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WO2024193965A1 - Acides nucléiques codant pour rsv-f - Google Patents

Acides nucléiques codant pour rsv-f Download PDF

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Publication number
WO2024193965A1
WO2024193965A1 PCT/EP2024/055127 EP2024055127W WO2024193965A1 WO 2024193965 A1 WO2024193965 A1 WO 2024193965A1 EP 2024055127 W EP2024055127 W EP 2024055127W WO 2024193965 A1 WO2024193965 A1 WO 2024193965A1
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Prior art keywords
rsv
seq
rna
protein
positions
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Inventor
Marco BIANCUCCI
Kambiz MOUSAVI
Nicholas John BARROWS
Xiaofeng Wang
Corey Mallett
Emily PHUNG
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Priority claimed from PCT/EP2023/066332 external-priority patent/WO2024041773A1/fr
Application filed by GlaxoSmithKline Biologicals SA filed Critical GlaxoSmithKline Biologicals SA
Priority to PCT/EP2024/066562 priority Critical patent/WO2024256637A1/fr
Priority to ARP240101539A priority patent/AR132981A1/es
Publication of WO2024193965A1 publication Critical patent/WO2024193965A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—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

  • RSV Respiratory syncytial virus
  • RSV A and RSV B two antigenically distinct subgroups
  • ribavirin is the only approved antiviral therapy for RSV treatment, but its use is restricted to severe hospitalized cases in infants and young children [3].
  • palivizumab Synagis
  • motavizumab two RSV-specific humanized monoclonal antibodies, palivizumab (Synagis) and motavizumab, are confirmed to be safe and effective in reducing RSV hospitalization rates and serious complications among high- risk children in multiple clinical settings [4, 5, 6, 7, 8].
  • Available treatment for RSV in older adults is generally supportive in nature, consisting of supplemental oxygen, intravenous fluids and bronchodilators.
  • RSV-F The RSV fusion protein (“RSV-F”) in the viral envelope is the most effective target of neutralising antibodies, such as motavizumab.
  • RSV-F structural biology have revealed changes in its antigenic characteristics that occur during the fusion process between the viral envelope and host cell membrane.
  • RSV-F adopts a metastable “pre-fusion” conformation in the viral envelope as a homotrimer, and then an irreversible and distinct “post-fusion” conformation during fusion with the host cell membrane (see Figure 2 of [9]).
  • the trimeric pre-fusion conformation is more immunogenic, and is bound by most RSV-F-specific neutralising antibodies in human sera.
  • pre-fusion RSV-F antigen designs which can be expressed at high levels from nucleic acids (and thus may be encoded into a nucleic acid-based vaccine).
  • the inventors have enhanced the cell-surface expression of trimeric, pre-fusion RSV-F protein when expressed from nucleic acids, e.g. through specific mutation of the protein’s C-terminal cytoplasmic Docket No.: 70280WO01 tail (CT).
  • Such mutation involves the deletion of C-terminal residues from the CT, such as at least 3, at least 5, at least 10, at least 15, or at least 20 residues, as opposed to deletion of the entire CT.
  • C-terminal residues from the CT
  • both 3 and 20 C-terminal residues were deleted from the CT of four different RSV-F protein designs.
  • Such deletion enhanced the in vitro cell-surface expression of trimeric pre-fusion RSV-F over a period of 96 hours for all antigen designs tested, compared to the corresponding RSV-F protein with both (i) a fully intact CT and (ii) the entire deletion of the CT (see e.g. Figure 2).
  • deletion of 5, 10 and 15 C-terminal residues also resulted in enhanced in vitro cell-surface expression of trimeric pre-fusion RSV-F protein, compared to the corresponding RSV-F protein with a fully intact CT (see, e.g. Example 5; Figure 5A and B).
  • 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 12; Figure 19A).
  • RNA constructs encoding various RSV-F proteins with either a fully intact CT, or comprising the deletion of 20 C-terminal residues (“ ⁇ CT20”) were used in murine immunisation studies.
  • constructs comprising a ⁇ CT20 generally elicited higher neutralising antibody titres against e.g. RSV of the A subtype, in comparison to their counterparts with a fully intact CT.
  • Example 13 also demonstrates the positive effects of the ⁇ CT20 deletion on neutralising antibody titres against RSV strains of both the A and B subtypes (comparing constructs “647” and “647 ⁇ CT20”).
  • Neutralising antibody titres generally correlate with inhibition of viral replication in the lungs and other respiratory sites, and thus protective efficacy in a subject.
  • a mutated CT as 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. reduced reactogenicity.
  • Protein subunit-based RSV vaccines are currently being pursued, for at least the older adult population [10], [11].
  • nucleic acid-based vaccines such as avoiding Docket No.: 70280WO01 the risks of pre- to post-fusion conformational change of a protein subunit during storage and transportation.
  • the modifications to the RSV-F protein disclosed herein may expand patient options to include nucleic acid-based vaccines which can elicit high and sustained antigen expression. Therefore, nucleic acids generated by the inventors (and proteins encoded thereby) may be useful, in particular in prophylactic vaccination against RSV.
  • the present disclosure provides: A recombinant nucleic acid encoding an RSV-F protein comprising a cytoplasmic tail; wherein, relative to a cytoplasmic tail according to SEQ ID NO: 3 or 4, 2-20 residues are deleted from the cytoplasmic tail of the RSV-F protein. The residues are preferably deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein.
  • the nucleic acid is preferably RNA.
  • the present disclosure provides an RSV-F protein that is encoded by a nucleic acid of the present disclosure.
  • the present disclosure provides a host cell comprising a nucleic acid of the present disclosure.
  • the present disclosure provides a carrier (preferably, a lipid nanoparticle) comprising a nucleic acid of the present disclosure.
  • a pharmaceutical composition comprising a nucleic acid (preferably RNA), RSV-F protein or carrier (preferably lipid nanoparticle) of the present disclosure.
  • the present disclosure provides a nucleic acid (preferably RNA), RSV- F protein, carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, for use in medicine.
  • the present disclosure provides a therapeutic method comprising administering an effective amount of the nucleic acid (preferably RNA), RSV-F protein, carrier (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure to a subject.
  • the present disclosure provides a method of inducing an immune response against RSV in a subject, the method comprising administering an effective amount of the RNA, carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure to a subject.
  • 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.
  • indirect immunofluorescence and imaging (10x objective) captures the individual cell nuclei (denoted ‘) and the cell surface RSV F (denoted “) variant F318 with 3 amino acids removed from the cytoplasmic tail (CT) in cells fixed approximately 8 (A’ & A”), 24 (B’ & B”), 48 (C’ & C”), 72 (D’ & D”) or 96 (E’ & E”) hours post transfection and labelled by using the primary antibody motavizumab.
  • the population distribution from High Content imaging (HCi) and analysis for BJ cells transfected and labelled corresponding to the representative images in panels A-E is shown at approximately 8 (F), 24 (G), 48 (H), 72 (I) and 96 (J) hours post transfection.
  • each plotted value expresses the average intensity of the Alexa647 signal for cells identified by automated image analysis from 9 imaged fields per well.
  • Each point on the line graph represents the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 biological replicates.
  • the area under the curve (AUC) for each line graph is shown (E) with 1 standard error of the mean (SEM).
  • the means, AUC and variability shown on the line and bar graphs were calculated by GraphPad Prism software.
  • Figure 3 Total expression of the RSV F protein increases for mRNA vaccine candidates with CT deletions.
  • the cell-surface expression of RSV F protein was evaluated by indirect immunofluorescent labelling using the primary anti-RSV F antibody motavizumab followed by quantification using HCi and analysis across a 4-day time course.
  • Each point on the line graph represents the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 biological replicates.
  • the area under the curve (AUC) for each line graph is shown (E) with 1 standard error of the mean (SEM).
  • the means, AUC and variability shown on the line and bar graphs were calculated by GraphPad Prism software.
  • Figure 4. In vitro validation of mRNAs for in vivo study. Select mRNAs encoding RSV F were forward transfected into primary BJ cell monolayers. The cell monolayers were fixed and RSV F protein expression was evaluated by indirect immunofluorescence coupled with HCi and image analysis.
  • the mRNAs encode RSV F variants including DS-CAV1, F(ii), F(iii) and F(i) proteins or the F318 and F319 protein constructs. Results for corresponding variants lacking the CT 20 amino acids ( ⁇ CT20) are also shown.
  • RSV F surface protein expression was quantified 1 day post infection by labelling cells using the anti-RSV F antibodies Motavizumab (A), D25 (E) or AM14 (I) or 3 days post transfection (Motavizumab (C), D25 (G) or AM14 (K)).
  • the average cell count for three imaged wells is shown and corresponds to the RSV F expression values for 1 day post infection (Motavizumab (B), D25 (F) or AM14 (J) or 3 days post transfection (motavizumab (D), D25 (H) or AM14 (L)).
  • Each graph depicts the mean ( ⁇ ) +/- 1 standard deviation ( ⁇ ) from 3 biological replicates as calculated by GraphPad Prism software.
  • Figure 5 A short, 5 amino acid CT (See Table 2, Row 6) for RSV F protein maximally enhanced RSV F protein expression both within the cell and at the cell surface.
  • RSV A neutralising antibody titres (ED60) on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either (A) 2 ⁇ g or (B) 0.2 ⁇ g of RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) Docket No.: 70280WO01 ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (where each point represents an individual animal).
  • Statistical comparisons of constructs are presented in (C)-(E).
  • 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 trimeric surface expression was detected by indirect immunofluorescent labelling (using AM14 antibody) followed by quantification using high content imaging and analysis.
  • hpt post-transfection
  • cell monolayers were fixed, then RSV-F was labelled and imaged using a 10x objective.
  • 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.
  • 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.
  • hpt 72 hours post-transfection
  • 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 10. 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.
  • Hydrogen bond between K228 and Y250 is depicted as a dashed line.
  • Figure 12. Zoomed in view of substitution 55T 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).
  • T55 is shown as sticks with a transparent surface. Residues forming the hydrophobic pocket and involved in van der Waals contacts with T55 are shown as sticks (including hydrophobic pocket).
  • Octet BLI of the minimal substitution designs bound to RSV-F antibodies (AM14, D25, RSB1, motavizumab), relative to DS-Cav1. Negative control (EXPIFECTAMINE and cell culture supernatant), F225 and F300 (wild-type) also shown.
  • Figure 16 Protein expression of RSV F in human skeletal muscle cells increased with ⁇ CT20 deletion. Primary human skeleton muscle cells (three donors) were transfected with mRNA using LIPOFECTAMINE3000 for 24 hours. Plot represents area under the curve (AUC) for integrated mean fluorescent average intensity (iMFI) of F(ii) parental protein (KM135) vs F(ii) ⁇ CT20 (KM140). Each dot represents three technical replicates.
  • AUC area under the curve
  • iMFI integrated mean fluorescent average intensity
  • RSV F-encoding mRNAs with different numbers of substitutions were screened in primary human BJ cells for their ability to express AM14-positive RSV-F antigen (see Table 8 for encoded proteins).
  • 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 19 The optimal length of the RSV F CT that supports cell-surface expression of the trimeric, pre-fusion RSV F protein includes CTs of at least 5, but not longer than 10, amino acids.
  • the cell- surface expression of trimeric, pre-fusion RSV F protein was evaluated by indirect immunofluorescent labelling using monoclonal antibody AM14 followed by quantification using high content imaging and analysis across a 4-day time course.
  • Primary, human fibroblast (BJ) cells were forward transfected in 96-well format with mRNAs encoding RSV F variant F(ii).
  • select CT variations are shown.
  • the parent (F(ii), solid line, solid box) was modified by deletion of the RNA sequence encoding the terminal 15 amino acids (F(ii) CTD ⁇ 15, solid line, solid circle), 16 amino acids ((F(ii) CTD ⁇ 16, dotted line, solid circle), 17 amino acids (F(ii) CTD ⁇ 17, dotted line, open circle), 20 amino acids (F(ii) CTD ⁇ 20, solid line, open circle), 21 amino acids (F(ii) CTD ⁇ 21, dotted line, solid box), or complete deletion of the CT domain (F(ii) CTD ⁇ 25, solid line, open box).
  • FIG. 20 The means, AUC and variability shown on the line and bar graphs were calculated by GraphPad Prism software.
  • Figure 20 As for Figure 19 but with D25 antibody binding being assessed.
  • Figure 21 (A) 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).
  • ED60 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).
  • nucleic acids encoding RSV-F proteins comprising deletions in the cytoplasmic tail
  • the present disclosure provides, in an independent aspect, a recombinant nucleic acid encoding an RSV-F protein comprising a cytoplasmic tail; wherein, relative to cytoplasmic tail according to SEQ ID NO: 3 or 4, 2-20 residues are deleted from the cytoplasmic tail of the RSV-F protein (preferably from the C-terminal end thereof).
  • a further independent aspect of the present disclosure provides a recombinant nucleic acid encoding an RSV-F protein comprising a cytoplasmic tail; wherein the cytoplasmic tail is 2-23 residues in length.
  • nucleic acids of the present disclosure and the RSV-F proteins which they encode, are respectively referred to herein as “nucleic acids of the present disclosure” and “RSV-F proteins of the present disclosure”.
  • the wild-type RSV-F sequences of SEQ ID NO: 1 (A2 subtype) and SEQ ID NO: 2 (B subtype strain 18537) and their wild-type cytoplasmic tails (SEQ ID NO: 3 and 4 respectively) are not “RSV-F proteins of the present disclosure”, as referred to herein.
  • a further independent aspect of the present disclosure is an RSV-F protein encoded by a nucleic acid of the present disclosure.
  • a further independent aspect of the present disclosure provides a multimer comprising protomers, wherein at least one protomer is an RSV-F protein of the present disclosure.
  • the multimer Docket No.: 70280WO01 is a trimer of RSV-F proteins of the present disclosure.
  • the trimer is a homotrimer (that is, comprising three RSV-F proteins of the present disclosure comprising or consisting of the same primary amino acid sequence).
  • an RSV-F protein having a “cytoplasmic tail” / “CT” 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 2 (Y in SEQ ID NO: 1 and SEQ ID NO: 2), when the F1 and transmembrane domains of the RSV-F protein is aligned with positions 137-549 of SEQ ID NO: 1 or 2. Accordingly, the cytoplasmic tail is positioned C-terminal to the transmembrane domain.
  • an RSV-F protein having a “cytoplasmic tail” / “CT” refers to the presence of residues (e.g.
  • the RSV-F construct referred to as ⁇ CT25 used in the examples does not comprise any residues C-terminal to the Y at position 549, and hence does not comprise a cytoplasmic tail.
  • Reference to e.g. deletion of 2-20 residues (and the like) from the CT refers to deletion of at least 2, and no more than 20, residues from the CT (relative to SEQ ID NO: 3 or 4), at any positions.
  • 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: 3 or 4 respectively, and at most the deletion of C-terminal residues TPVTLSKDQLSGINNIAFSN (SEQ ID NO: 142) or TPVTLSKDQLSGINNIAFSK (SEQ ID NO: 143) relative to SEQ ID NO: 3 or 4 respectively.
  • 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 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).
  • 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 [12]).
  • SEQ ID NO: 2 is the RSV-F sequence from B subtype strain 18537 (Uniprot ID: P13843).
  • SEQ ID NO:1, SEQ ID NO: 2, and any wild-type RSV-F sequence are referred to herein as “wild-type”.
  • Either of SEQ ID NO: 3 and 4 may also be referred to as a “wild type cytoplasmic tail”.
  • RSV-F proteins of the present disclosure may comprise mutations relative to SEQ ID NO: 1 or 2 found in RSV-F proteins from further strains and subtypes, both naturally-occurring and engineered (e.g. RSV-F proteins of further A subtype strains, or further B subtype strains).
  • RSV-F proteins of the present disclosure may be of the RSV-A or the RSV- B subtype.
  • RSV-F proteins of the present disclosure may also have a specific degree of sequence identity to SEQ ID NO: 1 or 2, e.g. as detailed in the embodiments below. Docket No.: 70280WO01 “Mutation” is used generally herein to encompasses substitution, insertion and deletion of residues.
  • Reference to a sequence / region of an RSV-F protein of the present disclosure “corresponding to positions x-y of SEQ ID NO: z” encompasses sequences / regions which align with positions x-y of SEQ ID NO: z (which, for the avoidance of doubt, includes positions x and y).
  • Corresponding residue positions e.g. position 549 of SEQ ID NO: 1, and so forth
  • 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 to a subject (e.g. via expression from nucleic acids), 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 a wild-type pre-fusion RSV-F, in particular one or more epitopes present on the (solvent-exposed) surface of the protein when in the pre-fusion conformation.
  • RSV-F proteins of the present disclosure may also be considered antigens (or, phrased differently, are antigenic) given their ability to be bound by antibodies AM14, D25 and motavizumab; in particular AM14 which recognises trimeric, pre-fusion RSV-F (heavy and light chain sequences of antibodies given below).
  • RSV-F proteins of the present disclosure may be considered as stabilised in the pre-fusion conformation, following expression from nucleic acids.
  • 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: 5 and 6 respectively, and SEQ ID NO: 7 and 8 respectively.
  • LC and HC light chain and a heavy chain
  • RSV-F proteins of the present disclosure e.g. in the ectodomain
  • Such mutations are in addition to those made to the CT.
  • RSV-F proteins of the present disclosure comprise an ectodomain (or at least a portion thereof), and Docket No.: 70280WO01 preferably comprise mutations in the ectodomain according to any of preferred classes (1)-(4) as detailed below, in addition to those made to the CT.
  • RSV-F proteins of the present disclosure comprise an ectodomain (or at least a portion thereof), and comprise mutations in the ectodomain according to any of preferred classes (1)-(3) as detailed below, in addition to those made to the CT.
  • the ectodomain of SEQ ID NO: 1 and 2 is positions 26-109 and 137-523 (positions 1-25 and 110-136 being removed in the mature protein via signal sequence cleavage and furin processing).
  • An ectodomain of an RSV-F protein of the present disclosure may be positions 26-109 and 137-523 of said RSV-F protein, e.g. with residues numbered according to SEQ ID NO: 1 or 2.
  • RSV-F proteins of the present disclosure comprise a cytoplasmic tail; wherein, relative to cytoplasmic tail according to SEQ ID NO: 3 or 4, 2-20 residues are deleted from the cytoplasmic tail of the RSV-F protein (preferably from the C-terminal end thereof). In some embodiments, 3-20 residues are deleted (preferably from said C-terminal end). In some embodiments, 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 (preferably from said C-terminal end). In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues are deleted (preferably 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, or 15-20 residues are deleted (preferably from said C-terminal end).
  • 2-5, such as 2-4, 2-3 or 3-4, and preferably 3, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according to SEQ ID NO: 3 or 4).
  • Example 2 deletion of the 3 C-terminal residues (“ ⁇ CT3”) enhanced cell-surface trimeric, pre-fusion RSV-F expression from nucleic acids (as measured by AM14 antibody binding) over a period of 96 hours post-transfection, relative to expression of the parental molecule with either an intact or a fully deleted CT.
  • This enhanced expression phenotype was observed for all four RSV-F constructs tested (F318, F319, F(i) and F(ii)). See also e.g. Example 5 ( Figure 5A), which uses the “ ⁇ CT5” construct.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-31 of SEQ ID NO: 69, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-29 of SEQ ID NO: 70, 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.: 70280WO01
  • 6-13 such as 7-13, 8-12, 9-11, 9-10 or 10-11, and preferably 10, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 3 or 4).
  • deletion of the 10 C-terminal residues (“ ⁇ CT10”) enhanced cell-surface trimeric, pre-fusion RSV-F expression from nucleic acids (as measured by AM14 antibody binding) over a period of 47 hours post-transfection, relative to expression of the parental molecule with either an intact or a fully deleted CT.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-24 of SEQ ID NO: 71, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • 14-16 such as 14-15 or 15-16, and preferably 15, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 3 or 4).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-19 of SEQ ID NO: 72, or (ii) an amino acid sequence at least 60%, 70%, 80% or 90% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • 16-20 such as 17-20, 18-20 or 19-20, and preferably 20, residues are deleted from the C-terminal end of the CT of the RSV-F protein (relative to a wild-type cytoplasmic tail according SEQ ID NO: 3 or 4).
  • deletion of the 20 C-terminal residues (“ ⁇ CT20”) enhanced cell-surface trimeric, pre-fusion RSV-F expression from nucleic acids (as measured by AM14 antibody binding) over a period of 96 hours post-transfection, relative to expression of the parental molecule with either an intact or a fully deleted CT.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-14 of SEQ ID NO: 73, or (ii) an amino acid sequence at least 60% or 80% identical to said positions and optionally the same length as Docket No.: 70280WO01 said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the deletions outlined above increase the cell-surface expression of RSV-F protein from RNA, relative to an RSV-F protein having the same amino acid sequence absent deletions, e.g. comprising a wild-type cytoplasmic tail, e.g. according to SEQ ID NO: 3 or 4 (e.g. over at least 24, 48, 72 or 96 hours; or e.g. over 24, 48, 72 or 96 hours).
  • the deletions outlined above increase the cell-surface expression of RSV-F protein in trimeric, pre-fusion form from RNA, relative to expression in such form of an RSV-F protein having the same amino acid sequence absent such deletions, e.g. comprising a wild-type cytoplasmic tail, e.g.
  • RSV-F proteins of the present disclosure comprise a cytoplasmic tail; wherein the cytoplasmic tail is 5-23 residues in length.
  • the cytoplasmic tail is 8-12, such as 9- 12, 10-12, 9-11, 9-10, 10-11, or preferably 10 residues in length.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-19 of SEQ ID NO: 72, or (ii) an amino acid sequence at least 60%, 70%, 80% or 90% identical to said positions and 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). See discussion of ⁇ CT15 in the preceding paragraphs.
  • the cytoplasmic tail is 10-18, such as 11-17, 12-16, 13-16, 14-15, or preferably 15 residues in length.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-24 of SEQ ID NO: 71, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and 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). See discussion of ⁇ CT10 in the preceding paragraphs.
  • the cytoplasmic tail is 18-23, 19-23, 20- 23, 21-23, 21-22, 22-23 or preferably 22 residues in length.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-31 of SEQ ID NO: 69, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii).
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-29 of SEQ ID NO: 70, or (ii) an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and the same length as said positions; and wherein Docket No.: 70280WO01 the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). See discussion of ⁇ CT5 in the preceding paragraphs.
  • the cytoplasmic tail 5-7, 5-6, 4-5, or more preferably 5 residues in length.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-14 of SEQ ID NO: 73, or (ii) an amino acid sequence at least 60% or 80% identical to said positions and 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). See discussion of ⁇ CT20 in the preceding paragraphs.
  • the cytoplasmic tail lengths outlined above increase the cell-surface expression of RSV-F protein from RNA, relative to an RSV-F protein having the same amino acid sequence but comprising a wild-type cytoplasmic tail, e.g. according to SEQ ID NO: 3 or 4 (e.g.
  • the cytoplasmic tail lengths outlined above increase the cell-surface expression of RSV-F protein in trimeric, pre-fusion form from RNA, relative to expression in such form of an RSV-F protein having the same amino acid sequence but comprising a wild-type cytoplasmic tail, e.g. according to SEQ ID NO: 3 or 4 (e.g. over at least 24, 48, 72 or 96 hours; or e.g. over 24, 48, 72 or 96 hours).
  • SEQ ID NO: 3 or 4 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: 5 and a heavy chain (HC) according SEQ ID NO: 6).
  • 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”).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising an F2 and an F1 domain, and the substitution relative to a wild-type RSV-F ectodomain (e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) of a residue for a C residue in both of the F2 and F1 domains.
  • RSV-F proteins of the present disclosure may comprise a C residue at both of positions 55 and 188 (not present in wild-type).
  • the C residue in the F1 domain may be within the fusion peptide of the RSV-F protein; optionally wherein the fusion peptide is the region corresponding to positions 137-157 of SEQ ID NO: 1 or 2.
  • the C residue may be within the region of the RSV-F protein corresponding to positions 143-153, 146-150 or 147-149 of SEQ ID NO: 1 or 2; and preferably at position 148 of the RSV-F protein.
  • the C residue in the F2 domain may be within the region of the RSV-F protein corresponding to positions 99-105, 100-104 or 102-104 of SEQ ID NO: 1 or 2; and preferably at position 103 of the RSV-F protein.
  • RSV-F proteins of the present disclosure may comprise the substitution (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) of one or more small aliphatic or small polar residues that are buried in the pre-fusion conformation (in wild-type), for larger aliphatic or larger aromatic residues.
  • Said small aliphatic or small polar residues may be, for example, a S, T, G, A, V, or R residue.
  • Said larger aliphatic or larger aromatic residues may be, for example, a I, Y, L, H, M or W residue.
  • RSV-F proteins of the present disclosure may, for example, comprise: (i) substitution at position 190, 55, 62, 155, or 290 for I, Y, L, H, or M; (ii) substitution at position 54, 58, 189, 219, or 397 for I, Y, L, H, or M; (iii) substitution at position 151 for A or H; (iv) substitution at position 147 or 298 for I, L, H, or M; (v) substitution at position 164, 187, 192, 207, 220, 296, 300, or 495 for I, Y, H; or (vi) substitution at position 106 for W; wherein substitutions at position 190 according to (i) are preferred; wherein substitution at position 190 for I is a preferred substitution at said position.
  • RSV-F proteins of the present disclosure may comprise substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) which reduce inter-protomer repulsive ionic interactions or increase inter-protomer attractive ionic interactions with E487 and D489 on an adjacent RSV-F protomer (when the RSV-F protein is in trimeric form).
  • substitutions generally further promote and/or stabilise the pre-fusion conformation.
  • RSV-F proteins of the present disclosure may comprise the substitution of a D or E residue for S, T, N, H, P, F, L or Q within the region of the RSV- F protein corresponding to positions 474-523 of SEQ ID NO: 1 or 2 (a.k.a. the heptad repeat B Docket No.: 70280WO01 (“HRB”) domain), such as D486S/H/N/T/P or E487Q/T/S/L/H.
  • HRB heptad repeat B Docket No.: 70280WO01
  • RSV-F proteins of the present disclosure may, for example, comprise: (vii) substitution at position 82, 92, or 487 for D, F, Q, T, S, L, or H; (viii) substitution at position 315, 394, or 399 for F, M, R, S, L, I, Q, or T; (ix) substitution at position 392, 486, or 489 for H, S, N, T, or P; and/or (x) substitution at position 106 or 339 for F, Q, N, or W; wherein substitutions at position 486 according to (ix) are preferred; wherein substitution at position 486 for S is a preferred substitution at said position.
  • RSV-F proteins of the present disclosure comprise the substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) 103C, 148C, 190I and 486S.
  • RSV-F proteins may be of the RSV-A or B subtype. See, e.g. construct F(i) as tested in the examples (in particular, Example 2; Figure 2 D).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-109 and 137-523 of SEQ ID NO: 17; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto and comprising the 103C, 148C, 190I, and 486S substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) a portion of positions 26-109 and 137-523 of SEQ ID NO: 17, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof and comprising the 103C, 148C, 190I, and 486S substitutions (relative to a wild-type RSV-F ectodomain, e.g.
  • positions 26-109 and 137-523 of SEQ ID NO: 1 or 2 ); or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to such a portion which comprises the 103C, 148C, 190I, and 486S substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • the region corresponding to positions 110- 136 of SEQ ID NO 1 or 2 may be absent due to furin processing.
  • nucleic acids of the present disclosure will typically encode an RSV-F protein of the present disclosure comprising the region corresponding to positions 110-136 of SEQ ID NO 1 or 2 (which may be positions 110-136 of the RSV-F protein). All of the above mutations in the subsection preferably promote and/or stabilise the pre-fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure may comprise the substitution of at least one residue in the ectodomain relative to a wild- type RSV-F ectodomain (e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2), at position 67 and/or 215 (typically both).
  • substitutions include: (i) substitution at position 67 for I; and/or (ii) substitution at position 215 for P; wherein a combination of substitutions according (i) and (ii) is a preferred set of substitutions.
  • RSV-F proteins of the present disclosure may comprise, in addition to the above substitutions(s), a substitution at position 486 or 487 for a residue without a negatively charged side chain. Examples of such substitutions include: (iii) substitution at position 486 for N; or (iv) substitution at position 487 for Q, N or I; wherein substitution at position 486 for N is a preferred substitution.
  • RSV-F proteins of the present disclosure may comprise, in addition to the above substitution(s), a linker sequence joining the F2 and F1 domains, e.g. wherein the linker sequence is from 1-10 amino acids (e.g. according to SEQ ID NO: 11, 12, 13 or 14, in particular according to SEQ ID NO: 14).
  • RSV-F proteins of the present disclosure preferably comprise combinations of the above substitutions, potentially in addition to further substitutions and/or use of a linker, such as: (v) 67I and 215P, optionally with a linker sequence joining the F2 and F1 domains (e.g. according to SEQ ID NO: 14); (vi) 67I, 215P and 487Q, optionally with a linker sequence joining the F2 and F1 domains (e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-109 and 137-523 of SEQ ID NO: 16; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto and comprising the N67I and S215P substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) a portion of positions 26-109 and 137-523 of SEQ ID NO: 16, such a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof and comprising the N67I and S215P substitutions (relative to a wild-type RSV-F ectodomain, e.g.
  • a further independent aspect of the present disclosure is a recombinant RNA encoding an RSV-F protein comprising an ectodomain according to preferred class (2).
  • the RSV-F protein (encoded by said RNA) does not comprise a cytoplasmic tail.
  • a further independent aspect of the present disclosure is an RSV-F protein encoded by said RNA.
  • deletion of the entire CT from construct F(ii) enhanced cell-surface trimeric, pre-fusion expression from nucleic acids (as measured by AM14 antibody binding) over a period of 96 hours post-transfection, relative to expression of the parental F(ii) construct with an intact CT.
  • nucleic acids and proteins are also referred to herein as nucleic acids and proteins “of the present disclosure”. All of the above mutations in the subsection preferably promote and/or stabilise the pre- fusion conformation of RSV-F.
  • RSV-F proteins of the present disclosure may comprise the substitution of at least two residues in the ectodomain relative to a wild- type RSV-F ectodomain (e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) for C residues, introducing a disulphide bond.
  • Said disulphide bond preferably promotes and/or stabilises the pre- fusion conformation.
  • Said disulphide bond is preferably an intra-protomer disulphide bond. Examples of positions which may be substituted for C residues to introduce said disulphide bond include 155C+290C (e.g. S155C + S290C).
  • RSV-F proteins of the present disclosure may comprise the substitution of at least two further residues in the ectodomain relative to a wild-type RSV-F ectodomain (e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) for C residues, introducing a further Docket No.: 70280WO01 disulphide bond
  • Said disulphide bond preferably promotes and/or stabilises the pre-fusion conformation.
  • Said disulphide bond is preferably an inter-protomer disulphide bond.
  • RSV-F proteins of the present disclosure may comprise the substitution of one or more residues in the ectodomain relative to a wild-type RSV-F ectodomain (e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2), wherein the residues have a side chain that is buried in a hydrophobic cavity in the pre-fusion conformation (in wild-type). Such substitutions may introduce residues with hydrophobic side chains (e.g.
  • RSV-F proteins of the present disclosure may comprise, in addition to the above substitution(s) / disulphide bonds, a linker sequence joining the F2 and F1 domains, e.g. wherein the linker sequence is from 1-10 amino acids (e.g. according to SEQ ID NO: 11, 12, 13 or 14, in particular according to SEQ ID NO: 13). In other embodiments of (3), the linker sequence is absent.
  • RSV-F proteins of the present disclosure preferably comprise combinations of the above substitutions, optionally in addition to the use of a linker, such as: (i) 149C, 155C, 190F, 207L, 290C and 458C, preferably with a linker sequence joining the F2 and F1 domains (e.g. according to SEQ ID NO: 13); (ii) 102A, 149C, 155C, 190F, 207L, 290C, 373R, 379V, 447V and 458C, preferably with a linker sequence joining the F2 and F1 domains (e.g.
  • the linker may replace wild-type residues 104-144 (i.e. there is ⁇ 104-144 deletion, which said positions being replaced by the linker, e.g. according to SEQ ID NO: 13).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-109 and 137-523 of SEQ ID NO: 15; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto and comprising the 155C, 190F, 207L and 290C substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising Docket No.: 70280WO01 or consisting of: (i) a portion of positions 26-109 and 137-523 of SEQ ID NO: 15, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof and comprising the S155C, S190F, V207L and S290C substitutions (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-103 and 106-485 of SEQ ID NO: 18; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto and comprising the 149C, 155C, 190F, 207L, 290C and 458C substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) a portion of positions 26-103 and 106-485 of SEQ ID NO: 18, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof and comprising the 149C, 155C, 190F, 207L, 290C and 458C substitutions (relative to a wild-type RSV-F ectodomain, e.g.
  • positions 26-109 and 137-523 of SEQ ID NO: 1 or 2 ); or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to such a portion which comprises the 149C, 155C, 190F, 207L, 290C and 458C substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-103 and 106-485 of SEQ ID NO: 18; (ii) or an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto and comprising the 102A, 149C, 155C, 190F, 207L, 290C, 373R, 379V, 447V and 458C substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) a portion of positions 26-103 and 106-485 of SEQ ID NO: 18, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof and comprising the 102A, 149C, 155C, 190F, 207L, 290C, 373R, 379V, 447V and 458C substitutions (relative to a wild-type RSV-F ectodomain, e.g.
  • a further independent aspect of the present disclosure is a recombinant RNA encoding an RSV-F protein comprising an ectodomain according to preferred class (3).
  • the RSV-F protein (encoded by said RNA) does not comprise a cytoplasmic tail.
  • a further independent aspect of the present disclosure is an RSV-F protein encoded by said RNA.
  • Such nucleic acids and proteins are also referred to herein as nucleic acids and proteins “of the present disclosure”.
  • RSV-F proteins of the present disclosure may comprise the substitution of residues in the ectodomain relative to a wild-type ectodomain (e.g.
  • positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) according to any of (a), (b) and (c) defined below: (a): substitution at position 55 for T, C, V, I; preferably T, C or V; preferably T or V; preferably T; (b): substitution at position 215 for A, P, V, I, or F; preferably A, V, I, or F; preferably A or P; preferably A; (c): substitution at position 228 for K, R, N, W, D, E, Q, H, S, T or Y; preferably K, R, W, N, Q, H, S, T or Y; preferably K, R, Q and N; preferably K, R or Q; preferably K or R, preferably K; Combinations of substitutions according to of (a), (b) or (c) will typically be present, such as: (a) and (b), (a) and (c), (b) and (c), or preferably (a), (b) and (c).
  • a minimal substitution screen revealed the 55T substitution to be a likely driver of the pre-fusion conformation (see Figure 15; design F308 – recombinant protein tested).
  • 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 12) appears to be accommodated well in the hydrophobic pocket discussed above, without generating significant steric clashes.
  • the addition of the CH3 group of T appears to provide new, energetically favourable VDW contacts of the type discussed above.
  • substitutions provided for position 55 by ROSETTA software include C and V (based on all amino acids being allowed (no evolutionary constraints) with energy thresholds of 0.0, -0.1 or -0.5 being used). Docket No.: 70280WO01 Furthermore, as detailed in Example 9, a minimal substitution screen revealed the 215A substitution to be a likely driver of the pre-fusion conformation (see Figure 15; design F309 – recombinant protein tested). Without wishing to be bound by this theory, 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 13).
  • 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.
  • Example 9 a minimal substitution screen revealed the 228K substitution alone to be able to achieve pre-fusion RSV-F (see Figure 15; design F310 – recombinant protein tested).
  • K at position 228 appears to result in an H bond with Y250 on the same protomer (see Figure 11, dashed line indicating hydrogen bond).
  • Said H bonding may stabilise Y250 to form a tertiary cation-pi-anion interaction between E232, Y250 and R235 (E232 and Y250 being on one protomer, with R235 being on an adjacent protomer).
  • E, Y and R are one of the dominant triads for such a tertiary cation-pi-anion interaction (see, e.g. [16]). Furthermore, residues with other H bond donors in their side chains (such as R or Q, in particular R) at position 228 may also provide this stabilising H bond with Y250. In addition, based on the proximity and orientation of the E232 side chain (see Figure 11), substitution for N may also provide a stabilising hydrogen bond with Y250. In all foregoing embodiments in this subsection, optionally 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
  • 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.
  • RSV-F proteins of the present disclosure may 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); Docket No.: 70280WO01 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); 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
  • RSV-F proteins of the present disclosure may 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; Docket No.: 70280WO01 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; 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.
  • 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 2 (a.k.a. the HRB domain) which form a disulphide bond.
  • the pair of C residues may be within the region corresponding to positions 474-513 of SEQ ID NO: 1 or 2.
  • this is an intra-protomer disulphide bond (i.e. linking two C residues within the same protomer).
  • 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 2, 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 2.
  • 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 2.
  • C residues in these positions Docket No.: 70280WO01 were 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 14 and/or are present in designs F528, F647, F651 and 2C (see e.g. Examples 8, 11 and 13) .
  • 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 2, 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 2.
  • 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, or 478 and 483 of SEQ ID NO: 1 or 2.
  • a first C residue of said pair is within a region of the RSV-F protein corresponding to positions 480-486 of SEQ ID NO: 1 or 2
  • a second C residue of said pair is within a region corresponding to positions 490-497 of SEQ ID NO: 1 or 2.
  • C residue pairs examples include those at positions: 486 and 490, 485 and 494, or 480 and 497 of SEQ ID NO: 1 or 2.
  • the pair of C residues is at positions 486 and 490 of SEQ ID NO: 1 or 2 (see inter alia, designs F528, F647, F651 and 2C in e.g. Examples 8, 11 and 13).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 21; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 210H, 211N, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 445D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 21, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 210H, 211N, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 445D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 22; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, S211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 22, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, S211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 23; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 215A, 228K, 241N, 315I, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 23, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 215A, 228K, 241N, 315I, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 24; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 215A, 228K, 315I, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 24, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 215A, 228K, 315I, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 25; (ii) or an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 215A, 228K, 315I, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 25, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 215A, 228K, 315I, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 26; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 445D, 455V and 459M (relative to a wild-type RSV-F Docket No.: 70280WO01 ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 26, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 445D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 27; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 27, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 28; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 28, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 29; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2). See e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or Docket No.: 70280WO01 consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 29, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild- type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of SEQ ID NO: 30; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 30, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 348N, 455V and 459M (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-109 and 137-523 of SEQ ID NO: 82; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V, 459M, 486C and 490C (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 26-109 and 137-523 of SEQ ID NO: 82, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V, 459M, 486C and 490C (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-472 of SEQ ID NO: 108; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 26-472 of SEQ ID NO: 108, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C (relative to a wild-type RSV-F Docket No.: 70280WO01 ectodomain, e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 26-109 and 137-523 of SEQ ID NO: 104; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto, preferably comprising the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may alternatively comprise an ectodomain comprising or consisting of a portion of positions 26-109 and 137-523 of SEQ ID NO: 104, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof.
  • Said portion preferably includes the substitutions 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C (relative to a wild-type RSV-F ectodomain, e.g.
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of: (i) an amino acid sequence according to positions 25-109 and 137-523 of any of SEQ ID NO: 31-37; or (ii) an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% identical thereto (preferably including all substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) present in the amino acid sequence according to any of SEQ ID NO: 31-37 where applicable).
  • RSV-F proteins of the present disclosure may comprise an ectodomain comprising or consisting of a portion of positions 25-109 and 137-523 of SEQ ID NO: 31-37, such as a portion at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% the length thereof (preferably including all substitutions (relative to a wild-type RSV-F ectodomain, e.g. positions 26-109 and 137-523 of SEQ ID NO: 1 or 2) present in the amino acid sequence according to any of SEQ ID NO: 31-37 where applicable). All of the above mutations in the subsection preferably promote and/or stabilise the pre-fusion conformation of RSV-F.
  • Nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure, preferably according to (1), (2), (3) or (4), 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%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity to positions 1-549 of SEQ ID NO: 1 or 2.
  • nucleic acids of the present disclosure encode an RSV-F protein of the present disclosure, preferably according to (1), (2), (3) or Docket No.: 70280WO01 (4), comprising or consisting of an amino acid sequence having at least 70% sequence identity to positions 26-549 SEQ ID NO: 1 or 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 26-549 SEQ ID NO: 1 or 2.
  • RSV-F proteins of the present disclosure comprise an E residue at position 66, and a P residue at position 101.
  • nucleic acids of the present disclosure encode an RSV-F protein of the present disclosure, preferably according to (1), (2), (3) or (4), comprising two domains (in the N-terminal to C-terminal direction, the “F2” and “F1” domains); the F2 domain comprising or consisting of an amino acid sequence having at least 70% sequence identity to positions 1-109 of SEQ ID NO: 1 or 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% or 99%, sequence identity to positions 1-109 of SEQ ID NO: 1 or 2; and the 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
  • nucleic acids of the present disclosure may encode an RSV-F protein of the present disclosure, preferably according to (1), (2), (3) or (4), comprising an F2 domain and an F1 domain; the 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 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% or 99%, sequence identity to positions 26-109 of SEQ ID NO: 1 or 2; and the 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 2, such as at least 75%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%
  • RSV-F proteins of the present disclosure preferably have such sequence identities (as defined in any of the three preceding paragraphs) according to SEQ ID NO: 1.
  • nucleic acids (preferably RNA) of the present disclosure, and the RSV-F proteins encoded thereby elicit a pre-fusion RSV-F-specific antibody response against RSV in vivo, e.g. an IgG antibody response (see, e.g. Example 6).
  • nucleic acids of the present disclosure, and the RSV-F proteins encoded thereby elicit a neutralising antibody response against RSV in vivo, e.g. against RSV-A (see, e.g. Example 6).
  • Said neutralising antibody response may inhibit replication of RSV in the respiratory system of a subject Docket No.: 70280WO01 (such as in the lungs). Said neutralising antibody response may yield protective immunity against RSV in a subject.
  • RSV-F proteins of the present disclosure are generally neither fused with, nor comprise, a green fluorescent protein (GFP). Nucleic acids of the present disclosure generally do not encode a GFP. In some embodiments, RSV-F proteins of the present disclosure do not comprise a S residue at position 552.
  • RSV-F proteins of the present disclosure do not comprise S residue at the position corresponding to position 552 of SEQ ID NO: 1 or 2, when the F1 and transmembrane domains of the RSV-F protein of the present disclosure is aligned with positions 137-549 of SEQ ID NO: 1 or 2.
  • Nucleic acids of the present disclosure generally do not comprise a Xba1 restriction site.
  • RSV-F proteins of the present disclosure generally neither comprise a cytoplasmic tail from (or derived from) vesicular stomatitis virus G protein (VSV-G), nor a portion of such a cytoplasmic tail.
  • VSV-G vesicular stomatitis virus G protein
  • Nucleic acids of the present disclosure generally encode neither a sequence of a cytoplasmic tail from (or derived from) VSV-G, nor a portion of such a cytoplasmic tail.
  • RSV-F proteins of the present disclosure are generally neither comprised within, nor located on the surface of, an RSV virion.
  • RSV-F proteins of the present disclosure are generally neither comprised within, nor located on the surface of, a Hep-2 cell.
  • nucleic acids of the present disclosure do not encode an RSV-F protein (e.g.
  • nucleic acids of the present disclosure do not encode an RSV-F protein (e.g.
  • nucleic acids of the present disclosure do not encode an RSV-F protein (e.g.
  • nucleic acids of the present disclosure do not encode an RSV-F protein (e.g. in the pre-fusion conformation when expressed) which is mutated relative to positions 1-513 of SEQ ID NO: 1 and comprises (a), (b) and (c): (a) (ai) at least one mutation relative to positions 1-513 of SEQ ID NO: 1 in a region corresponding to positions 38-60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (aii) at least one mutation relative to positions 1-513 of SEQ ID NO: 1 in a region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation relative to positions 1-513 of SEQ ID NO:
  • nucleic acids of the present disclosure do not encode an RSV-F protein (e.g. in the pre-fusion conformation when expressed) which is mutated relative to positions 1-513 of SEQ ID NO: 1 and comprises (a), (b) and (d): (a) (ai) at least one mutation relative to positions 1-513 of SEQ ID NO: 1 in a region corresponding to positions 38-60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and/or (aii) at least one mutation relative to positions 1-513 of SEQ ID NO: 1 in a region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and/or introduces, through substitution or insertion, a residue selected from M, F, I and V into the region; (b) at least one mutation
  • nucleic acids of the present disclosure do not encode a recombinant RSV-F protein in the pre-fusion conformation, comprising at least one mutation relative to positions 1-513 of SEQ ID NO: 1, wherein the at least one mutation introduces neither a disulphide bond nor a P residue into said positions.
  • Formats of nucleic acids 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; Docket No.: 70280WO01 and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g. those as detailed in the section entitled Preparing RSV-F proteins, above).
  • the nucleic acid is for the expression of an RSV-F protein of the present disclosure in vivo in a subject (i.e.
  • the nucleic acid is, or is part of, a nucleic acid-based vaccine).
  • the nucleic acid may comprise one or more heterologous sequences, such as a sequence encoding a further protein (e.g. as detailed below) and/or a control sequence, in particular a promoter or an internal ribosome entry site.
  • Nucleic acids of the present disclosure may be codon optimised. In some embodiments, nucleic acids of the present disclosure may be codon optimised for expression in human cells.
  • Codon optimisation refers to the use of specific codons, which, while not altering the sequence of the expressed protein (given genetic code redundancy), may increase translation efficacy and/or half- life of the nucleic acid.
  • codon optimised RNA are discussed in more detail in the subsection entitled RNA below.
  • nucleic acids of the present disclosure are in the form of a viral vector, such as a replicating or replication-deficient viral vector; including both DNA and RNA-based viral vectors.
  • viral vectors for encoding an RSV-F protein of the present disclosure include, for example: adenovirus vectors, such as replication-deficient or replication-competent adenovirus vectors; pox virus vectors, such as vaccinia virus vectors (e.g. modified vaccinia Ankara virus (MVA), NYVAC, avipox vectors, canarypox (e.g.
  • adenovirus vectors such as replication-deficient or replication-competent adenovirus vectors
  • pox virus vectors such as vaccinia virus vectors (e.g. modified vaccinia Ankara virus (MVA), NYVAC, avipox vectors, canarypox (e.g.
  • Alphavirus vectors such as Sindbis virus, Semlike Forest virus (SFV), Ross River virus, Venezuelan equine encephalitis (VEE) virus, and chimeras derived from Alphavirus vectors such as the foregoing; herpes virus vectors, such as cytomegalovirus (CMV)-derived vectors; arena virus vectors, such as lymphocytic choriomeningitis virus (LCMV) vectors; measles virus vectors; vesicular stomatitis virus vectors; pseudorabies virus vectors; adeno-associated virus vectors; retrovirus vectors; lentivirus vectors; and viral-like particles.
  • CMV cytomegalovirus
  • LCMV lymphocytic choriomeningitis virus
  • the nucleic acid is in the form of a DNA plasmid.
  • the viral vector is an adenovirus vector, such as a replication-incompetent adenovirus type 26 (“Ad26”) or a replication-incompetent chimpanzee-adenovirus-155 (“ChAd155”), preferably a replication- incompetent Ad26.
  • Ad26 replication-incompetent adenovirus type 26
  • ChoAd155 replication-incompetent chimpanzee-adenovirus-155
  • Ad26 replication-incompetent chimpanzee-adenovirus-155
  • Ad26 replication-incompetent chimpanzee-adenovirus-155
  • the adenovirus vector (preferably replication-incompetent Ad26) may also be co- formulated with an RSV-F protein (i.e. the protein per se) of the present disclosure, which may have the same, or a distinct, primary amino acid sequence to the RSV-F protein of the present disclosure encoded by the adenovirus.
  • RSV-F protein i.e. the protein per se
  • the adenovirus Docket No.: 70280WO01 vector (preferably replication-incompetent Ad26) may be co-formulated with a further RSV-F protein (i.e.
  • the protein per se that is not an RSV-F protein according to the present disclosure
  • an RSV-F protein with the p27 region deleted and optionally at least 2, 3, 4 or 5 mutations relative to wildtype RSV-F such as N67I and S215P; N67I, S215P and E487Q; or K66E, N67I, I76V, S215P and D486N; in particular the latter set of five mutations.
  • a particular patient group of interest in which the co-formulation may be used in therapy, in particular vaccination
  • is older adults see section entitled Medical uses and methods of treatment, below).
  • the co-formulation may be administered as, or as part of, a prime-boost regimen, in particular involving administration of the co-formulation as both prime administration(s) and boost administration(s).
  • the nucleic acid (preferably RNA) may encode an RSV-F protein of the present disclosure only (i.e. the nucleic acid encodes a single protein). Alternatively, the nucleic acid may encode multiple proteins, of which one is the RSV-F protein of the present disclosure. In some embodiments, the nucleic acid encodes at least (i) an RSV-F protein of the present disclosure; and (ii) at least one further protein.
  • the at least one further protein may be a nanoparticle, e.g. a ferritin nanoparticle (e.g.
  • the at least one further protein is an antigen; and as such may comprise, or may be, a viral, bacterial, fungal, parasitic, tumour, or allergenic (i.e. from, or derived from, an allergen) antigen; typically encoded by a separate open reading frame to the RSV-F protein of the invention.
  • the at least one further protein will typically be a pathogen antigen.
  • the at least one further protein will typically be an antigen that is a surface polypeptide e.g. a spike glycoprotein, a haemagglutinin, an adhesin or an envelope glycoprotein.
  • the at least one further protein is an antigen from, or derived from, a virus, in particular a virus causing respiratory disease, in particular a seasonal virus causing respiratory disease.
  • examples of such viruses include: Coronavirus, Orthomyxovirus, Pneumoviridae, Paramyxoviridae, Poxviridae, Picornavirus, Bunyavirus, Heparnavirus, Filovirus, Togavirus, Flavivirus, Pestivirus, Hepadnavirus, Rhabdovirus, Caliciviridae, Retrovirus, Reovirus, Parvovirus, Herpesvirus, Papovaviruses and Adenovirus.
  • the at least one further protein detailed above 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 is RNA encoding an RSV-F protein of the present disclosure in addition to an hMPV antigen (in particular, the F antigen).
  • a preferred patient group in which the RNA may be used in therapy, in Docket No.: 70280WO01 particular vaccination
  • 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 may be from, or derived from, the A or B subtype.
  • 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 / antigens; in particular selected from G (from or derived from the A subtype: “G A ”), G (from or derived from the B subtype: “G B ”) N and either M2-1 or M2-2; in particular G A, G B , N and either of M2-1 or M2-2.
  • the at least one further protein detailed above is a Coronavirus antigen.
  • Useful Coronavirus antigens can be from a SARS coronavirus, in particular SARS-CoV2.
  • Useful Coronavirus antigens include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein.
  • the Coronavirus antigen is a SARS- CoV2 spike protein.
  • Said SARS-CoV2 spike protein may be from any variant, e.g.
  • said SARS-CoV2 spike protein includes one or more mutations relative to the wild-type protein, in particular one or more (e.g. two) mutations to proline resides. Said one or more mutations may be introduced to stabilise said SARS-CoV2 spike protein in its pre-fusion conformation.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to a Coronavirus antigen, e.g. as detailed above.
  • a preferred patient group in which the RNA may be used in therapy, in particular vaccination
  • the at least one further protein detailed above is an Orthomyxovirus antigen.
  • Useful Orthomyxovirus antigens can be from an influenza A, B or C virus.
  • Useful Orthomyxovirus antigens include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin.
  • the Orthomyxovirus antigen is an influenza A virus haemagglutinin.
  • Said influenza A virus hemagglutinin may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.
  • the nucleic acid is RNA encoding an RSV-F protein of the present disclosure in addition to an Orthomyxovirus antigen, e.g. as detailed above.
  • a preferred patient group in which the RNA may be used in therapy, in particular vaccination
  • is older adults see section entitled Medical uses and methods of treatment, below).
  • the RNA may encode (i) an RSV-F protein of the present disclosure, (ii) a Coronavirus antigen, e.g. as detailed above, and (iii) an Orthomyxovirus antigen, e.g. as detailed above.
  • a plurality of nucleic acids of the present disclosure is, in particular, provided in purified or substantially purified form; that is, substantially free from other nucleic acids (e.g. free or substantially free from naturally-occurring nucleic acids, such as further nucleic acids expressed by a 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, below).
  • RNA in a preferred embodiment, the nucleic acid 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. In some embodiments, the RNA is self-replicating.
  • Self-replicating RNA can be produced using replication elements derived from, e.g., alphaviruses, and substituting sequences encoding the structural viral proteins with that encoding at least an RSV-F protein of the present disclosure.
  • a self- replicating RNA molecule is typically a positive-strand molecule which can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of the encoded protein (i.e.
  • the RSV-F protein of the present disclosure 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 Docket No.: 70280WO01 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).
  • EEEV eastern equine encephalitis virus
  • VEEV Venezuelan equine encephalitis virus
  • Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used for self-replicating RNA (see [18]).
  • 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, which may be added via enzymatic means or a non-enzymatic reaction.
  • the RNA may have the following exemplary 5’ caps: - a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge (also referred to as “Cap O”); - a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge, and wherein the first 5’ ribonucleotide comprises a 2’-methylated ribose (2’-O-Me) (also referred to as “Cap 1”); - a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotides by a triphosphate bridge, and wherein the first and second 5’ ribonucleotides comprise a 2’-methylated ribose (2’-O-Me) (also referred to as “Cap 2”); - or a 7-methylguanosine linked 5’-to-5
  • the 5’ cap is a 7-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleoside by a triphosphate bridge, and wherein the first 5’ ribonucleoside comprises a 2’- 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: Docket No.: 70280WO01 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 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 Docket No.: 70280WO01 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 (m6A); 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 m6A may be 0.1-5%, in particular 0.5- 2%, in particular 0.8-1.2%, such as about 1% (or 1%); in these embodiments the RNA may be circular RNA.
  • the percentage of standard Cs substituted with cytosine-substitutable modified nucleotide is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%.
  • the percentage of standard Gs substituted with G-substitutable modified nucleotide e.g.
  • the percentage of standard Us substituted with U-substitutable modified nucleotide is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%.
  • the percentage of standard Us substituted with U-substitutable modified nucleotide is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or preferably 100%; more preferably with 1m ⁇ and/or ⁇ (even more preferably 1m ⁇ ) .
  • the one or more modified ribonucleotides detailed above is, or comprise, 1m ⁇ and/or ⁇ , more preferably 1m ⁇ .
  • the RNA may comprise 1m ⁇ and/or ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. there are no standard U nucleotides, nor modified U ribonucleotides other than 1m ⁇ and/or ⁇ , in the RNA; i.e. 100% U substitution).
  • the RNA may comprise 1m ⁇ and/or ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e.
  • RNA may Docket No.: 70280WO01 comprise ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e. 100% U substitution with ⁇ ).
  • the RNA may comprise ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e. 100% U substitution with ⁇ with no other modified nucleotides being allowed).
  • the RNA comprises 1m ⁇ , and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e.100% U substitution with 1m ⁇ ). In an even more preferred embodiment, the RNA comprises 1m ⁇ , and neither standard U ribonucleotides nor other modified ribonucleotides (i.e.100% U substitution with 1m ⁇ with no other modified nucleotides being allowed).
  • “[may] comprise[s]... and neither [X]...nor [Y]” may be used interchangeably with the wording “[may] comprise[s]... and does not comprise... [X] and/or [Y] ”.
  • the RNA is codon-optimised.
  • 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 6
  • 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: - SEQ ID NO: 38 and 39, respectively, - SEQ ID NO: 40 and 41, respectively, - SEQ ID NO: 42 and 43, respectively, - SEQ ID NO: 44 and 45, respectively, Docket No.: 70280WO01 - SEQ ID NO: 46 and 47, respectively, and - RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 38, 40, 42, 44 or 46 (for the 5’ UTR) and RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 39, 41, 43, 45 or 47 (for the 3’ UTR) (in particular, the pairing of 5’ and 3’ UTRs having such identity to SEQ ID NO: 38 and 39, SEQ ID NO:
  • Both the 3’ and 5’ UTR may influence expression of the RSV-F protein of the present disclosure through a variety of mechanisms.
  • the 5’ UTR may affect the expression of at least the RSV-F protein of the present disclosure e.g. via pre-initiation complex regulation, closed-loop regulation, upstream open reading frame regulations (i.e. reinitiation), provision of internal ribosome entry sites, and provision of microRNA binding sites.
  • the 3’ UTR may affect the expression of at least the RSV-F protein of the present disclosure e.g. via providing regulation regions that post-transcriptionally influence expression; e.g.
  • the RNA is circular RNA.
  • the RNA fulfils at least two, at least three, at least four, or at least five 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 ⁇
  • the RNA fulfils all of criteria (a) – (f), above. Docket No.: 70280WO01 Generally, the RNA will comprise, in the 5’ to 3’ direction: 5’ Cap, 5’ UTR, open reading frame encoding at least an RSV-F protein of the present disclosure, 3’UTR, and 3’ poly-A tail (in particular, the 5’ Caps; 5’ UTRs, 3’UTRs and 3’ poly-A tails as detailed above throughout this subsection).
  • the RNA comprises or consists of the sequence: SEQ ID NO: 49; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, K315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 50; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, K315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 53; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 54; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 57 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 103C, 148C, 190I, 486S (relative to a wild-type RSV- F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 58 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present Docket No.: 70280WO01 disclosure comprising the substitutions 103C, 148C, 190I, 486S (relative to a wild-type RSV- F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 59 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 103C, 148C, 190I, 486S (relative to a wild-type RSV- F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 60 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P e.g. relative to (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 61 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P e.g. relative to (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 62 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P e.g. relative to (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 63 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P e.g. relative to (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 64 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P e.g. relative to (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 65 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, Docket No.: 70280WO01 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P e.g. relative to (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 SEQ ID NO: 117 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 2, preferably further comprising the deletion ⁇ 555-574 relative to (and numbered according to) SEQ ID NO: 1 or 2; SEQ ID NO: 140; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%
  • the present disclosure also provides, in a further independent aspect, a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising or consisting of: any of SEQ ID NO: 49, 50, 53, 54 or 57-66, or any of the foregoing sequences having sequence identity to any of SEQ ID NO: 49, 50, 53, 54 or 57-66.
  • a DNA construct preferably a DNA plasmid
  • RNA sequence comprising or consisting of: any of SEQ ID NO: 49, 50, 53, 54 or 57-66, or any of the foregoing sequences having sequence identity to any of SEQ ID NO: 49, 50, 53, 54 or 57-66.
  • the RNA comprises an open reading frame (ORF) comprising or consisting of the sequence of: positions 32-1744 of SEQ ID NO: 49; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, K315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • ORF open reading frame
  • SEQ ID NO: 1 or 2 positions 32-1693 of SEQ ID NO: 50; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 215A, 228K, K315I, 346Q, 348N, 445D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1744 of SEQ ID NO: 53; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1693 of SEQ ID NO: 54; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 55T, 152R, 210H, 215A, 228K, 241N, 315I, 346Q, 348N, 419D, 455V and 459M (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1744 of SEQ ID NO: 57 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, Docket No.: 70280WO01 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 103C, 148C, 190I, 486S (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1693 of SEQ ID NO: 58 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 103C, 148C, 190I, 486S (relative to a wild-type RSV-F sequence, e.g.
  • positions 32-1678 of SEQ ID NO: 59 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 103C, 148C, 190I, 486S (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1753 of SEQ ID NO: 60 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1744 of SEQ ID NO: 61 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1738 of SEQ ID NO: 62 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1723 of SEQ ID NO: 63 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 Docket No.: 70280WO01 positions 32-1708 of SEQ ID NO: 64 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1693 of SEQ ID NO: 65 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1678 of SEQ ID NO: 66 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions 67I and 215P (relative to a wild-type RSV-F sequence, e.g.
  • SEQ ID NO: 1 or 2 positions 32-1693 of SEQ ID NO: 117 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 2, preferably further comprising the deletion ⁇ 555-574 relative to (and numbered according to) SEQ ID NO: 1 or 2; positions 32-1693 of SEQ ID NO: 140 or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • the present disclosure also provides, in a further independent aspect, a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising an ORF; said ORF comprising or consisting of the sequence of: the respective positions of any of SEQ ID NO: 49, 50, 53, 54 or 57-66 as recited in the foregoing paragraphs, or any of the foregoing sequences having sequence identity to the respective positions of any of SEQ ID NO: 49, 50, 53, 54 or 57-66 as recited in the foregoing paragraphs.
  • the present disclosure also provides, in a further independent aspect, a vector comprising one or more RNAs of the present disclosure.
  • the present disclosure also provides, in a further independent aspect, a vector comprising a DNA construct encoding one or more RNAs of the present disclosure.
  • 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).
  • Docket No.: 70280WO01 Carriers comprising a nucleic acid Nucleic acids (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 to a positively charged polymer), cell-based (e.g. antigen presenting cells, such as dendritic cells loaded with the nucleic acid), or virus-based (e.g. viral replicon particles).
  • 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
  • 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 nucleic acids (preferably RNA) are encapsulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the present disclosure also provides an LNP encapsulating a nucleic acid (preferably RNA) which encodes an RSV-F protein of the present disclosure.
  • a plurality of such LNPs will be part of a composition (e.g.
  • a pharmaceutical composition as detailed in the section entitled Pharmaceutical compositions below) comprising free and/or encapsulated nucleic acid (preferably RNA), and in some embodiments the LNPs encapsulate at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least 100% of the total number of nucleic acid (preferably RNA) molecules in the composition.
  • At least 80% of the LNPs in the composition may be 20-200 nm, 40-190 nm, 60-180 nm or, in particular, 80-160 nm in diameter.
  • 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).
  • RNA molecules in general, an LNP may include 1-500 RNA molecules, e.g. ⁇ 200, ⁇ 100, ⁇ 50, ⁇ 20, ⁇ 10, ⁇ 5, or 1-4. Generally, an LNP includes fewer than 10 different species of RNA e.g. fewer than 5, 4, 3, or 2 different species. Preferably the LNP includes a single RNA species (i.e. all RNA molecules in the particle have the same sequence). LNPs according to the present disclosure may be formed from a single lipid (e.g.
  • the mixture comprises various classes of lipids, such as: (a) a mixture of cationic lipids and sterols, (b) a mixture of cationic lipids and neutral lipids, (c) a mixture of cationic lipids and polymer-conjugated lipids, (d) a mixture of cationic lipids, sterols and polymer-conjugated lipids, or (e) a mixture of cationic lipids, neutral lipids and polymer-conjugated lipids; or preferably: (f) a mixture of cationic lipids, sterols and neutral lipids; or more preferably: (g) a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids.
  • lipids such as: (a) a mixture of cationic lipids and sterols, (b) a mixture of cationic lipids and neutral lipids, (c)
  • 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 7.
  • 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- Docket No.: 70280WO01 methyl
  • 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:
  • the cationic lipid has the structure: (also referred to as lipid RV39).
  • the cationic lipid has the structure: Docket No.: 70280WO01
  • the cationic lipid has the structure:
  • the lipids in the LNP may comprise (in mole %) 20-80, 25-75, 30-70, or 35-65%, preferably 30-60, 40-55 or 40-50% cationic lipid; such as about 40% (or 40%), about 42% (or 42%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) cationic lipid.
  • the lipids in the LNP may comprise (in mole %) at least 20, 25 or at least 35%, or preferably at least 40% cationic lipid.
  • the lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60% or preferably no more than 50% cationic lipid.
  • the molar ratio of protonatable nitrogen atoms in the LNP’s cationic lipids to phosphates in the nucleic acid, preferably RNA 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 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 2.0
  • the PEGs of such PEGylated lipids may have a number average molecular weight of 0.5-11.0 kDa; such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0 or 1.0-3.5 kDa, preferably 1.0-3.0, Docket No.: 70280WO01 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, Docket No.: 70280WO01 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, or 1.7-2.3 kDa,
  • the PEGylated lipid may have the structure: Exemplary PEGylated lipids include 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, 1,2-dimyristoyl-sn-glycero-2- phosphoethanolamine-N-[methoxy(polyethylene glycol)] and 1,2-dimyristoyl-rac-glycerol-3- methoxypolyethylene glycol.
  • 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% 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, Docket No.: 70280WO01 lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9), 14-dien-3 ⁇ -ol (dihydro-FF-MAS-d6), 4,4-dimethyl(d6)- cholest-8(9)-en-3 ⁇ -ol (dihydro T-MAS-d6), zymostenol
  • the sterol is cholesterol or a cholesterol-based lipid (e.g. any of those provided in the foregoing paragraph).
  • the lipids in the LNP may comprise (in mole %) 20-80, 25-80, 30-70, 30-60, 35-60 or 40-60%, preferably 40-50% or 41-49% sterol; such as about 42% (or 42%), about 43% (or 43%), about 44% (or 44%), about 46% (or 46%), or about 48% (or 48%) sterol.
  • the lipids in the LNP may comprise (in mole %) at least 20, 30 or at least 35%, or preferably at least 40% or at least 41% sterol.
  • the lipids in the LNP may comprise (in mole %) no more than 80, 70 or no more than 60%, or preferably no more than 50% sterol. Docket No.: 70280WO01
  • the lipids in the LNP may have the following mole % in combination: 30-60% cationic lipid (such as 35-55%, or preferably 40-50%), 35-70% sterol (such as 40-55%, or preferably 41-49%), 0.8-4.0% polymer-conjugated lipid (such as 0.8-3.5%, or preferably 1.0-3.0%), and 0-15% neutral lipid (such as 6.0-12.0% or preferably 8.0-11.0%).
  • Such LNPs encapsulating nucleic acids may be formed by admixing a first solution comprising the nucleic acids with a second solution comprising lipids which form the LNP.
  • the admixing may be performed by any suitable means available to the skilled person, e.g. a T-mixer, microfluidics, or an impinging jet mixer. Admixing may be followed by filtration to obtain a desirable LNP size distribution (e.g. those as detailed above in this subsection).
  • the filtration may be performed by any suitable means available to the skilled person, e.g. tangential-flow filtration or cross-flow filtration.
  • the present disclosure provides a method of preparing an LNP encapsulating a nucleic acid (preferably RNA) of the present disclosure, comprising admixing a first solution comprising the nucleic acid and a second solution comprising lipids which form the LNP (e.g using the means as set out in the foregoing paragraph); and optionally filtering the obtained admixture (e.g. using the means as set out in the foregoing paragraph).
  • Pharmaceutical compositions also provides a pharmaceutical composition comprising a nucleic acid (preferably RNA), RSV-F protein and/or carrier (preferably lipid nanoparticle) of the present disclosure. Such compositions typically further comprise a pharmaceutically acceptable excipient.
  • compositions of the present disclosure are generally for immunising subjects against disease, preferably against RSV. Accordingly, pharmaceutical compositions of the present disclosure are generally considered vaccine compositions or immunogenic compositions.
  • Pharmaceutical compositions of the present disclosure may comprise the nucleic acid (preferably RNA), RSV-F protein and/or carrier (preferably lipid nanoparticle) in plain water (e.g. water for injection “w.f.i.”) or in a buffer e.g. a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will typically be included in the 5-20mM range.
  • 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).
  • Docket No.: 70280WO01 Pharmaceutical compositions of the present disclosure may include metal ion chelators (in particular, in embodiments wherein such compositions comprise RNA). These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis.
  • such compositions may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc..
  • Such 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. between 240-360 mOsm/kg, or between 290-310 mOsm/kg.
  • Pharmaceutical compositions of the present disclosure may include one or more preservatives, such as thiomersal or 2-phenoxyethanol.
  • Mercury-free compositions are preferred, and preservative-free vaccines can be prepared.
  • Pharmaceutical compositions of the present disclosure may be aseptic or sterile.
  • compositions of the present disclosure may be non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • Pharmaceutical compositions of the present disclosure may be gluten free.
  • Pharmaceutical compositions of the present disclosure may be prepared in unit dose form. In some embodiments a unit dose may have a volume of between 0.1 -1.0 mL e.g. about 0.5mL (or 0.5mL).
  • Pharmaceutical compositions of the present disclosure may be prepared as injectables, either as solutions or suspensions.
  • the composition may be prepared for pulmonary administration e.g. by an inhaler, using a fine spray.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops.
  • compositions of the present disclosure comprise an immunologically effective amount of RSV-F protein.
  • nucleic acid preferably RNA
  • carrier preferably lipid nanoparticle
  • effective amount and “immunologically effective amount” are used interchangeably.
  • 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.
  • RNA content will generally be expressed in terms of the amount of RNA per dose.
  • a preferred dose has ⁇ 120 ⁇ g RNA Docket No.: 70280WO01 e.g.
  • ⁇ 100 ⁇ g e.g.15-120 ⁇ g or 15-100 ⁇ g, such as 15 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g, or about 15 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g.
  • 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.
  • compositions of the present disclosure may further comprise an adjuvant (i.e. an agent that enhances an immune response in a non-specific manner).
  • Pharmaceutical compositions of the present disclosure (preferably when comprising a lipid nanoparticle comprising a nucleic acid of the present disclosure, preferably RNA) may be lyophilised.
  • pharmaceutical compositions of the present disclosure comprise (i) a nucleic acid (preferably RNA) encoding an RSV-F protein of the present disclosure, and (ii) a further nucleic acid (preferably RNA) encoding at least one further protein.
  • the nucleic acids of (i) and (ii) may be comprised within the same carrier (preferably lipid nanoparticle), or within separate carriers (preferably lipid nanoparticles).
  • the at least one further protein is an antigen; and as such may comprise, or may be, a viral, bacterial, fungal, parasitic, tumour, or allergenic (i.e. from, or derived from, an allergen) antigen.
  • the at least one further protein will typically be a pathogen antigen.
  • the at least one further protein will typically be an antigen that is a surface polypeptide e.g. a spike glycoprotein, a haemagglutinin, an adhesin or an envelope glycoprotein.
  • the at least one further protein is an antigen from, or derived from, a virus, in particular a virus causing respiratory disease, in particular a seasonal virus causing respiratory disease.
  • the at least one further protein is an antigen from, or derived from, a virus
  • examples of such viruses include: Coronavirus, Orthomyxovirus, Pneumoviridae, Paramyxoviridae, Poxviridae, Picornavirus, Bunyavirus, Heparnavirus, Filovirus, Togavirus, Flavivirus, Pestivirus, Hepadnavirus, Rhabdovirus, Caliciviridae, Retrovirus, Reovirus, Parvovirus, Herpesvirus, Papovaviruses and Adenovirus.
  • the at least one further protein encoded by the nucleic acid of (ii) is a further Pneumoviridae protein (in particular a Pneumoviridae antigen).
  • a further Pneumoviridae protein in particular a Pneumoviridae antigen.
  • Useful further Pneumoviridae proteins can be from an Orthopneumovirus or Metapneumovirus, in particular human RSV or human Metapneumovirus (hMPV).
  • Useful further hMPV antigens include e.g. the F, N, P, M, M2-1, and M2 antigens (in particular, the F antigen).
  • Such hMPV proteins (in particular, antigens) may be from, or derived from, the A or B subtype.
  • the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding an hMPV antigen (in particular, the F antigen).
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • is infants see section entitled Medical uses and methods of treatment, below.
  • Useful further human RSV antigens encoded by the nucleic acid of (ii) include e.g. the G, M1, M2-1, M2-2, Docket No.: 70280WO01 P, L, N, NS1, NS2 and SH antigens, in addition to further RSV-F antigens, i.e.
  • the at least one further protein encoded by the nucleic acid of (ii) is a Coronavirus antigen.
  • Useful Coronavirus antigens can be from a SARS coronavirus, in particular SARS-CoV2.
  • Useful Coronavirus antigens include the spike, M, E, HE, Nuclocapsid, Plpro and 3CLPro proteins, in particular spike protein.
  • the Coronavirus antigen is a SARS-CoV2 spike protein.
  • Said SARS-CoV2 spike protein may be from any variant, e.g. Omicron (such as Omicron BA.1, BA.2, BA3, BA.4 or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma, or Delta.
  • said SARS-CoV2 spike protein includes one or more mutations relative to the wild-type protein, in particular one or more (e.g. two) mutations to proline resides. Said one or more mutations may be introduced to stabilise said SARS- CoV2 spike protein in its pre-fusion conformation.
  • the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding a Coronavirus antigen, e.g. as detailed above.
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • the at least one further protein encoded by the nucleic acid of (ii) is an Orthomyxovirus antigen.
  • Useful Orthomyxovirus antigens can be from an influenza A, B or C virus.
  • Useful Orthomyxovirus antigens include the haemagglutinin, neuraminidase and matrix M2 proteins, in particular haemagglutinin.
  • the Orthomyxovirus antigen is an influenza A virus haemagglutinin.
  • Said influenza A virus hemagglutinin may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.
  • the nucleic acid of (i) is RNA encoding an RSV-F protein of the present disclosure and the nucleic acid of (ii) is RNA encoding an Orthomyxovirus antigen, e.g. as detailed above.
  • a preferred patient group in which the pharmaceutical composition may be used in therapy, in particular vaccination
  • the nucleic acid of (ii) may encode an RSV-F protein of the present disclosure
  • the nucleic acid of (ii) may encode an Orthomyxovirus antigen, e.g.
  • 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 a nucleic acid (preferably RNA), RSV-F protein or carrier (preferably lipid nanoparticle) of the present disclosure with a pharmaceutically acceptable excipient, to produce said composition.
  • a nucleic acid preferably RNA
  • RSV-F protein or carrier preferably lipid nanoparticle
  • said pharmaceutical composition has the features as detailed above throughout this section.
  • the present disclosure also provides a kit comprising a nucleic acid, RSV-F protein, carrier, pharmaceutical composition or delivery device of the present disclosure, and instructions for use.
  • the present disclosure also provides, in a further independent aspect, a nucleic acid (preferably RNA), RSV-F protein, carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, for use in medicine. Said use will generally be in a method for raising an immune response in a subject.
  • the present disclosure also provides, in a further independent aspect, the use of a nucleic acid (preferably RNA), RSV-F protein, carrier (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure, in the manufacture of a medicament. Said medicament will generally be for raising an immune response in a subject.
  • the present disclosure also provides, in a further independent aspect, a therapeutic method comprising the step of administering an effective amount of a nucleic acid (preferably RNA), RSV-F protein, 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 present disclosure provides a method of treatment of a subject comprising the step of administering an effective amount of the nucleic acid of the present disclosure to the subject.
  • the nucleic acid is RNA.
  • the present disclosure provides a method of treatment of a subject comprising the step of administering an effective amount of the RSV-F protein of the present disclosure.
  • the present disclosure disclosed a method of treatment of a subject comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.
  • the pharmaceutical composition comprises an adjuvant.
  • 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 nucleic acids, RSV-F proteins, carriers, or pharmaceutical compositions of the present disclosure may be for use in the prevention, reduction or treatment of infection or disease.
  • the nucleic acids, RSV-F proteins, carriers, or pharmaceutical compositions of the Docket No.: 70280WO01 present disclosure may be for use in the prevention, reduction or treatment of symptoms associated with infection or disease.
  • the infection is generally one by, and said disease is generally one associated with, a Pneumoviridae virus.
  • the Pneumoviridae virus is an Orthopneumovirus, which is more preferably RSV, and even more preferable human RSV (including both the A and B subtypes thereof).
  • the present disclosure also provides a nucleic acid, RSV-F protein, carrier or pharmaceutical composition of the present disclosure; for use in treating or preventing RSV (preferably a method of vaccination against RSV).
  • the present disclosure also provides the use of a nucleic acid, RSV-F protein, 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 nucleic acid, RSV-F protein, 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).
  • a vaccination regimen may involve the repeated administration of an immunologically identical protein antigen (in the form of, or delivered via, a nucleic acid, RSV-F protein, carrier, or pharmaceutical composition of the present disclosure), in particular in a prime-boost regimen.
  • the first administration (“prime”) may induce proliferation and maturation of B and/or T cell precursors specific to one or more immunogenic epitopes present on the delivered antigen (induction phase).
  • the second (and in some cases subsequent) administration (“boost”) may further stimulate and potentially select an anamnestic response of cells elicited by the prior administration(s).
  • the different administrations may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
  • the prime administration(s) and boost administration(s) will be temporally separated, e.g. by at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more months.
  • two prime administrations may be administered 3-9 weeks apart (e.g.4-9, 5-9, 6-9, 7-9 or 7-8 weeks apart, or about two months apart), followed by one or more boost administrations 4-14 months after the second prime administration (e.g.5-13, 6-13, 7-13, 8-13, 9-13, 10-13 or 11-13 months, or about one year).
  • prime administration is to a na ⁇ ve subject.
  • 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.
  • nucleic acid formats may be used, e.g. the protein antigen may be delivered via RNA (in particular via a carrier comprising RNA) Docket No.: 70280WO01 for the prime administration(s), and a via a viral vector (e.g.
  • an adenoviral vector for the boost administration(s), or vice versa.
  • the nucleic acids, RSV-F proteins, carriers, or pharmaceutical compositions of the present disclosure will generally be administered directly to the subject.
  • Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or to the interstitial space of a tissue).
  • Alternative delivery routes include rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • the nucleic acids, RSV-F proteins, carriers, or pharmaceutical composition of the present disclosure will be administered intramuscularly or intradermally (in particular via a needle such as a hypodermic needle), more preferably intramuscularly.
  • the nucleic acids, RSV-F proteins, lipid carriers, or pharmaceutical compositions of the present disclosure may be used to elicit systemic and/or mucosal immunity.
  • the subject of a method of vaccination according to the present disclosure may be a child (preferably an infant) or adult (preferably an older adult or pregnant female). Immunocompromised individuals may also be the subject of such vaccination (whether children or adults).
  • the nucleic acids, RSV-F proteins, carriers, or pharmaceutical compositions of the present disclosure are administered to infants (preferably human infants), as the subject of vaccination.
  • infants preferably human infants
  • the immune systems of infants are immature (see, e.g. [26]), hence this population is susceptible to RSV infection and resulting disease.
  • Infant vaccination may prevent lower respiratory tract infection (in particular, bronchiolitis and (broncho-)pneumonia).
  • the infant may be 0-12 months old.
  • the infant may be less than one year old, such as less than: 11, 10, 9, 8, 7, 6, 5, 4 or less than 3 months old.
  • the infant may be ⁇ one month old, such as ⁇ : 2, 3, 4, 5 or ⁇ 6 months old.
  • the infant is 2-6 months old (i.e.
  • the infant was born from a female to whom an RSV vaccine (such as a nucleic acid, RSV-F protein, carrier, or pharmaceutical composition of the present disclosure) was administered, preferably while pregnant with said infant.
  • an RSV vaccine such as a nucleic acid, RSV-F protein, 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 nucleic acids, RSV-F proteins, 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 Docket No.: 70280WO01 in, e.g. [27]), 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 nucleic acids, RSV-F proteins, 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).
  • Preparing RSV-F proteins 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.
  • Suitable recombinant host cells include, for example, insect cells (e.g. Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells); mammalian cells (e.g. Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g. HEK293, in particular Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells); avian cells (e.g. chicken embryonic fibroblasts and chicken embryonic germ cells); bacteria; and yeast cells.
  • HEK293 cells are preferred, more preferably Expi 293 cells (as were used in the examples).
  • the present disclosure also provides, in one independent aspect, a host cell (in particular, those detailed above) comprising a nucleic acid of the present disclosure (in particular, an expression vector as detailed above) 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 of the present disclosure (in particular, an expression vector as detailed above) 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 of the present disclosure (in particular, an expression vector as detailed above) encoding the RSV-F protein in a host cell (in particular, those detailed above). In an embodiment, the RSV-F protein is then purified.
  • RSV-F proteins of the present disclosure can be purified, following expression from a host cell, by routine methods, such as precipitation and chromatographic methods (e.g. hydrophobic interaction, ion exchange, affinity, chelating or size exclusion chromatography).
  • the RSV-F proteins of the present disclosure can include a tag that facilitates purification, such as an epitope tag or a histidine (HIS) tag, to facilitate purification e.g. by affinity chromatography.
  • HIS histidine
  • the word “or” is intended to include “and” unless the context clearly indicates otherwise.
  • the term “plurality” refers to two or more.
  • the term “at least one” refers to one or more. Unless specified otherwise, where a numerical range is provided, it is inclusive, i.e., the endpoints are included.
  • the terms “at least”, “no more than” and other such terms preceding a list of values are applicable to all members of said list (not merely the first member thereof), unless otherwise stated.
  • the term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
  • a recombinant nucleic acid encoding an RSV-F protein comprising a cytoplasmic tail; wherein the cytoplasmic tail is 5-23 residues in length.
  • nucleic acid of embodiment 1, wherein the cytoplasmic tail is 10-18, such as 11-17, 12- 16, 13-16, 14-15 or 15 residues in length. 6.
  • the nucleic acid of embodiment 1, wherein the cytoplasmic tail is 18-23, 19-23, 20-23, 21-23, 21-22, 22-23 or 22 residues in length. 7.
  • nucleic acid of any of embodiments 1-6 wherein cell-surface expression, optionally in human fibroblasts, optionally in human foreskin fibroblasts, optionally in human primary BJ cells, optionally the ATCC CRL-2522 cell line, of the RSV-F protein in trimeric, pre-fusion form from the nucleic acid is increased, relative to expression in such form of an RSV-F protein having the same amino acid sequence but comprising a wild-type cytoplasmic tail, such as according to SEQ ID NO: 3 or 4.
  • the nucleic acid of embodiment 7, wherein the increased cell surface expression is for a period of at least 24, 48, 72 or 96 hours.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-31 of SEQ ID NO: 69, 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.
  • cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-29 of SEQ ID NO: 70, 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.: 70280WO01 15.
  • nucleic acid of embodiment 11, wherein 14-16, such as 14-15 or 15-16, or 15 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein.
  • the cytoplasmic tail comprises or consists of (i) an amino acid sequence according to positions 10-19 of SEQ ID NO: 72, or (ii) an amino acid sequence at least 60%, 70%, 80% or 90% identical to said positions and optionally the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues C-terminal to the amino acid sequence of (i) or (ii). 19.
  • nucleic acid of embodiment 11 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.
  • 20 The nucleic acid of embodiment 19, wherein 20 residues are deleted from the C-terminal end of the cytoplasmic tail of the RSV-F protein. 21.
  • the nucleic acid of embodiment 22, wherein the increased cell surface expression is for a period of at least 24, 48, 72 or 96 hours. 24.
  • nucleic acid of any preceding embodiment wherein the cytoplasmic tail of the RSV-F protein comprises at least 5 residues that are C-terminal to position 549 of the RSV-F protein. Docket No.: 70280WO01 25.
  • the nucleic acid of embodiment 28, wherein the RSV-F protein comprises an ectodomain comprising substitutions as defined in preferred class (2).
  • 30. The nucleic of embodiment 28 or 29, wherein the ectodomain comprises the substitutions 67I and 215P.
  • 31. The nucleic acid of embodiment 28 or 29, wherein the ectodomain comprises the substitutions 66E, 67I, 76V, 215P and 486N. 32.
  • a wild-type RSV-F ectodomain such as positions 26-109 and 137-523 of SEQ ID NO: 1 or 2
  • the RSV-F protein comprises an ectodomain comprising substitutions as defined in preferred class (3).
  • nucleic acid of embodiment 32 or 33 wherein the ectodomain comprises the substitutions 149C, 155C, 190F, 207L, 290C and 458C; optionally with a linker joining the F2 and F1 domains, optionally replacing positions 104-144, optionally wherein the linker comprises or consists of an amino acid sequence according to SEQ ID NO: 13. 35.
  • the nucleic acid of embodiment 34 wherein the ectodomain comprises the substitutions 102A, 149C, 155C, 190F, 207L, 290C, 373R, 379V, 447V and 458C; optionally with a linker joining Docket No.: 70280WO01 the F2 and F1 domains, optionally replacing positions 104-144, optionally wherein the linker comprises or consists of an amino acid sequence according to SEQ ID NO: 13.
  • 36 The nucleic acid of embodiment 31 or 32, wherein ectodomain comprises the substitutions 155C, 190F, 207L and 290C. 37.
  • the nucleic acid of embodiment 1-24 or 37 comprising (a): substitution at position 55 for T, C, V, I; optionally T, C or V; optionally T or V; optionally T; (b): substitution at position 215 for A, P, V, I, or F; optionally A, V, I, or F; optionally A or P; optionally A; and/or, optionally and, (c): substitution at position 228 for K, R, N, W, D, E, Q, H, S, T or Y; optionally K, R, W, N, Q, H, S, T or Y; optionally K, R, Q and N; optionally K, R or Q; optionally K or R, optionally K.
  • nucleic acid of embodiment 1-24, 37 or 38 comprising the substitutions: (i) 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V, 459M, 486C and 490C; (ii) 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V, 459M, 486C and 490C; (iii) 55T, 152R, 215A, 228K, 315I, 346Q, 445D, 455V and 459M; (iv) 55T, 152R, 211N, 215A, 228K, 315I, 346Q, 348N, 445D, 455V and 459M; (v) 55T, 152R, 210H, 211N, 215A, 228K, 241N, 315I, 346Q, 348N, 419D
  • RNA of any preceding embodiment wherein, when expressed, the RSV-F protein is in the pre-fusion conformation. Docket No.: 70280WO01 41.
  • the nucleic acid of any preceding embodiment, wherein the RSV-F protein comprises 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 1-549 of SEQ ID NO: 1. 42.
  • the RSV-F protein comprises 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: 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 of SEQ ID NO: 1.
  • RSV-F protein comprises 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 of SEQ ID NO: 1.
  • RSV-F protein is of the A subtype.
  • RSV-F protein comprises 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 1-549 of SEQ ID NO: 2.
  • nucleic acid of any of embodiments 1-40, 45 or 46, wherein the RSV-F protein comprises 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: 2; and an F1 domain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, Docket No.: 70280WO01 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 of SEQ ID NO: 2.
  • the RSV-F protein of embodiment 53 in the form of a homotrimer.
  • the nucleic acid of embodiment 55, wherein the nucleic acid is RNA.
  • the RNA of embodiment 58 which is non-self-replicating RNA. 60.
  • the RNA of embodiment 58 which is self-replicating RNA.
  • the RNA of any of embodiments 58-60 comprising, in the 5’ to 3’ direction: a 5’ Cap, a 5’ UTR, an open reading frame encoding the RSV-F protein, a 3’UTR, and a 3’ poly-A tail.
  • RNA of embodiment 61 or 62, wherein the 3’ poly-A tail comprises a contiguous stretch of 100-500 A ribonucleotides.
  • 64. The RNA of embodiment 61 or 62, 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 Docket No.: 70280WO01 length respectively which are 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. 65.
  • RNA of any of embodiments 58-64 comprising a modified ribonucleotide.
  • 66. The RNA of embodiment 65, wherein the modified ribonucleotide is 1m ⁇ 67.
  • 68 The RNA of any of embodiments 58-67, having a GC content of 30-70%, 40-70%, 45-70%, 50-70%, or 55-70%. 69.
  • RNA of any of embodiments 58-67 having a GC content of 30-70%, 40-60%, 45-55%, 46-53%, 47-51%, or 48-50%.
  • 70. A carrier comprising nucleic acid of any of embodiments 1-52 or 58-69. 71.
  • the carrier of embodiment 70 which is a lipid nanoparticle.
  • 72. The lipid nanoparticle of embodiment 71, comprising a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids.
  • the lipid nanoparticle of embodiment 72, wherein the cationic lipid has a pKa of 5.0-8.0; optionally 5.0-7.6. 74.
  • 75. The lipid nanoparticle of any of embodiments 72-74, wherein the polymer-conjugated lipid is a PEGylated lipid; optionally wherein the PEG has a weight average molecular weight of 1-3 kDa.
  • 76. The lipid nanoparticle of any of embodiments 72-75, wherein the sterol is cholesterol or a cholesterol-based lipid. 77.
  • the lipid nanoparticle of any of embodiments 72-76 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. Docket No.: 70280WO01 78.
  • the lipid nanoparticle of any of embodiments 72-77, wherein the molar ratio of protonatable nitrogen atoms in the cationic lipid to phosphates in the RNA (“N:P ratio”) is 5.0-8.0, 5.5-7.0, 5.5-6.5 or 5.0-6.0.
  • a pharmaceutical composition comprising the nucleic acid of any of embodiments 1-52 or 55- 57, RSV-F protein of embodiment 53 or 54, RNA of any of embodiments 58-69 or carrier of any of embodiments 70-78; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant.
  • a vaccine composition comprising the nucleic acid of any of embodiments 1-52, 55 or 56, RSV-F protein of embodiment 53 or 54, RNA of any of embodiments 58-69, or carrier of any of embodiments 70-78; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant.
  • the composition of embodiment 79 or 80 for use in medicine. 82.
  • composition for use of embodiment 81 for use in a method of raising an immune response in a subject; optionally a protective immune response in a subject.
  • the composition for use of embodiment 82 for use in the treatment or prevention of RSV.
  • the composition for use of embodiment 83 for use in a method of vaccinating a subject against RSV; optionally wherein the vaccination is prophylactic.
  • the composition for use of any of embodiments 82-84 wherein the subject is a human infant; optionally 2-6 months old.
  • the composition for use of any of embodiments 82-84, wherein the subject is a human older adult; optionally ⁇ 50 years old, optionally ⁇ 60 years old. 87.
  • a method of inducing an immune response against RSV in a subject comprising administering to the subject an immunologically effective amount of the nucleic acid of any of embodiments 1-52 or 55-57, RSV-F protein of embodiment 53 or 54, RNA of any of embodiments 58-69, or carrier of any of embodiments 70-78. 89.
  • a method of enhancing the cell surface expression of RSV-F antigen in a subject comprising administering to the subject an immunologically effective amount of the nucleic acid of any of embodiments 1-52 or 55-57, RSV-F protein of embodiment 53 or 54, RNA of any of embodiments 58-69, or carrier of any of embodiments 70-78.
  • Docket No.: 70280WO01 90. Use of the nucleic acid of any of embodiments 1-52 or 55-57, RSV-F protein of embodiment 53 or 54, RNA of any of embodiments 58-69, or carrier of any of embodiments 70-78, in the manufacture of a medicament.
  • Use according to embodiment 90, wherein the medicament is for treating or preventing RSV. 92.
  • kits comprising the nucleic acid of any of embodiments 1-52 or 55-57, RSV-F protein of embodiment 53 or 54, RNA of any of embodiments 58--69, or carrier of any of embodiments 70-78, and instructions for use.
  • 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 monoclonal antibodies Materials & Methods Cloning and expression of RSV-F monoclonal antibodies (Example 1-5, 8, 11 and 12) Plasmids encoding RSV-F antibodies, AM14, D25 and Motavizumab were transiently transfected in Expi293F cells (THERMO FISHER SCIENTIFIC) according to manufacturer’s instructions and media was harvested 6-7 days post transfection.
  • the cell harvest media was passed over a MABSELECT SURE COLUMN (CYTIVA) and eluted with 0.1 M citrate pH 3 into 1 M Tris pH 9; buffer exchanged into 20 mM HEPES pH 7, 150 mM NaCl; followed by a final size exclusion chromatography step on a HILOAD 16/600 Superdex 30 pg column (CYTIVA) in 20 mM Hepes pH 7, 150 mM NaCl.
  • CYTIVA MABSELECT SURE COLUMN
  • the DNA gBLOCKS (INTEGRATED DNA TECHNOLOGIES) were amplified by PCR, and ligation into a vector with a polyA tail. Amino acid substitutions N67I and S215P (also known as design F(ii)) were incorporated DNA constructs and encoded in the eventual mRNA and protein. The additional variations (also known as DS-Cav1, F(iii), F(i), F318 and F319) and their amino acid substitutions are shown in the Table 1.
  • NEB Q5 Site-Directed Mutagenesis Kit (NEB # E0554) was used to generate 7 CT deletion constructs: FL (“full length”, or “reference CT”), ⁇ CT3, ⁇ CT5, ⁇ CT10, ⁇ CT15, ⁇ CT20 and ⁇ CT25 Table 2 – C-terminal, cytoplasmic tail (CT) variations CT description CT AA sequence 1 Reference RSV F CT, including AA 541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN additional transmembrane (TM) domain (SEQ ID NO: 68) residues N-terminal to CT start 2 ⁇ CT3 , including additional TM domain AA541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIA (SEQ residues N-terminal to CT start ID NO: 69) 3 ⁇ CT5, including additional TM domain AA 541 LIAVGLLLYCKARSTPVTLSKDQLSGINN (SEQ ID residues N-terminal to CT start NO: 70) Docke
  • the reverse primers were designed in which the 3’ end PCR annealing starting points are at 7 different positions: position d0, end of coding region; position d3, 3 amino acid residues upstream of the end of coding region; position ⁇ 5, 5 residues to the end; position ⁇ 10, 10 residues to the end; position ⁇ 15, 15 residues to the end; position ⁇ 20, 20 residues to the end; position ⁇ 25, 25 residues to the end, which is the entire CT region.
  • the PCR reaction was heated to 98 °C for 30 seconds, followed by 16 cycles at 98 °C for 10 seconds, 69 °C for 30 seconds, 72 °C for 30 seconds. and a final extension of 72 °C for 2 min.
  • the 7 PCR products were treated with KLD enzyme (NEB E0554) at room temperature for 5 minutes. Transformation with competent cells (NEB C3040H) was carried out by following manufacture instructions.24 hours after, colonies were screened to identify correct sequences.
  • the T7 promotor region and the UTRs were appended to 5’ and 3’ of the coding regions (5’ and 3’ “UTR4”) and a polyA tail is after 3’ UTR region.
  • the final plasmids were validated by Sanger sequencing and purified for mRNA production.
  • Example 8 the following mRNA constructs were tested: Table 5 – substitutions in mRNA-encoded protein designs tested in cell-based assay Designation mRNA construct Substitutions relative to wild-type RSV-F design in Figures designation 8-10 (x axis) and Ex 8 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.
  • Example 12 CT lengths tested using design F(ii) are set out in Table 9, below.
  • In vitro transcription to generate mRNA for RSV-F variations (Example 1-5, 8, 11 and 12)
  • the plasmids were linearized with the BspQI restriction enzyme (NEW ENGLAND BIOLABS) to produce the DNA templates for in vitro transcription.
  • mRNAs were produced by in vitro transcription with capping analogue (TRILINK CLEANCAP A/G) and 100% uridine replacement (with 1m ⁇ ), followed with DNase I, phosphatase treatments (NEW ENGLAND BIOLABS) and silica column purification (QIAGEN).
  • Newly synthesized mRNAs were validated by Tapestation (Agilent) and denaturing RNA gels.
  • Cell culture conditions (Example 1-5, 8, 11 and 12)
  • Primary BJ cells (ATCC, CRL-2522) were maintained by routine passaging in growth media (DMEM (LONZA 12-614F) supplemented with 10% FBS (CORNING 35-016-CV), antibiotic (GIBCO 15140- 122) and glutamine (GIBCO 25030-081)) and grown at 37°C, 5% CO 2 .
  • BJ cells were seeded in growth media at 1.5x105 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 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 Docket No.: 70280WO01 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 immunisation (Example 6) All recombinant RNA molecules were produced by in vitro transcription using N1-methyl pseudouridine to replace all uridines. All recombinant RNA molecules comprised a cap-1 5’ cap (TRILINK CLEANCAP) and a 3’ poly(A) tail. The mRNAs were purified and evaluated for mRNA integrity (by capillary and glyoxal denaturing gel electrophoresis).
  • RV39 LNP mRNA constructs were then formulated in LNPs comprising 40 mol% cationic lipid RV39; 2 mol% PEG-conjugated lipid; 48 mol% cholesterol; and 10 mol% 1,2-diastearoyl-sn-glycero- 3-phosphocholine (DSPC).
  • DSPC 1,2-diastearoyl-sn-glycero- 3-phosphocholine
  • F(iii) which includes a full cytoplasmic tail deletion (dCT), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20 (low dose only ), DS-Cav1 (high dose only), F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 into each mouse on day 0 and day 21 (see Table 4 for mRNA construct designations).
  • mice were anesthetized under isoflurane and terminally exsanguinated by cardiac stick to obtain an estimated 200 ⁇ L to 500 ⁇ L of whole blood, (100 ⁇ L of serum).
  • RSV pre-F IgG binding antibody titres and RSV A neutralising antibody titres were measured on day 21 and day 35 using the following methods.
  • LUMINEX microspheres (MAGPLEX microspheres, LUMINEX CORP from Austin, TX) were coupled with RSV preF antigen using sulfo- NHS and EDC, according to manufacturer’s instructions.
  • a 96-well plate 2,000 microspheres/well are added in a volume of 50 ⁇ l PBS with 1% BSA + 0.05% Na Azide (assay buffer) to 100 ⁇ l of mouse serum serial diluted.
  • the microspheres After incubation of the microspheres and serum on an orbital shaker, covered, at RT for 60 minutes, the microspheres are washed 2 times with 200 ⁇ l/well of PBS, 0.05% Tween-20 (wash buffer) on a plate washer using a magnet to allow settling of beads between washes. Following the wash, 50 ⁇ L/well of r-Phycoerythrin (r-PE) conjugated anti-mouse IgG (JACKSON IMMUNORESEARCH) was added, and plates are incubated, covered, on an orbital shaker at RT for 60 minutes.
  • r-PE r-Phycoerythrin conjugated anti-mouse IgG
  • RSV A neutralising antibody titre assay Heat-inactivated sera (incubated for 30 min at 56°C) were diluted 3-fold starting at 1/8 (for a final dilution of 1/16). A control serum (WYETH Human Reference Sera from WHO/NIBSC) was included at a starting dilution of 1/64 (1/128 final). For the serial dilutions, 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% CO 2 . 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. After incubation, plates were rinsed 3 times with 200 ⁇ L of PBS.
  • DS-Cav1 and RSV-F mutants were transiently expressed in Expi293 F cells (THERMO FISHER SCIENTIFIC).
  • Media was harvested after 4 days, and purified using affinity chromatography, either nickel affinity or strep-tag affinity. Briefly, for nickel affinity chromatography, cell harvest medium was passed over a HisTrap Excel column (CYTIVA) and eluted with a step gradient of imidazole.
  • the harvest medium was buffer exchanged into 50 mM Tris pH 8, 300 mM NaCl, passed over a StrepTrap HP column (CYTIVA) and eluted with elution buffer (100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA and 2.5 mM desthiobiotin). This was followed by a final size exclusion chromatography polishing step.
  • elution buffer 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA and 2.5 mM desthiobiotin.
  • HPLC High Performance Liquid Chromatography
  • the cell harvest media was passed over a MABSELECT SURE COLUMN (CYTIVA) and eluted with 0.1 M citrate pH 3 into 1 M Tris pH 9; buffer exchanged into 20 mM HEPES pH 7, 150 mM NaCl; followed by a final size exclusion chromatography step on a HILOAD 16/600 Superdex 30 pg column (CYTIVA) in 20 mM Hepes pH 7, 150 mM NaCl.
  • Initial Quantitation and Antigenicity using Biolayer Interferometry (Example 9) Quantitation experiments were performed on the unpurified cell harvest media of 6x His-tagged DS- Cav1 and RSV-F mutants using the Octet Red 384 instrument (SARTORIUS).
  • Purified DS-Cav1 diluted in EXPI293 expression media with 0.1% BSA, 0.05% Tween-20 was used to make a standard curve. BSA and Tween-20 were added to DS-Cav1 and RSV-F mutants unpurified cell harvest media to a final concentration of 0.1% and 0.05%, respectively.6x His-tagged purified DS-Cav1 and RSV-F mutant unpurified cell harvest media was captured on HIS2 biosensors for 2 min and the capture level was recorded. The concentrations were determined using unweighted 4 parameter logistics curve fitting in the manufacturer’s analysis software (Data Analysis HT 12.0.1.55).
  • AHC biosensors were washed in 1x PBS with 0.1% BSA and 0.05% Tween-20 for 30 sec, mAbs were loaded for 60 sec, and washed for 30 sec before capturing DS-Cav1 or RSV-F mutants from the unpurified cell harvest media. Binding and dissociation of DS-Cav1 and RSV-F mutants was measured for 180 sec each. The response of DS-Cav1 binding to each mAb was compared to the RSV-F mutants’ response to each mAb to determine yes or no binding.
  • Human skeletal muscle cells were maintained in media (RPMI1640, GIBCO) supplemented with IL4 (MILTENYI 130-093-922) and GM-CFS (MILTENYI 130-093-865) for 5 days.
  • RPMI was supplemented with 10% FBS (CORNING 35-016-CV), antibiotic (GIBCO 15140-122) and glutamine (GIBCO 25030-081)) and grown at 37°C, 5% CO2.
  • RNA transfection and high content imaging was performed as per Examples 1-5, 8, 11 and 12 (using D25 antibody). In vivo RNA immunisation (Example 13) All mRNA molecules were produced and formulated into LNPs as per Example 6.
  • mice 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 0.5 ⁇ g dose of 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 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 and post-F IgG binding antibody titres and RSV A neutralising antibody titres were measured on day 21 and day 35 using the following method.
  • RSV F IgG Binding A multiplex assay was performed to evaluate titres of RSV pre-F- and post-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 post-F and pre-F antigen by chemical coupling according to manufacturer instructions.
  • 2000 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.
  • the microspheres were washed two times with 200 ⁇ L/well of PBS with 0.05% Tween-20 (wash buffer) on a plate washer using a magnet to allow settling of beads between washes.
  • r-PE r-Phycoerythrin conjugated anti-mouse IgG
  • JACKSON IMMUNORESEARCH r-Phycoerythrin conjugated anti-mouse IgG
  • 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.
  • Neutralising antibody titres were measured on day 21 and day 35 as per Example 6.
  • Overview RSV-F protein (Table 1) is the primary target for a high quality vaccine to prevent severe illness and adverse outcomes from RSV infection..
  • mRNA-based vaccine designs encode a glycoprotein that is processed, folded and exported to the cell surface, resulting in a trimeric RSV F protein with Docket No.: 70280WO01 three distinct domains: the extracellular domain, transmembrane domain and CT residing on the cytoplasmic face of the cell surface. Furthermore, select truncation of the RSV F CT was used to produce a mRNA vaccine design with further unique features, as discussed in detail below.
  • Example 1 Human primary BJ cells are permissive for the cell-surface expression of RSV F protein encoded by exogenous mRNAs.
  • the steady-state, total cell-surface RSV F protein expression of the design, F318 CT ⁇ 20 is observed to increase from 8 hours post transfection (Figure 1A”) to 24 hours post transfection (Figure 1B”) in BJ cells and decay in the subsequent 3 days ( Figure 1, C”-E”). Quantification of RSV F levels using High Content imaging and image analysis in individual BJ cells in the transfected cell monolayer is shown ( Figure 1, F-J) and exhibits a corresponding shift in the population distribution indicates increasing RSV F levels over the first day and decay in the subsequent days.
  • Example 2 The RSV-F variant design F(ii) ( Figure 2A) expresses AM14-(+) RSV F protein, as does designs, F318 and F319 ( Figure 2B and 2C, respectively), and design F(i) ( Figure 2D). As shown by area under the curve (AUC), the four constructs perform similarly ( Figure 2E). Design F(ii), with CT deletions (in whole or in part), expresses AM14(+) RSV F to a greater degree than F(ii) parental molecule (i.e. absent CT deletions) ( Figure 2A).
  • the surface expression of immunogenic RSV F may include, but is not limited to, monomeric to multimeric F states and any abundant RSV F conformations. Total expression of RSV F protein at the cell surface can be captured using the monoclonal antibody motavizumab.
  • RSV F variant F(ii) is readily detected 24 hours post transfection, while 3 amino acid, 20 amino acid and complete CT deletion, respectively, engineered into F protein unambiguously increases expression (Figure 3A).
  • the RSV F variants F318, F319 and F(i) each demonstrate substantial increases for RSV F expression when carrying CT deletions ( Figure 3B, 4C & 4D, respectively).
  • Deletions in the CT universally increase RSV F expression ( Figure 3E)
  • Example 4 The RSV F protein variants F(ii) and DS-Cav1 (Table 1), were each modelled as their respective mRNA doppelgangers for an in vivo study (Example 6).
  • Example 6 In vivo immunisation RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319 or F319 ⁇ CT20 was administered to mice as set out in the Materials and Methods section.
  • Figure 6 displays the RSV pre-F IgG binding antibody geometric mean titres on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either 2 ⁇ g ( Figure 6A) or 0.2 ⁇ g ( Figure 6B) of RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (where each point represents an individual animal). There were no binding antibody responses in the saline control group (data not shown). On day 21, all constructs elicited measurable pre-F- specific IgG binding antibodies with a 2 ⁇ g dose.
  • a single dose of DS-Cav1 elicited the lowest pre-F- specific IgG binding antibodies compared to the other constructs. By day 35, all pre-F-specific IgG antibodies were boosted, and elicited similar antibody titres.
  • the two immunizations with a 2 ⁇ g dose of F318, F318 ⁇ CT20, F319, and F319 ⁇ CT20 boosted pre-F specific IgG antibodies to levels that were noninferior to the benchmark controls F(i), F(ii), F(iii) and DS-Cav1 ( Figures 6 A and C-F).
  • F318 ⁇ CT20 achieved noninferiority when compared to F(ii) and F(iii) at day 35 (2wp2) ( Figures 6B, D and F).
  • One 0.2 ⁇ g dose of F318 ⁇ CT20 or F319 ⁇ CT20 elicited significantly higher pre-F IgG titres compared to the non- ⁇ CT20 counterparts ( Figure 6B and G).
  • Figure 7 displays the RSV A neutralising antibody titres (ED60) on day 21 (3wp1) and day 35 (2wp2) in animals immunized with either ( Figure 7A) 2 ⁇ g or ( Figure 7B) 0.2 ⁇ g of RNA encoding F(iii), F(i), F(i) ⁇ CT20, F(ii), F(ii) ⁇ CT20, DS-Cav1, F318, F318 ⁇ CT20, F319, or F319 ⁇ CT20 (where each point represents an individual animal).
  • the saline group did not generate a measurable neutralisation Docket No.: 70280WO01 response to RSV A (data not shown).
  • F647 (SEQ ID NO: 104) 20) KM242 (SEQ ID ⁇ 555-574 R712d20 (SEQ NO: 115) ID NO: 116) 21) KM243 (SEQ ID D486C, A490C, ⁇ 555-574 R713d20 a.k.a NO: 117) F647d20 (SEQ ID NO: 118) 8A RSV F immunogenicity of mRNA vaccines may be improved by optimizing post-translational features of the RSV F antigen.
  • the parent construct (13, Table 5), includes a full-length C-terminal domain (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 5).
  • CT cytoplasmic tail
  • the F antigens (13 & 20) were evaluated in the context of two additional classes of post translational modifications.
  • S serine
  • N asparagine
  • 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 10A) & 72 ( Figure 10B) 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 9 Minimal substitution screen (recombinant protein) 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: 1). Substitutions were also individually added to RSV A2 WT background sequence to generate sequences F308 – F313 and F226 with one substitution each (see Table 7A). C-terminal sequences (positions 514 onwards) of all recombinant protein constructs were according to SEQ ID NO: 130.
  • sequence F310 containing substitution N228K had both protein expression and binding to AM14, D25, and RSB1 that was equivalent to DS-Cav1 ( Figures 14 & 15 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 7B, below). Long term stability of F310 was tested and is shown in Figure 23.
  • Increasing total expression, and specially cell surface expression, of the protein may be used to increase antigenicity.
  • RSV-F expression using the F(ii) construct was first assessed using human skeletal muscle cells. At 24 hours post-transfection (hpt), expression of pre-fusion RSV-F improved more than 3-fold through deletion of the C-terminal 20 amino acids ( Figure 16). RSV-F expression using the F(ii) and F647 constructs was also assessed in monocyte-derived dendritic cells. At 24 hpt, expression of pre-fusion RSV-F (both constructs) improved more than 5-fold through deletion of the C-terminal 20 amino acids ( Figure 17A and B).
  • F647code codon optimisation of F647-coding RNA
  • CT variations (incremental deletions) CT description CT AA sequence 1 Reference RSV F CT, including AA541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN additional transmembrane (TM) domain (SEQ ID NO: 68) residues N-terminal to CT start 2 ⁇ CT3, including additional TM domain AA541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIA (SEQ residues N-terminal to CT start ID NO: 69) 3 ⁇ CT5, including additional TM domain AA541 LIAVGLLLYCKARSTPVTLSKDQLSGINN (SEQ ID residues N-terminal to CT start NO: 70) 4 ⁇ CT10, including additional TM AA541 LIAVGLLLYCKARSTPVTLSKDQL (SEQ ID NO: domain residues N-terminal to CT start 71) Docket No.: 70280WO01 5 ⁇ CT15 , including additional TM AA541 LIAVGLLLYCKARSTPVTL (SEQ ID
  • 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 19B).
  • Figure 21 (A) 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 21 (B) 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 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.
  • day 35 all constructs generated comparable pre-F IgG binding antibody titres.
  • Figure 22B represents the post-F IgG binding antibody titres on day 21 and day 35. The post-F IgG binding titres were mostly comparable between all constructs tested on day 21 and day 35.
  • Example 14 Computational prediction of 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 11B, below) and F216 (SEQ ID NO: 141) were prepared by Docket No.: 70280WO01 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.
  • MOE Molecular Operating Environment software
  • Table 11A 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 11A (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.
  • SEQ ID NO:1 is herein referred to as wild-type.
  • SEQ ID NO:2 is herein referred to as wild-type.

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Abstract

La présente invention concerne entre autres un acide nucléique recombinant codant pour une protéine de fusion du virus respiratoire syncytial (VRS-F) comprenant une queue cytoplasmique. Par rapport à une queue cytoplasmique selon SEQ ID NO: 3 ou 4, 2 à 20 résidus subissent une délétion de la queue cytoplasmique de la protéine RSV-F.<i />
PCT/EP2024/055127 2023-03-17 2024-02-28 Acides nucléiques codant pour rsv-f Pending WO2024193965A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140190A1 (fr) * 2013-03-15 2014-09-18 Emergent Product Development Germany Gmbh Vaccins contre le vrs
US20180161458A1 (en) * 2015-06-12 2018-06-14 Vaxart, Inc. Formulations for small intestinal delivery of rsv and norovirus antigens
US20210170017A1 (en) * 2018-04-17 2021-06-10 Curevac Ag Novel rsv rna molecules and compositions for vaccination
WO2021155243A1 (fr) * 2020-01-30 2021-08-05 Modernatx, Inc. Compositions d'immunisation contre le virus respiratoire
WO2024041772A1 (fr) * 2022-08-22 2024-02-29 Glaxosmithkline Biologicals Sa Protéines rsv-f

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140190A1 (fr) * 2013-03-15 2014-09-18 Emergent Product Development Germany Gmbh Vaccins contre le vrs
US20180161458A1 (en) * 2015-06-12 2018-06-14 Vaxart, Inc. Formulations for small intestinal delivery of rsv and norovirus antigens
US20210170017A1 (en) * 2018-04-17 2021-06-10 Curevac Ag Novel rsv rna molecules and compositions for vaccination
WO2021155243A1 (fr) * 2020-01-30 2021-08-05 Modernatx, Inc. Compositions d'immunisation contre le virus respiratoire
WO2024041772A1 (fr) * 2022-08-22 2024-02-29 Glaxosmithkline Biologicals Sa Protéines rsv-f

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. KT992094
AWET, IMMUNOLOGY, vol. 120, no. 4, 2007, pages 435 - 46
CARBONELL-ESTRANY ET AL., PEDIATRICS, vol. 125, 2010, pages e35 - 51
CHEN ET AL., MOL CELL, vol. 76, no. 1, 2019, pages 96 - 109
EDGAR ET AL., BIORXIV. 2021. 06.20.449169
EDGAR, BMC BIOINFORMATICS., vol. 5, 2004, pages 113
FALSEY ET AL., N ENGL J MED., vol. 52, no. 17, 28 April 2005 (2005-04-28), pages 1749 - 59
FALSEYWALSH, CLIN MICROBIOL REV., vol. 13, 2000, pages 371 - 84
FELTES ET AL., PEDIATR RES, vol. 70, 2011, pages 186 - 91
GENNARO: "Remington: The Science and Practice of Pharmacy", 2000
GILMAN ET AL., PLOSPATHOGENS., vol. 11, no. 7, 2015, pages el005035
GROOTHUIS ET AL., ADV THER, vol. 28, 2011, pages 110 - 25
HSIEH ET AL., SCIENCE, vol. 369, 2020, pages 1501 - 1505
KRARUP ET AL., NAT COMMUN., vol. 6, 3 September 2015 (2015-09-03), pages 8143
LUCAS ET AL., CHEM SCI., vol. 7, 2016, pages 1038 - 1050
MCLELLAN ET AL., SCIENCE, vol. 340, no. 6136, 2013, pages 1113 - 7
O'BRIEN ET AL., LANCET INFECT DIS, vol. 15, 2015, pages 1398 - 408
PEDIATRICS, vol. 102, 1998, pages 531 - 537
RHA ET AL., PEDIATRICS, vol. 146, no. 1, July 2020 (2020-07-01), pages e20193611
SIEVERS ET AL., METHODS MOL BIOL., vol. 1079, 2014, pages 105 - 16
VAN DRUNNEN LITTLEVAN DEN HURK ET AL., REV MED VIROL., vol. 17, no. 1, 2007, pages 5 - 34
WHITEHEAD ET AL., JOURNAL OF VIROLOGY, vol. 72, no. 5, 1998

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