS-CoV2.
  • Useful Coronavirus antigens include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein.
  • the Coronavirus antigen is a SARS- CoV2 spike protein.
  • Said SARS-CoV2 spike protein may be from any variant, e.g.
  • said SARS-CoV2 spike protein includes one or more mutations relative to the wild-type protein, in particular one or more (e.g. two) mutations to proline resides. Said one or more mutations may be introduced to stabilise said SARS-CoV2 spike protein in its pre-fusion conformation.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to a Coronavirus antigen, e.g. as detailed above.
  • the at least one further protein detailed above is an Orthomyxovirus antigen.
  • Useful Orthomyxovirus antigens can be from an influenza A, B or C virus.
  • Useful Orthomyxovirus antigens include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin.
  • the Orthomyxovirus antigen is an influenza A virus haemagglutinin.
  • Said influenza A virus hemagglutinin may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to an Orthomyxovirus antigen, e.g. as detailed above.
  • a preferred patient group in which the RNA may be used in therapy, in particular vaccination
  • is older adults see section entitled Medical uses and methods of treatment, below).
  • the RNA may encode (i) an RSV-F protein of the present disclosure, (ii) a Coronavirus antigen, e.g. as detailed above, and (iii) an Orthomyxovirus antigen, e.g. as detailed above.
  • a plurality of nucleic acids of the present disclosure is, in particular, provided in purified or substantially purified form; that is, substantially free from other nucleic acids (e.g. free or substantially free from naturally-occurring nucleic acids, such as further nucleic acids expressed by a host cell).
  • Said plurality of nucleic acids is generally at least 50% pure (by weight), such as at least 60%, 70%, 80%, 90%, or 95% pure (by weight).
  • the present disclosure also provides, in a further independent aspect, a vector comprising one or more nucleic acids of the present disclosure.
  • Nucleic acids encoding an RSV-F protein of the present disclosure may be delivered naked, or preferably in conjunction with a carrier (e.g. as detailed in the section entitled Carriers comprising a nucleic acid encoding an RSV-F protein in the prefusion conformation, below).
  • a carrier e.g. as detailed in the section entitled Carriers comprising a nucleic acid encoding an RSV-F protein in the prefusion conformation, below.
  • nucleic acids (preferably RNA) of the present disclosure, and the RSV-F proteins encoded thereby elicit a pre-fusion RSV-F-specific antibody response in vivo, e.g. an IgG antibody response (see, e.g. Examples 6, 11 and 14).
  • nucleic acids (preferably RNA) of the present disclosure and the RSV-F proteins encoded thereby, elicit a neutralising antibody response against RSV (RSV A or B) in vivo, e.g. against RSV A (see, e.g. Examples 6, 11, 14, 15 and 16).
  • Said neutralising antibody response may inhibit replication of RSV (RSV A or B) in the respiratory system of a subject (such as in the lungs).
  • Said neutralising antibody response may yield protective immunity against RSV (such as RSV A or B) in a subject, e.g. against RSV A.
  • nucleic acids (preferably RNA) of the present disclosure elicit a cross-neutralising antibody response against RSV in vivo, e.g. against strains of both RSV A and B subtypes (see, e.g. Examples 6, 15 and 16). Said cross-neutralising antibody response may yield protective immunity against strains of both RSV A and B subtypes in a subject.
  • RNA in a preferred embodiment, the nucleic acid of the present disclosure (encoding an RSV-F protein of the present disclosure) is RNA.
  • RNA refers to an artificial (or, defined differently, recombinant) ribonucleic acid encoding an RSV-F protein of the present disclosure, which may be translated in a cell (i.e. mRNA).
  • the RNA is neither, nor comprised within, a viral vector or virus-based vaccine (such as a live-attenuated virus vaccine).
  • RNA molecules can have various lengths but are typically 500-20,000 ribonucleotides long e.g.1000- 20,000, 1000-15,000, 1000-10,000, 1000-5000, 1000-3000, 1000-2500, 1000-2500 or 1000-2000 ribonucleotides long.
  • the RNA can be non-self-replicating (also referred to as “conventional” RNA), or self-replicating; preferably non-self-replicating.
  • the RNA is self-replicating.
  • Self-replicating RNA can be produced using replication elements derived from, e.g., alphaviruses, and substituting sequences encoding the structural viral proteins with that encoding at least an RSV-F protein of the present disclosure.
  • a self- replicating RNA molecule is typically a positive-strand molecule which can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads Docket No.: 70348WO01 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 RNA may encode (i) an RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA and (ii) an RSV-F protein of the present disclosure.
  • the polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4.
  • alphavirus-based self-replicating RNA can use a replicase from, for example, a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus (EEEV), or a Venezuelan equine encephalitis virus (VEEV).
  • a self-replicating RNA encoding an RSV-F protein of the present disclosure may have two open reading frames.
  • the first (5') open reading frame encodes a replicase, in particular an alphavirus replicase (e.g. as detailed above); the second (3') open reading frame encodes the RSV-F protein of the present disclosure.
  • Further open reading frames may also be present, encoding (i) one or more further proteins (preferably one or more further antigens, e.g. as detailed above); and/or (ii) accessory polypeptides.
  • the RNA comprises a 5’ cap, such as a 7-methylguanosine (m 7 G / m7G), which may be added via enzymatic means or a non-enzymatic reaction.
  • the RNA may have the following exemplary 5’ caps: - a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge (also referred to as “Cap O”); - a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge, and wherein the first 5’ ribonucleotide comprises a 2’-methylated ribose (2’-O-Me) (also referred to as “Cap 1”); - a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotides by a triphosphate bridge, and wherein the first and second 5’ ribonu
  • the 5’ cap is a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleoside by a triphosphate bridge, and wherein the first 5’ ribonucleoside comprises a 2’- Docket No.: 70348WO01 methylated ribose (2’-O-Me), e.g. the 5’ end of the RNA has the structure m7G(5')ppp(5')(2'OMeA)pG.
  • this cap is added non-enzymatically through the use of the following reagent: Said reagent is sold as CLEANCAP Reagent AG (TRILINK BIOTECHNOLOGIES).
  • a cap may be added resulting in the 5’ end of the RNA having the structure m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.
  • This cap may be added non-enzymatically through the use of the following reagent: Said reagent is sold as CLEANCAP Reagent AG (3’OMe) (TRILINK BIOTECHNOLOGIES)
  • the RNA comprises a 3’ poly-adenosine (“poly-A”) tail, e.g. comprising 10-700 A ribonucleotides.
  • the poly-A tail may comprise at least two non-contiguous stretches of A Docket No.: 70348WO01 ribonucleotides (also referred to as a “split poly-A tail”), or a (in particular, only one) contiguous stretch of A ribonucleotides.
  • the total number of A ribonucleotides (“As”) in at least two non- contiguous stretches may be, for example, 10-700, such as 10-600, 10-500, 20-500, 50-500, 70-500, 100-500, 20-400, 30-300, 40-200, 50-150, 70-120, 100-120, or, in particular, 100-120.
  • the total number of As in a (in particular, only one) contiguous stretch may be, for example, 10-700; such as 10-600, 20-600 or in particular 40-600 (such as 50-600, 80-600, 80-550, 100-500; or 40-70, 50-65 or 55-65).
  • at least two non-contiguous stretches of As are used, these may be of differing length.
  • a first stretch may be 10-150 As in length, such as 10-100, 10-50, 15-50, 20-50, 20-40, 25-40, or, in particular 25-35 As in length.
  • a second stretch may be 10-150 As in length, such as 10-150, 20-120, 30-100, 40-90, 50-90, 60-90, 65-90, 70-90, or, in particular, 80-90 As in length.
  • the first stretch may be located 5’ or 3’ relative to the second stretch.
  • the first stretch is located 5’ relative to the second stretch.
  • the polyA tail comprises, in the 5’ to 3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 and 80-90 As in length respectively.
  • the polyA tail comprises, in the 5’-3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 and 65-90 As in length respectively.
  • the polyA tail comprises, in the 5’-3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 (e.g.28-32, 29- 31, about 30 or 30) and 25-45 (e.g. 25-40, 30-40, 35-40, 35-39, 36-38, about 37 or 37) As in length respectively.
  • the at least two non-contiguous stretches of As is from, or is part of, the 3’ untranslated region (UTR), e.g. as detailed below.
  • the RNA preferably comprises (in addition to any 5' cap structure) one or more modified ribonucleotides, i.e. ribonucleotides that are modified in structure relative to standard A, C, G or U ribonucleotides.
  • the RNA does not comprise modified ribonucleotides, i.e. the RNA contains standard A, C, G or U ribonucleotides only (except for any 5’ cap structure, if present, e.g. as detailed above).
  • said one or more modified ribonucleotides may be, or may comprise, N1-methylpseudouridine (“1m ⁇ ”); pseudouridine (“ ⁇ ”); N1-ethylpseudouridine; 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6- methyladenosine (m 6 A); N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1- methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenos
  • 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 Docket No.: 70348WO01 U nucleotides, nor modified U ribonucleotides other than 1m ⁇ and/or ⁇ , in the RNA; i.e. 100% U substitution).
  • the RNA may comprise 1m ⁇ and/or ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e. there are no standard U nucleotides, nor modified ribonucleotides of any type - A, C, G or U substitutable - other than 1m ⁇ and/or ⁇ , in the RNA; i.e. 100% U substitution with no other modified nucleotides being allowed).
  • the RNA may comprise ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. 100% U substitution with ⁇ ).
  • the RNA may comprise ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e. 100% U substitution with ⁇ with no other modified nucleotides being allowed). More preferably, the RNA comprises 1m ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e.100% U substitution with 1m ⁇ ). In an even more preferred embodiment, the RNA comprises 1m ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e.100% U substitution with 1m ⁇ with no other modified nucleotides being allowed). In the embodiments in this paragraph, “[may] comprise[s]...
  • the RNA is codon-optimised.
  • An example of a codon-optimised sequence is SEQ ID NO: 104 (codon-optimised version of SEQ ID NO: 83). Codon optimisation may provide an elevated GC content, relative to non-codon optimised RNA encoding the same protein(s).
  • the GC content (the percentage of all ribonucleotides (or, defined alternatively, all “nitrogenous bases”) in the RNA which are G or C) of the RNA may be at least 10%, such as at least 20%, 30%, 35% or at least 40%, preferably at least 45%, 46%, 47%, 48%, 49%, or at least 50%.
  • the GC content of the RNA may be 10-70%, such as 20-65%, 30-65% or 35-65%, preferably 40-60%, 45-55%, 46-53%, 47-51%, or 48-50%.
  • the GC content of the RNA may be 30-70%, such as 40-70%, 45-70%, 50-70%, or 55-70%.
  • Codon optimisation may provide an elevated C content relative to non-codon optimised RNA encoding the same protein(s).
  • the percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be least 30%, such as at least 40%, 50%, 55% or at least 60%, preferably at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% or at least 72%;
  • the percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be 30-80%, such as 40-90%, 45-90%, 50-80%, 55-80% or 60-80%, preferably 65-75%, 66-75%, 67-75%, 68-75%, 69-75%, 70-74%, 71-74% or 72-74%.
  • the RNA comprises a 5’ and/or a 3’ untranslated region (UTR), preferably both a 5’ and 3’ UTR; e.g. selected from the 5’and 3’ UTRs of RNA transcripts of the following genes (preferably the following human genes): beta-actin, albumin, ATP synthase beta subunit, fibroblast activation protein (“FAP”), H4 clustered histone 15 (“HIST2H4A”), glyceraldehyde-3-phosphate dehydrogenase, heat shock protein family A (Hsp70) member 8 gene,, interleukin-2 gene (“IL-2”), and transferrin.
  • UTR untranslated region
  • the RNA comprises a 5’ and a 3’ UTR selected from: Docket No.: 70348WO01 - SEQ ID NO: 94 and 95, respectively, - SEQ ID NO: 96 and 97, respectively, - SEQ ID NO: 98 and 99, respectively, - SEQ ID NO: 100 and 101, respectively, - SEQ ID NO: 102 and 103, respectively, and - 5’ and 3’ UTRs having at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 99.5% identity to SEQ ID NO: 94 and 95, SEQ ID NO: 96 and 97, SEQ ID NO: 98 and 99, SEQ ID NO: 100 and 101, and SEQ ID NO: 102 and 103, respectively); with RNA sequences according to SEQ ID NO: 94 and 95, SEQ ID NO: 96 and 97, SEQ ID NO: 98 and 99 (and RNA sequences having such identity thereto, preferably at least 9
  • 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 ribonucle
  • 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: 82; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C relative to (and numbered according to) SEQ ID NO: 1 or 3, preferably further comprising the deletion ⁇ 555-574 relative to (and numbered according to) SEQ ID NO: 1 or 3; SEQ ID NO: 104; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
  • SEQ ID NO: 86 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C, 490C relative to (and numbered according to) SEQ ID NO: 1 or 3, preferably further comprising a linker sequence joining the F1 and F2 domains of the RSV-F protein which is preferably a GS linker (SEQ ID NO: 18), preferably further comprising the deletion ⁇ 555-574 relative to (and numbered according to) SEQ ID NO: 1 or 3; or SEQ ID NO: 72; or an RNA
  • the RNA comprises or consists of any of SEQ ID NOs: 48, 50, 52, 54, 58, 60, 62, 64, 70, 74, 76, 78, 80, 84, 88, 90 or 92; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to any of said SEQ ID NOs, optionally encoding an RSV-F protein of the present Docket No.: 70348WO01 disclosure comprising the mutations relative to (and numbered according to) SEQ ID NO: 1 present in the protein encoded by any of said SEQ ID NOs respectively.
  • a DNA construct preferably a DNA plasmid
  • an RNA sequence comprising or consisting of: any of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, 62, 64, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 or 104; or any of the foregoing sequences having identity to said SEQ ID NOs.
  • the RNA comprises an open reading frame (ORF) comprising or consisting of the sequence of: positions 32-1693 of SEQ ID NO: 82; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C relative to (and numbered according to) SEQ ID NO: 1 or 3, preferably further comprising the deletion ⁇ 555-574 relative to (and numbered according to) SEQ ID NO: 1 or 3; positions 32-1693 of SEQ ID NO: 104; or an RNA sequence at least 90%, 91%, 92%
  • the RNA comprises an ORF comprising or consisting of the ORF from any of SEQ ID NOs: 48, 50, 52, 54, 58, 60, 62, 64, 70, 74, 76, 78, 80, 84, 88, 90 or 92; or an ORF at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to the ORF from any of said SEQ ID NOs, optionally encoding an RSV-F protein of the present disclosure comprising the mutations relative to (and numbered according to) SEQ ID NO: 1 present in the protein encoded by the ORF from any of said SEQ ID NOs respectively.
  • a DNA construct preferably a DNA plasmid
  • an RNA sequence comprising an ORF comprising or consisting of the sequence of: the ORF from any of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, 62, 64, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 or 104; or any of the foregoing ORFs having identity to the ORFs from said SEQ ID NOs.
  • Nucleic acid e.g.
  • RNA can conveniently be prepared by in vitro transcription (IVT).
  • IVT in vitro transcription
  • 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).
  • RNA-dependent RNA polymerase such as the bacteriophage T7, T3 or SP6 RNA polymerases
  • a DNA-dependent RNA polymerase can be used to transcribe the replicating RNA from a DNA template.
  • Appropriate capping and poly-A addition reactions can be used as required (although the poly-A tail is usually encoded within the DNA template).
  • Carriers comprising a nucleic acid encoding an RSV-F protein in the prefusion conformation Nucleic acid (especially RNA) by themselves and unprotected, may be degraded by the subject’s nucleases and may require a carrier to facilitate target cell entry.
  • the present disclosure also provides a carrier comprising a nucleic acid (preferably RNA) encoding an RSV-F protein of the present disclosure.
  • the carrier may be lipid-based (e.g. a lipid nanoparticle or cationic nanoemulsion), polymer-based (e.g. comprising polyamines, dendrimers and/or copolymers), peptide or protein-based (e.g. comprising protamine, a cationic cell-penetrating peptide, and/or an anionic peptide conjugated Docket No.: 70348WO01 to a positively charged polymer), cell-based (e.g.
  • the carrier is non-virion, i.e. free or substantially free of viral capsid.
  • lipid-based carriers provide a means to protect the nucleic acid (preferably RNA), e.g. through encapsulation, and deliver it to target cells for protein expression.
  • the lipid-based carrier is, or comprises, a cationic nano-emulsion (“CNE”). CNEs and methods for their preparation are described in, for example, [22].
  • the nucleic acid (preferably RNA) which encodes the RSV-F protein of the present disclosure is complexed with a CNE particle, in particular comprising an oil core and a cationic lipid.
  • the cationic lipid can interact with the negatively charged molecule, thereby anchoring the molecule to the emulsion particles.
  • a lipid-based carrier is a lipid inorganic nanoparticle (“LION”).
  • LNPs lipid inorganic nanoparticle
  • nucleic acids (preferably RNA) are encapsulated in a lipid nanoparticle (LNP).
  • the present disclosure also provides an LNP encapsulating a nucleic acid (preferably RNA) which encodes an RSV-F protein of the present disclosure.
  • a nucleic acid preferably RNA
  • a plurality of such LNPs will be part of a composition (e.g.
  • a pharmaceutical composition as detailed in the section entitled Pharmaceutical compositions below) comprising free and/or encapsulated nucleic acid (preferably RNA), and in some embodiments the LNPs encapsulate at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least 100% of the total number of nucleic acid (preferably RNA) molecules in the composition.
  • nucleic acid
  • At least 80% of the LNPs in the composition may be 20-200 nm, 40-190 nm, 60-180 nm or, in particular, 80-160 nm in diameter.
  • substantially all, or all, LNPs in the composition are 20-200 nm, 40-190 nm, 60-180 nm or, in particular, 80-160 nm in diameter.
  • the LNP can comprise multilamellar vesicles (MLV), small uniflagellar vesicles (SUV), or large unilamellar vesicles (LUV).
  • the amount of nucleic acid (preferably RNA) per LNP can vary, and the number of individual nucleic acid molecules per LNP can depend on the characteristics of the particle being used.
  • an LNP may include 1-500 RNA molecules, e.g. ⁇ 200, ⁇ 100, ⁇ 50, ⁇ 20, ⁇ 10, ⁇ 5, or 1-4.
  • an LNP includes fewer than 10 different species of RNA e.g. fewer than 5, 4, 3, or 2 different species.
  • the LNP includes a single RNA species (i.e. all RNA molecules in the particle have the same sequence).
  • LNPs according to the present disclosure may be formed from a single lipid (e.g.
  • a cationic lipid or, in particular, from a mixture of lipids.
  • the mixture comprises various classes of lipids, such as: Docket No.: 70348WO01 (a) a mixture of cationic lipids and sterols, (b) a mixture of cationic lipids and neutral lipids, (c) a mixture of cationic lipids and polymer-conjugated lipids, (d) a mixture of cationic lipids, sterols and polymer-conjugated lipids, or (e) a mixture of cationic lipids, neutral lipids and polymer-conjugated lipids; or preferably: (f) a mixture of cationic lipids, sterols and neutral lipids; or more preferably: (g) a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids.
  • lipids such as anionic lipids
  • the cationic lipid may have a pKa of 5.0-10.0, 5.0-9.0, 5.0-8.5, preferably 5.0-8.0, 5.0-7.9, or 5.0-7.8, 5.0-7.7, or more preferably 5.0-7.6.
  • the pKa of the cationic lipid is distinct to the pKa of the LNP as a whole (sometimes called “apparent pKa”).
  • pKa may be determined via any well-known method, such as via a toluene nitrosulphonic acid (TNS) fluorescence assay or acid base titration; preferably a TNS fluorescence assay; more preferably performed according to Example 10.
  • the cationic lipid preferably comprises a tertiary or quaternary amine group, more preferably a tertiary amine group.
  • Exemplary cationic lipids comprising tertiary amine groups include: 1,2-dilinoleyoxy- 3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1,2- dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DL
  • the cationic lipid has the structure of lipid RV28, RV31, RV33, RV37, RV39 RV42, RV44, RV73, RV75, RV81, RV84, RV85, RV86, RV88, RV91, RV92, RV93, RV94, RV95, RV96, RV97, RV99 or RV101, as disclosed in [24].
  • the cationic lipid has the structure: Docket No.: 70348WO01
  • the cationic lipid has the structure: (also referred to as lipid RV39).
  • the cationic lipid has the structure (referred to as Structure A): In another preferred embodiment, the cationic lipid has the structure: Docket No.: 70348WO01 In another preferred embodiment, the cationic lipid has the structure: (also referred to as lipid RV94).
  • the lipids in the LNP may comprise (in mole %) 20-80, 25-75, 30-70, or 35-65%, preferably 30-60, 40-55 or 40-50% cationic lipid; such as about 40% (or 40%), about 42% (or 42%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) cationic lipid.
  • the lipids in the LNP may comprise (in mole %) at least 20, 25 or at least 35%, or preferably at least 40% cationic lipid.
  • the lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60% or preferably no more than 50% cationic lipid.
  • the molar ratio of protonatable nitrogen atoms in the LNP’s cationic lipids to phosphates in the nucleic acid, preferably RNA (a.k.a “N:P” ratio), may be in the range of (including the endpoints) 1:1-20:1, 2:1-10:1, 3:1-9:1, or 4:1-8:1; preferably 4.5:1-7.5:1, 4.5:1-6.5:1 or 5.0:1-6.5:1.
  • the polymer-conjugated lipid is preferably a PEGylated lipid.
  • the PEGs of such PEGylated lipids may have an average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8- 6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0 (or 2.0 kDa).
  • the average molecular weight of such PEGs may be expressed as the median molecular weight.
  • the PEGs of such PEGylated lipids may have a weight average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8- 8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5- 2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0 (or 2.0 kDa).
  • 0.5-11.0 kDa such as 0.5-8.0, 0.8- 8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5- 2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0 (or
  • At least 80% of the PEGs of such PEGylated lipids may have a molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0, or 2.0 kDa.
  • 0.5-11.0 kDa such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa, or more preferably 1.8-2.2, 1.9-2.1, about 2.0,
  • the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.
  • the lipids in the LNP may comprise (in mole %) 0.1-8.0, 0.4-7.0, 0.6-6.0, 0.8-4.0 or 0.8-3.5%, preferably 1.0-3.0% polymer-conjugated lipid (preferably PEGylated lipid); such as about 1.0 (or 1.0%), about 1.5% (or 1.5%), about 2.0% (or 2.0%) or about 2.5% (or 2.5%) polymer-conjugated lipid (preferably PEGylated lipid).
  • the lipids in the LNP may comprise (in mole %) at least 0.1, 0.5 or at least 0.8%, or preferably at least 1% polymer-conjugated lipid (preferably PEGylated lipid).
  • the lipids in the LNP may comprise (in mole %) no more than 8.0, 6.0 or 4.0% or preferably no more than 3.0% polymer-conjugated lipid (preferably PEGylated lipid).
  • the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), although other neutral lipids available to the skilled person may also be used.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine
  • the lipids in the LNP may comprise (in mole %) 0-15.0, 0.1-15.0, 2.0-14.0, 5.0-13.0, 6.0-12.0 or 7.0- 11.0%, preferably 8.0-11.0% or 9.0-11.0% neutral lipid; such as about 9.4% (or 9.4%), about 9.6% (or 9.6%), about 9.8% (or 9.8%) or about 10.0% (or 10%) neutral lipid.
  • the lipids in the LNP may comprise (in mole %) at least 0.1, 5.0 or at least 7.0%, or preferably at least 8.0% or at least 9.0% Docket No.: 70348WO01 neutral lipid.
  • the lipids in the LNP may comprise (in mole %) no more than 15.0, 13.0 or no more than 12.0%, or preferably no more than 11.0% neutral lipid.
  • exemplary sterols include cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7- dehydrocholesterol, dihydrolanosterol, symosterol, lathosteriol, 14-demethyl-lanosterol, 8(9)- dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydrolanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9), 14-dien-3 ⁇ -ol (dihydro-FF-MAS-d6), 4,4-dimethyl
  • the sterol is cholesterol or a cholesterol-based lipid (e.g. any of those provided in the foregoing paragraph).
  • the lipids in the LNP may comprise (in mole %) 20-80, 25-80, 30-70, 30-60, 35-60 or 40-60%, preferably 40-50% or 41-49% sterol; such as about 42% (or 42%), about 43% (or 43%), about 44% Docket No.: 70348WO01 (or 44%), about 46% (or 46%), or about 48% (or 48%) sterol.
  • the lipids in the LNP may comprise (in mole %) at least 20, 30 or at least 35%, or preferably at least 40% or at least 41% sterol.
  • the lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60%, or preferably no more than 50% sterol.
  • the lipids in the LNP may have the following mole % in combination: 30-60% cationic lipid (such as 35-55%, or preferably 40-50%), 35-70% sterol (such as 40-55%, or preferably 41-49%), 0.8-4.0% polymer-conjugated lipid (such as 0.8-3.5%, or preferably 1.0-3.0%), and 0-15% neutral lipid (such as 6.0-12.0% or preferably 8.0-11.0%).
  • a preferred LNP comprises: (i) cationic lipid of Structure A (defined above), (ii) cholesterol, (iii) 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide and (iv) 1,2-distearoyl-sn-glycero-3- phosphocholine; wherein said LNP encapsulates RNA according to present disclosure (preferably according to SEQ ID NO: 104).
  • Such LNPs encapsulating nucleic acids may be formed by admixing a first solution comprising the nucleic acids with a second solution comprising lipids which form the LNP.
  • the admixing may be performed by any suitable means available to the skilled person, e.g. a T-mixer, microfluidics, or an impinging jet mixer. Admixing may be followed by filtration to obtain a desirable LNP size distribution (e.g. those as detailed above in this subsection).
  • the filtration may be performed by any suitable means available to the skilled person, e.g. tangential-flow filtration or cross-flow filtration.
  • the present disclosure provides a method of preparing an LNP encapsulating a nucleic acid (preferably RNA) encoding a RSV-F protein of the present disclosure, comprising admixing a first solution comprising the nucleic acid and a second solution comprising lipids which form the LNP (e.g using the means as set out in the foregoing paragraph); and optionally filtering the obtained admixture (e.g using the means as set out in the foregoing paragraph).
  • compositions in a further independent aspect, also provides a pharmaceutical composition comprising an RSV-F protein, nucleic acid (preferably RNA) and/or carrier (preferably lipid Docket No.: 70348WO01 nanoparticle) of the present disclosure.
  • Such compositions typically further comprise a pharmaceutically acceptable excipient.
  • Pharmaceutically acceptable excipients are well-known in the art, see, e.g. [25].
  • Such compositions are generally for immunising subjects against disease, preferably against RSV. Accordingly, pharmaceutical compositions of the present disclosure are generally considered vaccine compositions.
  • Pharmaceutical compositions of the present disclosure may comprise the RSV-F protein, nucleic acid (preferably RNA) and/or carrier (preferably lipid nanoparticle) in plain water (e.g.
  • compositions of the present disclosure may have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0.
  • Pharmaceutical compositions of the present disclosure compositions may include sodium salts (e.g. sodium chloride) to give tonicity.
  • a concentration of 10 ⁇ 2 mg/mL NaCl is typical, e.g. about 9 mg/mL (or 9 mg/mL).
  • compositions of the present disclosure may include metal ion chelators (in particular, in embodiments wherein such compositions comprise RNA). These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis.
  • such compositions may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc..
  • chelators are typically present at between 10-500 ⁇ e.g.0.1 mM.
  • a citrate salt, such as sodium citrate, can also act as a chelator, while advantageously also providing buffering activity.
  • Pharmaceutical compositions of the present disclosure may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g.
  • compositions of the present disclosure may include one or more preservatives, such as thiomersal or 2-phenoxyethanol.
  • Mercury-free compositions are preferred, and preservative-free vaccines can be prepared.
  • Pharmaceutical compositions of the present disclosure may be aseptic or sterile.
  • Pharmaceutical compositions of the present disclosure may be non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • Pharmaceutical compositions of the present disclosure may be gluten free.
  • Pharmaceutical compositions of the present disclosure may be prepared in unit dose form.
  • a unit dose may have a volume of between 0.1 -1.0 mL e.g. about 0.5mL (or 0.5mL).
  • Pharmaceutical compositions of the present disclosure may be prepared as injectables, either as solutions or suspensions.
  • the composition may be prepared for pulmonary administration e.g. by an Docket No.: 70348WO01 inhaler, using a fine spray.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops. Injectables for intramuscular administration are typical.
  • Pharmaceutical compositions of the present disclosure comprise an immunologically effective amount of RSV-F protein. nucleic acid (preferably RNA) and/or carrier (preferably lipid nanoparticle), as well as any other components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention, preferably prevention of RSV.
  • This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • RNA content will generally be expressed in terms of the amount of RNA per dose.
  • a preferred dose has ⁇ 120 ⁇ g RNA e.g. ⁇ 100 ⁇ g (e.g.10-120 ⁇ g or 10-100 ⁇ g, such as 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g, or about 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g), but expression can be seen at much lower levels e.g. ⁇ 1 ⁇ g/dose, ⁇ 100ng/dose, ⁇ 10ng/dose, ⁇ 1ng/dose, etc.
  • a further preferred dose has 1-100 ⁇ g RNA (e.g.1-90 ⁇ g, 1- 80 ⁇ g, 1-70 ⁇ g, 1-60 ⁇ g, 1-55 ⁇ g or 1-50 ⁇ g), with further preferred specific doses of 3 ⁇ g, 6 ⁇ g, 12.5 ⁇ g, 25 ⁇ g or 50 ⁇ g; in particular wherein said further preferred dose (or specific dose) is administered to a subject at least twice, separated by 1-3 months, e.g. about 2 months apart or 2 months apart.
  • Pharmaceutical compositions of the present disclosure may further comprise an adjuvant (i.e. an agent that enhances an immune response in a non-specific manner), in particular, but not exclusively, when comprising an RSV-F protein of the present disclosure.
  • an adjuvants include suspensions of minerals (e.g.
  • the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod.
  • the adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulphate.
  • the adjuvants described herein can be used singularly or in any combination, such as alum/TLR7 (also called AS37).
  • Pharmaceutical compositions of the present disclosure may comprise a saponin as an adjuvant, e.g. saponin fraction QS21 (see, e.g. [26]).
  • QS21 may be used in substantially pure form, e.g. at least 80% pure, such as at least 85, 90%, 95% or at least 98% pure.
  • a suitable QS-21 fraction is as described in [27].
  • compositions of the present disclosure may be lyophilised. Docket No.: 70348WO01
  • pharmaceutical compositions of the present disclosure comprise (i) a nucleic acid (preferably RNA) encoding an RSV-F protein of the present disclosure, and (ii) a further nucleic acid (preferably RNA) encoding at least one further protein.
  • the nucleic acids of (i) and (ii) may be comprised within the same carrier (preferably lipid nanoparticle), or within separate carriers (preferably lipid nanoparticles).
  • the at least one further protein is an antigen from, or derived from, a virus
  • examples of such viruses include: Coronavirus, Orthomyxovirus, Pneumoviridae, Paramyxoviridae, Poxviridae, Picornavirus, Bunyavirus, Heparnavirus, Filovirus, Togavirus, Flavivirus, Pestivirus, Hepadnavirus, Rhabdovirus, Caliciviridae, Retrovirus, Reovirus, Parvovirus, Herpesvirus, Papovaviruses and Adenovirus.
  • the at least one further protein encoded by the nucleic acid of (ii) is a further Pneumoviridae protein (in particular a Pneumoviridae antigen).
  • a further Pneumoviridae protein in particular a Pneumoviridae antigen.
  • Useful further Pneumoviridae proteins can be from an Orthopneumovirus or Metapneumovirus, in particular human RSV or human Metapneumovirus (hMPV).
  • Useful further hMPV antigens include e.g. the F, N, P, M, M2-1, and M2 antigens (in particular, the F antigen).
  • Such hMPV proteins (in particular, antigens) may be from, or derived from, the A or B subtype.
  • the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding an hMPV antigen (in particular, the F antigen).
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • is infants see section entitled Medical uses and methods of treatment, below.
  • Useful further human RSV antigens encoded by the nucleic acid of (ii) include e.g. the G, M1, M2-1, M2-2, P, L, N, NS1, NS2 and SH antigens, in addition to further RSV-F antigens, i.e.
  • the at least one further protein encoded by the nucleic acid of (ii) is a Coronavirus antigen.
  • Useful Coronavirus antigens can be from a SARS coronavirus, in particular SARS-CoV2.
  • Useful Coronavirus antigens include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein.
  • the Coronavirus antigen is a SARS-CoV2 spike protein.
  • Said SARS-CoV2 spike protein may be from any variant, e.g. Omicron (such as Omicron BA.1, BA.2, BA3, BA.4 or BA.5), Alpha, Epsilon, Eta, Theta, Docket No.: 70348WO01 Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma, or Delta.
  • said SARS-CoV2 spike protein includes one or more mutations relative to the wild-type protein, in particular one or more (e.g. two) mutations to proline resides.
  • the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding a Coronavirus antigen, e.g. as detailed above.
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • the at least one further protein encoded by the nucleic acid of (ii) is an Orthomyxovirus antigen.
  • Useful Orthomyxovirus antigens can be from an influenza A, B or C virus.
  • Useful Orthomyxovirus antigens include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin.
  • the Orthomyxovirus antigen is an influenza A virus haemagglutinin.
  • Said influenza A virus hemagglutinin may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.
  • compositions of the present disclosure comprise (i) a first plurality of RSV-F proteins and/or trimers according to the present disclosure, wherein the RSV-F proteins and/or trimers are of the A subtype; and (ii) a second plurality of RSV-F proteins and/or trimers according to the present disclosure, wherein the RSV-F proteins and/or trimers are of the B subtype.
  • compositions of the present disclosure comprise (i) a first plurality of nucleic acids (preferably RNAs) and/or carriers (preferably lipid nanoparticles) according to the present disclosure, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the present disclosure of the A subtype; and (ii) a second plurality of nucleic acids (preferably RNAs) and/or carriers (preferably lipid nanoparticles) according to the present disclosure, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the present disclosure of the B subtype.
  • a first plurality of nucleic acids preferably RNAs
  • carriers preferably lipid nanoparticles
  • 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.
  • a delivery device e.g. syringe, nebuliser, sprayer, inhaler, dermal patch, etc.
  • 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.
  • Said medicament will generally be for raising an immune response in a subject.
  • the present disclosure also provides, in a further independent aspect, a therapeutic method comprising the step of administering an effective amount of an RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure to a subject (preferably a subject in need of such administration).
  • Said method will generally be for raising an immune response in the subject.
  • the immune response is preferably protective and, preferably involves antibodies and/or cell-mediated immunity.
  • the subject is a vertebrate, preferably a mammal, more preferably a human or large veterinary mammal (e.g. horses, cattle, deer, goats, pigs), even more preferably a human.
  • the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure may be for use in the prevention, reduction or treatment of infection or disease.
  • the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure may be for use in the prevention, reduction or treatment of symptoms associated with infection or disease.
  • the infection is generally one by, and said disease is generally one associated with, a Pneumoviridae virus.
  • the Pneumoviridae virus is an Docket No.: 70348WO01 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.
  • the first administration may induce proliferation and maturation of B and/or T cell precursors specific to one or more immunogenic epitopes present on the delivered antigen (induction phase).
  • the second (and in some cases subsequent) administration (“boost”), may further stimulate and potentially select an anamnestic response of cells elicited by the prior administration(s).
  • the different administrations may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
  • the prime administration(s) and boost administration(s) will be temporally separated, e.g. by at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more months.
  • prime administrations may be administered 3-9 weeks apart (e.g.4-9, 5-9, 6-9, 7-9 or 7-8 weeks apart, or about two months apart), followed by one or more boost administrations 4-14 months after the second prime administration (e.g.5-13, 6-13, 7-13, 8-13, 9-13, 10-13 or 11-13 months, or about one year).
  • prime administration is to a na ⁇ ve subject.
  • the protein antigen may be delivered in the prime and boost administrations as, or via, different formats.
  • the protein antigen may be delivered as a protein for the prime administration(s), and via a nucleic acid (in particular RNA, in particular via a carrier comprising RNA) for the boost administration(s), or vice versa.
  • a nucleic acid in particular RNA, in particular via a carrier comprising RNA
  • different nucleic acid formats may be used, e.g. the protein antigen may be delivered via RNA (in particular via a carrier comprising RNA) for the prime administration(s), and a via a viral vector (e.g. an adenoviral vector) for the boost administration(s), or vice versa.
  • Docket No.: 70348WO01 The RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure will generally be administered directly to the subject.
  • the subject is administered (i) a first plurality of nucleic acids (preferably RNAs) and/or carriers (preferably lipid nanoparticles) according to the present disclosure, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the A subtype; and (ii) a second plurality of nucleic acids (preferably RNAs) and/or carriers (preferably lipid nanoparticles) according to the present disclosure, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the B subtype; wherein the first plurality and second plurality are administered simultaneously, separately or sequentially to one another (preferably simultaneously, e.g. in the same composition).
  • a first plurality of nucleic acids preferably RNAs
  • carriers preferably lipid nanoparticles
  • the subject is administered (i) a first plurality of nucleic acids (preferably RNAs) and/or carriers (preferably lipid nanoparticles) according to the present disclosure, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the present disclosure of the A subtype; and (ii) a second plurality of nucleic acids (preferably RNAs) and/or carriers (preferably lipid nanoparticles) according to the present disclosure, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the present disclosure of the B subtype; wherein the first plurality and second plurality are administered simultaneously, separately or sequentially to one another (preferably simultaneously, e.g.
  • the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure may be used to elicit systemic and/or mucosal immunity.
  • the subject of a method of vaccination according to the present disclosure may be a child (preferably an infant) or adult (preferably an older adult or pregnant female). Immunocompromised individuals may also be the subject of such vaccination (whether children or adults).
  • Infant vaccination In a preferred embodiment, the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure are administered to infants (preferably human infants), as the subject of vaccination.
  • the immune systems of infants are immature (see, e.g.
  • infant vaccination may prevent lower respiratory tract infection (in particular, bronchiolitis and (broncho-)pneumonia).
  • the infant may be less than one year old, such as less than: 11, 10, 9, 8, 7, 6, 5, 4 or less than 3 months old.
  • the infant may be ⁇ one month old, such as ⁇ : 2, 3, 4, 5 or ⁇ 6 months old.
  • Preferably the infant is 2-6 months old (i.e. within and including the ages of 2 and 6 months), more preferably 2-4 months old.
  • the infant was born from a female to whom an RSV vaccine (such as an RSV-F protein, nucleic acid, carrier, or pharmaceutical composition of the present disclosure) was administered, preferably while pregnant with said infant.
  • an RSV vaccine such as an RSV-F protein, nucleic acid, carrier, or pharmaceutical composition of the present disclosure
  • the combination of maternal and infant vaccination may advantageously provide passive transfer of maternal antibodies (i.e. via the placenta and/or breast milk) to, in addition to active immunity generated by, the infant.
  • the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure are administered to older adults (preferably human older adults), as the subject of vaccination. Older adults may suffer from age-related immunosenescence (reviewed in, e.g. [29]), hence this population is also susceptible to RSV infection and resulting disease.
  • Older adult vaccination may prevent lower respiratory tract infection (in particular, pneumonia).
  • the older adult may be ⁇ 50 years old, such as ⁇ : 55, 60, 65, 70, 75, 80, 85, 90, 95 or ⁇ 100 years old.
  • the older adult is ⁇ 60 or ⁇ 65 years old (such as 60-120 or 65-120 years old).
  • the RSV-F proteins, nucleic acids, carriers, or pharmaceutical compositions of the present disclosure are administered to pregnant females (preferably pregnant human females), as the subject of vaccination.
  • the primary object of maternal vaccination is to protect the infant from RSV infection when born, e.g. through passive transfer of antibodies via the placenta and/or breast milk.
  • the pregnant female may be in her first, second or third trimester of pregnancy, preferably third trimester.
  • the pregnant female may be ⁇ 20 weeks pregnant, such as ⁇ : 22, 24, 26, 28, 30, 32, 34, 36 or ⁇ 38 weeks pregnant.
  • the pregnant female is ⁇ 28 , ⁇ 29 or ⁇ 30 weeks pregnant (such as 28-43, 29-43 or 30-43 weeks pregnant).
  • General The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology.
  • the singular terms "a,” “an,” and “the” include plural referents unless context clearly indicates otherwise.
  • the word “or” is intended to include “and” unless the context clearly indicates otherwise.
  • the term “plurality” refers to two or more.
  • the term “at least one” refers to one or more. Docket No.: 70348WO01 Unless specified otherwise, where a numerical range is provided, it is inclusive, i.e., the endpoints are included.
  • the terms “at least”, “no more than” and other such terms preceding a list of values are applicable to all members of said list (not merely the first member thereof), unless otherwise stated.
  • the term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
  • An RSV-F protein comprising at least two mutations relative to SEQ ID NO: 1 or 3 within a region of the protein corresponding to positions 474-523 of SEQ ID NO: 1 or 3; wherein the at least two mutations introduce, through substitution or insertion, a pair of C residues into the region, which form a disulphide bond.
  • the RSV-F protein of embodiment 1 or 2 wherein the pair of C residues is within a region of the protein corresponding to positions 474-513 of SEQ ID NO: 1 or 3. 4.
  • the RSV-F protein of embodiment 3 wherein a first C residue of said pair is within a region of the protein corresponding to positions 478-501 of SEQ ID NO: 1 or 3. 5.
  • the RSV-F protein of embodiment 3 or 4 wherein a second C residue of said pair is within a region of the protein corresponding to positions 482-504 of SEQ ID NO: 1 or 3. 6.
  • the RSV-F protein of embodiment 7, 8 or 10 wherein a second C residue of said pair is within a region of the protein corresponding to positions 490-497 of SEQ ID NO: 1 or 3. 12.
  • the RSV-F protein of embodiment 10 or 11 wherein the pair of C residues is at positions 486 and 490, 485 and 494, or 480 and 497 of SEQ ID NO: 1 or 3. 13.
  • An RSV-F protein comprising a C residue at position 486 and a C residue at position 490; wherein the C residues form a disulphide bond. 15.
  • the RSV-F protein of any preceding embodiment comprising an aromatic residue at position 488, such as F, W, Y or H; optionally F, W or Y; optionally F.
  • the RSV-F protein of any preceding embodiment which is in the pre-fusion conformation. 29.
  • the RSV-F protein of any preceding embodiment comprising one or more further mutations, optionally one or more further substitutions, which stabilise and/or promote the pre-fusion conformation of RSV-F. 30.
  • the RSV-F protein of embodiment 29, comprising at least 2, 3, 4, 5, 6 or 7 further mutations, optionally substitutions, which stabilise and/or promote the pre-fusion conformation of RSV- F. 31.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 228 for K, R or Q, optionally K or R; and/or a substitution at position 232 for N. 32.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 55 for T, C, V, I or F; optionally T, C or V; optionally T or C. 35.
  • the RSV-F protein of embodiment 34 comprising a substitution at position 55 for T. 36.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 215 for A, P, V, I, or F; optionally A or P. 37.
  • the RSV-F protein of embodiment 36 comprising a substitution at position 215 for A. 38.
  • the RSV-F protein of any of embodiments 31-36 comprising a substitution at position 228 for K, a substitution at position 55 for T, and a substitution at position 215 for A.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 152 for R, L or W; optionally R or W. 40.
  • the RSV-F protein of embodiment 39 comprising a substitution at position 152 for R. 41.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 315 for I or V. 42.
  • the RSV-F protein of embodiment 41 comprising a substitution at position 315 for I. 43.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 346 for Q, D, H, K, N, R, S or W; optionally Q, D, H, K, N, R or S. 44.
  • the RSV-F protein of embodiment 43 comprising a substitution at position 346 for Q. 45.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 445 for D. 46.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 455 for V or I. 47.
  • the RSV-F protein of embodiment 46 comprising a substitution at position 455 for V. 48.
  • the RSV-F protein of any preceding embodiment comprising a substitution at position 459 for M. 49.
  • the RSV-F protein of any preceding embodiment comprising: a substitution at position 55 for T, a substitution at position 152 for R, a substitution at position 215 for A, a substitution at position 228 for K, a substitution at position 315 for I, a substitution at position 346 for Q, a substitution at position 445 for D, a substitution at position 455 for V, a substitution at position 459 for M, a substitution at position 486 for C, and a substitution at position 490 for C. Docket No.: 70348WO01 50.
  • the RSV-F protein of any preceding embodiment comprising (i) an F2 domain comprising or consisting of an amino acid sequence according to positions 26-109 of SEQ ID NO: 28 or positions 26-102 of SEQ ID NO: 32, and an F1 domain comprising or consisting of an amino acid sequence according to positions 137-513 of SEQ ID NO: 28 or positions 105-472 of SEQ ID NO: 32; or (ii) an F2 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to positions 26-109 of SEQ ID NO
  • the RSV-F protein of any preceding embodiment comprising or consisting of (i) the amino acid sequence of SEQ ID NO: 83, 69, 47, 57, 73 or 87; or (ii) an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% identity to any of SEQ ID NO: 83, 69, 47, 57, 73 or 87. 53.
  • RSV-F protein of any preceding embodiment which, when in the form of a homotrimer, is specifically bound by a pre-fusion mAb comprising a LC and HC according to SEQ ID NO: 7 and 8 respectively with a KD, as measured via SPR, of less than 1000, 900, 800, 700, 600, 500, 400, 350, 320, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25 or 20 pM. 54.
  • the RSV-F protein of any preceding embodiment which is specifically bound by a pre- fusion mAb comprising a LC and HC according SEQ ID NO: 9 and 10 respectively with a KD, as measured via SPR, of less than 5000, 4000, 3000, 2500, 2000, 1900, 1850, 1500, 1000, 800, 600, 400, 200, 100, 90, 80, 75, 70 or 65 pM. 55.
  • the RSV-F protein of any preceding embodiment which is specifically bound by a pre- fusion mAb comprising a LC and HC according SEQ ID NO: 11 and 12 respectively with a KD, as measured via SPR, of less than 1000, 500, 450, 400, 350, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 125, 120, 100, 90, 80, 70 or 65 pM. 56.
  • the RSV-F protein of any preceding embodiment having a T m 1 of at least 65.5, 66.0, 66.5, 67.0, 67.5, 68.0, 68.5, 69.0, 69.5, 70.0, 70.5, 71.0, 71.5, 72.0, 72.5, 73.0, 73.5, or 74.0 °C, when in the form of a homotrimer.
  • the RSV-F protein of embodiment 56 having a Tm1 of at least, or of, 65.7°C. Docket No.: 70348WO01 58.
  • the RSV-F protein of embodiment 56 having a T m 1 of at least, or of, 72.3°C. 59.
  • the RSV-F protein of embodiment 56 having a Tm1 of at least, or of, 74.4°C. 60.
  • the RSV-F protein of any preceding embodiment having a T m 2 of at least 78.0, 78.5, 79.0, 79.5.5, 80.0, 80.5, 81.0, 81.5, 82.0, 82.5, 83.0, or 83.5 °C, when in the form of a homotrimer.
  • the RSV-F protein of embodiment 60 having a T m 2 of at least, or of, 80.8 °C.
  • the RSV-F protein of embodiment 60 having a Tm2 of at least, or of, 79.4 °C. 63.
  • the RSV-F protein of embodiment 60 having a T m 2 of at least, or of, 80.7 °C. 64.
  • the RSV-F protein of any of embodiments 56-64 comprising a bacteriophage T4 fibritin foldon trimerisation domain at the C-terminus thereof, and/or C-terminal to the F1 domain, when the Tm1 and/or Tm2 is measured; optionally wherein RSV-F protein comprises a C- terminal domain comprising or consisting of positions 514-596 of SEQ ID NO: 2 or . 66.
  • the RSV-F protein of any preceding embodiment comprising a heterologous trimerisation domain on the C-terminus thereof and/or C-terminal to the F1 domain, optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain, optionally according to SEQ ID NO: 19.
  • the RSV-F protein of embodiments 1-65 comprising a transmembrane domain, and optionally a cytoplasmic tail C-terminal to said transmembrane domain.
  • the RSV-F protein of embodiment 68 or 69, wherein 2-5, such as 2-4, 2-3, 3-4 or 3 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein. 71.
  • the RSV-F protein of any of embodiments 68-70 wherein the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1-22 of SEQ ID NO: 5 or 6; or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). Docket No.: 70348WO01 72.
  • the RSV-F protein of embodiment 69 wherein 16-20, such as 17-20, 18-20 or 19-20 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein.
  • the RSV-F protein of embodiment 72 wherein 20 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein. 74.
  • the RSV-F protein of any preceding embodiment wherein a signal peptide is not present in the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO: 1 or 3. 78.
  • the RSV-F protein of any preceding embodiment comprising an F2 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to positions 26-109 of SEQ ID NO: 1; and an F1 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, sequence identity to positions 137-523 or 137-513 of SEQ ID NO: 1.
  • the RSV-F protein of any preceding embodiment comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Docket No.: 70348WO01 81.
  • the RSV-F protein of any of embodiments 1-78 comprising an F2 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to positions 1-109 of SEQ ID NO: 3; and an F1 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, sequence identity to positions 137-523 or 137-513 of SEQ ID NO: 3.
  • the RSV-F protein of any of embodiments 1-78 or 82 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3.
  • a trimer comprising three RSV-F proteins of any preceding embodiment.
  • a trimer according to embodiment 85 or 86 comprising an electrostatic repulsive ring comprising three negatively charged residues, each of which is in an HRB domain of an RSV-F protein in the trimer.
  • a trimer according to embodiment 87 wherein the negatively charged residues are at position 487 of each of the RSV-F proteins; optionally wherein the negatively-charged residue is E487 or D487; optionally wherein the negatively charged residue is E487.
  • 93. The nucleic acid of embodiment 91, wherein the nucleic acid is DNA; optionally wherein the DNA is a DNA plasmid. Docket No.: 70348WO01 94.
  • the nucleic acid of embodiment 91, wherein the nucleic acid is RNA. 95.
  • RNA of embodiment 97 wherein the 5’ cap comprises a 7’-methylguanosine linked 5’- to-5’ to the 5’ first ribonucleoside by a triphosphate bridge, and wherein the first 5’ ribonucleoside comprises a 2’-methylated ribose (2’-O-Me).
  • RNA of embodiment 97 or 98 wherein the 3’ poly-A tail comprises at least two non-contiguous stretches of A ribonucleotides; optionally: (a) 25-35 and 65-90 ribonucleotides in length respectively; optionally orientated in the 5’ to 3’ direction, or (b) 25-35 and 25-45 ribonucleotides in length respectively which are optionally orientated in the 5’ to 3’ direction.
  • 101. The RNA of any of embodiments 94-100, comprising a modified ribonucleotide.
  • 102 The RNA of embodiment 101, wherein the modified ribonucleotide is 1m ⁇ 103.
  • RNA of embodiment 102 wherein the RNA comprises 1m ⁇ and neither standard U ribonucleotides nor other modified U ribonucleotides; optionally wherein the RNA comprises 1m ⁇ and neither standard U ribonucleotides nor other modified ribonucleotides.
  • 104 The RNA of any of embodiments 94-103, having a GC content of 55-70%.
  • the RNA of any of embodiments 94-103 having a GC content of 40-60%. 106.
  • RNA of any of embodiments 94-105 comprising or consisting of (i) SEQ ID NO: 82, 104, 128, 68, 56, 46, 86 or 72; or (ii) an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to any of said sequences. 107.
  • RNA of embodiment 106 comprising or consisting of (i) SEQ ID NO: 104; or (ii) an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to SEQ ID NO: 104. Docket No.: 70348WO01 108.
  • RNA of any of embodiments 94-107 comprising an open reading frame (ORF) comprising or consisting of (i) positions 32-1693 of SEQ ID NO: 82, positions 32-1693 of SEQ ID NO: 104, positions 32-1693 of SEQ ID NO: 128, positions 32-1753 of SEQ ID NO: 68, positions 32-1693 of SEQ ID NO: 56, positions 32-1753 of SEQ ID NO: 46, positions 32-1693 of SEQ ID NO: 86, or positions 32-1753 of SEQ ID NO: 72; or (ii) an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to any of positions 32-1693 of SEQ ID NO: 82, positions 32-1693 of SEQ ID NO: 104, positions 32-1693 of SEQ ID NO:
  • a carrier comprising nucleic acid of any of embodiments 91 or 93-108. 110.
  • the carrier of embodiment 109 which is a lipid nanoparticle.
  • the lipid nanoparticle of embodiment 110 comprising a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. 112.
  • the lipid nanoparticle of embodiment 111, wherein the cationic lipid has a pKa of 5.0-8.0; optionally 5.0-7.6. 113.
  • the lipid nanoparticle of any of embodiments 111-116 comprising: (i) cationic lipid of Structure A; (ii) cholesterol; (iii) 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide; and (iv) 1,2-distearoyl-sn-glycero-3-phosphocholine; preferably wherein said LNP encapsulates RNA according to SEQ ID NO: 104. Docket No.: 70348WO01 118.
  • the lipid nanoparticle of embodiment 117 comprising (in mole %): (i) 30-60% cationic lipid of Structure A, such as 35-55%, or preferably 40-50%, 44-48%, 45-47%, about 46.2% or 46.2%; (ii) 35-70% cholesterol, such as 40-55%, or preferably 40-49%, 41-45%, 42-44%, about 42.6%, or 42.6%; (iii) 0.8-4.0% 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide, such as 0.8-3.5%, or preferably 1.0-3.0%, 1.6-2.0%, 1.7-1.9%, about 1.8%, or 1.8%; and (iv) 0-15% 1,2-distearoyl-sn-glycero-3-phosphocholine, such as 6.0- 12.0% or preferably 8.0-11.0%, 9.0-10.0%, about 9.4%, or 9.4%; preferably wherein said LNP encapsulates RNA according to SEQ ID
  • lipid nanoparticle of any of embodiments 111-116 comprising (in mole %) 30- 60% cationic lipid, 35-70% sterol, 0.8-4.0% polymer-conjugated lipid, and 0-15% neutral lipid; optionally 40-50% cationic lipid, 41-49% sterol, 1.0-3.0% polymer-conjugated lipid and 8.0-11.0% neutral lipid.
  • a pharmaceutical composition comprising the RSV-F protein of any of embodiments 1-84, trimer of any of embodiments 85-90, nucleic acid of any of embodiments 91-108, or carrier of any of embodiments 109-119; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant.
  • a vaccine composition comprising the RSV-F protein of any of embodiments 1-84, trimer of any of embodiments 85-90, nucleic acid of any of embodiments 91-108, or carrier of any of embodiments 109-119; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant. 122.
  • composition of embodiment 120 or 121 comprising (i) a first plurality of RSV-F proteins of any of embodiments 1-84 and/or trimers of any of embodiments 85-90, wherein the RSV-F proteins and/or trimers are of the A subtype; and (ii) a second plurality of RSV-F proteins of any of embodiments 1-84 and/or trimers of any of embodiments 85-90, wherein the RSV-F proteins and/or trimers are of the B subtype.
  • a first plurality of RSV-F proteins of any of embodiments 1-84 and/or trimers of any of embodiments 85-90 wherein the RSV-F proteins and/or trimers are of the A subtype
  • second plurality of RSV-F proteins of any of embodiments 1-84 and/or trimers of any of embodiments 85-90 wherein the RSV-F proteins and/or trimers are of the B subtype.
  • composition of embodiment 120 or 121 comprising (i) a first plurality of nucleic acids of any of embodiments 91-108 and/or carriers of any of embodiments 109-119, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the A subtype; and (ii) a second plurality of nucleic acids of any of embodiments 91-108 and/or carriers of any of embodiments 109-119, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the B subtype.
  • composition for use of embodiment 124 for use in a method of raising an immune response in a subject; optionally a protective immune response in a subject. Docket No.: 70348WO01 126.
  • the composition for use of embodiment 124 or 125 for use in the treatment or prevention of RSV. 127.
  • the composition for use of embodiment 126 for use in a method of vaccinating a subject against RSV; optionally wherein the vaccination is prophylactic. 128.
  • the composition for use of any of embodiments 125- 127, wherein the subject is a human infant; optionally 2-6 months old. 129.
  • the composition for use of any of embodiments 125- 127, wherein the subject is a human older adult; optionally ⁇ 50 or ⁇ 60 years old.
  • 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 1-84, trimer of any of embodiments 85-90, nucleic acid of any of embodiments 91-108, carrier of any of embodiments 109-119, pharmaceutical composition of embodiment 120, or vaccine composition of embodiment 121.
  • trimer of any of embodiments 85-90 comprising administering to the subject an immunologically effective amount of the RSV-F protein of any of embodiments 1-84, trimer of any of embodiments 85-90, nucleic acid of any of embodiments 91-108, carrier of any of embodiments 109-119, pharmaceutical composition of embodiment 120, or vaccine composition of embodiment 121.
  • RSV-F protein of any of any of embodiments the RSV-F protein of any of embodiments 1-84, trimer of any of embodiments 85-90, nucleic acid of any of embodiments 91-108, or carrier of any of embodiments 109-119, in the manufacture of a medicament.
  • the medicament is for treating or preventing RSV.
  • the medicament is a vaccine; optionally a prophylactic vaccine. 135.
  • compositions for use of any of embodiments 125-130, method of embodiment 131, or use of any of embodiments 132-134 wherein the subject is administered (i) a first plurality of nucleic acids of any of embodiments 91-108 and/or carriers of any of embodiments 109-119, wherein the nucleic acids encode, and/or the carriers comprise nucleic Docket No.: 70348WO01 acids encoding, an RSV-F protein of the A subtype; and (ii) a second plurality of nucleic acids of any of embodiments 91-108 and/or carriers of any of embodiments 109-119, wherein the nucleic acids encode, and/or the carriers comprise nucleic acids encoding, an RSV-F protein of the B subtype; wherein the first plurality and second plurality are administered simultaneously, separately or sequentially to one another.
  • a kit comprising the RSV-F protein of any of embodiments the RSV-F protein of any of embodiments 1-84, trimer of any of embodiments 85-90, nucleic acid of any of embodiments 91-108, carrier of any of embodiments 109-119, pharmaceutical composition of embodiment 120, or vaccine of embodiment 121, and instructions for use. 138.
  • RNA ribonucleic acid
  • RSV-F respiratory syncytial virus fusion
  • RNA ribonucleic acid
  • UTR untranslated region
  • poly-A poly-adenine
  • the RNA has a guanine-cytosine (GC) content of 30-70%
  • the encoded RSV-F protein comprises at least two mutations relative to SEQ ID NO: 1 or 3 within a region of the protein corresponding to positions 474-523 of SEQ ID NO: 1 or 3; wherein the at least two mutations introduce, through substitution or insertion, a pair of C residues into the region; and wherein, when the encoded RSV-F protein is expressed, the pair of C residues form a disulphide bond.
  • RNA of embodiment 138 wherein, when expressed, the encoded RSV-F protein is in the pre-fusion confirmation.
  • the RNA of embodiment 138 or 139, wherein the encoded RSV-F protein comprises one or more further substitutions, optionally at least 2, 3, 4, 5, 6 or 7 further substitutions.
  • RSV-F protein in the pre-fusion conformation comprising at least two mutations relative to SEQ ID NO: 1 or 3 within a region of the protein corresponding to positions 474- 523 of SEQ ID NO: 1 or 3; wherein the at least two mutations introduce, through substitution or insertion, a pair of C residues into the region, which form a disulphide bond; and wherein the RSV-F protein further comprises, relative to SEQ ID NO: 1 or 3: Docket No.: 70348WO01 (i) a substitution at position 228 for K, R or Q, and/or a substitution at position 232 for N; (ii) a substitution at position 55 for T, C, V, I or F; and/or (iii) a substitution at position 215 for A, P, V, I, or F.
  • RNA or RSV-F protein of any of embodiments 138-142 wherein the disulphide bond is an intra-protomer disulphide bond.
  • 144 The RNA or RSV-F protein of any of embodiments 138-143, wherein a first C residue of said pair is within a region of the encoded RSV-F protein or the RSV-F protein corresponding to positions 480-486 of SEQ ID NO: 1 or 3.
  • 145 The RNA or RSV-F protein of any of embodiments 138-144, wherein a second C residue of said pair is within a region of the encoded RSV-F protein or the RSV-F protein corresponding to positions 490-497 of SEQ ID NO: 1 or 3. 146.
  • RNA or RSV-F protein of embodiment 144 or 145 wherein the pair of C residues is at positions 486 and 490, 485 and 494, or 480 and 497 of SEQ ID NO: 1 or 3. 147.
  • RNA or RSV-F protein of any of embodiments 138-148 wherein the encoded RSV-F protein or wherein the RSV-F protein comprises, relative to SEQ ID NO: 1 or 3, a substitution at position 55 for T, C, V, I or F, optionally T, C or V, optionally T or C, optionally T. 150.
  • the RSV-F protein of any of embodiments 142-152 comprising a heterologous trimerisation domain on the C-terminus thereof and/or C-terminal to the F1 domain, optionally wherein the heterologous trimerisation domain is a T4 fibritin foldon domain, optionally according to SEQ ID NO: 19.
  • RNA or RSV-F protein of any of embodiments 138-150 wherein the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 1- 5 of SEQ ID NO: 5 or 6, 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). 156.
  • RNA or RSV-F protein of any of embodiments 138-155 wherein the encoded RSV-F protein or wherein the RSV-F protein comprises: an F2 domain comprising or consisting of an amino acid sequence having at least 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to positions 26-109 of SEQ ID NO: 1; and an F1 domain comprising or consisting of an amino acid sequence having at least 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, sequence identity Docket No.: 70348WO01 to positions 137-523 or 137-513 of SEQ ID NO: 1; optionally wherein the RSV-F protein is of the A
  • RNA of any of embodiments 138-141, 143-152 or 155-159 comprising or consisting of (i) SEQ ID NO: 82, 104, 68, 56, 46, 86 or 72; or (ii) an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 99.94% identical to any of SEQ ID NO: 82, 104, 68, 56, 46, 86 or 72. 161.
  • a lipid nanoparticle comprising the RNA of any of embodiments 138-141, 143-152 or 155-160.
  • a pharmaceutical composition comprising the RSV-F protein of any of embodiments 142-157, nucleic acid of embodiment 158, RNA of any of embodiments 138-141, 143-152 or 155-160 or lipid nanoparticle of embodiment 161; optionally for use in medicine. 163.
  • 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 142-157, nucleic acid of embodiment 158, RNA of any of embodiments 138-141, 143-152 or 155-160 or lipid nanoparticle of embodiment 161.
  • Docket No.: 70348WO01 EXAMPLES Many modifications and variations of the present disclosure are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, a skilled person in the art would recognise that the invention may be practiced otherwise than as specifically described. The illustrative embodiments and examples should not be construed as limiting the invention.
  • 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, F(i), F(ii), 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-F 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.
  • mAbs were diluted to 8 ⁇ g/mL in 1xPBS with 0.1% BSA and 0.05% Tween-20. BSA and Tween-20 was added to DS-Cav1 and RSV-F mutant un-purified cell harvest media to a final concentration of 0.1% and 0.05%, respectively.
  • AHC biosensors were regenerated in 10 mM Glycine pH 1.5 before and between experiments. AHC biosensors were washed in 1x PBS with 0.1% BSA and 0.05% Tween-20 for 30 sec, mAbs were loaded for 60 sec, and washed for 30 sec before capturing DS-Cav1, F(i), F(ii), or RSV-F mutants from the un-purified cell harvest media.
  • Binding and dissociation of DS-Cav1, F(i), F(ii), and RSV-F mutants was measured for 180 sec each. The binding response of DS-Cav1 binding to each mAb was compared to the RSV-F mutants’ binding response to each mAb to determine yes or no binding. Rapid Stability Assay using Biolayer Interferometry (Examples 1, 2 and 7) The rapid stability assay was performed on un-purified cell harvest media of DS-Cav1, F(i), F(ii), and RSV-F mutants to determine binding to AM14 and D25 mAbs using the OCTET Red 96 or 384 (SARTORIUS).
  • mAbs were diluted to 8 ⁇ g/mL in 1xPBS with 0.1% BSA and 0.05% Tween-20.
  • DS- Cav1 and RSV-F mutant unpurified cell harvest media were incubated at 50 or 60°C for 0, 30, 60, or 120 min and diluted 1:1 with 1xPBS with 1% BSA and 0.05% Tween-20.
  • AHC biosensors were regenerated in 10 mM Glycine pH 1.5 before and between experiments.
  • Binding Kinetics using BIACORE Single cycle kinetics experiments were performed in duplicate on a BIACORE 8K+ (CYTIVA) using a ligand capture method at 25°C. HBS-EP+ was used as both a running buffer and sample diluent. A blank run of buffer as the ligand was followed by runs with IgGs captured to 100-200 RUs in flow cell 2 on a Protein A chip, leaving flow cell 1 as a reference. DS-Cav1, F(i), F(ii), and RSV-F mutants were injected in both flow cells at 30 ⁇ L/min for 120 sec followed by 2400 sec dissociation. Antigen concentrations ranged from 0-10 nM.
  • QUANTIFOIL Cu 300 mesh grids Prior to sample loading, QUANTIFOIL Cu 300 mesh grids (AGAR SCIENTIFIC) were glow discharged (PELCO EASIGLOW) for 60s at 25 mA. 3.0 ⁇ l of complex mixture was applied to the grid and incubated on the grid for 10 sec at 10°C and ⁇ 99% relative humidity using a LEICA EM GP2 Plunge Freezer (LEICA MICROSYSTEMS). The grid was then plunged into liquid ethane at liquid nitrogen temperature after blotting for 3.5 sec. The grid was then transferred into a GLACIOS cryo-transmission electron microscope (THERMOFISHER SCIENTIFIC) equipped with a FALCON 3EC direct electron detector for data collection.
  • THERMOFISHER SCIENTIFIC GLACIOS cryo-transmission electron microscope
  • the grid was then plunged into Docket No.: 70348WO01 liquid ethane after blotting for 4.0s.
  • the grid was then transferred into a GLACIOS cryo-transmission electron microscope (THERMOFISHER SCIENTIFIC) equipped with Falcon 3EC direct electron detector for data collections.
  • a total of 3,470 micrographs were collected FALCON the EPU data collection software at 0.87 ⁇ /pixel with a total dose of 38.25 e-/ ⁇ 2.
  • Defocus targets cycled from -0.8 to -1.8 microns.
  • Cryo-EM Image Processing and Model Building of the F647:RSB1 complex (Example 3)
  • the collected data was processed in CRYOSPARC V4.0.3 (STRUCTURA BIOTECHNOLOGY INC.).
  • a total of 680,164 particles were picked by using blob picker, which were subjected to 2D classification resulting in the selection of 162,493 particles.
  • 137,515 particles were selected for non-uniform refinement, which resulted in a 3.47 ⁇ resolution map when C3 symmetry is applied.
  • This class was subjected to one more round of Ab-initio model building and 81,943 particles were selected for the final refinement, which then resulted in a map with a global resolution of 3.36 ⁇ , which was estimated using the Fourier shell correlation at 0.143 as the criterion.
  • UCSF ChimeraX [32] was used for structural visualization and analysis. Docket No.: 70348WO01 Cryo-EM sample preparation and data collection of the 2 nd Gen DS-Cav1:VHH-L66 nanobody complex (Example 12) Complex was formed by mixing ⁇ 5 ⁇ g of 2 nd Gen DS-Cav1 trimer with 1:3.5 ratio excess VHH-L66 nanobody with a final concentration of 0.4 mg/ml and incubated at 4°C for 2hrs. Prior to sample loading, the QUANTIFOIL Cu 300 mesh grids (AGAR SCIENTIFIC) were glow discharged (PELCO EASIGLOW) for 60s at 25 mA.
  • the ligation mixtures were transformed into competent cells (NEB C3040H) was carried out by following manufacture instructions.24 hours after, colonies were picked for sequencing validation to screen clones with correct sequences. The final plasmids of selected clones were validated by Sanger sequencing and purified to support mRNA production. In vitro transcription to generate mRNA for RSV-F variations (Example 4) The plasmids were linearized with the BspQ1 restriction enzyme (NEW ENGLAND BIOLABS) to produce the DNA templates for in vitro transcription.
  • mRNAs were produced by in vitro transcription with capping analogue (TRILINK CLEANCAP A/G) and 100% uridine replacement (with 1m ⁇ ), followed with DNase I and phosphatase treatments (NEW ENGLAND BIOLABS) and silica column purification (QIAGEN).
  • Cell culture conditions (Example 4) Primary BJ cells (ATCC, CRL-2522) are 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 .
  • BJ cells were seeded in growth media at 1.5x10 5 cells/mL onto 96-well, clear-bottom, black-walled imaging microwell plates (PERKIN ELMER 6055302). The following day, target mRNAs were complexed with TRANSIT mRNA transfection reagent (MIRUS mir2250) in OPTIMEM (GIBCO 31985-070). Each target mRNA was forward transfected into BJ cell monolayers using 0.35% transfection reagent (final concentration) with mRNAs diluted to 0.454ng/uL (final concentration), or water-only negative control. The transfected BJ cells were incubated according to the time-course assay.
  • 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 Docket No.: 70348WO01 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.
  • 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 a.k.a.
  • LKY750 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
  • An insulin syringe with a permanently attached needle was used to administer 50 ⁇ L (25 ⁇ L in each hindleg thigh muscle) of either saline or 0.5 ⁇ g dose of RNA encoding F528, F647, F647 ⁇ CT20, F651 ⁇ CT20, F(iii) which includes a full cytoplasmic tail deletion (dCT), F(i), F(ii), or DS-Cav1 into each mouse on day 0 and day 21.
  • dCT full cytoplasmic tail deletion
  • 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 Docket No.: 70348WO01 and terminally exsanguinated by cardiac stick to obtain an estimated 200 ⁇ L to 500 ⁇ L of whole blood, (minimum 100 ⁇ L of serum).
  • RSV pre-F IgG binding antibody titres and RSV neutralising antibody titres were measured on day 21 and day 35 using the following methods.
  • RSV A neutralising antibody titre assay (against RSV A and B strains): Heat-inactivated sera (incubated for 30 min at 56°C) were diluted 3-fold starting at 1/8 (for a final dilution of 1/16). A control serum (WYETH Human Reference Sera from WHO/NIBSC) was included at a starting dilution of 1/64 (1/128 final). For the serial dilutions, 30 ⁇ L of diluted serum was added on top of 60 ⁇ L of 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. After incubation, 50 ⁇ L of the serum-virus mixture was added on top of the vero cells (seeded the day before the test at a density of 15000 cells/well, to reach a minimum of 80% confluency) and incubated for 2 hours at 35°C 5% CO2. After incubation, serum-virus supernatant was removed and 200 ⁇ L of 0.5% carboxymethyl cellulose + RSV media was added on top of the cells.
  • Plates were incubated for 2 days (max of 42 hours) at 35°C 5% CO2. Plates were then washed 2 times with 100 ⁇ L of PBS and 50 ⁇ L of 1% paraformaldehyde was added per well. Plates were covered in aluminium and incubated overnight at 4°C. The next day, plates were rinsed 3 times with 150 ⁇ L of PBS.100 ⁇ L of blocking solution (2% milk + PBS) was added on top of the wells and incubated for 1 hour at 37°C.
  • RSV F IgG Binding A multiplex assay was performed to evaluate titers of RSV pre-F- specific antibodies in the serum of the mice immunized with new non replicating RSV mRNA vaccines. LUMINEX microspheres (MAGPLEX microspheres, LUMINEX from Austin, TX) were coupled with RSV pre-F antigen by chemical coupling according to manufacturer instructions.
  • microspheres/ well were added in a volume of 50 ⁇ L 1X PBS with 1% BSA + 0.05% Na Azide (assay buffer) to five-fold serial dilutions of mouse serum down each column. After incubation of the microspheres and serum on an orbital shaker, covered, at RT for 60 minutes, the microspheres were washed two times with 200 ⁇ L/well of PBS with 0.05% Tween-20 (wash buffer) on a plate washer Docket No.: 70348WO01 using a magnet to allow settling of beads between washes.
  • r-PE r- Phycoerythrin conjugated anti-mouse IgG
  • JACKSON IMMUNORESEARCH r-Phycoerythrin conjugated anti-mouse IgG
  • the raw data was analyzed using a SOFTMAX PRO template, where the serum sample binding potency was interpolated based on a five-parameter logistic fit of the standard curve.
  • Serum anti-RSV F binding was calculated in terms of ASSAY Units (AU) using a reference standard assigned to a concentration of 100 AU.
  • In vivo immunization (Example 11) mRNA production and LNP formulation was performed as per Example 6.
  • Female BALB/c mice were 7 - 8 weeks old at day 0 of the study.
  • An insulin syringe with a permanently attached needle was used to administer 50 ⁇ L (25 ⁇ L in each hindleg thigh muscle) of either saline or a 5-point, 3-fold dilution series starting at 1.5 ⁇ g of RNA encoding F647 ⁇ CT20, F647 ⁇ CT20 (codon optimized), F(iii), F(i) or F(i) ⁇ CT20 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, (minimum 100 ⁇ L of serum).
  • RSV Pre-F IgG binding and RSV A (long strain) neutralising antibody titres were analysed as per Example 6.
  • BD GOLGIPLUG a protein transport inhibitor containing Brefeldin A
  • HTS High Throughput Sampler
  • the raw .fcs data files were analyzed by the computational algorithm (a quality control process which identifies and removes aberrant events caused by anomalies in fluoresence signal across all acquired parameters or flow rate over time of data acquisition leaving only “good events” in the raw .fcs data file). Removal of aberrant events decreases the risk of reporting false data.
  • the initial data analysis was performed using FLOWJO v10.8.0. After defining gates identifying cytokine and CD107a expressing positive populations within the viable, Docket No.: 70348WO01 activated, CD4 and CD8 T cell populations, a Boolean Combination Gate tool was used to automatically generate all possible combinations (positive and negative) of cytokine and CD107a expressing cells.
  • CD4 and CD8 T cells For activated CD4 and CD8 T cells, all six analytes were included in the Boolean analysis generating a total of 64 multi-functional subsets.
  • the raw data was exported into MICROSOFT EXCEL for further analysis.
  • the response to peptide pool stimulation was determined by subtracting the response in the unstimulated media control for each sample. Negative values resulting from this subtraction were identified and changed to the number zero.
  • the multi-functional subsets were used to categorize the data into CD4 T helper (Th) and CD8 T cytotoxic (Tc) phenotypic subsets based on production of IFN- ⁇ , IL-13/IL-4, and IL-17F cytokines.
  • Th CD4 T helper
  • Tc cytotoxic
  • the cells were extracellularly stained with the following antibodies: CD3-BB700, CD4-BUV395, B220-BUV563, CD19-BUV737, IgD-BV510, IgM-BV786, GL7-PE, CD95-BV750, CD138-BV711, PD1-APCR700, CXCR5-BUV615, CD44-PeCy7, and F647-APC.
  • Cells were acquired on BD FACSYMPHONY A5 SORP cell analyzer and data analyzed with FLOWJO software v10.8.0 (BD BIOSCIENCES).
  • Statistical analysis of RSVA neutralization data in a preliminary step, the model to fit the data was selected stepwise.
  • the final model used to fit RSVA neutralization data is quadratic and includes as fixed effects: log10(dose), [log10(dose)]2, vaccine, time and all double and triple interactions except [log10(dose)]2 *vaccine*time.
  • the selected variance-covariance structure related to timepoints is Unstructured matrix.
  • Example 14 In vivo immunization (Example 14) mRNA production and LNP formulation was performed as per Example 6, except the following LNP formulation was used: 40 mol% cationic lipid RV94; 2 mol% PEG-conjugated lipid; 48 mol% cholesterol; and 10 mol% 1,2-diastearoyl-sn-glycero-3-phosphocholine (DSPC).
  • LNP formulation 40 mol% cationic lipid RV94; 2 mol% PEG-conjugated lipid; 48 mol% cholesterol; and 10 mol% 1,2-diastearoyl-sn-glycero-3-phosphocholine (DSPC).
  • serum samples were diluted two-fold in microplates starting from a 1:20 dilution and mixed with RSV A2 (ATCC VR-1540) virus (100 TCID50). After 1 hour of incubation at 37C, 100uL of vero cells were added to the 96-well serum-virus mixture. Plates were incubated for 3 days at 37C, 5% CO2 in humidified atmosphere, and then washed and fixed with Acetone. Primary and secondary commercial antibodies were added to the plate and the neutralizing effect was evaluated by a spectrophotometer after the addition of a substrate. The reciprocal of the highest serum dilution corresponding to a 50% protection of cell monolayer against viral infection represent the reported neutralizing antibody titer.
  • Pre-F IgG binding This LUMINEX assay is designed to detect the level of F647-specific IgG in rats based on a standard serum sample. LUMINEX microspheres were coupled with F647 antigen in Hepes/NaCl) at the target concentration of 10 ⁇ g/mL using sulfo-NHS and EDC, according to manufacturer’s instructions.
  • the assay was run using a standard 96-well plate configuration where 2,500 microspheres/well were added in a volume of 50 ⁇ l PBS with 1% BSA + 0.05% Na Azide (assay buffer) into 100 ⁇ l of two-fold serial dilutions of rat serum samples and serum internal control across each row. After incubation of the microspheres and serum on an orbital shaker, covered, at RT for 60 minutes, the microspheres were washed with 250 ⁇ l/well of PBS, 0.05% Tween-20 (wash buffer) on a plate washer using a magnet to allow settling of beads before washing.
  • the rat serum standard was assigned a value of 100 Assay Units/mL (AU/mL) and was used to calculate sample titers.
  • the standard and internal control is a pooled serum sample from, group 1, Day 42 (Rats immunized with RSV F647 d20 mRNA (codon optimized)).
  • In vivo immunization (Example 15) mRNA production and LNP formulation was performed as per Example 6.
  • Female BALB/c mice were 7 - 8 weeks old at day 0 of the study.
  • An insulin syringe with a permanently attached needle was used to administer 50 ⁇ L (25 ⁇ L in each hindleg thigh muscle) of either saline or 0.497 ⁇ g F647 into each mouse on day 0 and only Group 1 & 2 on day 21.
  • F647 also referred to as R713 in other examples.
  • 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 (minimum 100 ⁇ L of serum).
  • RSV A and B neutralization Neutralization assay was performed at VISMEDERI.
  • serum samples were diluted two-fold in microplates starting from a 1:20 dilution and mixed with RSV A2 (ATCC VR-1540) or RSV B WV/14617/85 (ATCC VR-1400) virus (100 TCID50). Remainder of method performed as per Example 14.
  • Female BALB/c mice were 7 - 8 weeks old at day 0 of the study.
  • an insulin syringe with a permanently attached needle was used to administer 50 ⁇ L (25 ⁇ L in each hindleg thigh muscle) of either: - (1) saline; - (2) 0.2 ⁇ g RNA encoding F647 ⁇ CT20 (codon optimised) – A subtype, A2 strain wildtype background sequence (“F647 A subtype RNA”, for the purpose of this example); Docket No.: 70348WO01 - (3) 0.2 ⁇ g RNA encoding F647 ⁇ CT20 (codon optimised) – B subtype, M16 strain wildtype background sequence (“F647 B subtype RNA”, for the purpose of this example); - (4) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-formulated; - (5) 0.2 ⁇ g of F647 A subtype RNA + 0.2 ⁇ g of F647 B subtype RNA, co-administered
  • the groups of animals, formulation lot numbers, stock concentrations, number of vials, and storage temperatures were as follows: Table 16A – RNA immunization study design (A and B subtype designs, co-formulation and co- administration) S t Storage T est Article ock C oncentration Number of vials supplied Temperature (°C) F647 A subtype RNA 41 ⁇ g/mL RV39 LNPs F647 B subtype R NA RV39 LNPs 42 ⁇ g/mL 3 vials (1 mL material per v ial) -80°C F647 A subtype RNA + F647 subtype RNA 41 ⁇ g/mL RV39 LNPs Table 16B – RNA immunization study design (A and B subtype designs, co-formulation and co- administration), continued Group Immunogen 1 st Dose 2 nd Dose Formulation 1 Saline Saline Saline N/A 2 F647 A subtype RNA 0.2 ⁇ g 0.2 ⁇ g RV39 3 F647
  • F647 also referred to as R713 in other examples.
  • 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 (minimum 100 ⁇ L of serum).
  • RSV neutralizing antibody titers performed as per Example 15. Detection of RSV-F-specific T cells by flow cytometry: Multi-parameter flow cytometry with intracellular cytokine staining (ICS) was used to assess RSV-specific CD4 + and CD8 + T cell responses.
  • ICS intracellular cytokine staining
  • RSV-F RSV Fusion
  • BD GOLGIPLUG a protein transport inhibitor containing Brefeldin A
  • HTS High Throughput Sampler
  • This automated quality control process identifies and removes aberrant events caused by anomalies in fluorescence signal across all acquired parameters or flow rates over data acquisition time, leaving only “good events” in the raw .fcs data file. Removal of aberrant events decreases the risk of reporting false data.
  • the initial data analysis was performed using FLOWJO v10.9.0. Gating was used to identify activated CD4 + and CD8 + T cells. After defining gates identifying cytokine and CD107a + expressing positive populations within the viable, activated, CD4 + and CD8 + T cell populations, the Boolean Combination Gate tool was used to automatically generate all possible combinations (positive and negative) of cytokine and CD107a + expressing cells.
  • CD4 + and CD8 + T cells For activated CD4 + and CD8 + T cells, all six analytes were included in the Boolean analysis, generating a total of 64 multi- functional subsets.
  • the raw data was exported into MICROSOFT EXCEL for further analysis.
  • the response to peptide pool stimulation was determined by subtracting each sample's response in the unstimulated media control. Negative values resulting from this subtraction were identified and changed to zero.
  • the multi-functional subsets were used to categorize the data into CD4 + T helper (Th) and CD8 + T cytotoxic (Tc) phenotypic subsets based on IFN- ⁇ , IL-13/IL-4, and IL-17F cytokine production. The four subsets are defined in Table 6.
  • Splenocytes from individual mice were plated in U-bottom 96 well tissue culture plates at 2x10 6 viable cells/well. Cells were stained with near IR live/dead cell stain (Invitrogen) for 20 min at room temperature and then incubated with Fc block (BD BIOSCIENCES) in FACS Wash Buffer (PBS plus 1% FBS, THERMO SCIENTIFIC) for 10 min at room temperature.
  • Fc block BD BIOSCIENCES
  • FACS Wash Buffer PBS plus 1% FBS, THERMO SCIENTIFIC
  • the cells were extracellularly stained with the following antibodies: CD3-BB700, CD4-BUV395, B220-BUV563, CD19-BUV737, IgD-BV510, IgM-BV786, GL7-PE, CD95-BV750, CD138-BV711, PD1-APCR700, CXCR5-BUV615, CD44-PeCy7, and F647- APC.
  • Cells were acquired on BD FACSYMPHONY A5 SORP cell analyzer, and data was analyzed with FLOWJO software v10.8.0 (BD BIOSCIENCES). Before analysis, the raw .fcs data files were analyzed using a computational algorithm.
  • This automated quality control process identifies and removes aberrant events caused by anomalies in fluorescence signal across all acquired parameters or flow rates over data acquisition time, leaving only “good events” in the raw .fcs data file. Removal of aberrant events decreases the risk of reporting false data.
  • the initial data analysis was performed using FLOWJO v10.9.0. and RSV F-specific B cells were identified according to the gating strategy. All graphs were generated with GRAPHPAD PRISM v9.5.1. Generation of B cell Antigen Probes: F647 was non-specifically biotinylated using the FLUORREPORTER Mini-Biotin-XX Protein Labeling Kit (THERMOFISHER) according to the manufacturer’s instructions.
  • biotinylated proteins were then conjugated to streptavidin-APC (INVITROGEN) in a 3:1 protein:streptavidin-APC ratio. Proteins and HEPES buffer were added, and the streptavidin-APC was added in 1/5 increments. Between each addition of streptavidin-APC, the mixture was rotated at 4°C for 20 minutes. The probes were titrated on frozen, RSV-immunized mouse splenocytes to confirm specificity and determine the optimal concentration.
  • Biolayer interferometry was used to test expression via affinity-based histidine tags, resulting in the identification of F528 (D486C:A490C; SEQ ID NO: 24), see Figure 1, which showed particularly high expression relative to DS-Cav1.
  • the designs were then tested for the presence of the pre-fusion specific epitope using three known antibodies: AM14 (quaternary epitope), D25 (site ⁇ ), and RSB1 (site V). F528 was able to bind all three antibodies, showing a similar binding response to DS-Cav1, see Figure 2. Finally, the designs were then tested for thermal heat resistance after incubation at 50 or 60°C by binding to AM14 and D25.
  • F528 showed higher antibody binding after heat stress compared to DS-Cav1, see Figure 3.
  • Table 3 – Round 5 Disulphide Bond Mutations (on top of F217 background substitutions*) Construct Mutations Construct Mutations F501 T50C-L456C F515 A153C-K461C F502 A74C-E218C F516 G184C-N428C F503 E92C-N254C F517 R235C-T249C F504 Q98C-N276C F518 V239C-P246C F505 T100C-S362C F519 G329C-D392C F506 G143C-S404C F520 G329C-S491C F507 G143C-S405C F521 G329C-S493C F508 V144C-V406C F522 N345C-N454C F509 G145C-Y457C F523 T369C-
  • Round 6 designs included inter alia, the addition of the D486C:A490C disulphide bond, identified in F528, and a GS linker ( ⁇ 103-145, substituted for GS, linking positions 102 and 146 – “GS102”; or ⁇ 104-144, substituted for GS, linking positions 103 and 145 – “GS103”).
  • GS linker ⁇ 103-145, substituted for GS, linking positions 102 and 146 – “GS102”; or ⁇ 104-144, substituted for GS, linking positions 103 and 145 – “GS103”.
  • Each of these mutations was individually added to the human RSV- Docket No.: 70348WO01 F A2 subtype wild-type ectodomain sequence (a.k.a “F300” – SEQ ID NO: 2) and the resulting designs were tested for expression and antibody binding.
  • Table 4 Thermostability as measured by nano-DSF of Round 6B designs C onstruct T m 1 T m 2 T m 3 DS-Cav1 55.5°C 67.2°C 82.0°C F(ii) 59.8°C 82.7°C F(i) 67.2°C 84.4°C F528 72.3°C 79.4°C F647 74.4°C 80.7°C F651 65.7°C 80.8°C F420 56.5°C 83.5°C F663 73.4°C 82.0°C F664 62.2°C 85.6°C Table 5 — Summary of biophysical characteristics of Round 6B designs Construct Thermostability Affinity (pM) T m 1, T m 2* Docket No.: 70348WO01 Protein AM14 D25 Motavizumab RSB1 Expression (mg) DS-Cav1 0.6 67.2°C, 82.0°C 13.7 18.4 58.8 61.8 F528 1.78 72.3°C, 79
  • Example 3 Structural characterisation of designs F528 and F647 by cryo-EM
  • the structures of F528 and F647 were solved as set out in Materials and Methods, above. See Figures 14 and 15 and associated description of figures, above, for results. Briefly, both F528 and F647 were in the pre-fusion conformation and were found to have an intra-protomer disulphide bond between cysteine residues at positions 486 and 490.
  • Figure 14A shows the cryo-EM structure of the RSV F528-AM14 Fab complex.
  • Figure 14B shows the presence of three intra-protomer disulphide bonds (per protomer) each linking 486C and 490C – electron density of the disulphide bond was clearly visible in the cryo-EM map.
  • Figure 14C shows an EM density map of one such disulphide bond.
  • Figure 15A shows the cryo-EM structure of the RSV F647-RSB1 Fab complex. This work generated a cryo-EM map of the F647 design with higher resolution than that of F528 (F647 at 3.3 ⁇ ), which provides clear density of not only the intra-protomer disulfide bonds but also the side chains of the stabilizing mutations.
  • Figure 15B shows a zoom view of the intra-protomer disulphide bonds captured by cryo-EM (EM density map shown in mesh).
  • Figures 15C and E shows the stabilised electrostatic repulsive ring in design F647.
  • Figure 15D and F shows the electrostatic repulsive ring in wild-type RSV F protein.
  • Example 4 RNA expression Constructs discussed in the following Example are presented below in Table 2 (a subset of Table 1 for ease of reference).
  • Table 2 substitutions in mRNA-encoded protein designs tested in cell-based assay (subset of Table 1) Docket No.: 70348WO01 Designation mRNA construct Protein substitutions relative to wild- RSV-F design in Figures designation type 16-18 (x axis) and Ex 4 n/a All mRNAs encode RSV-F proteins n/a having these mutations relative to WT (SEQ ID NO: 1). Further mutations in each RSV-F design are listed below.
  • the parent construct (13, Table 1), includes a full-length C-terminal domain Docket No.: 70348WO01 (CTD) and was compared to the F antigen with a cytoplasmic tail (CT) truncation of 20 amino acids from the C-terminus (see 20, Table 1).
  • CT cytoplasmic tail
  • the F antigens (13 & 20) were evaluated in the context of two additional classes of post translational modifications.
  • the addition of N-linked glycosylations was tested by mutating serine (S) 211 and 348 to asparagine (N) (see 1 & 2, Table 1).
  • the F antigen expression encoded by the eight candidate mRNAs was evaluated at the cell surface of primary human fibroblast (BJ) cells and readily quantified using High Content imaging.
  • the total expression of RSV F protein was assessed 24 ( Figure 18A) & 72 ( Figure 18B) hours post-transfection (hpt).
  • F antigens with a full-length CT were generally reduced in level at both time points, compared to corresponding F antigens with a truncated CTD, demonstrating the strong impact of the CT truncation on RSV F surface expression.
  • Example 5 Computational prediction of further intra-protomer disulphide bonds in the HRB domain Structures including pdb code 5ea4 and 5c69 as well as cryo-EM structures obtained for designs F21 (mutations vs SEQ ID NO: 1 in Table 7B, below) and F216 (SEQ ID NO: 122) were prepared by either cartesian refinement using ROSETTA Scripts and/or Quick Prep using the Molecular Operating Environment software (MOE; MOLSIS Inc., Japan). Once structures were optimised, residues within a C ⁇ -C ⁇ distance of 5 ⁇ were identified as having an optimal distance to form a disulphide bond. Residues that are within the same protomer (intra-protomer) were identified.
  • MOE Molecular Operating Environment software
  • Table 7A shows residue pairs in the HRB domain (residues 474-523) that have an optimal distance in at least one of the prepared structures and were predicted to form an intra-protomer disulphide bond, based on the 5 ⁇ distance criterion. Additionally, energy calculations were then performed using MOE and the Amber15 forcefield to predict the energy stabilization resulting from each of the disulphide substitutions. Table 7A (bolded entries) shows amino acids pairs in the HRB domain identified within a C ⁇ -C ⁇ distance of 5 ⁇ , and that were predicted to be stabilising of the pre-fusion conformation in at least one of the structures analysed. These include predicted disulphide pairs that would be expected to have similar stabilising effect as the C486:C490 disulphide.
  • Figure 23A presents the RSV A neutralising antibody titres (ED60) on day 21 (3wp1) and day 35 (2wp2) in animals immunized with 0.5 ⁇ g of F528, F647, F647 ⁇ CT20, F651 ⁇ CT20, F(iii), F(i), F(ii), or DS-Cav1 (where each point represents an individual animal).
  • the saline group did not generate a measurable neutralisation response to RSV A (data not shown).
  • F647 ⁇ CT20 elicited the highest RSV A-long neutralisation antibody titres with minimal variability within the group.
  • the neutralisation titres elicited from F647 ⁇ CT20 was higher than F(iii), F(i), F(iii), and DS-Cav1. Addition of a GS-linker (F651) did not substantially improve neutralisation titres.
  • RSV A neutralisation antibody titres from F647 d20 vaccination remained higher than F(iii), F(iii) , and DS- Cav1, and were comparable to the neutralisation titres elicited from vaccination with the F(i) antigen.
  • Figure 23B presents the RSV A and B day 35 (2wp2) cross-neutralisation titres to lab-adapted (RSV A-long and RSV B-18537) and clinical RSV strains (RSV A-Clinical 2015, RSV B-Clinical 2015 and Docket No.: 70348WO01 2017).
  • Cross-neutralisation was improved with the F647 antigen compared to F528 and was substantially higher compared to DS-Cav1. Similar to the RSV A neutralisation results, the addition of GS-linker (F651) did not improve neutralisation titres.
  • F647 ⁇ CT20 elicited consistent cross-neutralisation to all RSV A and B strains tested.
  • F647 ⁇ CT20 generated the highest pre-F IgG antibody titres compared to F528, F647, F651 ⁇ CT20, F(iii), F(ii) and DS-Cav1, and the magnitude of F647 d20-elicited pre-F IgG binding antibodies were comparable to F(i) construct.
  • all constructs generated comparable pre-F IgG binding antibody titres.
  • Example 7 – Minimal substitution screen Constructs F301 – F307 were generated as recombinant proteins with 6 substitutions each against the RSV A2 WT background sequence (positions 1-513 of SEQ ID NO: 2).
  • sequence F310 containing substitution N228K had both protein expression and binding to AM14, D25, and RSB1 that was equivalent to DS-Cav1 ( Figures 25 & 26 respectively), indicating that this substitution has a significant contribution to the stabilisation of pre-fusion RSV F, and is able to stabilise the pre-fusion conformation independently.
  • F301-F307 were further characterized and showed optimal biophysical properties including thermostability similar to F225 by nano-DSF (See Table 8B, below). Long term stability of F310 was tested and is shown in Figure 30.
  • Table 8B Thermostability measured by nano-DSF Sample Onset Tm1 Tm2 Tm3 DS-Cav1 49.5°C 56.3°C 67.9°C 80.9°C F225 46.8°C 53.4°C 81.3°C F301 47.2°C 52.6°C 81.2°C F302 45.7°C 53.2°C 81.4°C F303 51.4°C 57.2°C 81.7°C F304 49.9°C 54.7°C 80.8°C F305 46.5°C 52.6°C 81.2°C Docket No.: 70348WO01 F306 45.9°C 53.0°C 81.6°C F307 46.3°C 52.9°C 81.4°C F310 44.0°C 56.0°C 84.2°C
  • CT AA sequence 1 Reference RSV F CT, including AA 541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN additional transmembrane (TM) domain (portion of SEQ ID NO: 1) residues N-terminal to CT start 2 ⁇ CT3, including additional TM domain AA 541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIA residues N-terminal to CT start (portion of SEQ ID NO: 1) 3 ⁇ CT5, including additional TM domain AA 541 LIAVGLLLYCKARSTPVTLSKDQLSGINN (portion residues N-terminal to CT start of SEQ ID NO: 1) 4 ⁇ CT10, including additional TM AA 541 LIAVGLLLYCKARSTPVTLSKDQL (portion of SEQ domain residues N-terminal to CT start ID NO: 1) 5 ⁇ CT15, including additional TM AA 541 LIAVGLLLYCKARSTPVTL
  • the peak cell-surface, trimeric, prefusion RSV F expression is specific to variants using the CTD length at least 5 amino acids long, and in contrast, CTD lengths less than 5 amino acids are associated with reduced F protein expression (Figure 29B).
  • Example 10 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 plate, which has a clear bottom; (3) admixing 400
  • F647 ⁇ CT20 (codon optimised) generated the highest pre-F IgG titers at low doses, but pre-F IgG binding titers were comparable across groups at high doses ( Figures 31B and 32B).
  • F647 ⁇ CT20 (codon optimised) elicited statistically significantly higher neutralisation titers compared to F647 ⁇ CT20, F(iii), and F(i) almost over the entire dose range, except below 0.057 ⁇ g (geometric mean ratio (GMR); data not shown).
  • F647 ⁇ CT20 (codon optimised) elicited statistically significantly higher neutralisation titers over the entire dose range compared to F(iii) and below 0.5 ⁇ g compared to F647 ⁇ CT20 and F(i) (GMR; data not shown).
  • splenocytes from the saline 0.5 ⁇ g and 1.5 ⁇ g F647 ⁇ CT20 (codon optimised) dose groups were harvested two weeks after immunization (day 35) for flow cytometry staining and acquisition.
  • the disulphide bond acts as a kink that restricts the rearrangement of residues 485-492 into an extended alpha helix, a necessary step for transition to the post fusion conformation (Figure 35).
  • Such restriction increases the energy barrier for the conformational change to the post-fusion conformation.
  • the 486:490 disulphide repositions wild-type residue F488 to form a pi-pi stacking interaction with wild-type residue F137 (the latter being within the fusion peptide) – see Figures 36A, C and D.
  • residue F137 forms a cation-pi stacking interaction with K339 (see Figure 36B).
  • the 486:490 disulphide repositions the side chain of F488 away from the trimeric center (see, e.g. Figure Docket No.: 70348WO01 36A) and enables a pi-pi stacking interaction between the F488 and F137 in addition to the cation-pi stacking interaction between the F137 and K339 (see, e.g. Figure 36D).
  • This cation-pi-pi trio-stacking further restricts the movement of the fusion peptide, thereby helping to stabilise the pre-fusion conformation (see, e.g. Figures 36C and D).
  • F651 carries all the mutations as in F647, but with a GS linker modification that replaces the p27 domain and fusion peptide; 2 nd generation DS-Cav1 has a different set of mutations and a similar GS linker (see Figure 37D for sequence alignments). Both F651 and 2 nd generation DS-Cav1 were found to have a broader trimer base compared to F647, which is most obvious at the ⁇ 10 helices ( Figure 37A-C).
  • the CD8 + Tc1-skewed frequency was similar for all RNA-administered groups except for low dose F647 A subtype RNA, which appeared to have a lower frequency ( Figure 42B). Additionally, on day 35, the F647-specific germinal center B cell response was examined. A similar frequency of total germinal center B cells was observed for both the lower and higher dose of F647 A subtype RNA and F647 B subtype RNA, and a slight increase observed in the co-formulated and co- administered RNA groups ( Figure 43B).
  • SEQ ID NO: 1 Full length amino acid (AA) sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094.
  • AA Full length amino acid sequence of wild-type RSV-F (A2 strain) containing substitutions K66E and Q101P relative to GenBank Accession number KT992094.

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Abstract

La présente divulgation propose, entre autres, une protéine VRS-F comprenant au moins deux mutations par rapport à SEQ ID NO : 1 ou 3 dans une région de la protéine correspondant aux positions 474-523 de SEQ ID NO : 1 ou 3 ; lesdites au moins deux mutations introduisant, par substitution ou insertion, une paire de résidus C dans la région, qui forment un pont disulfure.
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