RSV-F proteins

Abstract

The present disclosure provides, inter alia, a respiratory syncytial virus fusion (RSV-F) protein in a pre-fusion conformation that is mutated relative to the wild-type RSV-F according to SEQ ID NO: 1 and comprises the following (a), (b) and (c): (ai) at least one mutation relative to the wild-type in the region corresponding to positions 38-60 of SEQ ID NO: 1, wherein the at least one mutation increases hydrophobicity of the region relative to positions 38-60 of SEQ ID NO: 1; and / or (aii) at least one mutation relative to the wild type in the region corresponding to the 296-318 position of SEQ ID NO: 1 wherein the at least one mutation increases the hydrophobicity of the region relative to the 296-318 position of SEQ ID NO: 1 and / or a residue selected from the group consisting of M, F, I and V is introduced into the region by substitution or insertion; (b) at least one mutation relative to the wild type in the region corresponding to the 208-216 position of SEQ ID NO: 1 wherein the at least one mutation increases the hydrophobicity of the region relative to the 208-216 position of SEQ ID NO: 1 and / or introduces a P residue into the region by substitution or insertion; and (c) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO: 1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion.

Description

Technical Field

[0001] The present disclosure is in the field of vaccinology, particularly structure-based protein design of vaccine antigens. Background Art

[0002] Respiratory syncytial virus (“RSV”) is a ribonucleic acid virus of the family Pneumoviridae, which exists in two antigenically distinct subgroups, called RSV A and RSV B.

[0003] RSV is a major cause of hospitalization and death in infants and the elderly. RSV causes approximately 58,000 hospitalizations and 100 - 500 deaths in children under five years old in the United States each year [1], while 177,000 hospitalizations and 14,000 deaths in adults 65 years old and above [2]. Therefore, the development of a safe and effective vaccine to prevent severe diseases and hospitalizations caused by RSV is a high priority.

[0004] The antiviral drug ribavirin is the only approved antiviral therapy for RSV treatment, but in infants and young children, its use is limited to severe hospitalization cases [3]. In addition, two RSV-specific humanized monoclonal antibodies, palivizumab (Synagis) and motavizumab, have been confirmed to be safe and effective in reducing RSV hospitalization rates and severe complications in high-risk children in a variety of clinical settings [4,5,6,7,8]. RSV treatment available in the elderly is generally supportive and consists of supplemental oxygen, intravenous fluids, and bronchodilators. In May 2023, the first RSV vaccine was approved by the FDA (AREXVY, for the elderly). However, there is clearly still a need for other safe and effective RSV prophylactic vaccines.

[0005] Structure-based antigen design may be the key to developing such a vaccine. The RSV fusion protein (“RSV-F”) in the viral envelope is the most effective target for neutralizing antibodies such as motavizumab. Recent advances in the structural biology of RSV-F have revealed changes in its antigenic characteristics that occur during the fusion process between the viral envelope and the host cell membrane. RSV-F adopts a metastable “pre-fusion” conformation as a homotrimer in the viral envelope and then an irreversible and unique “post-fusion” conformation during fusion with the host cell membrane (see Figure 2 ) of [9]. This pre-fusion conformation is more immunogenic and is bound by most RSV-F-specific neutralizing antibodies in human serum. However, the native pre-fusion conformation is not energetically favorable. Therefore, the pre-fusion RSV-F antigen for vaccination purposes needs to be stabilized to prevent irreversible folding into the post-fusion conformation.

[0006] Structure-based antigen design strategies have previously been used in attempts to stabilize the prefusion conformation. However, there remains a need for a prefusion RSV-F protein design that can be used as a vaccine antigen and that, particularly when expressed from nucleic acid, is amenable to high expression yields. SUMMARY OF THE INVENTION

[0007] The inventors have generated a novel RSV-F protein in the prefusion conformation.

[0008] Using a computational model of wild-type prefusion RSV-F (strain A2), the inventors first identified an in silico residue substitution landscape that enhances the expression and stability of trimeric prefusion RSV-F (see, e.g., Example 2). This strategy utilizes a combination of sequence-based evolutionary bioinformatics and structure-based thermodynamic design. Successive rounds of expression, characterization, and validation of the prefusion structure then narrowed this landscape to identify a smaller set of substitutions that enable the prefusion conformation (see, e.g., Examples 3-5). Then, in vitro screening by the inventors revealed individual substitutions that drive this prefusion conformation (see, e.g., Example 6).

[0009] The introduction of disulfide bonds and / or proline (P) residues is a common stabilization strategy in structure-based antigen design and has previously been applied to RSV-F (see, e.g., [9,10]). However, the RSV-F proteins generated by the inventors retain their prefusion conformation without the introduction of further (artificial) disulfide bonds and (in some embodiments) P residues into the wild-type sequence. Additionally, in some embodiments, the RSV-F proteins generated by the inventors involve the stabilization of multiple domains and the folding of RSV-F. Further, exemplary RSV-F proteins according to the present disclosure exhibit higher expression yields in vitro than DS-Cav1 of reference

[10] (see, e.g., Examples 4 and 6; Figure 8 and Figure 18 ). Exemplary RSV-F proteins according to the present disclosure also exhibit greater long-term stability than DS-Cav1 (see, e.g., Example 9; Figures 32A - 32C ).

[0010] In the in vivo context, when administered in a murine model, exemplary RSV-F proteins according to the present disclosure elicit a prefusion RSV-F specific antibody response and also elicit a neutralizing antibody response against, for example, RSV A (see, e.g., Examples 10, 11, and 13; Figure 34A 、 Figures 34B - 3 7、 Figures 43A - 43G and Figures 44A - 44E ).

[0011] In view of the above, the RSV-F protein generated by the present inventor can be used as a vaccine antigen, i.e., for prophylactic vaccination against RSV.

[0012] Accordingly, in a first independent aspect, the present disclosure provides:

[0013] An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises the following (a), (b) and (c):

[0014] (a) (ai) At least one mutation relative to the wild-type in the 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

[0015] (aii) At least one mutation relative to the wild-type in the 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 a residue selected from M, F, I and V into the region by substitution or insertion;

[0016] (b) At least one mutation relative to the wild-type in the region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion; and

[0017] (c) At least one mutation relative to the wild-type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion.

[0018] In a further independent aspect, the present disclosure provides a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure.

[0019] In a further independent aspect, the present disclosure provides a host cell comprising the nucleic acid of the present disclosure.

[0020] In a further independent aspect, the present disclosure provides an in vitro method for producing the RSV-F protein of the present disclosure, which comprises expressing the nucleic acid (preferably an expression vector) of the present disclosure in a host cell and optionally purifying the RSV-F protein.

[0021] In a further independent aspect, the present disclosure provides a vector (preferably a lipid nanoparticle) comprising the nucleic acid of the present disclosure.

[0022] In a further independent aspect, the present disclosure provides a pharmaceutical composition comprising the RSV-F protein, nucleic acid (preferably RNA), or vector (preferably lipid nanoparticle) of the present disclosure.

[0023] In a further independent aspect, the present disclosure provides the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure for medical use.

[0024] In a further independent aspect, the present disclosure provides a method of treatment comprising the step of administering an effective amount of the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure to a subject (preferably a subject in need of such administration).

[0025] Further independent aspects of the present disclosure are provided throughout the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A - 1B . A consensus design based on sequence and structure to stabilize the pre-fusion conformation of RSV-F. (A) The ROSETTA protein design suite was used to search for combinatorial substitutions at different computational simulation energy thresholds, generating 12 sequences ranging from -0.5 kcal / mol to -6 kcal / mol (in 0.5 kcal / mol increments) relative to the wild type; (B) The subsequent substitution panorama is shaded to illustrate the sequence diversity in the potential design relative to the known epitope positions (sites I, II, III, IV, V) (darker shading represents greater sequence diversity).

[0027] Figure 2 . Expression of the "first round" design relative to DS-Cav1 (from reference

[10] ). Biolayer interferometry (BLI) of the histidine tag sequence indicated that designs F21 and F28 (referred to as 21 and 28) were expressed in mammalian cells when compared to spent media (confirmed by subsequent experiments, data not shown).

[0028] Figure 3 . Binding affinity (K D ) of pre-fusion RSV-F-specific antibodies to "first round" RSV-F mutants. The pre-fusion conformations of two designs, F21 and F28, were tested by biolayer interferometry (BLI) against the antibodies AM14 (quaternary epitope), D25 (site ), RSB1 (site V), and motavizumab (site II).

[0029] Figures 4A - 4D.(A) AM14 (Quaternary epitope), (B) D25 (site ), (C) motavizumab (site II), and (D) RSB1 (site V) Fab binding models to wild-type RSV-F. For clarity, only a single copy of Fab binding to each pre-F trimer is shown.

[0030] Figure 5 .Three-dimensional structure and substitutions relative to wild-type in the "Round 1" design F21 (the trimer is shown, with a single protomer highlighted in dark gray, and the substitutions relative to wild-type are shown as spheres).

[0031] Figure 6. Design of the "Round 2" constructs, using the substitution panorama from "Round 1" for the human RSV-F A2 subtype sequence for (A) all residue designs (including solvent-accessible) or (B) buried-only designs. The localization of the mutations included in each design scheme is shown in dark coloring.

[0032] Figure 7 .Octet BLI of 15 "Round 2" sequences (F212 - F226) that bind to RSV-F antibodies (AM14, D25, RSB1, motavizumab), or for which expression was quantified using a histidine tag (the "F" prefix is not used in the figure). The reaction was normalized to DS-Cav1.

[0033] Figure 8 .Quantification of protein yields from 80 ml cultures for 5 "Round 2" consensus designs compared to DS-Cav1 (the "F" prefix is not used in the figure).

[0034] Figure 9 .Substitutions relative to WT in 5 designs from "Round 2".

[0035] Figure 10 .Overview of the expression, thermal stability, binding affinity to RSV-F mAbs, and antigenicity of the "Round 2" RSV-F antigen. The second column provides the protein expression yields from 80 ml cultures.

[0036] Figure 11 .Three-dimensional structure and substitutions relative to wild-type in the "Round 2" design F224. F225 has the same set of substitutions except for A241N (position 241 is A as in wild-type).

[0037] Figure 12 .Three-dimensional structure and substitutions relative to wild-type in the "Round 2" design F216.

[0038] Figures 13A - 13B. (A) Categories of 2D cryo-EM observations of Design F21 (“Round 1”, 31 permutations). (B) Design F21 in complex with AM14 Fab cryo-EM density map.

[0039] Figure 14 . cryo-EM density map of Design F216 (“Round 2”, 14 permutations) in complex with AM14 Fab cryo-EM density map.

[0040] Figure 15 . cryo-EM density map of Design F224 (“Round 2”, 8 permutations) in complex with AM14 Fab cryo-EM density map.

[0041] Figure 16 . Cryo-EM parameters for 3D structure analysis of Designs F21, F216, F224, and F310.

[0042] Figure 17 . Round 3 minimal permutation screening - study design. Single reverse permutations were made in the F225 sequence (7 mutations from WT, fewest mutations in the successful Round 2 constructs), such that each sample would have 6 mutations relative to the WT sequence (left column). Additionally, single permutations from the F225 sequence were added to WT individually (right column).

[0043] Figure 18 . (A) Protein yields of Round 3 minimal permutation designs relative to DS-Cav1. Also shown are negative controls (EXPIFECTAMINE and cell culture supernatant), F225, and F300 (wild type). F225 showed comparable yields in Round 3 (white bars) and Round 2 (dark bars).

[0044] Figure 19 . Octet BLI of “Round 3” minimal permutation designs in complex with RSV-F antibodies (AM14, D25, RSB1, motavizumab) relative to DS-Cav1. Also shown are negative controls (EXPIFECTAMINE and cell culture supernatant), F225, and F300 (wild type).

[0045] Figure 20 . Study design for Round 3 (mRNA)-only epitope rescue experiment.

[0046] Figure 21.The percentage of positive cells from the "second round" and "third round" RSV-F designs and controls (DS-Cav1, positive control RSV-F construct, and negative control JW27 (NCBI:txid65840)) expressed from mRNA was detected by RSB1, AM14, motavizumab, and 4D7 antibodies.

[0047] Figure 22 .An enlarged view of the substituted S55T from the cryo-EM structure of F21. Thr55 is shown as a stick with a transparent surface. Residues that form a hydrophobic pocket and participate in van der Waals contacts with Thr55 are shown as sticks (including the hydrophobic pocket).

[0048] Figure 23 .An enlarged view of the substituted S215A from the F21 cryo-EM structure (including the proximal α-helix). A215 is depicted as a stick with a transparent surface. Residues that form a hydrophobic region and may participate in van der Waals contacts with A215 are shown as sticks.

[0049] Figures 24A - 24B .(A) The position of the N348 glycan in design F216; (A)(1) An image of the F216 trimer highlighting the position of the N348 glycan (shown as a sphere). The enlarged view shows the position of the N348 glycan and the proximal K419D substitution on the neighboring protomer. (B) The position of the N348 glycan in design F224; (B)(1) An image of the F224 trimer highlighting the position of the N348 glycan (shown as a sphere). The enlarged view depicts the position of the N348 glycan in design F224 and the proximal K419 residue (not substituted relative to the wild type) on the neighboring protomer.

[0050] Figure 25 .An enlarged view of the substituted N228K from the cryo-EM structure of F21. K228 and the surrounding residues are depicted as sticks. The hydrogen bond between K228 and Y250 is depicted as a dashed line.

[0051] Figure 26 .The cryo-EM density map of design F310 ("third round", 1 substitution) bound to AM14 Fab.

[0052] Figure 27 .The HPLC chromatograms evaluating the monodispersity of F310 or F310_v2 (removing the 2x Strep tag relative to F310) after purification, incubation overnight at 4 °C, or one freeze / thaw cycle.

[0053] Figure 28. After nickel affinity purification from 90 mL of cell harvest medium, protein yields of the 3rd round epitope recovery design relative to DS-Cav1 are shown. Protein yields from the 2nd and 3rd round purifications of DS-Cav1 and the 2nd round are shown.

[0054] Figure 29 . Octet BLI of the 3rd round epitope recovery design binding to RSV-F antibodies (AM14, D25, RSB1, motavizumab) relative to DS-Cav1 is also shown. Negative controls from the 2nd round (EXPIFECTAMINE and cell culture supernatant), F216, and F217 (discontinuous lines around the bars, data from Figure 7 ).

[0055] Figures 30A - 30E . Binding of (A) DS Cav-1, (B) F216, (C) F217, (D) F318, and (E) F319 to RSV F antibodies (AM14 and D25) was determined using BIACORE at 50 °C or 60 °C for 30, 60, or 120 min. Results were reported as the response relative to the control (time 0) sample.

[0056] Figure 31 . No changes in long-term stability were observed as determined by changes in thermal stability as determined by nano-DSF after incubation at 4 °C or 25 °C for up to 21 days. Curves of the melting temperature (Tm) of unincubated or samples incubated at 4 °C or 21 °C for 21 days were plotted in the bar graph.

[0057] Figures 32A - 32C . Binding of the 2nd round designs F216, F217, F224 (and DS-Cav1 control) incubated at 4 °C or 25 °C for 21 days to RSV F antibodies (AM14, D25, and RSB1) was evaluated using BIACORE. Results were reported as the binding relative to the unincubated protein.

[0058] Figures 33A - 33B. (A) The total levels of RSV prefusion protein-specific IgG binding antibodies from mice immunized with a 3 μg dose were measured using Luminex assays. Anti-RSV prefusion IgG antibodies from immunized mice measured in absorbance units per mL are shown at days 21 and 35. The geometric mean titer (GMT) and 95% confidence intervals are represented (bars). The GMT values at days 21 and 35 are shown below. (B) The geometric mean ratio (GMR) and 90% confidence intervals comparing the constructs to DS-Cav1 were calculated. Data at day 21 (bottom) and day 35 (top) are shown. The raw numbers for GMR, lower limit (LL), and upper limit (UP) are shown on the right. At day 21, F224 was statistically similar to DS-Cav1. At day 35, F216 was statistically similar to DS-Cav1. "PreF Design 16" = F216, "PreF Design 17" = F217, "PreF Design 24" = F224, "PreF Design 25" = F225.

[0059] Figures 34A - 34B . (A) The total levels of RSV prefusion protein-specific IgG binding antibodies from mice immunized with a 0.3 μg dose were measured using Luminex assays. Anti-RSV prefusion IgG antibodies from immunized mice measured in absorbance units per mL are shown at days 21 and 35. The geometric mean titer (GMT) and 95% confidence intervals are represented (bars). The GMT values at days 21 and 35 are shown below. The limit of detection (LOD) is represented by the dashed line. (B) The geometric mean ratio (GMR) and 90% confidence intervals comparing the constructs to DS-Cav1 were calculated. Data at day 21 (bottom) and day 35 (top) are shown. The raw numbers for GMR, lower limit (LL), and upper limit (UP) are shown on the right. At day 21, F216 and F217 were statistically similar to DS-Cav1. "PreF Design 16" = F216, "PreF Design 17" = F217, "PreF Design 24" = F224, "PreF Design 25" = F225.

[0060] Figures 35A - 35B. (A) On day 35, RSV neutralizing antibody titers were measured by neutralization assay in mice immunized with 3.0 or 0.3 μg doses. Group 1 = saline; Group 2 = DS-CAV1 3 μg; Group 3 = DS-CAV1 0.3 μg; Group 4 = F216 3 μg; Group 5 = F216 0.3 μg; Group 6 = F217 3 μg; Group 7 = F217 0.3 μg; Group 8 = F224 3 μg; Group 9 = F224 0.3 μg; Group 10 = F225 3 μg; Group 11 = F225 0.3 μg. Neutralizing antibody levels are shown as circles. GMTs (bars) with 95% confidence intervals are shown. (B) On day 35, RSV neutralizing antibody titers were measured by neutralization assay in mice immunized with 3.0 or 0.3 μg doses. GMRs with 90% confidence intervals were calculated. At the 0.3 μg dose, F216, F217, F224, and F225 were statistically similar to DS-Cav1. At the 3 μg dose, F217 was statistically similar to DS-Cav1. "PreF design 16" = F216, "PreF design 17" = F217, "PreF design 24" = F224, "PreF design 25" = F225.

[0061] Figures 36A - 36C . RSV pre-F IgG-binding antibody geometric mean titers at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of RNA encoding F(ii) constructs, DS-Cav1, F216, F217, F317, or F319. Each point represents an individual animal. (C) Statistical analysis: geometric mean ratios, upper confidence intervals (UCI), and lower confidence intervals (LCI) of the 2 μg dose results.

[0062] Figure 37. RSV A neutralizing antibody titers (ED60) at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319. Each point represents an individual animal. (C) Statistical analysis: GMR, UCI, and LCI of the 2 μg dose results.

[0063] Figure 38A- Figure 38J. Human primary BJ cells supported the surface expression of RSV F protein from candidate mRNAs. Representative images from a 4-day time-course assay are shown. As depicted, at approximately 8 hours (A’ and A”), 24 hours (B’ and B”), 48 hours (C’ and C”), 72 hours (D’ and D”), or 96 hours (E’ and E”) post-transfection, cells were fixed and labeled with the primary antibody motavizumab, and individual cell nuclei (denoted as ‘) and cell surface RSV F (denoted as “) of variant F318, with 3 amino acids removed from the cytoplasmic tail (CT), were captured by indirect immunofluorescence and imaging (10x objective). Population distributions corresponding to the representative images in columns A - E and analysis of transfected and labeled BJ cells at approximately 8 hours (F), 24 hours (G), 48 hours (H), 72 hours (I), and 96 hours (J) post-transfection were shown from high-content imaging (HCi). As a representative of non-specific staining, population distributions of BJ cells processed as described above and fixed 1 hour post-transfection were shown in each column (F - J) for reference. Population distributions were binned and plotted using the cell-specific RSV F mean intensity values from high-content imaging (HCi) and analysis by GraphPad Prism.

[0064] Figures 39A - 39E . Deletion of the RSV F CT increased the cell surface expression of pre-fusion RSV F trimers. Indirect immunofluorescence labeling with the monoclonal antibody AM14, followed by high-content imaging and analysis, was performed over a 4-day time-course to evaluate the cell surface expression of the RSV F trimer protein. Primary human BJ cells in a 96-well format were transfected forward with mRNAs encoding RSV F variants F(ii) (A), F318 (B), F319 (C), or F(i) (D) (solid dots, solid lines) or their respective CT deletion variants CTΔ3 (solid dots, dashed lines), ΔCT20 (open circles, dashed lines), or ΔCT (i.e., whole CT deletion – open circles, solid lines). Cell monolayers were fixed at specific time points (hours post-transfection), then RSV F was labeled and imaged using a 10x objective. For the line graphs, each plotted value represents the mean intensity of the Alexa647 signal of cells identified from 9 imaging fields per well by automated image analysis. Each point on the line graph represents the mean (μ) + / − 1 standard deviation (σ) from 3 biological replicates. The area under the curve (AUC) (E) and the standard error of the mean (SEM) are shown for each line graph. Means, AUCs, and variabilities shown on the line graphs and bar graphs were calculated by GraphPad Prism software.

[0065] Figures 40A - 40E. For mRNA vaccine candidates with CT deletions, the total expression of RSV F protein increases. The cell surface expression of RSV F protein was evaluated by indirect immunofluorescence labeling with the primary anti-RSV F antibody motavizumab, followed by HCi and analysis, and quantification was performed over a 4-day time course. Primary human BJ cells in 96-well format were transfected with mRNA encoding the RSV F variants F(ii)(A), F318(B), F319(C), or F(i)(D) (solid dots, solid lines) or their respective CT-deleted variants CTΔ3 (solid dots, dashed lines), ΔCT20 (open circles, dashed lines), or ΔCT (open circles, solid lines). The cell monolayer was fixed at specific time points (hours post-transfection), followed by labeling and imaging using a 10x objective. For the line graphs, each plotted value represents the mean intensity of the Alexa647 signal of cells identified from 9 imaging fields per well by automated image analysis. Each point on the line graph represents the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates. The area under the curve (AUC) (E) and the standard error of the mean (SEM) of the mean are shown for each line graph. The means, AUCs, and variabilities shown on the line graphs and bar graphs were calculated using GraphPad Prism software.

[0066] Figures 41A - 41L . In vitro validation of mRNA for in vivo studies. Selected mRNAs encoding RSV F were transfected into primary BJ cell monolayers. The cell monolayers were fixed, and RSV F protein expression was evaluated by indirect immunofluorescence in combination with HCi and image analysis. The mRNAs encoded RSV F variants, including DS-CAV1, F(ii), F(iii), and F(i) proteins or F318 and F319 protein constructs. Results for the corresponding variants lacking 20 amino acids of CT (ΔCT20) are also shown. RSV F surface protein expression was quantified by labeling cells with the anti-RSV F antibodies motavizumab (A), D25 (E), or AM14 (I) 1 day post-transfection, or 3 days post-transfection (motavizumab (C), D25 (G), or AM14 (K)). The mean cell count of three imaging wells is shown and corresponds to the RSV F expression values 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 (μ) + / - 1 standard deviation (σ) from 3 biological replicates as calculated using GraphPad Prism software.

[0067] Figures 42A - 42B. The short 5 - amino acid CT of the RSV F protein (see line 6 of Table 8) maximally enhanced RSV F protein expression both intracellularly and at the cell surface. In vitro - transcribed mRNAs encoding F(ii) CT length variants (0, 5, 10, 15, 20, 22 amino acids, and full - length) were transfected forward into a primary BJ cell monolayer. The cell monolayer was fixed at time points 20 hours or 47 hours post - transfection. After immunolabeling of the fixed BJ cells, surface - exposed trimeric RSV F ( Figure 42A ) or pre - fusion RSV F in whole cells ( Figure 42B ) was quantified by high - content imaging. Trimeric pre - fusion RSV F (identified by AM14) or pre - fusion F (identified by D25) was quantified. Each graph depicts the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates as calculated by GraphPad Prism software. Background staining represents the median from 6 wells treated simultaneously as experimental non - transfected wells.

[0068] Figures 43A - 43G . RSV pre - F IgG - binding antibody geometric mean titers at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of constructs encoding F(iii), F(i), F(i)ΔCT20, F(ii), F(ii)ΔCT20, DS - Cav1, F318, F318ΔCT20, F319, or F319ΔCT20 (where each point represents an individual animal). Statistical comparisons of the constructs (GMR and 90% CI) are presented in (C) - (G).

[0069] Figures 44A - 44E . RSV A neutralizing antibody titers (ED60) at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of constructs encoding F(iii), F(i), F(i)ΔCT20, F(ii), F(ii)ΔCT20, DS - Cav1, F318, F318ΔCT20, F319, or F319ΔCT20 (where each point represents an individual animal). Statistical comparisons of the constructs (GMR and 90% CI) are presented in (C) - (E).

[0070] Figures 45A - 45C.The optimal length of RSV F CT supporting the cell surface expression of RSV F protein before trimerization fusion includes a CT of at least 5 but not longer than 10 amino acids. Indirect immunofluorescence labeling was performed using monoclonal antibody AM14, followed by high-content imaging and analysis, and quantification was performed across a 4-day time course to evaluate the cell surface expression of RSV F protein before trimerization fusion. Primary human fibroblasts (BJ) in a 96-well format were forward-transfected with mRNA encoding RSV F variant F (ii). In (A), selected CT variations were shown. The parent (F(ii), solid line, solid box) was modified by deletion of RNA sequences encoding the terminal 15 amino acids (F(ii)CTDΔ15, solid line, solid circles), 16 amino acids (F(ii)CTDΔ16, dashed line, solid circles), 17 amino acids (F(ii)CTDΔ17, dashed line, open circles), 20 amino acids (F(ii)CTDΔ20, solid line, open circles), 21 amino acids (F(ii)CTDΔ21, dashed line, solid boxes), or complete deletion of the CT domain (F(ii)CTDΔ25, solid line, open boxes). In (B), the area under the curve as calculated from the line graph in (A) is shown and expanded to include additional CT deletions, and (C), the same data as in B are depicted as line graphs. Cell monolayers were fixed at specific time points (hours after transfection) and RSV F was labeled and imaged using a 10x objective. For the line graph, each plotted value represents the average intensity of the Alexa647 signal of the cells identified from 9 imaging fields per well by automated image analysis. Each point on the line graph represents the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates. (B) shows the area under the curve (AUC) and 1 standard error of the mean (SEM). The mean, AUC, and variability shown on the line graph and bar graph were calculated by GraphPad Prism software.

[0071] Figures 46A - 46C As in Figure 45, but D25 antibody binding was assessed.

[0072] Throughout this specification, in FIG. 38- Figure 42A , Figure 42B , Figures 45A - 45C and Figures 46A - 46C Reference to "CTD" (cytoplasmic tail domain) is equivalent to reference to "CT" (cytoplasmic tail). Thus, "CTDΔ20" is equivalent to "ΔCT20", and so on. DETAILED DESCRIPTION

[0073] RSV - F protein in the pre - fusion conformation

[0074] As noted above, in a first independent aspect, the present disclosure provides an RSV-F protein in a pre-fusion conformation that is mutated relative to wild-type RSV-F according to SEQ ID NO:1 and comprises the following (a), (b), and (c):

[0075] (a) (ai) at least one mutation relative to wild-type in the 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

[0076] (aii) at least one mutation relative to wild-type in the 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 a residue selected from M, F, I, and V into the region by substitution or insertion;

[0077] (b) at least one mutation relative to wild-type in the region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion; and

[0078] (c) at least one mutation relative to wild-type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion.

[0079] In a second independent aspect, the present disclosure further provides an RSV-F protein in a pre-fusion conformation that is mutated relative to wild-type RSV-F according to SEQ ID NO:1 and comprises (a) and (b) as defined above, and:

[0080] (d) at least one mutation relative to wild-type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0081] In a third independent aspect, the present disclosure further provides an RSV-F protein in a pre-fusion conformation that is mutated relative to wild-type RSV-F according to SEQ ID NO:1 and comprises (a) as defined above.

[0082] In a fourth independent aspect, the present disclosure also provides an RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises (b) as defined above.

[0083] In a fifth independent aspect, the present disclosure also provides an RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises (c) as defined above.

[0084] In a sixth independent aspect, the present disclosure also provides an RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises (d) as defined above.

[0085] In a seventh independent aspect, the present disclosure also provides an RSV-F protein in a pre-fusion conformation, which comprises at least one mutation relative to the wild-type RSV-F according to SEQ ID NO:1, wherein the at least one mutation does not introduce a disulfide bond or a P residue into the wild-type protein.

[0086] In an eighth independent aspect, the present disclosure also provides a multimer, which comprises protomers, wherein at least one protomer comprises or consists of the RSV-F protein of the present disclosure (i.e., according to any one of the first to seventh independent aspects detailed above).

[0087] To avoid doubt, the RSV-F protein according to the first to seventh independent aspects and the protomers of the RSV-F protein according to the eighth independent aspect are the "RSV-F protein of the present disclosure" as mentioned herein. The wild-type RSV-F (A2 subtype) sequences of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:84, and 107 are not the "RSV-F protein of the present disclosure" as mentioned herein. The wild-type RSV-F sequence of SEQ ID NO:108 (B subtype strain 18537) is also not the "RSV-F protein of the present disclosure" as mentioned herein.

[0088] As used herein, "mutation" encompasses substitutions, insertions, and deletions of residues, although substitutions and insertions are preferred and substitutions are more preferred according to all aspects of the present disclosure. A mutation that "introduces" a given residue by substitution or insertion may be interchangeably referred to as a "substitution or insertion" of the residue.

[0089] The RSV-F proteins of the present disclosure and the mutations relative to SEQ ID NO:1 generally contained therein are all "engineered". Thus, to the knowledge of the inventors, the RSV-F proteins of the present disclosure are not naturally occurring. In the sense that the mutations contained therein are generally "engineered" in that such mutations may occur individually in nature but have been deliberately selected and introduced into the protein in order to stabilize the pre-fusion conformation. The RSV-F proteins of the present disclosure may also be considered "recombinant" (in this context, "engineered" and "recombinant" may be used interchangeably). The RSV proteins of the present disclosure generally contain engineered mutations relative to SEQ ID NO:1, as defined throughout the present disclosure. SEQ ID NO:1 is the RSV-F sequence from a human RSV strain of subtype A2, which contains two mutations (K66E and Q101P) relative to GenBank accession number KT992094 and is referred to herein as "wild-type". To the knowledge of the inventors, the F protein substitutions K66E and Q101P arose by passage of the A2 strain deposited under GenBank accession number KT992094 (also wild-type), see for example

[11] . Thus, for the purposes of the present disclosure, SEQ ID NO:1 (which contains these two substitutions) is referred to as "wild-type" (in accordance with for example

[12] ). References to "wild-type RSV-F according to SEQ ID NO:1" and "SEQ ID NO:1" may be used interchangeably herein. SEQ ID NO:1 does not contain the trimerization domain, transmembrane domain or cytoplasmic domain at the C-terminus, as the domain(s) contained at the C-terminus may vary depending on the format of the RSV-F protein when used as a vaccine antigen (such as an RSV-F protein-based vaccine or a nucleic acid-based vaccine encoding RSV-F). The RSV proteins of the present disclosure may also contain mutations relative to SEQ ID NO:1 that are present in RSV-F proteins from other naturally occurring and engineered strains and subtypes (such as RSV-F proteins from other subtype A or subtype B strains). Thus, the RSV-F proteins of the present disclosure may be of subtype A or subtype B.

[0090] "wherein the at least one mutation increases the hydrophobicity of the [given sequence / region] relative to the wild-type [corresponding sequence / region]" means that as a result of the at least one mutation, the sum hydrophobicity of all residues in the region is increased relative to the corresponding wild-type region. For example, considering a single substitution in a given sequence / region, S can be substituted with a residue selected from I, V, L, F, C, M, A, G, T, and W (all of which are more hydrophobic than S). Combinations of mutations (preferably, substitutions) that individually increase hydrophobicity and individually decrease hydrophobicity are also within the scope of the present disclosure, provided that the sum hydrophobicity of all residues in the sequence / region is increased relative to the corresponding wild-type sequence / region. For the purposes of the present disclosure, hydrophobicity can be measured using the Kyte and Doolittle scale

[13] , see Table 2, "Hydrophilicity Index", as set forth below (larger values indicate greater hydrophobicity).

[0091] Isoleucine (I) 4.5

[0092] Valine (V) 4.2

[0093] Leucine (L) 3.8

[0094] Phenylalanine (F) 2.8

[0095] Cysteine (C) 2.5

[0096] Methionine (M) 1.9

[0097] Alanine (A) 1.8

[0098] Glycine (G) -0.4

[0099] Threonine (T) -0.7

[0100] Tryptophan (W) -0.9

[0101] Serine (S) -0.8

[0102] Tyrosine (Y) -1.3

[0103] Proline (P) -1.6

[0104] Histidine (H) -3.2

[0105] Glutamic acid (E) -3.5

[0106] Glutamine (Q) -3.5

[0107] Aspartic acid (D) -3.5

[0108] Asparagine (N) -3.5

[0109] Lysine (K) -3.9

[0110] Arginine (R)-4.5

[0111] References to the sequence / region of the RSV-F protein of the present disclosure that "corresponds to positions x-y of SEQ ID NO:1" cover the sequence / region aligned with positions x-y of SEQ ID NO:1 (for the avoidance of doubt, which includes positions x and y). However, in a preferred embodiment, the at least one mutation (as defined throughout the present disclosure) is introduced within positions x-y of SEQ ID NO:1 (likewise, including positions x and y). The alignment can be performed visually or by any well-known algorithm; for example, using the NCBI BLAST algorithm, such as "blastp", for example using the default settings (available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins), or for example using the "ClustalOmega" algorithm (see, for example

[14] ), for example using the default settings; the Clustal Omega algorithm is preferred. The corresponding residue positions (such as positions 55, 215, and 348 of SEQ ID NO:1, etc.) are readily identifiable to the person skilled in the art and can be identified by aligning amino acid sequences using any well-known method (visually or algorithmically, for example, as detailed above).

[0112] As used herein, the "heptad repeat A" domain ("HRA") refers to positions 149-206 of SEQ ID NO:1, the "heptad repeat B" domain ("HRB") refers to positions 474-523 of SEQ ID NO:1, and the "heptad repeat C" domain ("HRC") refers to positions 53-100 of SEQ ID NO:1.

[0113] The RSV-F protein of the present disclosure is preferably an antigen (or, in other words, is antigenic). Thus, the RSV-F protein of the present disclosure preferably elicits an immune response when administered in vivo, i.e., against RSV. The immune response can include an antibody response (usually including IgG) and / or a cell-mediated immune response, particularly an antibody response. The immune response will generally recognize the three-dimensional structure of the corresponding wild-type pre-fusion RSV-F, particularly one or more epitopes present on the surface of the protein (exposed to the solvent) in the pre-fusion conformation.

[0114] In view of the fact that the RSV-F protein of the present disclosure can be bound by antibodies AM14, D25, RSB1, and motavizumab (especially AM14, D25, and RSB1, especially AM14), it can also be considered an antigen (or, in other words, antigenic). For example, as measured by SPR, its dissociation constant (K D ) is less than 10 nM, such as 1 pM - 10 nM, as detailed below.

[0115] In the RSV-F protein of the present disclosure, it is contemplated to incorporate both naturally and non-naturally occurring amino acids, although naturally occurring amino acids are preferred.

[0116] Generally, the RSV-F protein of the present disclosure elicits a pre-fusion RSV-F specific antibody response against RSV in vivo, such as an IgG antibody response (see, for example, Examples 10, 11, and 13).

[0117] Generally, the RSV-F protein of the present disclosure elicits a neutralizing antibody response against RSV in vivo, such as against RSV A (see, for example, Examples 10, 11, and 13). The neutralizing antibody response can inhibit the replication of RSV in the respiratory system of a subject (such as in the lungs). The neutralizing antibody response can generate protective immunity against RSV in a subject.

[0118] Pre - fusion conformation

[0119] Generally, the RSV-F protein of the present disclosure can be considered to be stabilized in the pre-fusion conformation.

[0120] The pre-fusion conformation of the RSV-F protein of the present disclosure can be confirmed by the binding of a pre-fusion RSV-F specific monoclonal antibody (“pre-fusion mAb”). For example, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising a light chain and a heavy chain (LC and HC) selected from the group consisting of SEQ ID NO:2 and 3, SEQ ID NO:4 and 5, and SEQ ID NO:8 and 9, respectively. The foregoing are the LC and HC sequences of pre-fusion mAbs AM14, D25, and RSB1, respectively; see, for example, [15, 16, 17].

[0121] (One or more) specific binding (or lack thereof) of the pre-fusion mAb can be determined by surface plasmon resonance (“SPR”) or biolayer interferometry (“BLI”), however, SPR is preferred. SPR can be performed using a BIACORE system, preferably as described in the examples (see subsection “Binding Kinetics Using BIACORE”). Generally, the RSV-F protein of the present disclosure can be specifically bound by any of the above pre-fusion mAbs, as measured by SPR, with a dissociation constant (K D ) less than 10 nM, such as 1 pM - 10 nM; particularly less than 1 nM (1000 pM), such as 1 - 1000 pM.

[0122] For determining the pre-fusion conformation by antibody binding, AM14 is preferred. Different from other pre-fusion mAbs, AM14 is specific for RSV-F in the pre-fusion conformation when in the intact trimer. The antibody motavizumab (see, for example

[18] ) is also used in the examples (LC and HC of SEQ ID NOs: 6 and 7, respectively), but also binds to the post-fusion conformation and is therefore not preferred for confirming the pre-fusion conformation.

[0123] In a specific embodiment, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising an LC and an HC according to SEQ ID NOs: 2 and 3, respectively (or differently defined as antibody AM14), as measured by SPR, with a K D less than 1000, 900, 800, 700, 650 or 600 pM; or, in some embodiments, less than 550 pM; or, in some embodiments, less than 100, 90, 80, 70, 60, 50 or 40 pM; or, in some embodiments, less than 35 pM. A lower K D (such as those K D ) are preferred embodiments. By way of example, RSV-F proteins according to the present disclosure named F216, F217, F224 and F225 are specifically bound by this mAb, as measured by SPR, with K D being 598, 546, 37.8 and 30.2 pM, respectively (see, for example, Example 4, Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising an LC and an HC according to SEQ ID NOs: 2 and 3, respectively (or differently defined as antibody AM14), as measured by SPR, with a K Din the range of 1 - 1000, 1 - 900, 1 - 800, 1 - 700, 1 - 650, 1 - 600, 1 - 550, 1 - 100, 1 - 90, 1 - 80, 1 - 70, 1 - 60, 1 - 50 or 1 - 40 pM; such as 10 - 1000, 10 - 900, 10 - 800, 10 - 700, 10 - 650, 10 - 600, 10 - 550, 10 - 100, 10 - 90, 10 - 80, 10 - 70, 10 - 60, 10 - 50 or 10 - 40 pM; such as 20 - 1000, 20 - 900, 20 - 800, 10 - 700, 20 - 650, 20 - 600, 20 - 550, 20 - 100, 20 - 90, 20 - 80, 20 - 70, 20 - 60, 20 - 50 or 20 - 40 pM. In the foregoing embodiments in this paragraph, the RSV-F protein of the present disclosure is generally assembled in a trimeric form as a homotrimer.

[0124] In a specific embodiment, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 4 and 5, respectively (or alternatively defined as antibody D25), as measured by SPR, the K D less than 200, 180, 160, 140 or 130 pM; or, in certain embodiments, less than 100, 95, 90 or 85 pM; or, in certain embodiments, less than 80 pM; or, in certain embodiments, less than 70 pM. For example, the RSV-F proteins according to the present disclosure named F216, F217, F224 and F225 are specifically bound by such mAb, as measured by SPR, the K D are 119, 75.2, 67.8 and 83.6 pM, respectively (see, for example, Example 4; Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 4 and 5, respectively (or alternatively defined as antibody D25), as measured by SPR, the K DRanging from 1 - 200, 1 - 180, 1 - 160, 1 - 140, 1 - 130, 1 - 100, 1 - 95, 1 - 90, 1 - 85, 1 - 80, or 1 - 70 pM; such as 20 - 200, 20 - 180, 20 - 160, 20 - 140, 20 - 130, 20 - 100, 20 - 95, 20 - 90, 20 - 85, 20 - 80, or 20 - 70 pM; such as 40 - 200, 40 - 180, 40 - 160, 40 - 140, 40 - 130, 40 - 100, 40 - 95, 40 - 90, 40 - 85, 40 - 80, or 40 - 70 pM; such as 50 - 200, 50 - 180, 50 - 160, 50 - 140, 50 - 130, 50 - 100, 50 - 95, 50 - 90, 50 - 85, 50 - 80, or 50 - 70 pM.

[0125] In a specific embodiment, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NO: 8 and 9 respectively (or alternatively defined as antibody RSB1), as measured by SPR, with a K D Less than 150, 120, 110, 100, 105, 95, or 90 pM; or, in some embodiments, less than 80, 75, or 70 pM; or, in some embodiments, less than 60, 55, or 50 pM; or, in some embodiments, less than 45 pM. For example, the RSV-F proteins according to the present disclosure named F216, F217, F224, and F225 are specifically bound by this mAb, as measured by SPR, with a K D Of 85.6, 67.6, 40.4, and 46.5 pM respectively (see, for example, Example 4, Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NO: 6 and 7 respectively (or alternatively defined as antibody RSB1), as measured by SPR, with a K D Ranging from 1 - 150, 1 - 120, 1 - 110, 1 - 100, 1 - 105, 1 - 95, 1 - 80, 1 - 75, 1 - 70, 1 - 60, 1 - 55, 1 - 50, or 1 - 45 pM; such as 10 - 150, 10 - 120, 10 - 110, 10 - 100, 10 - 105, 10 - 95, 10 - 80, 10 - 75, 10 - 70, 10 - 60, 10 - 55, 10 - 50, or 10 - 45 pM; such as 20 - 150, 20 - 120, 20 - 110, 20 - 100, 20 - 105, 20 - 95, 20 - 80, 20 - 75, 20 - 70, 20 - 60, 20 - 55, 20 - 50, or 20 - 45 pM.

[0126] In a preferred embodiment, the RSV-F protein of the present disclosure is specifically bound by:

[0127] (i) A prefusion mAb (or alternatively defined as antibody AM14) comprising an LC and an HC according to SEQ ID NOs: 2 and 3, respectively, having a K D less than 1000, 900, 800, 700 or less than 650 pM (such as 1 - 1000, 1 - 900, 1 - 800, 1 - 700 or 1 - 650 pM);

[0128] (ii) A prefusion mAb (or alternatively defined as antibody D25) comprising an LC and an HC according to SEQ ID NOs: 4 and 5, respectively, having a K D less than 300, 250, 200, 150 or less than 130 pM; optionally less than 100 or 80 pM (such as 1 - 300, 1 - 250, 1 - 200, 1 - 150 or 1 - 130 pM; optionally 1 - 100 or 1 - 80 pM); and / or

[0129] (iii) A prefusion mAb (or alternatively defined as antibody RSB1) comprising an LC and an HC according to SEQ ID NOs: 8 and 9, respectively, having a K D less than 200, 150, 100 or 90 pM (such as 1 - 200, 1 - 150, 1 - 100 or 1 - 90 pM);

[0130] wherein the K according to (i) - (iii) D is measured by SPR. Preferably, the RSV-F protein of the present disclosure meets 2 of the criteria (i), (ii) and (iii), or more preferably all 3. For example, proteins F216 and F217 meet all of the said criteria (see, for example, Example 4, Figure 10 ), where F217 meets the optional criteria set forth in (ii). Optionally, such an RSV-F protein can be bound by an antibody comprising an LC and an HC according to SEQ ID NOs: 6 and 7, respectively (or alternatively defined as motavizumab), as measured by SPR, having a K D less than 200, 150, 100 or less than 80 pM (such as 1 - 200, 1 - 150, 1 - 100 or 1 - 80 pM).

[0131] In a preferred embodiment, the RSV-F protein of the present disclosure is specifically bound by:

[0132] (iv) A prefusion mAb (or alternatively defined as antibody AM14) comprising an LC and an HC according to SEQ ID NOs: 2 and 3, respectively, having a K DLess than 200, 150, 100, 80, 60 or 40 pM (such as 1 - 200, 1 - 150, 1 - 100, 1 - 80, 1 - 60 or 1 - 40 pM);

[0133] (v) A pre - fusion mAb (or alternatively defined as antibody D25) comprising an LC and an HC according to SEQ ID NO:4 and 5 respectively, whose K D Less than 200, 150, 100, 90 or 85 pM; optionally less than 70 pM (such as 1 - 200, 1 - 150, 1 - 100, 1 - 90 or 1 - 85 pM; optionally 1 - 70 pM); and / or

[0134] (vi) A pre - fusion mAb (or alternatively defined as antibody RSB1) comprising an LC and an HC according to SEQ ID NO:8 and 9 respectively, whose K D Less than 200, 100, 80, 60 or 50 pM (such as 1 - 200, 1 - 100, 1 - 80, 1 - 60 or 1 - 50 pM);

[0135] Wherein the K according to (iv) - (vi) D Is measured by SPR. Preferably, the RSV - F proteins of the present disclosure meet 2 of the criteria (iv), (v) and (vi), or more preferably all 3. By way of example, proteins F224 and F225 meet all of the said criteria (see, for example, Example 4, Figure 10 ), where F224 meets the optional criterion set forth in (v). Optionally, such RSV - F proteins can be bound by an antibody (or alternatively defined as motavizumab) comprising an LC and an HC according to SEQ ID NO:6 and 7 respectively, as measured by SPR, whose K D Less than 40 pM (such as 1 - 40 pM).

[0136] Generally, the RSV-F protein of the present disclosure can be bound by a pre-fusion mAb (in particular, any one of the above definitions) for a time period such as at least: 24 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks, 7 weeks or 8 weeks; for example, where the RSV-F protein is stored in buffer at 4°C or 25°C for the said (one or more) period(s), and then assayed to determine whether specific binding of a pre-fusion mAb (in particular AM14 or D25) or an antigen-binding fragment thereof (such as its Fab fragment) is present. The binding over a period of time can be determined, for example, by SPR or BLI. The buffer can be a HEPES buffer, for example 20 mM HEPES containing 150 mM NaCl. Thermal stability can also be evaluated (for example using Nano-DSF, for example as carried out in the examples). Aggregation of the protein can also be evaluated (for example by high performance liquid chromatography (“HPLC”), for example as carried out in the examples).

[0137] In addition to the above, the RSV-F protein of the present disclosure can also be bound by an antibody comprising an LC and HC according to SEQ ID NOs: 6 and 7 respectively (or alternatively defined as motavizumab), as measured by SPR, with a K D less than 200, 180, 160, 140 or 120 pM; or, in certain embodiments, less than 110, 100 or 95 pM; or, in certain embodiments, less than 80, 70, 60 or 55 pM; or, in certain embodiments, less than 50, 45 or 40 pM. By way of example, RSV-F proteins according to the present disclosure named F216, F217, F224 and F225 are specifically bound by such mAb, as measured by SPR, with K D values of 74.8, 117, 38.6 and 52.8 pM respectively (see, for example, Example 4, Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb (or alternatively defined as motavizumab) comprising an LC and HC according to SEQ ID NOs: 6 and 7 respectively, as measured by SPR, with a K DRanging from 1 - 200, 1 - 180, 1 - 160, 1 - 140, 1 - 120, 1 - 110, 1 - 100, 1 - 95, 1 - 80, 1 - 70, 1 - 55, 1 - 50, 1 - 45 or 1 - 40 pM; such as 10 - 200, 10 - 180, 10 - 160, 10 - 140, 10 - 120, 10 - 110, 10 - 100, 10 - 95, 10 - 80, 10 - 70, 10 - 55, 10 - 50, 10 - 45 or 10 - 40 pM, such as 20 - 200, 20 - 180, 20 - 160, 20 - 140, 20 - 120, 20 - 110, 20 - 100, 20 - 95, 20 - 80, 20 - 70, 20 - 55, 20 - 50, 20 - 45 or 20 - 40 pM.

[0138] In an alternative and more preferred method of mAb binding, the pre - fusion conformation of the RSV - F protein of the present disclosure can be confirmed by single - particle analysis of cryo - electron microscopy (“cryo - EM” – see, for example, Example 4, Figure 13A , Figures 13B - 16 ), preferably when the protein is complexed with the antigen - binding fragment of the pre - fusion mAb. Preferably, such cryo - EM includes the following steps:

[0139] Complex the RSV - F protein of the present disclosure with the antigen - binding fragment of the pre - fusion mAb (such as a Fab fragment) (preferably the antigen - binding fragment of AM14, preferably the Fab fragment of AM14) to form a complex;

[0140] Isolate (e.g., by gel filtration) and concentrate the complex;

[0141] Deposit the complex on an electron microscopy grid and vitrify the complex and the grid (e.g., by plunge - freezing into liquid ethane);

[0142] Image by electron microscopy; and

[0143] Resolve the structure of the complex by single - particle analysis.

[0144] More preferably, such cryo - EM is performed as in the examples (see subsection “Cryo - electron microscopy of RSV - F designed F21, F216 and F224”).

[0145] (a)

[0146] The RSV - F protein of the present disclosure comprises (according to the first, second, and third independent aspects) or may comprise (according to the fourth, fifth, sixth, seventh, and eighth independent aspects):

[0147] (ai) at least one mutation relative to the wild type in the 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

[0148] (aii) at least one mutation relative to the wild type in the 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 a residue selected from M, F, I, and V into the region by substitution or insertion.

[0149] In the embodiment of (a) below, the mutation according to (ai) is preferred.

[0150] Preferably, the region where the at least one mutation according to (ai) is located contains or consists of a beta-sheet, and the at least one mutation increases the hydrophobicity of the beta-sheet relative to the wild-type beta-sheet (i.e., positions 38-60 of SEQ ID NO:1). Preferably, the region where the at least one mutation according to (aii) is located contains or consists of a beta-sheet, and the at least one mutation increases the hydrophobicity of the beta-sheet relative to the wild-type beta-sheet (i.e., positions 296-318 of SEQ ID NO:1).

[0151] As noted above, "corresponding to..." encompasses the sequences / regions of the RSV-F proteins of the present disclosure aligned with the corresponding wild-type sequences / regions. Thus, the RSV-F proteins of the present disclosure can comprise: (ai) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 38-60 of SEQ ID NO:1 (preferably which forms a beta-sheet), 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 SEQ ID NO:1 in the region of the protein aligned with positions 296-318 of SEQ ID NO:1 (preferably which forms a beta-sheet), 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 a residue selected from M, F, I, and V into the region. However, in a preferred embodiment, the RSV-F proteins of the present disclosure comprise: (ai) at least one mutation relative to SEQ ID NO:1 within positions 38-60 of SEQ ID NO:1, wherein the at least one mutation results in an increase in hydrophobicity relative to said positions; and / or (aii) at least one mutation relative to SEQ ID NO:1 within positions 296-318 of SEQ ID NO:1, wherein the at least one mutation results in an increase in hydrophobicity relative to said positions and / or introduces a residue selected from M, F, I, and V into said positions.

[0152] In the wild-type RSV-F sequence, positions 38-60 and 296-318 form two beta-sheets which form at least part of a largely hydrophobic pocket at the interface between the F1 domain (positions 137-513 of SEQ ID NO:1) and the heptad repeat A ("HRA") domain, see Figure 22 . Without wishing to be bound by theory, increasing the hydrophobicity of one or both of the corresponding beta-sheets in the RSV-F proteins of the present disclosure (relative to the wild-type) can provide new energetically favorable van der Waals (VDW) contacts within the largely hydrophobic pocket. Introducing M, F, I, and V into the beta-sheet corresponding to positions 296-318 (which have relatively large and / or hydrophobic side chains) can also provide such VDW contacts. Such VDW contacts can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0153] The at least one mutation according to (ai) may comprise, consist of, or consist essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 mutations (preferably substitutions) relative to positions 38 - 60 of SEQ ID NO:1; particularly only 1, 2, 3, 4, 5, 6, 7, or 8 such mutations (preferably substitutions); particularly only 1, 2, 3, 4, or 5 such mutations (preferably substitutions); particularly only 1 or 2 such mutations (preferably substitutions); preferably only 1 such mutation (preferably substitution).

[0154] The at least one mutation according to (aii) may comprise, consist of, or consist essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 mutations (preferably substitutions) relative to positions 296 - 318 of SEQ ID NO:1; particularly only 1, 2, 3, 4, 5, 6, 7, or 8 such mutations (preferably substitutions); particularly only 1, 2, 3, 4, or 5 such mutations (preferably substitutions); particularly only 1 or 2 such mutations (preferably substitutions); preferably only 1 such mutation (preferably substitution).

[0155] In the RSV - F protein of the present disclosure, the region corresponding to positions 38 - 60 of SEQ ID NO:1 (preferably forming a beta - sheet) may have at least 50%, 60%, 70%, 80% sequence identity with positions 38 - 60 of SEQ ID NO:1, or preferably at least 85%, 90%, or 95% sequence identity. In the RSV - F protein of the present disclosure, the region corresponding to positions 296 - 318 of SEQ ID NO:1 (preferably forming a beta - sheet) may have at least 50%, 60%, 70%, 80% sequence identity with positions 296 - 318 of SEQ ID NO:1, or preferably at least 85%, 90%, or 95% sequence identity.

[0156] In some embodiments of the RSV-F protein of the present disclosure, one or more S residues in the wild-type β-sheet at positions 38-60 of SEQ ID NO:1 (e.g., at positions 38, 41, 46, and / or 55) can be replaced with a residue that is more hydrophobic than S (e.g., I, V, L, F, C, M, A, G, T, or W). For example, positions 38, 41, 46, and / or 55 of SEQ ID NO:1 can be replaced with T, C, V, I, or F, particularly T, C, or V, preferably T. Additionally or alternatively, in some embodiments, the RSV-F protein of the present disclosure can comprise a substitution at position 301 with a residue selected from M, F, and I; and / or a substitution at position 303 with a residue selected from V, M, F, and I; in particular, such substitutions are present at both positions 301 and 303. In the wild type, the V301 and L303 side chains point into the largely hydrophobic pocket discussed above (see Figure 22 ). Thus, without being bound by theory, introducing relatively large and / or hydrophobic side chains at these positions can, in particular, provide energetically favorable VDW contacts within the pocket.

[0157] In a specific embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 55 (S) of SEQ ID NO:1 with a more hydrophobic residue (e.g., I, V, L, F, C, M, A, G, T, or W, which is more hydrophobic than S at wild-type position 55). Optionally, in such embodiments, the RSV-F protein of the present disclosure can comprise a substitution at position 301 with a residue selected from M, F, and I; and / or a substitution at position 303 with a residue selected from V, M, F, and I; in particular, such substitutions are present at both positions 301 and 303.

[0158] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T, C, V, I, or F for S at position 55 of SEQ ID NO:1. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T, C, or V for S at position 55 of SEQ ID NO:1. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T or V for S at position 55 of SEQ ID NO:1. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T for S at position 55 of SEQ ID NO:1. Such preferred substitutions at position 55 may be the only mutations according to (ai); and optionally the only mutations in the region corresponding to positions 38-60 of SEQ ID NO:1. Such preferred substitutions at position 55 may be the only mutations according to (ai), where (aii) no mutations are present; and optionally the only mutations in the region corresponding to positions 38-60 of SEQ ID NO:1.

[0159] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S55T mutation may be a driver of the pre-fusion conformation (designated F308). Without wishing to be bound by this theory, replacing S with T at position 55 provides a slightly larger residue which (from computer-simulated three-dimensional structural analysis, see Figure 22 ) appears to fit well within the hydrophobic pocket discussed above without creating significant steric clashes. Additionally, adding the CH3 group of T appears to provide new energetically favorable VDW contacts of the type discussed above. Additionally, alternative substitutions provided by the ROSETTA software for position 55 include C and V (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0, -0.1, or -0.5).

[0160] Generally, a mutation according to (a) (preferably a substitution, preferably such a substitution at position 55 as detailed above) can stabilize the interface between the F1 domain (amino acids 137 - 513 of SEQ ID NO:1) and the heptad repeat A ("HRA") domain. Such stabilization can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, a mutation according to (a) (preferably such a substitution, preferably the substitution at position 55 as detailed above) can provide energetically favorable VDW contacts within the hydrophobic pocket of RSV-F, at the interface between the F1 domain and the HRA domain. Such contacts can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, a mutation according to (a) (preferably a substitution, preferably the substitution at position 55 as detailed above) can inhibit the refolding of the HRA and HRC domains. Such refolding can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, a mutation according to (a) (preferably a substitution, preferably the substitution at position 55 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0161] (b)

[0162] The RSV-F protein of the present disclosure comprises (according to the first, second, and fourth independent aspects) or can comprise (according to the third, fifth, sixth, seventh, and eighth independent aspects):

[0163] At least one mutation relative to the wild type in the region corresponding to amino acids 208 - 216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to amino acids 208 - 216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion.

[0164] Preferably, the region in which the at least one mutation according to (b) is located comprises or consists of a loop (more preferably a loop connecting two α-helices), and the at least one mutation increases the hydrophobicity of the loop relative to the wild type hinge (i.e., amino acids 208 - 216 of SEQ ID NO:1), and / or introduces at least one P residue into the hinge. As mentioned herein, a "loop" can also be referred to as a "loop region" or a "flexible loop", or, if a part of the protein rotates about the loop during a conformational change (especially as in the case of amino acids 208 - 216), as a "hinge loop", "hinge", or "hinge region".

[0165] As mentioned above, "corresponding to" encompasses the sequences / regions of the RSV-F proteins of the present disclosure aligned with the corresponding wild-type sequences / regions. Thus, in some embodiments, the RSV-F proteins of the present disclosure may comprise: (b) at least one mutation relative to SEQ ID NO:1 in the protein sequence aligned with positions 208-216 of SEQ ID NO:1 (preferably forming a loop, more preferably a loop connecting two α-helices), wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region. However, in a preferred embodiment, the RSV-F proteins of the present disclosure comprise: (b) at least one mutation relative to SEQ ID NO:1 within positions 208-216 of SEQ ID NO:1; wherein the at least one mutation results in an increase in hydrophobicity relative to said positions, and / or introduces a P residue into said positions.

[0166] In the wild-type RSV-F sequence, positions 208-216 form a loop connecting two α-helices (α4 helix and α5 helix in the wild-type), see Figure 23 . Without being bound by this theory, increasing the hydrophobicity of the loop and / or introducing a P residue into the loop may stabilize or rigidify the loop, and / or facilitate packing away from the RSV-F surface. Such stabilization, rigidification, and / or packing may inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, by inhibiting the relative movement of two α-helices adjacent to the loop, generally the α4 helix and α5 helix of RSV-F).

[0167] The at least one mutation according to (b) may comprise or consist of 1, 2, 3, 4, 5, 6, 7, or 8 substitutions or insertions (preferably substitutions) relative to positions 208-216 of SEQ ID NO:1; particularly only 1, 2, 3, or 4 such substitutions or insertions (preferably substitutions), particularly only 1, 2, or 3 such substitutions or insertions (preferably substitutions), particularly only 1 or 2 such substitutions or insertions (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).

[0168] In the RSV-F proteins of the present disclosure, the region corresponding to positions 38-60 of SEQ ID NO:1 (preferably forming a loop, more preferably a loop connecting two α-helices) may have at least 50% or 60% sequence identity with positions 208-216 of SEQ ID NO:1, or preferably at least 75% or 85% sequence identity.

[0169] In some embodiments of the RSV-F protein of the present disclosure, one or more S residues in the wild-type loop at positions 208-216 of SEQ ID NO:1 (e.g., at positions 211, 213, and / or 215) can be replaced with a residue more hydrophobic than S (e.g., I, V, L, F, C, M, A, G, T, or W). For example, the wild-type residues at positions 211, 213, and / or 215 of SEQ ID NO:1 can be replaced with an A residue or a P residue, preferably A.

[0170] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A, P, V, I, or F. In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A, V, I, or F. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A or P. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A. Such preferred substitutions at position 215 can be the only mutations according to (b); and optionally the only mutations in the region corresponding to positions 208-216 of SEQ ID NO:1.

[0171] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S215A mutation may be a driving factor for the pre-fusion conformation (designated F309). Without being bound by this theory, removing the hydrophilic OH group (since S is replaced with A) may favor the stacking and rigidity of this loop (see Figure 23 ). Additionally, the A residue at position 215 can provide energetically favorable VDW contacts with residues at positions 79, 206 (I residue in the wild-type), L203, and / or T219. Such stacking, rigidification, and / or VDW contacts can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, inhibit the relative movement of two α-helices adjacent to this loop, generally the α4 helix and α5 helix of RSV-F, or alternatively defined as inhibiting the refolding of the HRC domain and the HRA domain). Additionally, the side chains of P, V, I, or F can also reduce the conformational freedom of this loop and thus also favor the stacking and rigidification of this loop.

[0172] Generally, a mutation according to (b) (preferably such a substitution, preferably such a substitution at position 215 as detailed above) can stabilize or rigidify the loop corresponding to positions 208-216 of SEQ ID NO:1. This stabilization or rigidification can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, by inhibiting the relative movement of two α-helices adjacent to this loop (generally the α4 helix and α5 helix of RSV-F)). Generally, such a mutation according to (b) (preferably such a substitution, preferably such a substitution at position 215 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, by inhibiting the relative movement of two α-helices adjacent to this loop (generally the α4 helix and α5 helix of RSV-F), or alternatively defined as by inhibiting the refolding of the HRC structure and the HRA domain).

[0173] To avoid doubt, the embodiments of (b) in this subsection in which a P residue is introduced into the region corresponding to positions 208-216 of SEQ ID NO:1 are not applicable to the seventh independent aspect of the present disclosure. The same applies to such embodiments of (b) in the subsections titled (a), (b), and ((c) or (d)) below.

[0174] (c)

[0175] The RSV-F protein of the present disclosure comprises (according to the first independent aspect and the fifth independent aspect) or can comprise (according to the second independent aspect, the third independent aspect, the fourth independent aspect, the sixth independent aspect, the seventh independent aspect, and the eighth independent aspect):

[0176] At least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into this region by substitution or insertion.

[0177] Preferably, the region where the at least one mutation according to (c) is located comprises a β-sheet and a loop (and optionally, at least a part of other β-sheets), and the at least one mutation introduces a glycosylation site into this region.

[0178] As described above, "corresponding to..." encompasses the sequences / regions of the RSV-F protein of the present disclosure aligned with the corresponding wild-type sequences / regions. Thus, in some embodiments, the RSV-F protein of the present disclosure may comprise: (c) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 345-352 of SEQ ID NO:1 (preferably comprising a beta-sheet and a loop); wherein the at least one mutation introduces a glycosylation site into the region. However, in a preferred embodiment, the RSV-F protein of the present disclosure comprises: (c) at least one mutation relative to SEQ ID NO:1 within positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into said positions. For the avoidance of doubt, in the wild-type, positions 348-352 of SEQ ID NO:1 form a beta-sheet, positions 346-347 of SEQ ID NO:1 form a loop, and position 345 is the C-terminal residue of another beta-sheet.

[0179] Preferably, the at least one mutation according to (c) results in glycosylation of the residues within the region (preferably comprising a beta-sheet and a loop); or alternatively defined as, the at least one mutation according to (c) results in the introduction of a glycan linked to a residue within the region (preferably comprising a beta-sheet and a loop).

[0180] Without wishing to be bound by this theory, glycosylation of the positions within the above region may provide a stabilizing trans-protomer interaction with the charged patch at approximately positions 416-422 of SEQ ID NO:1 (especially K419; see Figures 24A - 24B ), thereby inhibiting, at least partially inhibiting, or completely inhibiting the transition of RSV-F from the pre-fusion to the post-fusion conformation. As discussed below, this interaction can be maintained or enhanced by introducing further mutations at positions 416-422 of SEQ ID NO:1.

[0181] The at least one mutation according to (c) may comprise, or consist of, one or more substitutions or insertions (preferably substitutions) relative to positions 345-352 of SEQ ID NO:1; especially only 1, 2, or 3 such substitutions or insertions (preferably substitutions), especially only 1 or 2 such mutations (preferably substitutions), especially only 1 such substitution or insertion (preferably substitution).

[0182] In the RSV-F protein of the present disclosure, the region corresponding to positions 345-352 of SEQ ID NO:1 (preferably comprising a beta-sheet and a loop) may have at least 50% or 60% sequence identity with positions 345-352 of SEQ ID NO:1, or preferably at least 75% or 85% sequence identity.

[0183] Glycosylation sites / glycosylation can be introduced into this region (preferably comprising beta-sheets and loops) by introducing at least one N residue (resulting in N-linked glycosylation) or at least one S and / or T residue (resulting in O-linked glycosylation) by mutation, or the glycan can be linked to this region. Preferably, at least one N residue is introduced by substitution, resulting in the generation of an NXT or NXS motif (as required for N-linked glycosylation), where X is any amino acid other than P (as required for N-linked glycosylation).

[0184] Generally, the glycosylation / glycan will comprise a core structure containing or consisting of N-acetylglucosamine (GlcNAc). Generally, the glycosylation / glycan will comprise GlcNAc or consist of it.

[0185] In a preferred embodiment of the RSV-F protein of the present disclosure, the glycosylation site / glycosylation is introduced into the beta-sheet corresponding to positions 348 - 352 of SEQ ID NO:1 by mutation, or the glycan is linked to the residues in the beta-sheet. In an even more preferred embodiment of the RSV-F protein of the present disclosure, the glycosylation site / glycosylation is introduced into the said beta-sheet by substituting the S at position 348 of SEQ ID NO:1 with N, or the glycan is linked to the said beta-sheet. The glycosylation site can be conserved by maintaining the wild-type residue S at position 350 or substituting S350 with T. Such a preferred substitution at position 348 can be the only mutation according to (c); and optionally the only mutation in the region corresponding to positions 345 - 352 of SEQ ID NO:1.

[0186] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S348N mutation may be a driver of the pre-fusion conformation (designated F311). Additionally, cryo-EM studies of designs F216 and F225 (see, for example, Example 5) revealed the presence of a glycan linked to the N residue at position 348 (determined by observing additional electron density protruding from the N348 position). This glycan may form a trans-protomer hydrogen bond with a charged local region (particularly the K419 position) at approximately positions 416 - 422 of SEQ ID NO:1 (see Figure 24B )).

[0187] In a specific embodiment, the RSV-F protein of the present disclosure further comprises at least one mutation (preferably a substitution) in the region corresponding to positions 416-422 of SEQ ID NO:1 (preferably forming a loop), wherein the at least one mutation increases the negative charge of the region (e.g., by introducing D and / or E residues). Preferably, the at least one mutation is a substitution at position 419 of SEQ ID NO:1 with D or E. In an even more preferred embodiment, the at least one mutation is a substitution at position 419 of SEQ ID NO:1 with D (as in the design of F216, see Figure 24A ). Such mutations in the region corresponding to positions 416-422 of SEQ ID NO:1 (preferably forming a loop) can result in trans-protomer interactions, thereby contributing to the inhibition of the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0188] Generally, the mutations according to (c) (preferably such substitutions, preferably such substitutions at position 348 as detailed above) can stabilize or rigidify the loop region corresponding to positions 346-347 of SEQ ID NO:1 in the F1 domain. This stabilization or rigidification can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutations according to (c) (preferably such substitutions, preferably such substitutions at position 348 as detailed above) can provide trans-protomer interactions (such as hydrogen bonds). Such trans-protomer interactions can occur with one or more charged residues, including, for example, at position 419 of SEQ ID NO:1 (e.g., K, E, or D residues at position 419). Such trans-protomer interactions can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutations according to (c) (preferably such substitutions, preferably such substitutions at position 348 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0189] (d)

[0190] The RSV-F protein of the present disclosure comprises (according to the second and sixth independent aspects) or may comprise (according to the first, third, fourth, seventh, and eighth independent aspects):

[0191] At least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0192] Preferably, the region in which the at least one mutation according to (d) is located contains a beta-sheet and a loop (and optionally, at least a portion of other beta-sheets). More preferably, the at least one mutation according to (d) is introduced into the beta-sheet corresponding to positions 348-352 of SEQ ID NO:1.

[0193] As noted above, "corresponding to..." encompasses two sequences / regions being aligned (one being the sequence / region of the RSV-F protein of the present disclosure and the other being the sequence / region of the wild type). Thus, in some embodiments, the RSV-F protein of the present disclosure may comprise: (d) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 345-352 of SEQ ID NO:1 (preferably which contains a beta-sheet and a loop), wherein the at least one mutation introduces at least one residue selected from D, F, H, K, L, N, Q, R, T, W, and Y into the region. However, in a preferred embodiment, the RSV-F protein of the present disclosure comprises: (d) at least one mutation relative to SEQ ID NO:1 within positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from D, F, H, K, L, N, Q, R, T, W, and Y into said positions.

[0194] The at least one mutation according to (d) may comprise or consist of 1, 2, 3, 4, 5, 6, 7, or 8 substitutions or insertions (preferably substitutions) relative to positions 345-352 of SEQ ID NO:1; in particular only 1, 2, 3, 4, 5 such substitutions or insertions (preferably substitutions), in particular only 1, 2, or 3 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such mutations (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).

[0195] In the RSV-F protein of the present disclosure, the region corresponding to positions 345-352 of SEQ ID NO:1 (preferably containing a beta-sheet and a loop) may have at least 50% or 60% sequence identity with positions 345-352 of SEQ ID NO:1, or preferably at least 75% or 85% sequence identity.

[0196] In some embodiments of the RSV-F protein of the present disclosure, one or more S residues (e.g., at positions 348 and / or 350) in the wild-type beta-sheet corresponding to positions 348-352 of SEQ ID NO:1 may be substituted with N, D, F, H, K, L, Q, R, T, W, or Y. For example, positions 348 and / or 350 of SEQ ID NO:1 may be substituted with N, F, H, K, N, Q, R, T, W, or Y, in particular N, F, R, W, or Y, preferably N.

[0197] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N, D, F, H, K, L, Q, R, T, W, or Y. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N, F, H, K, N, Q, R, T, W, or Y. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N, F, R, W, or Y. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N. Such preferred substitutions at position 348 may be the only mutations according to (d); and optionally the only mutations in the region corresponding to positions 345 - 352 of SEQ ID NO:1.

[0198] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S348N mutation may be a driver of the prefusion conformation (design F311). Additionally, alternative substitutions provided by the ROSETTA software for position 348 include: D, F, H, K, L, Q, R, T, W, and Y (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0 or -0.1), F, H, K, N, Q, R, T, W, and Y (using the same parameters, except for an energy threshold of -0.5), and F, R, W, or Y (using the same parameters, except for an energy threshold of -2).

[0199] A mutation according to (d) introducing an N residue or a T residue may introduce a glycosylation site into the above region (preferably comprising β - sheets and loops); preferably resulting in glycosylation of residues within the region, or alternatively defined as resulting in the introduction of a glycan linked to residues within the region. In such embodiments, the glycosylation site may be conserved by maintaining the wild - type residue (S) at position 350 or by replacing S350 with T.

[0200] In a specific embodiment, the RSV-F protein of the present disclosure further comprises at least one mutation (preferably a substitution) in the region corresponding to positions 416-422 of SEQ ID NO:1 (preferably forming a loop), wherein the at least one mutation increases the negative charge of the region (e.g., by introducing D and / or E residues). Preferably, the mutation is a substitution at position 419 of SEQ ID NO:1 with D or E. In a more preferred embodiment, the mutation is a substitution at position 419 of SEQ ID NO:1 with D. Such mutations (in the presence or absence of glycosylation) in the region corresponding to positions 416-422 of SEQ ID NO:1 (preferably forming a loop) can enhance the trans-protomer interaction, thereby contributing to the inhibition of the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0201] Generally, the mutation according to (d) (preferably such a substitution, preferably the substitution at position 348 as detailed above) can stabilize or rigidify the loop region corresponding to positions 346-347 of SEQ ID NO:1 in the F1 domain. This stabilization or rigidification can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutation according to (d) (preferably such a substitution, preferably the substitution at position 348 as detailed above) can provide trans-protomer interaction. This trans-protomer interaction can occur with one or more charged residues, including, for example, at position 419 of SEQ ID NO:1 (e.g., K, E, or D residues at position 419). This trans-protomer interaction can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutation according to (d) (preferably such a substitution, preferably the substitution at position 348 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0202] (a), (b) and the combination of ((c) or (d))

[0203] In preferred embodiments according to all independent aspects of the present disclosure, the RSV-F protein of the present disclosure relative to SEQ ID NO:1 comprises:

[0204] (a) a substitution at position 55 (S) of SEQ ID NO:1 with T, C, V, I, preferably T, C, or V, preferably T or V, more preferably T;

[0205] (b) a substitution at position 215 (S) of SEQ ID NO:1 with A, P, V, I, or F, preferably A, V, I, or F, preferably A or P, more preferably A; and

[0206] (c) A substitution at position 348 (S) of SEQ ID NO:1 with N or T, more preferably N; optionally, wherein the glycan is linked to said N or T at position 348.

[0207] The foregoing substitutions are preferably the only mutations relative to SEQ ID NO:1 according to (a), (b), and (c).

[0208] In a further preferred embodiment, the RSV-F protein of the present disclosure comprises, relative to SEQ ID NO:1:

[0209] (a) A substitution at position 55 (S) of SEQ ID NO:1 with T, C, V, I, preferably T, C, or V, preferably T or V, more preferably T;

[0210] (b) A substitution at position 215 (S) of SEQ ID NO:1 with A, P, V, I, or F, preferably A, V, I, or F, preferably A or P, more preferably A; and

[0211] (d) A substitution at position 348 (S) of SEQ ID NO:1 with N, D, F, H, K, L, Q, R, T, W, or Y, preferably N, F, H, K, N, Q, R, T, W, or Y, preferably N, F, R, W, or Y, more preferably N.

[0212] The foregoing substitutions are preferably the only mutations relative to SEQ ID NO:1 according to (a), (b), and (d).

[0213] In a more preferred embodiment, the RSV-F protein of the present disclosure comprises substitutions S55T, S215A, and S348N relative to SEQ ID NO:1; optionally, wherein the glycan is linked to said N at position 348; optionally, except for further mutations (preferably substitutions) as detailed below. The foregoing mutations are preferably the only mutations relative to SEQ ID NO:1 according to (a), (b), and ((c) or (d)).

[0214] Further mutations

[0215] According to all independent aspects of the present disclosure, except for (a), (b), and / or ((c) or (d)) as detailed above, the RSV-F protein of the present disclosure may comprise at least one further mutation, preferably at least one further substitution, relative to SEQ ID NO:1.

[0216] In some embodiments, the at least one further substitution is selected from (numbering and original residue according to SEQ ID NO:1):

[0217] A74R; substitution at position 152 (V) with R, L, or W; S169E; S180E; S190I; substitution at position 210 (Q) with H, A, F, K, N, W, or Y; S211N; E218; K226L; substitution at position 228 (N) with K, R, Q, N, or A; A241N; M251L; S275L; M289L; V296I; L305I; substitution at position 315 (K) with I or V; T326D, substitution at position 346 (A) with Q, D, H, K, N, R, S, or W; S350I, K359I; V384K; substitution at position 419 (K) with D, N, S, or T; K445D; substitution at position 455 (T) with V or I; V459M; F477R; E487Q and Q501K. Except for (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or at least 50 of the foregoing substitutions; such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the foregoing substitutions. In a specific embodiment, except for (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 of the foregoing substitutions, such as no more than 14, such as no more than 11, such as no more than 8, such as no more than 5, such as no more than 4 of the foregoing substitutions.

[0218] In a specific embodiment, the substitutions V152R and / or A346Q may be present in the RSV-F protein of the present disclosure. Such substitutions may enhance the expression of the RSV-F protein. V152R and A346Q are surface-exposed substitutions present in two designs, F216 and F217, which show higher levels of in vitro expression from mRNA than F224 and F225 (only buried substitutions), see, for example, Example 7; Figure 21 .

[0219] In a specific embodiment, the substitutions S211N and / or K445D (in particular, both) may be present in the RSV-F protein of the present disclosure. As illustrated in Example 7, the presence of S211N and K445D is shown to improve the stability of the protein after heat stress (see Figures 30A - 30E , comparing F217 (with both substitutions) with F318 (F217 lacking S211N), and comparing F216 (with both substitutions) with F319 (lacking both substitutions)).

[0220] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure further comprises a substitution at position 228 (N) of SEQ ID NO:1 with K, R, Q, N, or A (preferably K, R, Q, or N, or K, R, or Q; more preferably K or R; even more preferably K). The minimal substitution screenings conducted by the inventors revealed that the N228K substitution alone is capable of achieving pre-fusion RSV-F (see, for example, Example 6; Figure 19 , designing F310). Thus, this substitution is a driver of the pre-fusion conformation and, when incorporated into the RSV-F protein of the present disclosure, can provide, for example, long-term stability of the conformation. Without wishing to be bound by this theory, based on three-dimensional structure analysis, substituting N with K at position 228 is shown to result in the formation of a hydrogen bond with Y250 on the same protomer (see Figure 25 , the dotted line indicates the hydrogen bond). The hydrogen bond can stabilize Y250 to form trans-protomer interactions, such as hydrogen bonds or tertiary cation-pi-anion interactions, between, for example, E232, Y250, and R235 (E232 and Y250 are on one protomer, and R235 is on an adjacent protomer). E, Y, and R are one of the main triads of such tertiary cation-pi-anion interactions (see, for example,

[19] ). Additionally, based on the proximity and orientation of the E232 side chain (see Figure 25 ), substituting R or Q at position 228 can also provide a stabilizing hydrogen bond with Y250. Alternative residues provided by the ROSETTA software (based on allowing all amino acids (without evolutionary constraints), using an energy threshold of 0.0, -0.1, or -0.5) include A.

[0221] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0222] Replacement at position 152 (V) with R, L or W, preferably R or W, preferably R;

[0223] Replacement at position 210 (Q) with H, A, F, K, N, W or Y, preferably H, F, K, N, W or Y, preferably H, F or Y; preferably H;

[0224] Optionally, replacement at position 211 (S) with N;

[0225] Replacement at position 228 (N) with K, R, Q, N or A; preferably K, R, Q or N; preferably K, R or Q; preferably K or R; preferably K;

[0226] Replacement at position 241 (A) with N;

[0227] Replacement at position 315 (K) with I or V, preferably I;

[0228] Replacement at position 346 (A) with Q, D, H, K, N, R, S or W, preferably Q, D, H, K, N, R or S, preferably Q;

[0229] Replacement at position 419 (K) with D, N, S or T, preferably D or T, preferably D;

[0230] Optionally, replacement at position 445 (K) with D;

[0231] Replacement at position 455 (T) with V or I, preferably V; and

[0232] Replacement at position 459 (V) with M.

[0233] Except for alternative residues provided by the ROSETTA software (which allows all amino acids (without evolutionary constraints) with an energy threshold of 0.0) and / or visual analysis of the three-dimensional structure, the foregoing substitutions at positions 152, 210, 211, 228, 241, 315, 346, 419, 445, 455, and 459 include the substitutions present in Design F216 (F216 also has S55T, S215A, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis was used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V, and V459M relative to SEQ ID NO:1, and optionally S211N and / or K445D (all of these substitutions are present in Design F216, see, for example, Example 4); optionally wherein the glycan is N-linked at position 316; optionally there are no further mutations relative to SEQ ID NO:1.

[0234] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, 3, 4, 5, 6, or at least 7 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0235] A substitution of V at position 152 with R, L, or W, preferably R or W, preferably R;

[0236] Optionally, a substitution of S at position 211 with N;

[0237] A substitution of N at position 228 with K, R, Q, N, or A; preferably K, R, Q, or N; preferably K, R, or Q; preferably K or R; preferably K;

[0238] A substitution of K at position 315 with I or V, preferably I;

[0239] A substitution of A at position 346 with Q, D, H, K, N, R, S, or W, preferably Q, D, H, K, N, R, or S, preferably Q;

[0240] Optionally, a substitution of K at position 445 with D;

[0241] A substitution of T at position 455 with V or I, preferably V; and

[0242] A substitution of V at position 459 with M.

[0243] Except for alternative residues suggested by ROSETTA software (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0) and / or visual analysis of the three-dimensional structure, the foregoing substitutions at positions 152, 211, 228, 315, 346, 445, 455, and 459 include the substitutions present in Design F217 (F217 also has S55T, S215A, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis was used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V, and V459M relative to SEQ ID NO:1, and optionally S211N and / or K445D (e.g., as present in Design F217, see, for example, Example 4); optionally wherein the glycan is N-linked at position 348; and optionally there are no further mutations relative to SEQ ID NO:1.

[0244] In a preferred embodiment, except for (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, 3, or at least 4 (e.g., 1, 2, 3, 4, or 5) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0245] A substitution of N at position 228 to K, R, Q, N, or A; preferably K, R, Q, or N; preferably K, R, or Q; preferably K or R; preferably K.

[0246] A substitution of K at position 315 to I or V, preferably I.

[0247] A substitution of A at position 241 to N.

[0248] A substitution of T at position 455 to V or I, preferably V; and

[0249] A substitution of V at position 459 to M.

[0250] Except for alternative residues provided by ROSETTA software (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0) and / or visual analysis of the three-dimensional structure, the foregoing substitutions at positions 228, 241, 315, 455, and 459 include the substitutions present in Design F224 (F224 also has S55T, S215A, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis are used to generate the subset. In a preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V, and V459M relative to SEQ ID NO:1 (e.g., as present in Design F224, see, for example, Example 4); optionally wherein the glycan is N-linked at position 348; optionally there are no further mutations relative to SEQ ID NO:1.

[0251] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2 or at least 3 (e.g., 1, 2, 3, or 4) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0252] A substitution of N at position 228 with K, R, Q, N, or A; preferably K, R, Q, or N; preferably K, R, or Q; preferably K or R; preferably K.

[0253] A substitution of K at position 315 with I or V, preferably I.

[0254] A substitution of T at position 455 with V or I, preferably V; and

[0255] A substitution of V at position 459 with M.

[0256] Except for alternative residues provided by ROSETTA software (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0) and / or visual analysis of the three-dimensional structure, the foregoing substitutions at positions 228, 315, 455, and 459 include the substitutions present in Design F225 (F225 also has S55T, S215A, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis are used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, S215A, N228K, K315I, S348N, T455V, and V459M relative to SEQ ID NO:1 (as present in Design F225, see, for example, Example 4); optionally wherein the glycan is N-linked at position 348; optionally there are no further mutations relative to SEQ ID NO:1.

[0257] Generally, further mutations as detailed above in this subsection (preferably such substitutions, preferably such substitutions at positions 152, 210, 211, 228, 241, 315, 346, 419, 445, 455, and / or 459 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the prefusion to the postfusion conformation.

[0258] General sequence features of RSV - F protein (the protein itself) in the pre - fusion conformation

[0259] When considering the protein itself (e.g., the mature furin-processed protein), the RSV-F protein of the present disclosure generally has two domains (in the N-terminal to C-terminal direction, the "F2" domain and the "F1" domain), which may or may not be linked by a peptide bond (although in the wild-type protein they are not so linked; the linkage usually occurs via a disulfide bond). The F2 domain may have at least 70% sequence identity with positions 26-108 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 26-108; and the F1 domain may have at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1. The F2 domain may have at least 70% sequence identity with positions 26-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with positions 26-109; and the F1 domain may have at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.2% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0260] In a preferred embodiment, the signal peptide is absent from the RSV-F protein of the present disclosure, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is positions 1-25 of SEQ ID NO:1.

[0261] In some embodiments, the RSV-F protein of the present disclosure comprises an E residue at position 66 of SEQ ID NO:1 and a P residue at position 101.

[0262] The RSV-F protein of the present disclosure may have at least 70% sequence identity with SEQ ID NO:13, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:13. The RSV-F protein of the present disclosure may have at least 70% sequence identity with SEQ ID NO:84, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:84. SEQ ID NO:13 and 84 are the mature furin-processed sequences of wild-type RSV-F from the A2 subtype (i.e., SEQ ID NO:1 without the signal sequence and p27).

[0263] In the embodiments of the previous paragraph and all embodiments presented below in this subsection, positions 84 (R) and 85 (F) of SEQ ID NOs: 13, 28-38, 50-59 and 84-106 (positions 109 and 137 of SEQ ID NO:1, respectively) are generally discontinuous and may or may not (preferably not) be connected by an intervening amino acid sequence (such as a linker sequence). That is, positions 1-84 of SEQ ID NOs: 13, 28-38, 50-59 and 84-106 form (in whole or in part) the F2 domain, and positions 85-461 of said sequences form (in whole or in part) the F1 domain, wherein the F2 domain and the F1 domain may or may not (preferably not) be connected by an intervening amino acid sequence (such as a linker sequence) between positions 84 and 85 of said sequences. In the mature furin-processed protein, the p27 peptide may still be present as a result of furin cleavage occurring at only one site, for example the p27 peptide is connected to one of the F2 domain or the F1 domain by a peptide bond.

[0264] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 28 or 85, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 28 and 85.

[0265] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 29 or 86, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 29 and 86.

[0266] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 30 or 87, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 30 and 87.

[0267] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 31 or 88, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses all or part of the F2 domain and the F1 domain (i.e., at least part of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, S215A, N228K, K315I, S348N, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 31 and 88.

[0268] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 32 or 89, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least part of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, S215A, and S348N (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 32 and 89.

[0269] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 33 or 90, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least part of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 33 and 90.

[0270] In a preferred embodiment, the RSV-F protein of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO: 34 or 91 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 34 and 91.

[0271] In a preferred embodiment, the RSV-F protein of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO: 35 or 92 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 35 and 92.

[0272] In a preferred embodiment, the RSV-F protein of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO: 36 or 93 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 36 and 93.

[0273] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 37 or 94 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 37 and 94.

[0274] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 38 or 95 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 38 and 95.

[0275] In a further embodiment, the RSV-F protein of the present disclosure can comprise, consist of, or consist essentially of the amino acid sequence according to any one of SEQ ID NOs: 50 - 59 or a portion of any of the foregoing (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain compared to its full-length sequence). The portion preferably contains all substitutions relative to SEQ ID NO: 13 that are present in the amino acid sequence according to any one of SEQ ID NOs: 50 - 59 (as applicable).

[0276] In a further embodiment, the RSV-F protein of the present disclosure can comprise, consist of, or consist essentially of an amino acid sequence according to any one of SEQ ID NOs: 96-105 or a portion (such as at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length) of any of the foregoing. The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains all of the substitutions relative to SEQ ID NO: 84 that are present in the amino acid sequence according to any one of SEQ ID NOs: 96-105 (where applicable).

[0277] In embodiments where the F2 domain and the F1 domain are connected by a peptide bond (e.g., those peptide bonds interposing an amino acid sequence), they can be connected by a linker sequence. The linker sequence will connect the C-terminal and N-terminal regions / residues of the F2 domain and the F1 domain. The linker sequence can be glycine-serine rich or consist of G and S residues, such as GSGSG (SEQ ID NO: 10), GSGSGRS (SEQ ID NO: 11), or GS (SEQ ID NO: 12). In a specific embodiment, the "F2" domain and the "F1" domain can be connected by a linker comprising or consisting of SEQ ID NO: 11 (or a linker having at least 55%, 75%, or 85% identity thereto). In an alternative specific embodiment, the "F2" domain and the "F1" domain can be connected by a linker comprising or consisting of SEQ ID NO: 12 (or G or S residues). In embodiments where the "F2" domain and the "F1" domain are not connected by a peptide bond, they can be connected by at least one disulfide bond (usually two such bonds, which are usually naturally occurring, e.g., as in the wild-type protein).

[0278] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13; in particular, it can have at least 75% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 80% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 85% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 90% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 95% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 99% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 99.4% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 99.5% sequence identity with SEQ ID NO: 13 over at least 80% of SEQ ID NO: 13, at least 75% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 80% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 85% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 90% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 95% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 99% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 99.4% sequence identity with SEQ ID NO: 13 over at least 90% of SEQ ID NO: 13, at least 99.5% sequence identity, having at least 75% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 80% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 85% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 90% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 95% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 99% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 99.4% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, or having at least 99.5% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13..

[0279] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:13, such as having 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 with SEQ ID NO:13 over 100% of SEQ ID NO 13.

[0280] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84; in particular, it can have at least 75% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 80% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 85% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 90% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 95% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 99% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 99.4% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 99.5% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 75% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 80% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 85% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 90% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 95% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 99% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 99.4% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 99.5% sequence identity, having at least 75% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 80% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 85% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 90% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 95% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 99% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 99.4% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, or having at least 99.5% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84..

[0281] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:84, such as having 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 with SEQ ID NO:84 over 100% of SEQ ID NO 84.

[0282] When considering the protein itself, in a preferred embodiment, the RSV-F protein of the present disclosure comprises a heterotrimerization domain at its C-terminus ("heterologous" meaning not native to the viral protein). Additionally, or alternatively, the trimerization domain may be located at the C-terminus of the F1 domain. The trimerization domain is a sequence that promotes the assembly of the RSV-F protein of the present disclosure (i.e., a single protomer) into a trimer, i.e., particularly by associating with other trimerization domains (i.e., those on other protomers). In some embodiments, the trimerization domain may fold into a coiled coil. Exemplary trimerization domains include: the T4 minor fiber protein (fibritin) foldon domain; the yeast GCN4 leucine zipper, e.g., according to SEQ ID NO:39 (or an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% identical thereto, particularly having trimerization function); TRAF2 (GENBANK accession number Q12933 [gi:23503103]; amino acids 299 - 348); thrombospondin 1 (accession number PO7996 [gi:135717]; amino acids 291 - 314); Matrilin-4 (accession number 095460 [gi:14548117]; amino acids 594 - 618); CMP (matrilin-1) (accession number NP_002370 [gi:4505111]; amino acids 463 - 496); HSF1 (accession number AAX42211 [gi:61362386]; amino acids 165 - 191); Cubilin (accession number NP_001072 [gi:4557503]; amino acids 104 - 138); the trimerization domain from influenza hemagglutinin; the trimerization domain from the SARS spike protein, the trimerization domain from HIV gp41; NadA; and aspartate transcarbamoylase (ATCase). Preferably, the trimerization domain is the T4 minor fiber protein foldon domain, more preferably comprising the amino acid sequence according to SEQ ID NO:14 (or an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% identical thereto, preferably having trimerization function), or consisting thereof. The trimerization domain is preferably linked to the C-terminus (i.e., the F1 domain) of the RSV-F protein of the present disclosure via a linker sequence. The linker sequence preferably comprises the amino acid sequence according to SEQ ID NO:60 (or an amino acid sequence that is at least 50% or 75% identical thereto), or consists thereof.

[0283] As noted above, an eighth independent aspect of the present disclosure is a multimer that includes protomers, where at least one protomer is the RSV-F protein of the present disclosure. Preferably, the multimer is a trimer of the RSV-F protein of the present disclosure. Preferably, the trimer is a homotrimer (i.e., includes three RSV-F proteins of the present disclosure that include or are composed of the same primary amino acid sequence).

[0284] Preparation of RSV - F protein in the pre - fusion conformation

[0285] The RSV-F protein of the present disclosure can be prepared by conventional methods, such as by expression in a recombinant host system using a nucleic acid expression vector (e.g., an expression vector as detailed in the section titled "Nucleic Acids Encoding the RSV-F Protein" below).

[0286] 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, especially Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells); avian cells (e.g., chicken embryo fibroblasts and chicken embryo germ cells); bacteria; and yeast cells. HEK293 cells are preferred, and Expi 293 cells (as used in the examples) are more preferred. Thus, in an independent aspect, the present disclosure also provides a host cell (specifically, those host cells detailed above) that includes a nucleic acid encoding the RSV-F protein of the present disclosure (specifically, an expression vector as detailed below). In a further independent aspect, the present disclosure also provides a host cell (specifically, those host cells detailed above) that includes and / or expresses the RSV-F protein of the present disclosure. In a further independent aspect, the present invention also provides a composition that includes a host cell (specifically, those host cells detailed above) and (i) a nucleic acid encoding the RSV-F protein of the present disclosure (specifically, an expression vector as detailed below), and / or (ii) the RSV-F protein of the present disclosure. In a further independent aspect, the present disclosure also provides an in vitro method for producing the RSV-F protein of the present disclosure, which includes expressing a nucleic acid encoding the RSV-F protein (specifically, an expression vector as detailed below) in a host cell (specifically, those host cells detailed above), and optionally purifying the RSV-F protein.

[0287] The RSV-F protein of the present disclosure can be purified by conventional methods after expression from host cells, such as precipitation and chromatography methods (e.g., hydrophobic interaction, ion exchange, affinity, chelation, or size exclusion chromatography). The RSV-F protein of the present disclosure can contain tags that facilitate purification, such as epitope tags or histidine (HIS) tags, to facilitate purification, for example, by affinity chromatography.

[0288] Nucleic acid encoding RSV - F protein in the pre - fusion conformation

[0289] In a further independent aspect, the present disclosure also provides a nucleic acid encoding the RSV-F protein of the present disclosure.

[0290] General sequence features of RSV - F protein in the pre - fusion conformation when encoded by nucleic acid (such as RNA)

[0291] The nucleic acid of the present disclosure can encode the RSV-F protein of the present disclosure, which has at least 70% sequence identity with SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:1. SEQ ID NO:1 is the sequence of wild-type RSV-F from subtype A2, which contains a signal sequence (positions 1-25 of SEQ ID NO:1), and a p27 peptide (positions 109-136 or 110-136 of SEQ ID NO:1), which is cleaved by furin processing in the mature protein.

[0292] The nucleic acid of the present disclosure can encode the RSV-F protein of the present disclosure, which contains an F2 domain having at least 70% sequence identity with positions 26-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with positions 26-109 of SEQ ID NO:1; and an F1 domain having at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.2% or 99.5% sequence identity

[0293] In some embodiments, the RSV-F protein of the present disclosure comprises an E residue at position 66 of SEQ ID NO:1 and a P residue at position 101.

[0294] In a specific embodiment, the signal peptide (positions 1-25 of SEQ ID NO:1) is not considered in the above sequence identity assessment. Thus, in some embodiments, the nucleic acid of the present disclosure encodes the RSV-F protein of the present disclosure, which has at least 70% sequence identity with SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identity with positions 26-513 of SEQ ID NO:1.

[0295] In a preferred embodiment, the nucleic acid of the present disclosure encodes an RSV-F protein comprising the amino acid sequence according to SEQ ID NO:17; or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of it. The portion preferably comprises the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M present in SEQ ID NO:17.

[0296] In a preferred embodiment, the nucleic acid of the present disclosure encodes an RSV-F protein comprising the amino acid sequence according to SEQ ID NO:18; or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of it. The portion preferably comprises the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M present in SEQ ID NO:18.

[0297] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 19; or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M present in SEQ ID NO: 19.

[0298] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 20; or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M present in SEQ ID NO: 20.

[0299] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 21; or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, S215A and S348N present in SEQ ID NO: 21.

[0300] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 22 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M present in SEQ ID NO: 22.

[0301] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 23 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M present in SEQ ID NO: 23.

[0302] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 24 or a portion thereof (such as a portion of at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M present in SEQ ID NO: 24.

[0303] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 25 or a portion thereof (such as a portion of at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M present in SEQ ID NO: 25.

[0304] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 26 or a portion thereof (such as a portion of at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M present in SEQ ID NO: 26.

[0305] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 27 or a portion thereof (such as a portion of at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consists of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M present in SEQ ID NO: 27.

[0306] In a further embodiment, the nucleic acid of the present disclosure can encode an RSV-F protein comprising the amino acid sequence according to any one of SEQ ID NOs: 40-49 or a portion of any of the foregoing (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting thereof. The portion preferably comprises all substitutions relative to SEQ ID NO: 1 present in the amino acid sequence according to any one of SEQ ID NOs: 40-49 (if applicable).

[0307] Two furin cleavage sites are present between positions 108 and 137 of SEQ ID NO: 1 (positions 109-136 or 110-136 of SEQ ID NO: 1 defining the "p27" peptide). In some embodiments, the nucleic acid of the present disclosure encodes the RSV-F protein of the present disclosure, wherein the p27 peptide is artificially absent (i.e., at the level of the encoding nucleic acid, there is an artificial absence of the p27 peptide, for example, by recombinant means). In such embodiments, the fusion peptide (positions 137-157 of SEQ ID NO: 1) can also be artificially absent. In some embodiments, the p27 peptide (and optionally, the fusion peptide) can be replaced with a linker sequence encoded by the nucleic acid. The linker sequence can be glycine-serine rich (or consisting of G residues and S residues), such as GSGSG (SEQ ID NO: 10), GSGSGRS (SEQ ID NO: 11) or GS (SEQ ID NO: 12). In a specific embodiment, the p27 peptide (or at least 80%, 85%, 90% or 95% of its residues) is artificially absent and replaced with a linker comprising or consisting of SEQ ID NO: 11 (or a linker having at least 55%, 75% or 85% identity thereto). In an alternative specific embodiment, both the p27 peptide and the fusion peptide (or at least 80%, 85%, 90% or 95% of their residues) are artificially absent and replaced with a linker comprising or consisting of SEQ ID NO: 12 (or G or S residues).

[0308] In embodiments in which the p27 peptide is absent (including embodiments in which the fusion peptide is also absent), the nucleic acids of the present disclosure can encode an RSV-F protein of the present disclosure comprising two domains (in the N-terminal to C-terminal direction, an "F2" domain and an "F1" domain); the F2 domain has at least 70% sequence identity with positions 1-108 or 1-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 1-108 or 1-109 of SEQ ID NO:1; and the F1 domain has at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0309] Alternatively, in embodiments in which the p27 peptide is absent (including embodiments in which the fusion peptide is also absent), the nucleic acids of the present disclosure can encode an RSV-F protein of the present disclosure comprising two domains (in the N-terminal to C-terminal direction, an "F2" domain and an "F1" domain); the F2 domain has at least 70% sequence identity with positions 26-108 or 26-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 26-108 or 26-109; and the F1 domain has at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0310] The nucleic acids of the present disclosure can also encode the RSV-F proteins of the present disclosure, which have at least 70% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1; in particular, having at least 75% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 80% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 85% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 90% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 95% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 99% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 99.4% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 99.5% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 75% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 80% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 85% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 90% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 95% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 99% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 99.4% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 99.5% sequence identity, having at least 75% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 80% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 85% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 90% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 95% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 99% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 99.4% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, or having at least 99.5% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1.

[0311] The nucleic acids of the present disclosure preferably encode an RSV-F protein of the present disclosure having at least 70% sequence identity with SEQ ID NO:1, such as having 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 with SEQ ID NO:1 over 100% of SEQ ID NO 1.

[0312] The nucleic acids of the present disclosure preferably encode an RSV-F protein comprising a transmembrane domain and, optionally, a cytoplasmic domain linked (directly or indirectly) to its C-terminus at the C-terminus of the transmembrane domain (i.e., at the C-terminus of position 513 of SEQ ID NO:1, or alternatively defined as at the C-terminus of the F1 domain). In some embodiments, there is no complete cytoplasmic domain. Preferably, the transmembrane domain comprises (or consists of) the amino acid sequence according to SEQ ID NO:15 (or a sequence at least 80%, 85%, 90% or 95% identical thereto). Preferably, the cytoplasmic domain (if present) comprises (or consists of) the amino acid sequence according to SEQ ID NO:16, 109 or 110 (or a sequence at least 80%, 85%, 90%, 95% or 95% identical thereto).

[0313] Nucleic acid encoding RSV - F protein with cytoplasmic tail deletion

[0314] The terms "cytoplasmic domain" and "cytoplasmic tail" are used interchangeably herein (including in the appended numbered embodiments and claims).

[0315] When expressed from nucleic acid in vitro, cell surface expression of the pre-fusion RSV-F trimer has been enhanced by deleting residues from the C-terminal cytoplasmic tail (see, e.g., Example 12). Additionally, surprisingly, deletion of 15, 16, 17, and 20 C-terminal residues resulted in higher pre-fusion RSV-F trimer expression at 72 and 96 hours post-transfection compared to deletion of 21 C-terminal residues (see, e.g., Example 14; Figure 45A ). Further, in vivo, at the lower of two different nucleic acid doses tested, RSV-F constructs containing a cytoplasmic tail deletion generally elicited higher neutralizing antibody titers against, for example, RSV of subtype A compared to their counterparts with a fully intact cytoplasmic tail (see, e.g., Example 13; Figure 44B ). Neutralizing antibody titers are generally associated with inhibition of viral replication in the lungs and other respiratory sites and are thus associated with protective efficacy in a subject. Thus, without wishing to be bound by theory, the cytoplasmic tail deletions disclosed herein may allow for protective efficacy against RSV to be achieved at lower doses of nucleic acid-based vaccines, leading to further possible benefits such as reduced reactogenicity.

[0316] In embodiments in which the RSV-F protein comprises a cytoplasmic tail deletion (as defined in this subsection and the appended numbered embodiments and claims), when the F1 domain and transmembrane domain of the RSV-F protein are aligned with positions 137 - 549 of SEQ ID NO:107 or 108, an RSV-F protein having a "cytoplasmic tail" refers to the presence of residues (e.g., 5 residues) at the C-terminus of the residue aligned with position 549 (Y) of SEQ ID NO:107 or 108. Thus, the cytoplasmic tail is at the C-terminus of the transmembrane domain. Preferably, an RSV-F protein having a "cytoplasmic tail" refers to the presence of residues (e.g., 5 residues) at the C-terminus of position 549 of the RSV-F protein. For example, the RSV-F construct designated ΔCT25 used in the examples (see, e.g., Table 8) does not contain any residues at the C-terminus of the Y at position 549 and thus does not contain a cytoplasmic tail. Reference to, for example, deletion of 2 - 20 residues from the C-terminus of the CT (or the like) (relative to SEQ ID NO:109 or 110) refers to the deletion of at least these two and no more than these 20 most C-terminal residues from the CT. That is, relative to SEQ ID NO:109 or 110, respectively, at least the C-terminal residues SN or SK are deleted, and relative to SEQ ID NO:109 or 110, respectively, no more than the C-terminal residues TPVTLSKDQLSGINNIAFSN or TPVTLSKDQLSGINNIAFSK are deleted.

[0317] In some embodiments, the nucleic acids of the present disclosure encode an RSV-F protein comprising a cytoplasmic tail; wherein, relative to the cytoplasmic tail according to SEQ ID NO: 109 or 110, 2-20 residues are deleted from the C-terminus of the cytoplasmic tail of the RSV-F protein. In some embodiments, 3-20 residues are deleted from the C-terminus. 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 from the C-terminus. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues are deleted from the C-terminus. In some embodiments, 2-5, 3-5, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20 residues are deleted from the C-terminus.

[0318] In a preferred embodiment, 2-5, such as 2-4, 2-3, or 3-4, and preferably 3 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12B (see Figures 39A - 39E ), relative to the expression of the parental molecule with a full-length CD or complete CD deletion, the deletion of these 3 C-terminal residues (“ΔCT3”) enhanced the expression of cell surface trimeric pre-fusion RSV-F from the nucleic acid (as measured by AM14 antibody binding) during the 96-hour period after transfection. This enhanced expression phenotype was observed for all four RSV-F constructs (F318, F319, F(i), and F(ii)) tested. See also, for example, Example 12E ( Figure 42A) which uses the "ΔCT5" construct. In a preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-31 of SEQ ID NO: 134, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii). In another embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-29 of SEQ ID NO: 135, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0319] In a preferred embodiment, 6-13, such as 7-13, 8-12, 9-11, 9-10 or 10-11, and preferably 10 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12E (see Figures 42A - 42B ), within a period of 47 hours post-transfection, deletion of these 10 C-terminal residues ("ΔCT10") enhanced the expression of cell surface pre-fusion RSV-F from nucleic acid (as measured by AM14 antibody binding) relative to the expression of the parental molecule with an intact CT or a completely deleted CT. In a preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-24 of SEQ ID NO: 136, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0320] In a preferred embodiment, 14-16, such as 14-15 or 15-16, and preferably 15 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12E (see Figures 42A - 42B) As described, within the 47-hour period after transfection, deletion of these 15 C-terminal residues (“ΔCT15”) enhanced the expression of cell surface trimeric prefusion RSV-F from nucleic acid (as measured by AM14 antibody binding) relative to the expression of parental molecules with intact CT or completely lacking CT. In a preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-19 according to SEQ ID NO:137, or (ii) an amino acid sequence that is at least 60%, 70%, 80% or 90% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0321] In a more preferred embodiment, 16-20, such as 17-20, 18-20 or 19-20, and preferably 20 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO:109 or 110). As described, for example, in Example 12B (see Figures 39A - 39E ) within the 96-hour period after transfection, deletion of these 20 C-terminal residues (“ΔCT20”) enhanced the expression of cell surface trimeric prefusion RSV-F from nucleic acid (as measured by AM14 antibody binding) relative to the expression of parental molecules with intact CT or completely lacking CT. This expression was also enhanced compared to the “ΔCT3” construct, and this phenotype was observed for all four RSV-F constructs tested (F318, F319, F(i) and F(ii)). See also, for example, Example 12E ( Figure 42A ), where the highest trimeric prefusion expression was observed for the ΔCT20 construct. See also, for example, Example 13, where at low doses of RNA (0.2 μg), constructs with ΔCT20 tended to elicit a more potent neutralizing antibody response in vivo compared to their parental molecules with a completely intact CT (see, for example, Figure 44B ). Thus, deletion of 16-20, such as 17-20, 18-20 or 19-20 C-terminal residues, and especially deletion of these 20 C-terminal residues, is more preferred than deletion of other numbers of residues from the C-terminus. In another more preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-14 according to SEQ ID NO:138, or (ii) an amino acid sequence that is at least 60% or 80% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not comprise any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0322] Generally, relative to an RSV-F protein having the same amino acid sequence but lacking the deletion (e.g., comprising a wild-type cytoplasmic tail, e.g., according to SEQ ID NO: 109 or 110), the deletions outlined above increase the cell surface expression of the RSV-F protein from RNA (e.g., within at least 24, 48, 72, or 96 hours; or e.g., within 24, 48, 72, or 96 hours). Generally, relative to the expression of an RSV-F protein having the same amino acid sequence but lacking such deletions (e.g., comprising a wild-type cytoplasmic tail, e.g., according to SEQ ID NO: 109 or 110) in this form, the deletions outlined above increase the cell surface expression of the RSV-F protein in the pre-fusion trimeric form from RNA (e.g., within at least 24, 48, 72, or 96 hours; or e.g., within 24, 48, 72, or 96 hours).

[0323] When determining the effect of the cytoplasmic tail deletion, AM14 antibody binding (or alternatively defined as, the binding of an antibody comprising the light chain (LC) according to SEQ ID NO: 2 and the heavy chain (HC) according to SEQ ID NO: 3) is typically used to evaluate pre-fusion trimeric RSV-F expression. AM14 antibody binding can be assayed using indirect immunofluorescence labeling, e.g., using the protocol in the Examples (see subsection “Indirect Immunofluorescence Labeling and Detection of Surface-Expressed RSV F”). Cell surface expression can be evaluated in fibroblasts, preferably human fibroblasts, preferably human foreskin fibroblasts, preferably human primary BJ cells, preferably the CRL-2522 cell line (deposited with the American Type Culture Collection (ATCC) under the said accession number and publicly available).

[0324] General features of nucleic acid

[0325] The nucleic acids of the present disclosure can be DNA or RNA (including their hybrids), preferably RNA. DNA and RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases, are within the scope of the present disclosure. The nucleic acid can be linear, circular, and / or branched, but will generally be linear. Typically, the nucleic acid will be in recombinant form, i.e., a form not found in nature.

[0326] The nucleic acid can be used to express the RSV-F protein of the present disclosure from a host cell in vitro (i.e., the nucleic acid is an expression vector or a part thereof). Suitable nucleic acid expression vectors (in particular, DNA 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 transcriptional control elements (e.g., promoters, enhancers or terminators), and / or one or more translation signals; and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., those sequences as detailed above in the section entitled "Preparation of RSV-F Protein"). In a preferred alternative embodiment, the nucleic acid is used to express the RSV-F protein of the present disclosure in vivo in a subject (i.e., the nucleic acid is a nucleic acid-based vaccine or a part thereof). In such preferred embodiments, in addition to the sequence encoding the RSV-F protein of the present disclosure, the nucleic acid can comprise one or more heterologous sequences, such as sequences encoding other proteins (e.g., as detailed below) and / or control sequences, in particular promoters or internal ribosome entry sites.

[0327] The nucleic acids of the present disclosure can be codon optimized. In some embodiments, the nucleic acids of the present disclosure can be codon optimized for expression in human cells. Codon optimization refers to the use of specific codons that can increase the translation efficiency and / or half-life of the nucleic acid while not changing the sequence of the expressed protein (taking into account genetic code redundancy). Embodiments of codon-optimized RNA are discussed in more detail in the subsection entitled "RNA" below.

[0328] In some embodiments, the nucleic acid of the present disclosure is in the form of a viral vector, such as a replicating viral vector or a replication-deficient viral vector; including viral vectors based on both DNA and RNA. Suitable examples of viral vectors for encoding the RSV-F protein of the present disclosure include, for example: adenoviral vectors, such as replication-deficient or replicable adenoviral vectors; poxviral vectors, such as vaccinia virus vectors (e.g., modified vaccinia Ankara virus (MVA), NYVAC, avipox vectors, canarypox (e.g., ALVAC) and fowl pox virus (FPV)); alphaviral vectors, such as Sindbis virus, Semlike Forest virus (SFV), Ross River virus, Venezuelan equine encephalitis (VEE) virus, and chimeras derived from alphaviral vectors, such as the aforementioned viruses; herpesviral vectors, such as vectors derived from cytomegalovirus (CMV); arenaviral vectors, such as lymphocytic choriomeningitis virus (LCMV) vectors; measles virus vectors; vesicular stomatitis virus vectors; pseudorabies virus vectors; adeno-associated virus vectors; retroviral vectors; lentiviral vectors; and virus-like particles. In other embodiments, the nucleic acid is in the form of a DNA plasmid.

[0329] In embodiments in which the nucleic acids of the present disclosure are viral vectors, preferably the viral vector is an adenoviral vector, such as a replication-incompetent adenovirus type 26 (“Ad26”) or a replication-incompetent chimpanzee adenovirus 155 (“ChAd155”), preferably replication-incompetent Ad26. In such adenoviral vector embodiments, a particular group of patients of interest (in which the adenovirus can be used for therapy, particularly vaccination) is infants and the elderly (see the section entitled “Medical Uses and Methods of Treatment” below). In such adenoviral vector embodiments, the adenoviral vector (preferably replication-incompetent Ad26) can also be co-formulated with the RSV-F protein of the present disclosure (i.e., the protein itself), which can have the same or a different primary amino acid sequence as the RSV-F protein of the present disclosure encoded by the adenovirus. In such adenoviral vector embodiments, alternatively, the adenoviral vector (preferably replication-incompetent Ad26) can be co-formulated with other RSV-F proteins (i.e., the protein itself, which is not the RSV-F protein according to the present disclosure), such as an RSV-F protein with a p27 region deletion (or without a p27 region deletion), and optionally having at least 2, 3, 4, or 5 mutations relative to wild-type RSV-F (such as N67I and S215P; N67I, S215P, and E487Q; or K66E, N67I, I76V, S215P, and D486N; particularly the latter group of five mutations). In such co-formulation embodiments, a particular group of patients of interest (in which the co-formulation can be used for therapy, particularly vaccination) is the elderly (see the section entitled “Medical Uses and Methods of Treatment” below). In such elderly patients, the co-formulation can be administered as an initial prime-boost regimen or as part thereof, particularly involving administering the co-formulation as both an (one or more) initial administration and an (one or more) boost administration.

[0330] The nucleic acid (preferably RNA) can encode only the RSV-F protein of the present disclosure (i.e., the nucleic acid encodes a single protein). Alternatively, the nucleic acid can encode multiple proteins, one of which is the RSV-F protein of the present disclosure. In some embodiments, the nucleic acid encodes at least (i) the RSV-F protein of the present disclosure; and (ii) at least one other protein. The at least one other protein can be a nanoparticle, such as a ferritin nanoparticle (e.g., which, together with the RSV-F protein of the present disclosure, is encoded by a single open reading frame, resulting in the expression of a single polypeptide). In a preferred embodiment, the at least one other protein is an antigen; and thus can comprise or can be a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or an allergenic (i.e., derived from or originating from an allergen) antigen; typically encoded by a separate open reading frame of the RSV-F protein of the present invention. The at least one other protein will generally be a pathogen antigen. The at least one other protein will generally be an antigen that is a surface polypeptide, such as a spike glycoprotein, a hemagglutinin, an adhesin, or an envelope glycoprotein. In a specific embodiment, the at least one other protein is an antigen from or derived from a virus (especially a virus that causes a respiratory disease, especially a seasonal virus that causes a respiratory disease). In embodiments where the at least one other 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.

[0331] In a preferred embodiment, the at least one other protein detailed above is a paramyxoviridae protein (especially a paramyxoviridae antigen). Useful paramyxoviridae proteins (especially, antigens) can be from orthoparamyxovirus or metaparamyxovirus, especially human RSV or human metaparamyxovirus (hMPV). Useful other hMPV antigens include, for example, F, N, P, M, M2-1, and M2 antigens (especially, the F antigen). Such hMPV proteins (especially, antigens) can be from or derived from subtype A or B. In a preferred embodiment, the nucleic acid is an RNA that encodes the RSV-F protein of the present disclosure in addition to an hMPV antigen (especially, the F antigen). In such RNA embodiments, the preferred patient group (wherein the RNA can be used for therapy, especially vaccination) is infants (see the section entitled "Medical Use and Treatment Methods" below). In addition to other RSV-F antigens (i.e., having a different amino acid sequence from the RSV-F protein of the present disclosure encoded by the nucleic acid), useful other human RSV antigens include, for example, G, M1, M2-1, M2-2, P, L, N, NS1, NS2, and SH antigens. Such other human RSV proteins (especially, antigens; especially, the F antigen) can be from or derived from subtype A or B. In a specific embodiment, the nucleic acid is a viral vector (especially, a poxvirus vector, especially an MVA vector) that encodes the RSV-F protein of the present disclosure in addition to a plurality of other RSV proteins (especially, antigens; especially at least 2, 3, or 4 other RSV proteins / antigens; especially selected from G (from or derived from subtype A: "G A "), G (from or derived from subtype B: "G B "), N, and either M2-1 or M2-2; especially G A ), G B ), N, and either M2-1 or M2-1). In such viral vector embodiments, the specific patient group (wherein the viral vector can be used for therapy, especially vaccination) is the elderly (see the section entitled "Medical Use and Treatment Methods" below).

[0332] In a preferred embodiment, the at least one other protein detailed above is a coronavirus antigen. Useful coronavirus antigens can be from the SARS coronavirus, particularly SARS-CoV2. Useful coronavirus antigens (preferably SARS-CoV2 antigens) include spike, M, E, HE, nucleocapsid, Plpro, and 3CLPro proteins, particularly the spike protein. Preferably, the coronavirus antigen is the SARS-CoV2 spike protein. The SARS-CoV2 spike protein can be from any variant, such as Omicron (e.g., Omicron BA.1, BA.2, BA.3, BA.4, or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma, or Delta. Preferably, the SARS-CoV2 spike protein comprises one or more mutations relative to the wild-type protein, particularly mutations of one or more (e.g., two) proline residues. The one or more mutations can be introduced to stabilize the SARS-CoV2 spike protein in the pre-fusion conformation. In a preferred embodiment, the nucleic acid is an RNA that encodes the RSV-F protein of the present disclosure in addition to the coronavirus antigen, e.g., as detailed above. In such RNA embodiments, the preferred patient group (wherein the RNA can be used for therapy, particularly vaccination) is the elderly (see the section titled "Medical Use and Methods of Treatment" below).

[0333] In another preferred embodiment, the at least one other protein detailed above is an orthomyxovirus antigen. Useful orthomyxovirus antigens can be from influenza A, B, or C viruses. Useful orthomyxovirus antigens (particularly influenza A, B, or C virus antigens) include hemagglutinin, neuraminidase, and matrix M2 protein, particularly hemagglutinin. Preferably, the orthomyxovirus antigen is the hemagglutinin of influenza A virus. The hemagglutinin of influenza A virus can be from any subtype, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In a preferred embodiment, the nucleic acid is an RNA that encodes the RSV-F protein of the present disclosure in addition to the orthomyxovirus antigen, e.g., as detailed above. In such RNA embodiments, the preferred patient group (wherein the RNA can be used for therapy, particularly vaccination) is the elderly (see the section titled "Medical Use and Methods of Treatment" below). In such RNA embodiments, the RNA can encode (i) the 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.

[0334] In particular, the various nucleic acids of the present disclosure are provided in purified or substantially purified form; that is, substantially free of other nucleic acids (e.g., free or substantially free of naturally occurring nucleic acids, such as other nucleic acids expressed by a host cell). The various nucleic acids are generally at least 50% pure (by weight), such as at least 60%, 70%, 80%, 90% or 95% pure (by weight).

[0335] In a further independent aspect, the present disclosure also provides a vector comprising one or more nucleic acids of the present disclosure.

[0336] The nucleic acid encoding the RSV-F protein of the present disclosure can be delivered naked or, preferably, in combination with a vector (e.g., as detailed in the section entitled "Vectors Comprising Nucleic Acids Encoding RSV-F Proteins in the Pre-Fusion Conformation" below).

[0337] Generally, the nucleic acids of the present disclosure (preferably RNA) and the RSV-F proteins encoded thereby elicit a pre-fusion RSV-F specific antibody response against RSV in vivo, such as an IgG antibody response (see, e.g., Examples 11 and 13).

[0338] Generally, the nucleic acids of the present disclosure (preferably RNA) and the RSV-F proteins encoded thereby elicit a neutralizing antibody response against RSV in vivo, such as a neutralizing antibody response against RSV-A (see, e.g., Examples 11 and 13). The neutralizing antibody response can inhibit the replication of RSV in the respiratory system of a subject (such as in the lungs). The neutralizing antibody response can generate protective immunity against RSV in a subject.

[0339] RNA

[0340] In a preferred embodiment, the nucleic acid of the present disclosure (encoding the RSV-F protein of the present disclosure) is RNA.

[0341] In the context of the section entitled "RNA" herein, "RNA" refers to an artificial (or alternatively defined as recombinant) ribonucleic acid encoding the RSV-F protein of the present disclosure, which can be translated in a cell (i.e., mRNA). Preferably, the RNA is neither a viral vector or virus-based vaccine (such as a live attenuated virus vaccine), nor does it contain one therein.

[0342] RNA molecules can have different lengths, but are typically 500 - 20,000 ribonucleotides long, such as 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.

[0343] In some embodiments, the RNA is self-replicating. Self-replicating RNA can be produced using replication elements derived from, for example, alphaviruses and replacing the sequences encoding structural viral proteins with sequences encoding at least one RSV-F protein of the present disclosure. Self-replicating RNA molecules are typically positive-strand molecules, 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. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, as well as the collinear subgenomic transcripts themselves, can be translated to provide in situ expression of the encoded protein (i.e., the RSV-F protein of the present disclosure); or can be transcribed to provide other transcripts with the same sense strand as the delivered RNA, which are translated to provide in situ expression of the encoded protein. The overall result of this transcriptional sequence is a significant amplification of the number of introduced RNAs, and thus the encoded RSV-F protein of the present disclosure (potentially in addition to other proteins as detailed above) becomes the major polypeptide product of the cell.

[0344] In such embodiments where the RNA self-replicates, the RNA can encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA, and (ii) the RSV-F protein of the present disclosure. The polymerase can be an alphavirus replicase, such as comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Such alphavirus-based self-replicating RNAs can use replicases from, for example, Sindbis virus, Semliki Forest virus, eastern equine encephalitis virus (EEEV), or Venezuelan equine encephalitis virus (VEEV). Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used for self-replicating RNAs (see

[20] ). Thus, the self-replicating RNA encoding the RSV-F protein of the present disclosure can have two open reading frames. The first (5') open reading frame encodes the replicase, particularly an alphavirus replicase (e.g., as detailed above); the second (3') open reading frame encodes the RSV-F protein of the present disclosure. There can also be other open reading frames encoding (i) one or more other proteins (preferably one or more other antigens, e.g., as detailed above); and / or (ii) accessory polypeptides.

[0345] Generally, the RNA comprises a 5' cap, such as 7'-methylguanosine (also known as 7-methylguanosine / m 7 G / m7G), which can be added by enzymatic means or non-enzymatic reactions. The RNA can have the following exemplary 5' caps:

[0346] - 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge (also known as "CapO");

[0347] - 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge, and wherein the first 5' ribonucleotide comprises 2'-methylated ribose (2'-O-Me) (also known as "Cap 1");

[0348] - 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge, and wherein the first 5' ribonucleotide and the second 5' ribonucleotide comprise 2'-methylated ribose (2'-O-Me) (also known as "Cap 2");

[0349] - or 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge, and wherein the first, second, and third 5' ribonucleotides comprise 2'-methylated ribose (2'-O-Me).

[0350] In a preferred embodiment, the 5' cap is a 7'-methylguanosine linked 5' to 5' via a triphosphate bridge to the 5' first ribonucleoside, 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. Preferably, such a cap is added non-enzymatically using the following reagents:

[0351]

[0352] The reagents are sold as CLEANCAP Reagent AG (TRILINK BIOTECHNOLOGIES).

[0353] In other embodiments, a cap can be added such that the 5' end of the RNA has the structure m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. Such a cap can be added non-enzymatically using the following reagents:

[0354]

[0355] The reagents are sold as CLEANCAP Reagent AG (3'OMe) (TRILINK BIOTECHNOLOGIES).

[0356] Generally, the RNA comprises a 3'-polyadenosine ("polyA") tail, for example, comprising 10 - 700 A ribonucleotides. The polyA tail can comprise at least two stretches of contiguous A ribonucleotides (also referred to as "split polyA tail") that are non - contiguous, or (in particular, only one) a stretch of contiguous A ribonucleotides. The total number of A ribonucleotides ("A") in at least two non - contiguous stretches can 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 A in (in particular, only one) contiguous stretch can be, for example, 10 - 700; such as 10 - 600, 20 - 600, or in particular 40 - 600 (such as 50 - 600, 80 - 600, 80 - 550, 100 - 500; or 40 - 70, 50 - 65 or 55 - 65). Where at least two non - contiguous stretches of A are used, these stretches can have different lengths. For example, the length of the first stretch can be 10 - 150 A, such as 10 - 100, 10 - 50, 15 - 50, 20 - 50, 20 - 40, 25 - 40, or in particular a length of 25 - 35 A. For example, the length of the second stretch can be 10 - 150 A, such as 10 - 150, 20 - 120, 30 - 100, 40 - 90, 50 - 90, 60 - 90, 65 - 90, 70 - 90, or in particular a length of 80 - 90 A. The first stretch can be located 5' or 3' relative to the second stretch. However, in a specific embodiment, the first stretch is located 5' relative to the second stretch. In a further specific embodiment, the polyA tail comprises a first non - contiguous stretch of A and a second non - contiguous stretch of A in the 5' to 3' direction, with lengths of 25 - 35 and 80 - 90 A respectively. In a further specific embodiment, the polyA tail comprises a first non - contiguous stretch of A and a second non - contiguous stretch of A in the 5' to 3' direction, with lengths of 25 - 35 and 65 - 90 A respectively. In some embodiments, the at least two non - contiguous stretches of A are from or are part of the 3' untranslated region (UTR), as detailed below, for example.

[0357] The RNA preferably comprises (in addition to any 5' cap structure) one or more modified ribonucleotides, i.e., ribonucleotides that are structurally modified relative to the standard A, C, G, or U ribonucleotides. In other embodiments, the RNA does not comprise modified ribonucleotides, i.e., the RNA contains only standard A, C, G, or U ribonucleotides (except for any 5' cap structure (if present), e.g., as detailed above). In preferred embodiments in which one or more modified ribonucleotides are used, the one or more modified ribonucleotides can be or can comprise N1-methylpseudouridine ("1mΨ"); pseudouridine ("Ψ"); N1-ethylpseudouridine; 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine (m 6A); N6-Threonylcarbamoyladenosine; 1,2’-O-Dimethyladenosine; 1-Methyladenosine; 2’-O-Methyladenosine; 2’-O-Ribosyladenosine (phosphate); 2-Methyladenosine; 2-Methylthio-N6-isopentenyladenosine; 2-Methylthio-N6-hydroxy-norvalylcarbamoyladenosine; 2’-O-Methyladenosine; 2’-O-Ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-Hydroxyisopentenyl)adenosine; N6,2’-O-Dimethyladenosine; N6,2’-O-Dimethyladenosine; N6,N6,2’-O-Trimethyladenosine; N6,N6-Dimethyladenosine; N6-Acetyladenosine; N6-Hydroxy-norvalylcarbamoyladenosine; N6-Methyl-N6-threonylcarbamoyladenosine; 2-Methyladenosine; 2-Methylthio-N6-isopentenyladenosine; 7-Deazaadenosine; N1-Methyladenosine; N6,N6(Dimethyl)adenine; N6-cis-Hydroxyisopentenyladenosine; α-Thioadenosine; 2(Amino)adenine; 2(Aminopropyl)adenine; 2(Methylthio)N6(isopentenyl)adenine; 2-(Alkyl)adenine; 2-(Aminoalkyl)adenine; 2-(Aminopropyl)adenine; 2-(Halogenated)adenine; 2-(Halogenated)adenine; 2-(Propyl)adenine; 2’-Amino-2’-deoxy-ATP; 2’-Azido-2’-deoxy-ATP; 2’-Deoxy-2’-α-aminoadenosine TP; 2’-Deoxy-2’-α-azidoadenosine TP; 6(Alkyl)adenine; 6(Methyl)adenine; 6-(Alkyl)adenine; 6-(Methyl)adenine; 7(Deaza)adenine; 8(Alkenyl)adenine; 8(Alkynyl)adenine; 8(Amino)adenine; 8(Thioalkyl)adenine; 8-(Alkenyl)adenine; 8-(Alkyl)adenine; 8-(Alkynyl)adenine; 8-(Amino)adenine; 8-(Halogenated)adenine; 8-(Hydroxy)adenine; 8-(Thioalkyl)adenine; 8-(Mercapto)adenine; 8-Azidoadenosine; Azidoadenine; Deazaadenine; N6(Methyl)adenine; N6-(Isopentyl)adenine; 7-Deaza-8-azidoadenosine; 7-Methyladenine; 1-Deazaadenosine TP; 2’-Fluoro-N6-Bz-deoxyadenosine TP; 2’-OMe-2-Amino-ATP; 2’O-Methyl-N6-Bz-deoxyadenosine TP; 2’-α-Ethynyladenosine TP; 2-Aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2’-α-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2’-β-Ethynyladenosine TP; 2-Bromoadenosine TP; 2’-β-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2’-Deoxy-2’,2’-difluoroadenosine TP; 2’-Deoxy-2’-α-mercaptoadenosine TP; 2’-Deoxy-2’-α-thiomethoxyadenosine TP; 2’-Deoxy-2’-β-aminoadenosine TP; 2’-Deoxy-2’-β-azidoadenosine TP;2'-Deoxy-2'-β-bromoadenosine TP; 2'-Deoxy-2'-β-chloroadenosine TP; 2'-Deoxy-2'-β-fluoroadenosine TP; 2'-Deoxy-2'-β-iodoadenosine TP; 2'-Deoxy-2'-β-mercaptoadenosine TP; 2'-Deoxy-2'-β-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-Methoxyadenine; 2-Methylthioadenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP; 8-Bromoadenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-Aminopurine; 7-Deaza-2,6-diaminopurine; 7-Deaza-8-aza-2,6-diaminopurine; 7-Deaza-8-aza-2-aminopurine; 2,6-Diaminopurine; 7-Deaza-8-azaadenine, 7-Deaza-2-aminopurine; 2-Thiocytidine; 3-Methylcytidine; 5-Formylcytidine; 5-Hydroxymethylcytidine; 5-Methylcytidine; N4-Acetylcytidine; 2'-O-Methylcytidine; 2'-O-Methylcytidine; 5,2'-O-Dimethylcytidine; 5-Formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-Dimethylcytidine; N4-Acetyl-2'-O-methylcytidine; N4-Methylcytidine; N4,N4-Dimethyl-2'-OMe-cytidine TP; 4-Methylcytidine; 5-Azacytidine; Pseudoisocytidine; Pyrrolocytidine; α-Thiocytidine; 2-(Thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxyCTP; 2'-Deoxy-2'-α-aminocytidine TP; 2'-Deoxy-2'-α-azidocytidine TP; 3(Deaza)5(Azido)cytosine; 3(Methyl)cytosine; 3-(Alkyl)cytosine; 3-(Deaza)5(Azido)cytosine; 3-(Methyl)cytidine; 4,2'-O-Dimethylcytidine; 5(Halo)cytosine; 5(Methyl)cytosine; 5(Propargyl)cytosine; 5(Trifluoromethyl)cytosine; 5-(Alkyl)cytosine; 5-(Alkynyl)cytosine; 5-(Halo)cytosine; 5-(Propargyl)cytosine; 5-(Trifluoromethyl)cytosine; 5-Bromocytidine; 5-Iodocytidine; 5-Propargylcytosine; 6-(Azido)cytosine; 6-Azacytidine; Azidocytosine; Deazacytosine; N4(Acetyl)cytosine; 1-Methyl-1-deazapseudoisocytidine; 1-Methylpseudoisocytidine; 2-Methoxy-5-methylcytidine; 2-Methoxycytidine; 2-Thio-5-methylcytidine; 4-Methoxy-1-methylpseudoisocytidine; 4-Methoxypseudoisocytidine; 4-Thio-1-methyl-1-azapseudoisocytidine; 4-Thio-1-methylpseudoisocytidine;4-Thio-pseudoisocytidine; 5-Aza-zebularine; 5-Methyl-zebularine; Pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2’-Anhydrocytidine TP Hydrochloride; 2’-Fluoro-N4-Bz-cytidine TP; 2’-Fluoro-N4-Acetylcytidine TP; 2’-O-Methyl-N4-Acetylcytidine TP; 2’O-Methyl-N4-Bz-cytidine TP; 2’-α-Ethynylcytidine TP; 2’-α-Trifluoromethylcytidine TP; 2’-β-Ethynylcytidine TP; 2’-β-Trifluoromethylcytidine TP; 2’-Deoxy-2’,2’-Difluorocytidine TP; 2’-Deoxy-2’-α-Mercaptocytidine TP; 2’-Deoxy-2’-α-Thiomethoxy-cytidine TP; 2’-Deoxy-2’-β-Aminocytidine TP; 2’-Deoxy-2’-β-Azacytidine TP; 2’-Deoxy-2’-β-Bromocytidine TP; 2’-Deoxy-2’-β-Chlorocytidine TP; 2’-Deoxy-2’-β-Fluorocytidine TP; 2’-Deoxy-2’-β-Iodocytidine TP; 2’-Deoxy-2’-β-Mercaptocytidine TP; 2’-Deoxy-2’-β-Thiomethoxy-cytidine TP; 2’-O-Methyl-5-(1-Propynyl)cytidine TP; 3’-Ethynylcytidine TP; 4’-Azacytidine TP; 4’-Carbocyclic Cytidine TP; 4’-Ethynylcytidine TP; 5-(1-Propynyl)Ara-C TP; 5-(2-Chlorophenyl)-2-Thiocytidine TP; 5-(4-Aminophenyl)-2-Thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynyl Ara-C TP; 5-Ethynylcytidine TP; 5’-Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethylcytidine TP; N4-Aminocytidine TP; N4-Benzoyl Cytidine TP; Pseudoisocytidine; 7-Methylguanosine; N2,2’-O-Dimethylguanosine; N2-Methylguanosine; Wyosine; 1,2’-O-Dimethylguanosine; 1-Methylguanosine; 2’-O-Methylguanosine; 2’-O-Ribosylguanosine (Phosphate); 2’-O-Methylguanosine; 2’-O-Ribosylguanosine (Phosphate); 7-Aminomethyl-7-Deazaguanosine; 7-Cyano-7-Deazaguanosine; Archaeosine; Methylwyosine; N2,7-Dimethylguanosine; N2,N2,2’-O-Trimethylguanosine; N2,N2,7-Trimethylguanosine; N2,N2-Dimethylguanosine; N2,7,2’-O-Trimethylguanosine; 6-Thioguanosine; 7-Deazaguanosine; 8-Oxoguanosine; N1-Methylguanosine; α-Thioguanosine; 2(Propyl)Guanine; 2-(Alkyl)Guanine; 2’-Amino-2’-Deoxy-GTP; 2’-Azido-2’-Deoxy-GTP; 2’-Deoxy-2’-α-Aminoguanosine TP; 2’-Deoxy-2’-α-Azaguanosine TP; 6(Methyl)Guanine; 6-(Alkyl)Guanine; 6-(Methyl)Guanine;6-Methylguanosine; 7(alkyl)guanine; 7(denitrified)guanine; 7(methyl)guanine; 7-(alkyl)guanine; 7-(denitrified)guanine; 7-(methyl)guanine; 8(alkyl)guanine; 8(alkynyl)guanine; 8(halogenated)guanine; 8(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halogenated)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; Azaguanine; Denitrified guanine; N(methyl)guanine; N-(methyl)guanine; 1-Methyl-6-thioguanosine; 6-Methoxyguanosine; 6-Thio-7-denitrified-8-azaguanosine; 6-Thio-7-denitrified guanosine; 6-Thio-7-methylguanosine; 7-Denitrified-8-azaguanosine; 7-Methyl-8-oxoguanosine; N2,N2-Dimethyl-6-thioguanosine; N2-Methyl-6-thioguanosine; 1-Me-GTP; 2’-Fluoro-N2-isobutylguanosine TP; 2’O-Methyl-N2-isobutylguanosine TP; 2’-α-Ethynylguanosine TP; 2’-α-Trifluoromethylguanosine TP; 2’-β-Ethynylguanosine TP; 2’-β-Trifluoromethylguanosine TP; 2’-Deoxy-2’,2’-difluoroguanosine TP; 2’-Deoxy-2’-α-mercapto guanosine TP; 2’-Deoxy-2’-α-thiomethoxyguanosine TP; 2’-Deoxy-2’-β-aminoguanosine TP; 2’-Deoxy-2’-β-azidoguanosine TP; 2’-Deoxy-2’-β-bromoguanosine TP; 2’-Deoxy-2’-β-chloroguanosine TP; 2’-Deoxy-2’-β-fluoroguanosine TP; 2’-Deoxy-2’-β-iodoguanosine TP; 2’-Deoxy-2’-β-mercapto guanosine TP; 2’-Deoxy-2’-β-thiomethoxyguanosine TP; 4’-Azidoguanosine TP; 4’-Carbocyclic guanosine TP; 4’-Ethynylguanosine TP; 5’-Homo-guanosine TP; 8-Bromoguanosine TP; 9-Denitrified guanosine TP; N2-Isobutylguanosine TP; 1-Methylinosine; Inosine; 1,2’-O-Dimethylinosine; 2’-O-Methylinosine; 7-Methylinosine; 2’-O-Methylinosine; Epoxyqueuosine; Galactosyl queuosine; Mannosyl queuosine; Queuosine; Allylaminothymidine; Azidothymidine; Denitrified thymidine; Deoxythymidine; 2’-O-Methyluridine; 2-Thiouridine; 3-Methyluridine; 5-Carboxymethyluridine; 5-Hydroxyuridine; 5-Methyluridine; 5-Taurinomethyl-2-thiouridine; 5-Taurinomethyluridine; Dihydrouridine; (3-(3-Amino-3-carboxypropyl)uridine; 1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-Methylpseudouridine; 1-Methylpseudouridine; 2’-O-Methyluridine; 2’-O-Methylpseudouridine; 2’-O-Methyluridine; 2-Thio-2’-O-methyluridine;3-(3-Amino-3-carboxypropyl)uridine; 3,2’-O-dimethyluridine; 3-methylpseudouridine TP; 4-thiouridine; 5-(carboxymethyl)uridine; 5-(carboxymethyl)uridine methyl ester, 5,2’-O-dimethyluridine; 5,6-dihydrouridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2’-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2’-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2’-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-oxoacetic acid uridine TP; 5-oxoacetic acid methyl ester uridine TP; N1-methylpseudouridine; N1-ethylpseudouridine; uridine 5-oxoacetic acid; uridine 5-oxoacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2’-O-methyluridine TP; 5-(isopentenylaminomethyl)uridine TP; 5-propynyluracil; α-thiouridine; 1(aminoalkylaminocarbonylvinyl)-2(thio)-pseudouridine; 1(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-4(thio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-pseudouridine; 1(aminocarbonylvinyl)-2(thio)-pseudouridine; 1(aminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminocarbonylvinyl)-4(thio)pseudouridine; 1(aminocarbonylvinyl)-pseudouridine; 1-substituted 2(thio)-pseudouridine; 1-substituted 2,4-(dithio)pseudouridine; 1-substituted 4(thio)pseudouridine; 1-substituted pseudouridine; 1-(aminoalkylaminocarbonylvinyl)-2-(thio)-pseudouridine; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine UTP; 1-methylpseudouridine UTP; 2(thio)pseudouridine; 2’-deoxyuridine; 2’-fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2’-methyl, 2’-amino, 2’-azido, 2’-fluoro-guanosine; 2’-amino-2’-deoxy-UTP; 2’-azido-2’-deoxy-UTP; 2’-azido-deoxyuridine TP; 2’-O-methylpseudouridine; 2’-deoxyuridine; 2’-fluorouridine; 2’-deoxy-2’-a-aminouridine TP;2'-Deoxy-2'-α-azidouridine TP; 2-methylpseudouridine; 3(3-amino-3-carboxypropyl)uracil; 4(thio)pseudouridine; 4-(thio)pseudouridine; 4-(thio)uracil; 4-thiouridine; 5(1,3-diazol-1-yl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidylalkyl)uracil; 5(methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5(methyl)2(thio)uracil; 5(methyl)2,4(dithio)uracil; 5(methyl)4(thio)uracil; 5(methylaminomethyl)-2(thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5(propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouridine; 5-(alkyl)-2,4(dithio)pseudouridine; 5-(alkyl)-4(thio)pseudouridine; 5-(alkyl)pseudouridine; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidylalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazol-1-yl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2(thio)uracil; 5-(methyl)2,4(dithio)uracil; 5-(methyl)4(thio)uracil; 5-(methyl)-2-(thio)pseudouridine; 5-(methyl)-2,4(dithio)pseudouridine; 5-(methyl)-4(thio)pseudouridine; 5-(methyl)pseudouridine; 5-(methylaminomethyl)-2(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyluridine; 5-bromouridine; 5-iodouridine; 5-uracil; 6(aza)uracil; 6-(aza)uracil; 6-azauridine; allylaminouridine; azauracil; deazauracil; N3(methyl)uracil; pseudouridine-1-2-acetic acid; pseudouridine; 4-thiopseudouridine; 1-carboxymethylpseudouridine; 1-methyl-1-deazapseudouridine; 1-propynyluridine; 1-tauromethyl-1-methyluridine; 1-tauromethyl-4-thiouridine; 1-tauromethylpseudouridine; 2-methoxy-4-thiopseudouridine; 2-thio-1-methyl-1-deazapseudouridine; 2-thio-1-methylpseudouridine; 2-thio-5-azauridine; 2-thiodihydropseudouridine; 2-thiodihydrouridine; 2-thiopseudouridine;4-Methoxy-2-thiouridine; 4-Methoxyuridine; 4-Thio-1-methyluridine; 4-Thiouridine; 5-Azacytidine; Dihydrouridine; (.+-.)1-(2-Hydroxypropyl)uridine TP; (2R)-1-(2-Hydroxypropyl)uridine TP; (2S)-1-(2-Hydroxypropyl)uridine TP; (E)-5-(2-Bromovinyl)arabinouridine TP; (E)-5-(2-Bromovinyl)uridine TP; (Z)-5-(2-Bromovinyl)arabinouridine TP; (Z)-5-(2-Bromovinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)uridine TP; 1-(2,2-Diethoxyethyl)uridine TP; 1-(2,4,6-Trimethylbenzyl)uridine TP; 1-(2,4,6-Trimethylbenzyl)pseudo-UTP; 1-(2,4,6-Trimethylphenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Aminoethyl)pseudo-UTP; 1-(2-Hydroxyethyl)uridine TP; 1-(2-Methoxyethyl)uridine TP; 1-(3,4-Bis(trifluoromethoxy)benzyl)uridine TP; 1-(3,4-Dimethoxybenzyl)uridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Aminopropyl)pseudo-UTP; 1-(3-Cyclopropylprop-2-ynyl)uridine TP; 1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Aminobenzyl)uridine TP; 1-(4-Aminobutyl)pseudo-UTP; 1-(4-Aminophenyl)pseudo-UTP; 1-(4-Azidobenzyl)uridine TP; 1-(4-Bromobenzyl)uridine TP; 1-(4-Chlorobenzyl)uridine TP; 1-(4-Fluorobenzyl)uridine TP; 1-(4-Iodobenzyl)uridine TP; 1-(4-Methanesulfonylbenzyl)uridine TP; 1-(4-Methoxybenzyl)uridine TP; 1-(4-Methoxybenzyl)pseudo-UTP; 1-(4-Methoxyphenyl)pseudo-UTP; 1-(4-Methylbenzyl)uridine TP; 1-(4-Methylbenzyl)pseudo-UTP; 1-(4-Nitrobenzyl)uridine TP; 1-(4-Nitrobenzyl)pseudo-UTP; 1-(4-Nitrophenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)uridine TP; 1-(4-Trifluoromethoxybenzyl)uridine TP; 1-(4-Trifluoromethylbenzyl)uridine TP; 1-(5-Aminopentyl)pseudo-UTP; 1-(6-Aminohexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]uridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}uridine TP; 1-Acetyluridine TP; I-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)-pseudo-UTP;1-alkyl-6-allyl pseudouridine triphosphate; 1-alkyl-6-ethynyl pseudouridine triphosphate; 1-alkyl-6-homoallyl pseudouridine triphosphate; 1-alkyl-6-vinyl pseudouridine triphosphate; 1-allyl pseudouridine triphosphate; 1-aminomethyl pseudouridine triphosphate; 1-benzoyl pseudouridine triphosphate; 1-benzyloxymethyl pseudouridine triphosphate; 1-benzyl pseudouridine triphosphate; 1-biotinyl-PEG2-pseudouridine triphosphate; 1-biotinyl pseudouridine triphosphate; 1-butyl pseudouridine triphosphate; 1-cyanomethyl pseudouridine triphosphate; 1-cyclobutylmethyl pseudouridine triphosphate; 1-cyclobutyl pseudouridine triphosphate; 1-cycloheptylmethyl pseudouridine triphosphate; 1-cycloheptyl pseudouridine triphosphate; 1-cyclohexylmethyl pseudouridine triphosphate; 1-cyclohexyl pseudouridine triphosphate; 1-cyclooctylmethyl pseudouridine triphosphate; 1-cyclooctyl pseudouridine triphosphate; 1-cyclopentylmethyl pseudouridine triphosphate; 1-cyclopentyl pseudouridine triphosphate; 1-cyclopropylmethyl pseudouridine triphosphate; 1-cyclopropyl pseudouridine triphosphate; 1-ethyl pseudouridine triphosphate; 1-hexyl pseudouridine triphosphate; 1-homoallyl pseudouridine triphosphate; 1-hydroxymethyl pseudouridine triphosphate; 1-isopropyl pseudouridine triphosphate; 1-Me-2-thio pseudouridine triphosphate; 1-Me-4-thio pseudouridine triphosphate; 1-Me-α-thio pseudouridine triphosphate; 1-methanesulfonylmethyl pseudouridine triphosphate; 1-methoxymethyl pseudouridine triphosphate; 1-methyl-6-(2,2,2-trifluoroethyl) pseudouridine triphosphate; 1-methyl-6-(4-morpholinyl)-pseudouridine triphosphate; 1-methyl-6-(4-thiomorpholinyl)-pseudouridine triphosphate; 1-methyl-6-(substituted phenyl) pseudouridine triphosphate; 1-methyl-6-amino pseudouridine triphosphate; 1-methyl-6-azido pseudouridine triphosphate; 1-methyl-6-bromo pseudouridine triphosphate; 1-methyl-6-butyl pseudouridine triphosphate; 1-methyl-6-chloro pseudouridine triphosphate; 1-methyl-6-cyano pseudouridine triphosphate; 1-methyl-6-dimethylamino pseudouridine triphosphate; 1-methyl-6-ethoxy pseudouridine triphosphate; 1-methyl-6-ethylcarboxylate pseudouridine triphosphate; 1-methyl-6-ethyl pseudouridine triphosphate; 1-methyl-6-fluoro pseudouridine triphosphate; 1-methyl-6-formyl pseudouridine triphosphate; 1-methyl-6-hydroxyamino pseudouridine triphosphate; 1-methyl-6-hydroxy pseudouridine triphosphate; 1-methyl-6-iodo pseudouridine triphosphate; 1-methyl-6-isopropyl pseudouridine triphosphate; 1-methyl-6-methoxy pseudouridine triphosphate; 1-methyl-6-methylamino pseudouridine triphosphate; 1-methyl-6-phenyl pseudouridine triphosphate; 1-methyl-6-propyl pseudouridine triphosphate; 1-methyl-6-tert-butyl pseudouridine triphosphate; 1-methyl-6-trifluoromethoxy pseudouridine triphosphate; 1-methyl-6-trifluoromethyl pseudouridine triphosphate; 1-morpholinylmethyl pseudouridine triphosphate; 1-pentyl-pseudouridine triphosphate; 1-phenyl pseudouridine triphosphate; 1-pivaloyl pseudouridine triphosphate; 1-propynyl pseudouridine triphosphate; 1-propyl pseudouridine triphosphate; 1-propynyl pseudouridine; 1-p-tolyl pseudouridine triphosphate; 1-tert-butyl pseudouridine triphosphate; 1-thiomethoxymethyl pseudouridine triphosphate; 1-thiomorpholinylmethyl pseudouridine triphosphate; 1-trifluoroacetyl pseudouridine triphosphate; 1-trifluoromethyl pseudouridine triphosphate; 1-vinyl pseudouridine triphosphate;2,2’-anhydrouridine TP; 2’-bromodeoxyuridine TP; 2’-F-5-methyl-2’-deoxy-UTP; 2’-OMe-5-Me-UTP; 2’-OMe-pseudo-UTP; 2’-α-ethynyluridine TP; 2’-α-trifluoromethyluridine TP; 2’-β-ethynyluridine TP; 2’-β-trifluoromethyluridine TP; 2’-deoxy-2’,2’-difluorouridine TP; 2’-deoxy-2’-α-mercaptouridine TP; 2’-deoxy-2’-α-thiomethoxyuridine TP; 2’-deoxy-2’-β-aminouridine TP; 2’-deoxy-2’-β-azidouridine TP; 2’-deoxy-2’-β-bromouridine TP; 2’-deoxy-2’-β-chlorouridine TP; 2’-deoxy-2’-β-fluorouridine TP; 2’-deoxy-2’-β-iodouridine TP; 2’-deoxy-2’-β-mercaptouridine TP; 2’-deoxy-2’-β-thiomethoxyuridine TP; 2-methoxy-4-thiouridine; 2-methoxyuridine; 2’-O-methyl-5-(1-propynyl)uridine TP; 3-alkylpseudo-UTP; 4’-azidouridine TP; 4’-carbocyclic uridine TP; 4’-ethynyluridine TP; 5-(1-propynyl)arauridine TP; 5-(2-furyl)uridine TP; 5-cyano uridine TP; 5-dimethylamino uridine TP; 5’-homo uridine TP; 5-iodo-2’-fluorodeoxyuridine TP; 5-phenylethynyluridine TP; 5-trideuteriomethyl-6-deutero uridine TP; 5-trifluoromethyluridine TP; 5-vinylarauridine TP; 6-(2,2,2-trifluoroethyl)-pseudo-UTP; 6-(4-morpholinyl)-pseudo-UTP; 6-(4-thiomorpholinyl)-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-aminopseudo-UTP; 6-azidopseudo-UTP; 6-bromopseudo-UTP; 6-butylpseudo-UTP; 6-chloropseudo-UTP; 6-cyanopseudo-UTP; 6-dimethylaminopseudo-UTP; 6-ethoxypseudo-UTP; 6-carboxyethylpseudo-UTP; 6-ethylpseudo-UTP; 6-fluoropseudo-UTP; 6-formylpseudo-UTP; 6-hydroxyaminopseudo-UTP; 6-hydroxypseudo-UTP; 6-iodopseudo-UTP; 6-isopropylpseudo-UTP; 6-methoxypseudo-UTP; 6-methylaminopseudo-UTP; 6-methylpseudo-UTP; 6-phenylpseudo-UTP; 6-phenylpseudo-UTP; 6-propylpseudo-UTP; 6-tert-butylpseudo-UTP; 6-trifluoromethoxypseudo-UTP; 6-trifluoromethylpseudo-UTP; α-thiopseudo-UTP; pseudouridine 1-(4-methylbenzenesulfonate) TP; pseudouridine 1-(4-methylbenzoate) TP; pseudouridine TP 1-[3-(2-ethoxy)]propionate; pseudouridine TP1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionate; pseudouridine TP 1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy)-ethoxy}-ethoxy]-ethoxy}]propionate;Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP diethyl 1-methylphosphonate; Pseudouridine TP-N1-3-propionic acid; Pseudouridine TP-N1-4-butyric acid; Pseudouridine TP-N1-5-valeric acid; Pseudouridine TP-N1-6-caproic acid; Pseudouridine TP-N1-7-enanthic acid; Pseudouridine TP-N1-methyl-p-benzoic acid; Pseudouridine TP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowybutosine; Peroxywybutosine; Hypomodified hydroxywybutosine; 4-Demethylisowybutosine; 2,6-(Diamino)purine; 1-(Aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diazabenzene)-2-(oxo)-phenothiazin-1-yl; 1,3-(Diazabenzene)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(Triaza)-2,6-(dioxo)-naphthalene; 2(Amino)purine; 2,4,5-(Trimethyl)phenyl; 2'-Methyl,2'-amino,2'-azido,2'-fluoro-cytidine; 2'-Methyl,2'-amino,2'-azido,2'-fluoro-adenine; 2'-Methyl,2'-amino,2'-azido,2'-fluoro-uridine; 2'-Amino-2'-deoxyribose; 2-Amino-6-chloropurine; 2-Azidoinosine; 2'-Azido-2'-deoxyribose; 2'-Fluoro-2'-deoxyribose; 2'-Fluoro-modified base; 2'-O-Methylribose; 2-Oxo-7-aminopyridopyrimidin-3-yl; 2-Oxo-pyridopyrimidin-3-yl; 2-Pyridone; 3-Nitropyrrole; 3-(Methyl)-7-(propynyl)isoquinolinyl; 3-(Methyl)isoquinolinyl; 4-(Fluoro)-6-(methyl)benzimidazole; 4-(Methyl)benzimidazole; 4-(Methyl)indolyl; 4,6-(Dimethyl)indolyl; 5-Nitroindole; 5-Substituted pyrimidine; 5-(Methyl)isoquinolinyl; 5-Nitroindole; 6-(Aza)pyrimidine; 6-(Aza)thymine; 6-(Methyl)-7-(aza)indolyl; 6-Chloropurine; 6-Phenylpyrrolopyrimidin-2-one-3-yl; 7-(Aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(Aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(Aminoalkylhydroxy)-1,3-(diazabenzene)-2-(oxo)-phenoxazin-1-yl; 7-(Aminoalkylhydroxy)-1,3-(diazabenzene)-2-(oxo)-phenothiazin-1-yl; 7-(Aminoalkylhydroxy)-1,3-(diazabenzene)-2-(oxo)-phenoxazin-1-yl; 7-(Aza)indolyl; 7-(Guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinyl;7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl; 7-(guanidylalkyl-hydroxy)-1,3-(diaz)-2-(oxo)-phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl; 7-(propargyl)isoquinolyl; 7-(propargyl)isoquinolyl, propargyl-7-(aza)indolyl; 7-deazainosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidazopyridyl; aminoindolyl; anthryl; bis-o-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl; bis-o-substituted-6-phenylpyrrolopyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridyl; inosinyl; isoquinolyl; isoguanosine; N2-substituted purine; N6-methyl-2-aminopurine; N6-substituted purine; N-alkylated derivative; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; Nubularine; O6-substituted purine; O-alkylated derivative; o-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl; o-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl; Oxoformycin TP; p-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl; p-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl; pentacenyl; Phenanthracenyl; phenyl; propargyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolopyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrazinyl; Stilbenzyl; substituted 1,2,4-triazole; Tetracenyl; Tubercidine; xanthine; xanthosine-5'-TP; 2-thiozebularine; 5-azathiozebularine; 7-deaza-2-aminopurine; pyridin-4-one ribonucleoside; 2-aminoriboside-TP; formycin A TP; formycin B TP; Pyrrolosine TP; 2'-OH-arabinosyladenosine TP; 2'-OH-arabinosylcytidine TP; 2'-OH-arabinouridine TP; 2'-OH-arabinosylguanosine TP; 5-(2-methoxycarbonylviny)uridine TP;or N6-(19-aminopentadecaoxanonadecyl)adenosine TP;

[0358] In some embodiments, the percentage of canonical A substituted by a nucleotide modified with a substitutable A (such as those nucleotides above) is at least: 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9% or 100%. In some embodiments, the percentage of canonical A substituted by m 6 A can be 0.1 - 5%, particularly 0.5 - 2%, particularly 0.8 - 1.2%, such as about 1% (or 1%); in these embodiments, the RNA can be circular RNA. The low substitution level of canonical A substituted by m 6 A (such as 1%) has been shown to inhibit innate immune activation

[21] . In some embodiments, the percentage of canonical C substituted by a nucleotide modified with a substitutable cytosine (such as those nucleotides above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9% or 100%. In some embodiments, the percentage of canonical G substituted by a nucleotide modified with a substitutable G (such as those nucleotides above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9% or 100%. In preferred embodiments, the percentage of canonical U substituted by a nucleotide modified with a substitutable U (such as those nucleotides above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or preferably 100%; more preferably substituted by 1mΨ and / or Ψ (even more preferably 1mΨ).

[0359] In a preferred embodiment, the one or more modified ribonucleotides detailed above are or comprise 1mΨ and / or Ψ, more preferably 1mΨ. In such embodiments, the RNA may comprise 1mΨ and / or Ψ and not comprise standard U ribonucleotides or other modified U ribonucleotides (i.e., there are no standard U ribonucleotides or modified U ribonucleotides other than 1mΨ and / or Ψ in the RNA; i.e., 100% U substitution). In particular, the RNA may comprise 1mΨ and / or Ψ and not comprise standard U ribonucleotides or other modified ribonucleotides (i.e., there are no standard U nucleotides or any type of modified ribonucleotide other than 1mΨ and / or Ψ (capable of substituting A, C, G, or U) in the RNA; i.e., 100% U substitution and no other modified nucleotides are allowed). The RNA may comprise Ψ and not comprise standard U ribonucleotides or other modified U ribonucleotides (i.e., 100% U is substituted by Ψ). In particular, the RNA may comprise Ψ and not comprise standard U ribonucleotides or other modified ribonucleotides (i.e., 100% U is substituted by Ψ and no other modified nucleotides are allowed). More preferably, the RNA comprises 1mΨ and not comprise standard U ribonucleotides or other modified U ribonucleotides (i.e., 100% U is substituted by 1mΨ). In an even more preferred embodiment, the RNA comprises 1mΨ and not comprise standard U ribonucleotides or other modified ribonucleotides (i.e., 100% U is substituted by 1mΨ and no other modified nucleotides are allowed). In the embodiments of this paragraph, "[may] comprise... and not comprise [X]... or [Y]" may be used interchangeably with the phrase "[may] comprise... and not comprise... [X] and / or [Y]".

[0360] Preferably, the RNA is codon-optimized. Codon optimization can provide an increased GC content relative to non-codon-optimized RNA encoding the same protein(s). The GC content (percentage of all ribonucleotides that are G or C in the RNA (alternatively defined as all "nitrogenous bases")) can 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 can 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 can be 30 - 70%, such as 40 - 70%, 45 - 70%, 50 - 70% or 55 - 70%. Codon optimization can provide an increased C content relative to non-codon-optimized RNA encoding the same protein(s). As a result of codon optimization, the percentage of C-optimisable codons in the RNA that have been replaced with codons having a greater C content (while encoding the same amino acid) can be at 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%; as a result of codon optimization, the percentage of C-optimisable codons in the RNA that have been replaced with codons having a greater C content (while encoding the same amino acid) can be 30 - 80%, such as 40 - 90%, 45 - 90%, 50 - 80%, 55 - 80% or 60 - 80%, preferably 65 - 75%, 66 - 75%, 67 - 75%, 68 - 75%, 69 - 75%, 70 - 74%, 71 - 74% or 72 - 74%.

[0361] Generally, the RNA comprises a 5' and / or 3' untranslated region (UTR), preferably both a 5' UTR and a 3' UTR; for example, the 5' UTR and 3' UTR of an RNA transcript selected from the following genes (preferably the following human genes): beta-actin, albumin, ATP synthase beta subunit, fibroblast activation protein ("FAP"), histone cluster 1 H4 family member 15 ("HIST2H4A"), glyceraldehyde-3-phosphate dehydrogenase, heat shock protein family A (Hsp70) member 8 gene, interleukin-2 gene ("IL-2") and transferrin. In some preferred embodiments, the RNA comprises a 5' UTR and a 3' UTR selected respectively from:

[0362] - SEQ ID NO:61 and 62,

[0363] - SEQ ID NO:63 and 64,

[0364] - SEQ ID NOs: 65 and 66,

[0365] - SEQ ID NOs: 67 and 68,

[0366] - SEQ ID NOs: 69 and 70, and

[0367] - RNA sequences that are at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 61, 63, 65, 67 or 69 (for the 5’ UTR), and RNA sequences that are at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 62, 64, 66, 68 or 70 (for the 3’ UTR) (in particular, pairs of 5’ UTR and 3’ UTR that have such identity with SEQ ID NO: 61 and 62, SEQ ID NO: 63 and 64, SEQ ID NO: 65 and 66, SEQ ID NO: 67 and 68, and SEQ ID NO: 69 and 70 respectively);

[0368] wherein the RNA sequences according to SEQ ID NO: 61 and 62, SEQ ID NO: 67 and 68, SEQ ID NO: 69 and 70 (and RNA sequences having such identity therewith, preferably at least 95% or higher) are more preferred; and the RNA sequences according to SEQ ID NO: 61 and 62 (and RNA sequences having such identity therewith, preferably at least 95% or higher) are even more preferred.

[0369] Both the 3’ and 5’ UTRs can affect the expression of the RSV-F protein of the present disclosure through multiple mechanisms. Without being bound by this theory, the 5’ UTR can at least affect the expression of the RSV-F protein of the present disclosure, for example, through pre-initiation complex regulation, closed-loop regulation, upstream open reading frame regulation (i.e., re-initiation), providing internal ribosome entry sites, and providing microRNA binding sites. Without being bound by this theory, the 3’ UTR can at least affect the expression of the RSV-F protein of the present disclosure, for example, by providing regulatory regions that affect expression post-transcriptionally; for example, affecting translation efficiency, the localization of the RNA, the stability of the RNA, polyadenylation, and the circularization of the RNA.

[0370] In a specific embodiment, the RNA is circular RNA.

[0371] In a preferred embodiment, the RNA meets any 2, 3, 4 or 5 of the following criteria (e.g., (a), (b), (d) and (f); (a), (b), (c), (d) and (f); or (a), (b), (d), (e) and (f)):

[0372] (a) is non-self-replicating;

[0373] (b) is single-stranded;

[0374] (c) contains a 5' cap which is 7'-methylguanosine linked 5' to 5' by a triphosphate bridge to the 5' first ribonucleotide, and wherein the first 5' ribonucleotide contains 2'-O-methylated ribose (2'-O-Me);

[0375] (d) contains a 3' polyA tail;

[0376] (e) contains 1mΨ and does not contain standard U ribonucleotides or other modified ribonucleotides;

[0377] (f) contains 5' and 3' UTRs.

[0378] More preferably, the RNA meets all of the above criteria (a)-(f).

[0379] Generally, the RNA will contain, in the 5' to 3' direction: a 5' cap, a 5' UTR, an open reading frame encoding at least the RSV-F protein of the present disclosure, a 3' UTR and a 3' polyA tail (in particular, the 5' cap as detailed above throughout this subsection; the 5' UTR, 3' UTR and 3' polyA tail).

[0380] In a preferred embodiment, the RNA comprises the following sequence or consists of the following sequence:

[0381] SEQ ID NO:71; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded according thereto);

[0382] SEQ ID NO:142; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0383] SEQ ID NO:72; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0384] SEQ ID NO:143; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0385] SEQ ID NO:73; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0386] SEQ ID NO:74; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0387] SEQ ID NO:75; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0388] SEQ ID NO:76; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0389] SEQ ID NO:77; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0390] SEQ ID NO:144; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0391] SEQ ID NO:78; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0392] SEQ ID NO:115; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0393] SEQ ID NO:116; or an RNA sequence that is 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 the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0394] SEQ ID NO:117; or an RNA sequence that is 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 substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0395] SEQ ID NO:79; or an RNA sequence that is 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 substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0396] SEQ ID NO:145; or an RNA sequence that is 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 substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0397] SEQ ID NO:118; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0398] SEQ ID NO:119; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0399] SEQ ID NO:120; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby); or

[0400] SEQ ID NO:80; or an RNA sequence that is 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 substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby).

[0401] In a further independent aspect, the present disclosure also provides a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising any one of SEQ ID NOs:71 - 80, or any of the foregoing sequences having identity with any one of SEQ ID NOs:71 - 80.

[0402] In a preferred embodiment, the RNA comprises an open reading frame (ORF) comprising or consisting of the following sequence:

[0403] positions 32 - 1753 of SEQ ID NO:71; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0404] Positions 32 - 1753 of SEQ ID NO:142; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0405] Positions 32 - 1753 of SEQ ID NO:72; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0406] Positions 32 - 1753 of SEQ ID NO:143; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0407] positions 32 - 1753 of SEQ ID NO:73; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0408] positions 32 - 1753 of SEQ ID NO:74; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0409] positions 32 - 1753 of SEQ ID NO:75; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0410] positions 32 - 1753 of SEQ ID NO:76; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the said positions, preferably encoding an RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0411] positions 32 - 1753 of SEQ ID NO:77; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the said positions, preferably encoding an RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0412] positions 32 - 1753 of SEQ ID NO:144; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the said positions, preferably encoding an RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0413] positions 32 - 1753 of SEQ ID NO:78; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said position, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0414] positions 32 - 1744 of SEQ ID NO:115; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said position, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0415] positions 32 - 1693 of SEQ ID NO:116; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said position, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0416] Positions 32-1678 of SEQ ID NO:117; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said position, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0417] Positions 32-1753 of SEQ ID NO:79; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said position, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0418] Positions 32-1753 of SEQ ID NO:145; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said position, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0419] positions 32 - 1744 of SEQ ID NO:118; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0420] positions 32 - 1693 of SEQ ID NO:119; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0421] positions 32 - 1678 of SEQ ID NO:120; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby); or

[0422] Positions 32 - 1753 of SEQ ID NO:80; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding the RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby).

[0423] In a further independent aspect, the present disclosure also provides a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising an ORF; said ORF comprising or consisting of the following sequence: positions 32 - 1753 of any one of SEQ ID NO:71 - 80, or any of the foregoing sequences having identity with positions 32 - 1753 of any one of SEQ ID NO:71 - 80.

[0424] Nucleic acid (e.g., RNA) alignments can be performed by, for example, visual inspection or any well-known algorithm; for example, using the NCBI BLAST algorithm, such as "megablast", for example using default settings (e.g., available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch); or for example using the "Muscle" algorithm (see, e.g.,

[22] ,

[23] ), for example using default settings; wherein the Muscle algorithm is preferred. The corresponding nucleotide or ribonucleotide positions are readily identifiable to the skilled person and can be identified by aligning the nucleotide or ribonucleotide sequences using any well-known method (e.g., visual inspection or algorithm, such as the methods detailed above).

[0425] The RNA can be conveniently prepared by in vitro transcription (IVT). IVT can use a (DNA) template that is produced and propagated in bacteria in plasmid form, or is produced synthetically (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). For example, a DNA-dependent RNA polymerase (such as phage T7, T3 or SP6 RNA polymerase) can be used to transcribe the replicated RNA from the DNA template. Appropriate capping and polyA addition reactions can be used as needed (although the polyA tail is usually encoded within the DNA template).

[0426] Vector containing nucleic acid encoding RSV - F protein in the pre - fusion conformation

[0427] Nucleic acids (especially RNA) are not protected per se, may be degraded by the subject's nucleases, and may require a vector to facilitate entry into target cells. Accordingly, the present disclosure also provides a vector that comprises a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure. The vector can be lipid-based (such as lipid nanoparticles or cationic nanoemulsions), polymer-based (such as comprising polyamines, dendrimers, and / or copolymers), peptide- or protein-based (such as comprising protamine, cationic cell-penetrating peptides, and / or anionic peptides conjugated to a positively charged polymer), cell-based (such as antigen-presenting cells, such as dendritic cells loaded with the nucleic acid), or virus-based (such as virus replicon particles). In a specific embodiment, the vector is non-viral particle, i.e., free or substantially free of a viral capsid.

[0428] In particular, lipid-based vectors provide a means of protecting the nucleic acid (preferably RNA), e.g., by encapsulation, and delivering it to target cells for protein expression. In certain embodiments, the lipid-based vector is or comprises a cationic nanoemulsion ("CNE"). CNE and methods for their preparation are described, for example, in

[24] . For CNE, the nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure is complexed with CNE particles, especially 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 particle. In a specific embodiment, the lipid-based vector is a lipid inorganic nanoparticle ("LION").

[0429] LNP

[0430] In a preferred embodiment, the nucleic acid (preferably RNA) is encapsulated in a lipid nanoparticle (LNP). Accordingly, in a preferred embodiment, the present invention also provides an LNP that encapsulates a nucleic acid (preferably RNA) encoding the RSV-F protein of the present invention.

[0431] Multiple such LNPs will be part of a composition (e.g., a pharmaceutical composition as detailed in the section titled "Pharmaceutical Compositions" below) that contains free and / or encapsulated nucleic acids (preferably RNA), and in some embodiments, these 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. The diameter of at least 80% of these LNPs in the composition can be 20 - 200 nm, 40 - 190 nm, 60 - 180 nm, or particularly 80 - 160 nm. In a specific embodiment, the diameter of substantially all or all of the LNPs in the composition is 20 - 200 nm, 40 - 190 nm, 60 - 180 nm, or particularly 80 - 160 nm.

[0432] The LNP can include multilamellar vesicles (MLV), small unilamellar vesicles (SUV), or large unilamellar vesicles (LUV).

[0433] The amount of nucleic acid (preferably RNA) per LNP can vary, and the number of individual nucleic acid molecules per LNP can depend on the characteristics of the particles used. For RNA molecules, generally, an LNP can contain 1 - 500 RNA molecules, such as <200, <100, <50, <20, <10, <5 or 1 - 4. Generally, an LNP contains fewer than 10 different RNA species, such as fewer than 5, 4, 3, or 2 different species. Preferably, the LNP contains a single RNA species (i.e., all RNA molecules in the particle have the same sequence).

[0434] The LNPs according to the present disclosure can be formed from a single lipid (e.g., a cationic lipid), or particularly, from a mixture of lipids. In particular, the mixture contains various classes of lipids, such as:

[0435] (a) A mixture of a cationic lipid and a sterol,

[0436] (b) A mixture of a cationic lipid and a neutral lipid,

[0437] (c) A mixture of cationic lipids and polymer-conjugated lipids,

[0438] (d) A mixture of cationic lipids, sterols and polymer-conjugated lipids, or

[0439] (e) A mixture of cationic lipids, neutral lipids and polymer-conjugated lipids;

[0440] Or preferably:

[0441] (f) A mixture of cationic lipids, sterols and neutral lipids;

[0442] Or more preferably:

[0443] (g) A mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids.

[0444] Other classes of lipids, such as anionic lipids, may also be present in the mixture of lipids.

[0445] The pKa of the cationic lipid can be 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 different from the pKa of the entire LNP (sometimes referred to as the "apparent pKa"). The pKa can be determined by any well-known method, such as by fluorescence assay with toluene nitrosulfonic acid (TNS) or acid-base titration; preferably by TNS fluorescence assay; more preferably according to Example 8.

[0446] The cationic lipid preferably comprises a tertiary amine or quaternary amine group, more preferably a tertiary amine group. Exemplary cationic lipids comprising a tertiary amine group include: 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinolenylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linolenyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinolenyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinolenyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinolenylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinolenyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinolenyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinolenyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA), dilinolenylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA); or MC3 (see, e.g.,

[25] ).

[0447] In some embodiments, the cationic lipid has the structure of the following lipids: RV28, RV31, RV33, RV37, RV39, RV42, RV44, RV73, RV75, RV81, RV84, RV85, RV86, RV88, RV91, RV92, RV93, RV94, RV95, RV96, RV97, RV99 or RV101, as disclosed in

[26] . In further embodiments, the cationic lipid has the following structure:

[0448]

[0449] In preferred embodiments, the cationic lipid has the following structure:

[0450]

[0451] (also known as lipid RV39).

[0452] In another preferred embodiment, the cationic lipid has the following structure:

[0453]

[0454] In another preferred embodiment, the cationic lipid has the following structure:

[0455]

[0456] The lipids in the LNP can comprise (in mol%) 20 - 80, 25 - 75, 30 - 70 or 35 - 65%, preferably 30 - 60, 40 - 55 or 40 - 50% cationic lipid; 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 can comprise (in mol%) at least 20, 25 or at least 35%, or preferably at least 40% cationic lipid. The lipids in the LNP can comprise (in mol%) no more than 80, 70 or no more than 60% or preferably no more than 50% cationic lipid.

[0457] The molar ratio of the protonatable nitrogen atoms in the cationic lipid of the LNP to the phosphate in the nucleic acid (preferably RNA), also known as the "N:P" ratio, can be in the following ranges (including the endpoints): in the range of 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.

[0458] The polymer-conjugated lipid is preferably a PEGylated lipid. In the LNP, the average molecular weight of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 (or 2.0 kDa). The average molecular weight of such PEG can be expressed as the median molecular weight. In the LNP, the weight-average molecular weight of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 (or 2.0 kDa). Alternatively, in the LNP, the number-average molecular weight of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 (or 2.0 kDa). Alternatively, in the LNP, the molecular weight of at least 80% of the PEG of such PEGylated lipid can be 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.

[0459] The PEGylated lipid can have the following structure:

[0460]

[0461] Exemplary PEGylated lipids include 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000, 1,2-dimyristoyl-sn-glycero-2-phosphoethanolamine-N-[methoxy(polyethylene glycol)], and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol. Preferably, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

[0462] The lipids in the LNP can comprise (in mol %) 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); 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 can comprise (in mol %) 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 can comprise (in mol %) no more than 8.0%, 6.0%, or 4.0%, or preferably no more than 3.0% polymer-conjugated lipid (preferably PEGylated lipid).

[0463] Preferably, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), although other neutral lipids available to those skilled in the art can also be used.

[0464] The lipids in the LNP can comprise (in mol %) 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; 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 can comprise (in mol %) 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 can comprise (in mol %) no more than 15.0, 13.0, or no more than 12.0%, or preferably no more than 11.0% neutral lipid.

[0465] Exemplary sterols include cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, coprosterol, campesterol, 14-demethyl lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydro lanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholesta-8(9),14-dien-3β-ol (dihydro-FF-MAS-d6), 4,4-dimethyl(d6)-cholesta-8(9)-en-3β-ol (dihydro T-MAS-d6), ergosterol, sitostanol, campestanol, campestanol, 7-dehydrodesmosterol, pregnenolone, 4,4-dimethyl-cholesta-8(9)-en-3β-ol (dihydro T-MAS), Δ5-avenasterol, brassicasterol, dihydro FF-MAS, 24-methylenecholesterol, oxysterol, deuterated sterol, fluorinated sterol, sulfated sterol, phosphorylated sterol, A-ring substituted sterol, cholest-5-ene-3β,4β-diol, 5α-cholestan-3β-ol, 4-cholesten-3-one, cholesta-8(9),24-dien-3-one, cholesta-8(9),24-dien-3-one, 2,2,3,4,4-pentadeuterio-5α-cholestan-3β-ol, cholesteryl phosphocholine, cholesterol-d7 pentadecanoate, cholesterol-d7 palmitate, B-ring substituted sterol, cholestanol, 5β,6β-epoxy-d7, 3β-hydroxy-5-cholesten-7-one, 6α-hydroxy-5α-cholestane, cholestanol, 5α,6α-epoxy, cholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, cholestanol, 5α,6α-epoxy-d7, Δ5,7-cholestenol, cholest-5,8(9)-dien-3β-ol, cholest-5,8(14)-dien-3β-ol, 7α-hydroxy-4-cholesten-3-one, ergosterol-d7, ergosterol, 7-dehydrodesmosterol, 3β,5α-dihydroxy-cholestan-6-one, D-ring substituted sterol, 3β-hydroxy-5α-cholesta-8(14)-en-15-one, 3β-hydroxy-5α-cholestan-15-one, 5α-cholesta-8(14)-ene-3β,15α-diol, 5α-cholesta-8(14)-ene-3β,15β-diol, lanosterol-95, 5α-7,24-cholestadiene, 14-dehydroergosterol, ergosta-5,7,9(11),22-tetraen-3β-ol, cholest-5-ene-3β,25-diol, cholest-(25R)-5-ene-3β,27-diol, 24(R / S),25-epoxycholesterol, 24(S),25-epoxycholesterol, 24(R / S),25-epoxycholesterol-d6, cholest-5-ene-3β,22(S)-diol, cholest-5-ene-3β,22(R)-diol, cholest-5-ene-3β,24(S)-diol, cholest-5-ene-3β,24(R)-diol, 27-hydroxy-4-cholesten-3-one, campestanol, N,N-dimethyl-3β-hydroxy cholenoamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3β-hydroxy cholenoamide, 25,27-dihydroxycholesterol, 5-cholestene-3β,20α-diol, 24S,25-epoxy-5α-cholest-8(9)-en-3β-ol, 24(S / R),25-epoxylanost-8(9)-en-3β-ol, 7-keto-27-hydroxycholesterol, 7α,27-dihydroxy-4-cholesten-3-one, 7α,27-dihydroxycholesterol, 7β,27-dihydroxycholesterol, 5α,6β-dihydroxy campestanol, 7α,25-dihydroxycholesterol, 7β,25-dihydroxycholesterol, 7α,24(S)-dihydroxycholesterol, 7α,24(S)-dihydroxy-4-cholesten-3-one, 7-keto-25-hydroxycholesterol, 7α,24S,27-trihydroxycholesterol, dihydrotestosterone, testosterone, estrone, estrogen, estradiol, corticosterone, cortisol or 24S,27-dihydroxycholesterol.,

[0466] Preferably, the sterol is cholesterol or a cholesterol-based lipid (such as any of the lipids provided in the previous paragraph).

[0467] The lipids in the LNP can comprise (in mol %) 20 - 80, 25 - 80, 30 - 70, 30 - 60, 35 - 60 or 40 - 60%, preferably 40 - 50% or 41 - 49% sterol; 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 can comprise (in mol %) at least 20, 30 or at least 35%, or preferably at least 40% or at least 41% sterol. The lipids in the LNP can comprise (in mol %) no more than 80, 70 or no more than 60%, or preferably no more than 50% sterol.

[0468] The lipids in the LNP can have the following combinations in mol %: 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%).

[0469] Such LNPs encapsulating nucleic acids (preferably RNA) can be formed by admixing a first solution containing the nucleic acid with a second solution containing the lipids that form the LNP. The admixing can be carried out by any suitable method available to the person skilled in the art, such as a T-mixer, a microfluidic or an impinging jet mixer. Filtration can be carried out after admixing to obtain the desired LNP size distribution (e.g., those distributions detailed above in this subsection). The filtration can be carried out by any suitable method available to the person skilled in the art, such as tangential flow filtration or cross-flow filtration.

[0470] According to a further independent aspect, the present disclosure provides a method for preparing an LNP encapsulating a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure, comprising admixing a first solution containing the nucleic acid and a second solution containing the lipids that form the LNP (e.g., using the methods described in the foregoing paragraph); and optionally filtering the obtained admixture (e.g., using the methods described in the foregoing paragraph).

[0471] Pharmaceutical composition

[0472] In a further independent aspect, the present disclosure also provides a pharmaceutical composition comprising the RSV-F protein, nucleic acid (preferably RNA) and / or carrier (preferably lipid nanoparticle) of the present disclosure. Such compositions generally further comprise a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known in the art, see, for example,

[27] . Such compositions are generally used to immunize a subject against a disease, preferably against RSV. Thus, the pharmaceutical compositions of the present disclosure are generally regarded as vaccine compositions.

[0473] The pharmaceutical compositions of the present disclosure can comprise the RSV-F protein, nucleic acid (preferably RNA) and / or carrier (preferably lipid nanoparticle) in pure water (e.g., "w.f.i") or in a buffer (e.g., phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer or citrate buffer). The buffer salts will generally be included in the range of 5 - 20 mM.

[0474] The pH value of the pharmaceutical compositions of the present disclosure can be between 5.0 and 9.5, for example between 6.0 and 8.0.

[0475] The pharmaceutical compositions of the present disclosure can comprise sodium salts (e.g., sodium chloride) to provide tonicity. A concentration of 10 ± 2 mg / mL NaCl is typical, for example about 9 mg / mL (or 9 mg / mL).

[0476] The pharmaceutical compositions of the present disclosure may comprise metal ion chelators (in particular, in embodiments where such compositions comprise RNA). These chelators can extend RNA stability by removing ions that can accelerate phosphodiester hydrolysis. Thus, such compositions may comprise one or more of the following: EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typically present at 10 - 500 μM (e.g., 0.1 mM). Citrates (such as sodium citrate) can also act as chelators and advantageously also provide buffering activity.

[0477] The osmolality of the pharmaceutical compositions of the present disclosure can be between 200 mOsm / kg and 400 mOsm / kg, such as between 240 - 360 mOsm / kg, or between 290 - 310 mOsm / kg.

[0478] The pharmaceutical compositions of the present disclosure may comprise one or more preservatives, such as thimerosal or 2 - phenoxyethanol. Mercury - free compositions are preferred, and vaccines can be prepared without preservatives.

[0479] The pharmaceutical compositions of the present disclosure can be aseptic or sterile.

[0480] The pharmaceutical compositions of the present disclosure can be pyrogen - free, for example containing < 1 EU (endotoxin unit, a standard measure) per dose, and preferably < 0.1 EU per dose.

[0481] The pharmaceutical compositions of the present disclosure can be gluten - free.

[0482] The pharmaceutical compositions of the present disclosure can be prepared in unit - dose form. In some embodiments, the volume of a unit dose can be between 0.1 - 1.0 mL, such as about 0.5 mL (or 0.5 mL).

[0483] The pharmaceutical compositions of the present disclosure can be prepared as injectable formulations, as solutions or suspensions. The composition can be prepared for pulmonary administration, for example by an inhaler, using a fine spray. The composition can be prepared for nasal, ear, or eye administration, for example as a spray or drops. Injectable formulations for intramuscular injection are typical.

[0484] The pharmaceutical composition of the present disclosure comprises an immunologically effective amount of RSV-F protein, nucleic acid (preferably RNA) and / or a carrier (preferably a lipid nanoparticle), and any other components as required. "Immunologically effective amount" means that administering this amount to an individual (administered as a single dose or as part of a series) is effective for treatment or prevention (preferably prevention of RSV). This amount varies depending on the health and physical condition of the individual to be treated, age, taxonomic group of the individual to be treated (such as non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection required, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. It is expected that this amount will fall within a relatively wide range that can be determined by routine testing. In embodiments where the pharmaceutical composition of the present disclosure comprises RNA, the RNA content will generally be expressed as the amount of RNA per dose. Preferred doses have ≤120 μg RNA, such as ≤100 μg (e.g., 10 - 120 μg or 10 - 100 μg, such as 10 μg, 25 μg, 50 μg, 75 μg or 100 μg, or about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg), but expression can be seen at much lower levels, such as ≤1 μg / dose, ≤100 μg / dose, ≤10 μg / dose, ≤1 μg / dose, etc.

[0485] The pharmaceutical composition of the present disclosure may further comprise an adjuvant (i.e., a reagent that enhances the immune response in a non-specific manner), particularly, but not limited to, when comprising the RSV-F protein of the present disclosure. Common adjuvants include: suspensions of minerals (such as alum, aluminum hydroxide, aluminum phosphate) that can adsorb RSV-F protein; emulsions, including water-in-oil and oil-in-water (and their variants, including double emulsions and reversible emulsions); liposaccharide, lipopolysaccharide, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll receptor agonists (particularly, TLR2, TLR4, TLR7 / 8 and TLR9 agonists), and various combinations of such components. In some embodiments, the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod. In some embodiments, the adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. The adjuvants described herein can be used alone or in any combination, such as alum / TLR7 (also known as AS37). The pharmaceutical composition of the present disclosure may comprise a saponin as an adjuvant, such as saponin fraction QS21 (see, for example

[28] ). QS21 can be used in a substantially pure form, such as at least 80% pure, such as at least 85%, 90%, 95% or at least 98% pure. Suitable QS-21 fractions are described in

[29] .

[0486] The pharmaceutical composition of the present disclosure (preferably when comprising lipid nanoparticles containing the nucleic acid (preferably RNA) of the present disclosure) can be lyophilized.

[0487] In some embodiments, the pharmaceutical composition of the present disclosure comprises (i) a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure, and (ii) other nucleic acids (preferably RNA) encoding at least one other protein. The nucleic acids of (i) and (ii) may be contained within the same vector (preferably a lipid nanoparticle) or within separate vectors (preferably lipid nanoparticles). In a preferred embodiment, the at least one other protein is an antigen; and thus may comprise or may be a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or an allergenic (i.e., derived from or originating from an allergen) antigen. The at least one other protein will typically be a pathogen antigen. The at least one other protein will typically be an antigen that is a surface polypeptide, such as a spike glycoprotein, hemagglutinin, adhesin, or envelope glycoprotein. In a specific embodiment, the at least one other protein is an antigen from or derived from a virus (especially a virus that causes respiratory diseases, especially a seasonal virus that causes respiratory diseases). In embodiments where the at least one other protein is an antigen from or derived from a virus, examples of such viruses include: coronaviruses, orthomyxoviruses, pneumoviridae, paramyxoviridae, poxviridae, picornaviruses, bunyaviruses, hepadnaviruses, filoviruses, togaviruses, flaviviruses, pestiviruses, hepadnaviruses, rhabdoviruses, caliciviridae, retroviruses, reoviruses, parvoviruses, herpesviruses, papovaviruses, and adenoviruses.

[0488] In a preferred embodiment, the at least one other protein encoded by the nucleic acid of (ii) is a other Paramyxoviridae protein (especially a Paramyxoviridae antigen). Useful other Paramyxoviridae proteins (especially, antigens) can be from orthoparamyxoviruses or metaparamyxoviruses, especially human RSV or human metapneumovirus (hMPV). Useful other hMPV antigens include, for example, the F, N, P, M, M2-1, and M2 antigens (especially, the F antigen). Such hMPV proteins (especially, antigens) can be from or derived from subtype A or B. In a preferred embodiment, the nucleic acid of (i) is an RNA encoding the RSV-F protein of the present disclosure, and the nucleic acid of (ii) is an RNA encoding an hMPV antigen (especially, the F antigen). In such RNA embodiments, the preferred group of patients (wherein the pharmaceutical composition can be used for therapy, especially vaccination) is infants (see the section entitled "Medical Use and Methods of Treatment" below). In addition to other RSV-F antigens (i.e., having a different amino acid sequence from the RSV-F protein of the present disclosure encoded by the nucleic acid), useful other human RSV antigens encoded by the nucleic acid of (ii) include, for example, the G, M1, M2-1, M2-2, P, L, N, NS1, NS2, and SH antigens. Such other human RSV proteins (especially, antigens, especially the F antigen) can be from or derived from subtype A or B, especially subtype B.

[0489] In a preferred embodiment, the at least one other protein encoded by the nucleic acid of (ii) is a coronavirus antigen. Useful coronavirus antigens can be from the SARS coronavirus, particularly SARS-CoV2. Useful coronavirus antigens (preferably SARS-CoV2 antigens) include spike, M, E, HE, nucleocapsid, Plpro, and 3CLPro proteins, particularly the spike protein. Preferably, the coronavirus antigen is the SARS-CoV2 spike protein. The SARS-CoV2 spike protein can be from any variant, such as Omicron (e.g., Omicron BA.1, BA.2, BA3, BA.4, or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma, or Delta. Preferably, the SARS-CoV2 spike protein comprises one or more mutations relative to the wild-type protein, particularly mutations of one or more (e.g., two) proline residues. The one or more mutations can be introduced to stabilize the SARS-CoV2 spike protein in the pre-fusion conformation. In a preferred embodiment, the nucleic acid of (i) is an RNA encoding the RSV-F protein of the present disclosure, and the nucleic acid of (ii) is an RNA encoding a coronavirus antigen, e.g., as detailed above. In such RNA embodiments, the preferred patient group (wherein the pharmaceutical composition can be used for therapy, particularly vaccination) is the elderly (see the section entitled "Medical Use and Methods of Treatment" below).

[0490] In another preferred embodiment, the at least one other protein encoded by the nucleic acid of (ii) is an orthomyxovirus antigen. Useful orthomyxovirus antigens can be from influenza A, B, or C viruses. Useful orthomyxovirus antigens (especially influenza A, B, or C virus antigens) include hemagglutinin, neuraminidase, and matrix M2 protein, especially hemagglutinin. Preferably, the orthomyxovirus antigen is influenza A virus hemagglutinin. The influenza A virus hemagglutinin can be from any subtype, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In a preferred embodiment, the nucleic acid of (i) is RNA encoding the RSV-F protein of the present disclosure, and the nucleic acid of (ii) is RNA encoding an orthomyxovirus antigen, for example, as detailed above. In such RNA embodiments, the preferred group of patients (wherein the pharmaceutical composition can be used in therapy, especially vaccination) is the elderly (see the section titled "Medical Use and Treatment Methods" below). In such RNA embodiments, the nucleic acid of (i) can encode the RSV-F protein of the present disclosure, the nucleic acid of (ii) can encode an orthomyxovirus antigen, for example, as detailed above, and a third nucleic acid can be present in the pharmaceutical composition, which can encode a coronavirus antigen, for example, as detailed in the foregoing paragraph above.

[0491] In a further independent aspect, the present disclosure also provides a delivery device (such as a syringe, nebulizer, atomizer, inhaler, skin patch, etc.) comprising the pharmaceutical composition of the present disclosure. This device can be used to administer the composition to a vertebrate subject.

[0492] In a further independent aspect, the present disclosure also provides a method of preparing a pharmaceutical composition, which includes formulating the RSV-F protein, nucleic acid (preferably RNA), or carrier (preferably lipid nanoparticle) of the present disclosure together with a pharmaceutically acceptable excipient to produce the composition. In particular, the pharmaceutical composition has the characteristics as detailed throughout this section above.

[0493] In a further independent aspect, the present disclosure also provides a kit comprising the RSV-F protein, nucleic acid, carrier, pharmaceutical composition, or delivery device of the present disclosure, as well as instructions for use.

[0494] Medical uses and treatment methods

[0495] In a further independent aspect, the present disclosure also provides the RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure for medical use. The use will generally be in a method for enhancing the immune response of a subject.

[0496] In a further independent aspect, the present disclosure also provides the use of the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure in the manufacture of a medicament. The medicament will generally be used to enhance the immune response of a subject.

[0497] In a further independent aspect, the present disclosure also provides a method of treatment comprising the step of administering to a subject (preferably a subject in need thereof) an effective amount of the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure. The method will generally be used to enhance the immune response of a subject.

[0498] The immune response is preferably protective and preferably involves antibody and / or cell-mediated immunity. Generally, the subject is a vertebrate, preferably a mammal, more preferably a human or a large veterinary mammal (such as a horse, cow, deer, goat, pig), and even more preferably a human.

[0499] The RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure can be used to prevent, alleviate or treat an infection or disease. Additionally or alternatively, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure can be used to prevent, alleviate or treat symptoms associated with an infection or disease. The infection is generally an infection caused by a virus of the family Pneumoviridae, and the disease is generally a disease associated with a virus of the family Pneumoviridae. In a preferred embodiment, the virus of the family Pneumoviridae is a orthopneumovirus, more preferably RSV, and even more preferably human RSV (including both its A and B subtypes).

[0500] Accordingly, the present disclosure also provides the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure; for treating or preventing RSV (preferably a method of vaccinating against RSV). The present disclosure also provides the use of the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating or preventing RSV (preferably wherein the medicament is a vaccine). The present disclosure also provides a method of inducing an immune response against RSV in a subject (preferably a method of vaccinating a subject against RSV) comprising administering to the subject an immunologically effective amount of the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure.

[0501] Vaccination according to the present disclosure can be prophylactic (i.e., preventing infection) or therapeutic (i.e., treating infection), but will typically be prophylactic. Such vaccination methods can include single-dose administration. Alternatively, such vaccination methods can include vaccination regimens (i.e., administration of multiple doses). Such regimens can involve repeated administration of immunologically identical protein antigens (in the form of or delivered by the RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure), particularly in the initial prime-boost regimen. In a prime-boost regimen, the first administration ("prime") can induce the proliferation and maturation of B and / or T cell precursors specific for one or more immunogenic epitopes present on the delivered antigen (induction phase). The second (and in some cases subsequent) administration ("boost") can further stimulate and potentially select the cellular recall response triggered by the previous administration(s). The different administrations can be given by the same or different routes, such as parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. The (one or more) prime administrations and the (one or more) boost administrations will be temporally separated, for example, separated by at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 months or more. In some embodiments, two prime administrations can be administered at intervals of 3-9 weeks (e.g., intervals of 4-9, 5-9, 6-9, 7-9, or 7-8 weeks, or an interval of about two months), followed by one or more boost administrations 4-14 months (e.g., 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, or 11-13 months, or about one year) after the second prime administration. In some embodiments, the prime administration is administered to an initial subject. In some embodiments, the protein antigen can be delivered in different formats or by different formats in the prime and boost administrations. For example, the protein antigen can be delivered as a protein for the (one or more) prime administrations and delivered by nucleic acid (particularly RNA, particularly by a vector comprising RNA) for the (one or more) boost administrations, and vice versa. Alternatively, different nucleic acid formats can be used, such as the protein antigen can be delivered by RNA (particularly by a vector comprising RNA) for the (one or more) prime administrations and also delivered by a viral vector (such as an adenovirus vector) for the (one or more) boost administrations, and vice versa.

[0502] The RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure will generally be administered directly to a subject. Direct delivery can be accomplished by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal or injection into the interstitial space of tissue). Alternative delivery routes include rectal, oral (e.g., tablets, sprays), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, otic, pulmonary or other mucosal administration. Preferably, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure will be administered intramuscularly or intradermally (especially by needle, such as a subcutaneous injection needle), more preferably intramuscularly.

[0503] The RSV-F protein, nucleic acid, lipid vector or pharmaceutical composition of the present disclosure can be used to elicit systemic and / or mucosal immunity.

[0504] Subjects according to the vaccination method of the present disclosure can be children (preferably infants) or adults (preferably the elderly or pregnant women). Immunocompromised individuals can also be subjects of such vaccination (whether children or adults).

[0505] Infant vaccination

[0506] In a preferred embodiment, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure is administered to an infant (preferably a human infant) as a subject of vaccination. The immune system of an infant is immature (see, e.g.,

[30] ), and thus this group is vulnerable to RSV infection and resulting diseases. Infant vaccination can prevent lower respiratory tract infections (especially bronchiolitis and (bronchial) pneumonia).

[0507] The infant can be less than one year old, such as less than: 11, 10, 9, 8, 7, 6, 5, 4 or less than 3 months of age. The infant can be ≥ one month old, such as ≥: 2, 3, 4, 5 or ≥ 6 months of age. Preferably, the infant is 2-6 months of age (i.e., within 2 and 6 months of age, and including 2 and 6 months of age), more preferably 2-4 months of age.

[0508] In a preferred embodiment, the infant is born to a female who has been administered an RSV vaccine (such as the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure) (preferably during pregnancy of the infant). In addition to the active immunity generated by the infant, the combination of maternal and infant vaccination can also advantageously provide passive transfer of maternal antibodies to the infant (i.e., via the placenta and / or breast milk).

[0509] Elderly vaccination

[0510] In another preferred embodiment, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure is administered to the elderly (preferably elderly humans) as a subject for vaccination. The elderly may suffer from age-related immunosenescence (reviewed in, for example,

[31] ), and thus this group of people is also vulnerable to RSV infection and resulting diseases. Vaccination of the elderly can prevent lower respiratory tract infections (in particular, pneumonia).

[0511] The elderly may be ≥50 years old, such as ≥: 55, 60, 65, 70, 75, 80, 85, 90, 95 or ≥100 years old. Preferably, the elderly are ≥60 or ≥65 years old (such as 60 - 120 or 65 - 120 years old).

[0512] Pregnant women vaccination

[0513] In another preferred embodiment, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure is administered to pregnant women (preferably pregnant human women) as a subject for vaccination. The primary purpose of maternal vaccination is to protect the infant from RSV infection at birth, for example, through the passive transfer of antibodies across the placenta and / or in breast milk.

[0514] The pregnant woman may be in the first trimester, second trimester or third trimester of her pregnancy, preferably the third trimester. The pregnant woman may be ≥20 weeks pregnant, such as ≥: 22, 24, 26, 28, 30, 32, 34, 36 or ≥38 weeks pregnant. Preferably, the pregnant woman is ≥28, ≥29 or ≥30 weeks pregnant (such as 28 - 43, 29 - 43 or 30 - 43 weeks pregnant).

[0515] Generally

[0516] Unless otherwise indicated, the practice of the present disclosure will employ conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology.

[0517] Unless the context clearly dictates otherwise, the singular terms "a", "an" and "the" include plural referents. Similarly, unless the context clearly dictates otherwise, the word "or" is intended to include "and". The term "plurality" refers to two or more. The term "at least one" refers to one or more.

[0518] Unless otherwise specified, when a numerical range is provided, it is inclusive, i.e., it includes the endpoints.

[0519] Unless otherwise indicated, the terms "at least", "not exceeding", and other such terms before a list of values apply to all members of the said list (and not only its first member).

[0520] The term "comprising" encompasses "including" as well as "consisting of", e.g., a composition "comprising" X can consist of only X or can include additional things, e.g., X + Y.

[0521] The term "about" associated with a numerical value x is optional and means, for example, x ± 10%.

[0522] The word "substantially" does not exclude "completely", e.g., a composition "substantially free of" Y can be completely free of Y. Where necessary, the word "substantially" can be omitted from the definitions of the present disclosure.

[0523] References to charges, cations, anions, etc. are taken at pH 7.

[0524] Embodiments

[0525] The present disclosure also provides the following numbered embodiments. The combinations of features of the present disclosure presented below are exemplary and should not be construed as exhaustive.

[0526] 1. An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises the following (a):

[0527] (ai) at least one mutation relative to the wild-type in the region corresponding to positions 38 - 60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38 - 60 of SEQ ID NO:1; and / or (aii) at least one mutation relative to the wild-type in the 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 residues selected from M, F, I, and V are introduced into the region by substitution or insertion.

[0528] 2. The RSV-F protein according to embodiment 1, which comprises (b) at least one mutation relative to the wild-type in the region corresponding to positions 208 - 216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208 - 216 of SEQ ID NO:1, and / or a P residue is introduced into the region by substitution or insertion.

[0529] 3. The RSV-F protein according to Embodiment 1 or 2, which comprises (c) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion; or (d) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0530] 4. An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises (b) at least one mutation relative to the wild type in the region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion.

[0531] 5. The RSV-F protein according to Embodiment 4, which comprises (c) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion; or (d) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0532] 6. An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises (c) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion; or (d) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0533] 7. The RSV-F protein according to any one of Embodiments 1-3, wherein the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 each comprise a beta sheet.

[0534] 8. The RSV-F protein according to any one of Embodiments 1-3 or 7, wherein the region corresponding to positions 38-60 of SEQ ID NO: 1 has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with positions 38-60 of SEQ ID NO: 1; and / or the region corresponding to positions 296-318 of SEQ ID NO: 1 has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with positions 296-318 of SEQ ID NO: 1.

[0535] 9. The RSV-F protein according to any one of Embodiments 1-3, 7 or 8, which comprises (a) a substitution at position 55 of SEQ ID NO: 1 with T, C, V, I or F; optionally T, C or V; optionally T or C.

[0536] 10. The RSV-F protein according to any one of Embodiments 1-3, 7, 8 or 9, which comprises (a) a substitution at position 55 of SEQ ID NO: 1 with T.

[0537] 11. The RSV-F protein according to any one of Embodiments 2-5 or 7-10, wherein the region corresponding to positions 208-216 of SEQ ID NO: 1 contains a loop.

[0538] 12. The RSV-F protein according to any one of Embodiments 2-5 or 7-11, wherein the region corresponding to positions 38-60 of SEQ ID NO: 1 has at least 50%, 60%, 75% or 85% sequence identity with positions 208-216 of SEQ ID NO: 1.

[0539] 13. The RSV-F protein according to any one of Embodiments 2-5, 7-11 or 12, which comprises (b) a substitution at position 215 of SEQ ID NO: 1 with A, P, V, I or F; optionally A or P.

[0540] 14. The RSV-F protein according to any one of Embodiments 2-5 or 7-13, which comprises (b) a substitution at position 215 of SEQ ID NO: 1 with A.

[0541] 15. The RSV-F protein according to any one of Embodiments 10-20, wherein the region corresponding to positions 345-352 of SEQ ID NO: 1 contains a beta sheet and a loop.

[0542] 16. The RSV-F protein according to any one of embodiments 3, 5, 6, or 7 - 15, wherein the region corresponding to positions 345 - 352 of SEQ ID NO:1 has at least 50%, 60%, 75%, or 85% sequence identity with positions 345 - 352 of SEQ ID NO:1.

[0543] 17. The RSV-F protein according to any one of embodiments 3, 5, 6, 7 - 15, 16, wherein it comprises a substitution of (c) position 348 of SEQ ID NO:1 with T or N.

[0544] 18. The RSV-F protein according to any one of embodiments 3, 5, 6, 7 - 15, 16, 17, wherein it comprises a substitution of (c) position 348 of SEQ ID NO:1 with N.

[0545] 19. The RSV-F protein according to embodiment 17 or 18, which comprises a glycan linked to position 348 of SEQ ID NO:1; optionally, wherein the glycan comprises N-acetylglucosamine.

[0546] 20. The RSV-F protein according to any one of embodiments 3, 5, 6, or 7 - 16, which comprises a substitution of (d) position 348 of SEQ ID NO:1 with N, D, F, H, K, L, N, Q, R, T, W, or Y; optionally N, F, H, K, N, Q, R, T, W, or Y; optionally N, F, R, W, or Y.

[0547] 21. The RSV-F protein according to embodiment 20, which comprises a substitution of (d) position 348 of SEQ ID NO:1 with N.

[0548] 22. The RSV-F protein according to any one of embodiments 1 - 19, which comprises:

[0549] (a) a substitution of position 55 of SEQ ID NO:1 with T, C, V, I, or F; optionally T, C, or V; optionally T or V;

[0550] (b) a substitution of position 215 of SEQ ID NO:1 with A, P, V, I, or F; optionally A or P; and

[0551] (c) a substitution of position 348 of SEQ ID NO:1 with T or N.

[0552] 23. The RSV-F protein according to embodiment 22, which comprises:

[0553] (a) a substitution of position 55 of SEQ ID NO:1 with T;

[0554] (b) A substitution at position 215 of SEQ ID NO:1 with A; and

[0555] (c) A substitution at position 348 of SEQ ID NO:1 with N; wherein the N at position 348 is linked to a glycan; which optionally contains N-acetylglucosamine.

[0556] 24. The RSV-F protein according to any one of embodiments 1-16, 20 or 21, comprising:

[0557] (a) A substitution at position 55 of SEQ ID NO:1 with T, C, V, I or F; optionally T, C or V; optionally T or V;

[0558] (b) A substitution at position 215 of SEQ ID NO:1 with A, P, V, I or F; optionally A or P; and

[0559] (d) A substitution at position 348 of SEQ ID NO:1 with N, D, F, H, K, L, N, Q, R, T, W or Y; optionally N, F, H, K, N, Q, R, T, W or Y; optionally N, F, R, W or Y.

[0560] 25. The RSV-F protein according to embodiment 24, comprising:

[0561] (a) A substitution at position 55 of SEQ ID NO:1 with T;

[0562] (b) A substitution at position 215 of SEQ ID NO:1 with A; and

[0563] (d) A substitution at position 348 of SEQ ID NO:1 with N.

[0564] 26. The RSV-F protein according to any one of the preceding embodiments, comprising an F2 domain having at least 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity to positions 26-108 or 26-109 of SEQ ID NO:1.

[0565] 27. The RSV-F protein according to any one of the foregoing embodiments, which comprises an F1 domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity to positions 137-513 of SEQ ID NO:1.

[0566] 28. The RSV-F protein according to any one of the foregoing embodiments, which comprises a heterotrimerization domain at its C-terminus and / or at the C-terminus of the F1 domain; optionally wherein the heterotrimerization domain is the T4 minor fiber protein foldon domain.

[0567] 29. The RSV-F protein according to embodiments 1-27, which comprises a transmembrane domain at its C-terminus and / or at the C-terminus of the F1 domain, and optionally comprises a cytoplasmic domain at the C-terminus of the transmembrane domain.

[0568] 30. The RSV-F protein according to any one of embodiments 1-27 or 29, which comprises a cytoplasmic domain; wherein, relative to the cytoplasmic domain according to SEQ ID NO:109 or 110, 2-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0569] 31. The RSV-F protein according to embodiment 30, wherein 3-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0570] 32. The RSV-F protein according to embodiment 30 or 31, wherein 2-5, such as 2-4, 2-3, 3-4 or 3 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0571] 33. The RSV-F protein according to any one of embodiments 30-32, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence of positions 10-31 of SEQ ID NO:134 or (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the positions, and optionally having the same length as the positions; and wherein the cytoplasmic domain does not comprise any residues at the C-terminus of the amino acid sequence in (i) or (ii).

[0572] 34. The RSV-F protein according to any one of embodiments 30 - 32, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence from positions 10 - 29 of SEQ ID NO: 135, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic domain does not include any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0573] 35. The RSV-F protein according to embodiment 31, wherein 6 - 13, such as 7 - 13, 8 - 12, 9 - 11, 9 - 10, 10 - 11 or 10 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0574] 36. The RSV-F protein according to any one of embodiments 30, 31 or 35, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence from positions 10 - 24 of SEQ ID NO: 136 or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic domain does not include any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0575] 37. The RSV-F protein according to embodiment 31, wherein 14 - 16, such as 14 - 15 or 15 - 16 or 15 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0576] 38. The RSV-F protein according to any one of embodiments 30, 31 or 37, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence from positions 10 - 19 of SEQ ID NO: 137, or (ii) an amino acid sequence that is at least 60%, 70%, 80% or 90% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic domain does not include any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0577] 39. The RSV-F protein according to embodiment 31, wherein 16 - 20, such as 17 - 20, 18 - 20 or 19 - 20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0578] 40. The RSV-F protein according to embodiment 39, wherein 20 residues are deleted from the C-terminus of the cytoplasmic tail of the RSV-F domain.

[0579] 41. The RSV-F protein according to any one of embodiments 30, 31, 39 or 40, wherein the cytoplasmic tail comprises or consists of the following: (i) the amino acid sequence of positions 10-14 of SEQ ID NO: 138, or (ii) an amino acid sequence that is at least 60% or 80% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0580] 42. The RSV-F protein according to any one of embodiments 30-41, wherein optionally in human fibroblasts, optionally in human foreskin fibroblasts, optionally in primary human BJ cells, optionally the ATCC CRL-2522 cell line, relative to the RSV-F protein having the same amino acids but lacking such deletions (such as the RSV-F protein comprising the cytoplasmic domain according to SEQ ID NO: 109 or 110) in the pre-fusion trimeric form of expression, the deletion increases the cell surface expression of the RSV-F protein in this form from the nucleic acid.

[0581] 43. The RSV-F protein according to embodiment 42, wherein the increased cell surface expression persists for a period of at least 24, 48, 72 or 96 hours.

[0582] 44. The RSV-F protein according to any one of the preceding embodiments, wherein the signal peptide is absent from the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO: 1.

[0583] 45. The RSV-F protein according to any one of the preceding embodiments, wherein the p27 peptide is absent from the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO: 1.

[0584] 46. The RSV-F protein according to any one of the preceding embodiments, wherein the RSV-F protein comprises an E residue at position 66 and a P residue at position 101 of SEQ ID NO: 1.

[0585] 47. The RSV-F protein according to any one of the preceding embodiments, which further comprises relative to SEQ ID NO: 1:

[0586] A substitution at position 152 with R, L or W; optionally R or W; optionally a substitution with R;

[0587] Substitution at position 210 with H, A, F, K, N, W or Y; optionally H, F, K, N, W or Y; optionally H, F or Y; optionally substitution with H;

[0588] Optionally, substitution at position 211 with N;

[0589] Substitution at position 228 with K, R, Q, N or A; optionally K, R, Q or N; optionally K, R or Q; optionally K or R; optionally substitution with K;

[0590] Substitution at position 241 with N

[0591] Substitution at position 315 with I or V; optionally substitution with I;

[0592] Substitution at position 346 with Q, D, H, K, N, R, S or W; optionally Q, D, H, K, N, R or S; optionally substitution with Q;

[0593] Substitution at position 419 with D, N, S or T; optionally D or T; optionally substitution with D;

[0594] Optionally, substitution at position 445 with D;

[0595] Substitution at position 455 with V or I; optionally substitution with V;

[0596] And / or, optionally and,

[0597] Substitution at position 459 with M.

[0598] The RSV-F protein according to any one of the preceding embodiments, further comprising, relative to SEQ ID NO:1:

[0599] Substitution at position 152 with R, L or W; optionally R or W; optionally substitution with R;

[0600] Optionally, substitution at position 211 with N;

[0601] Substitution at position 228 with K, R, Q, N or A; optionally K, R, Q or N; optionally K, R or Q; optionally K or R; optionally substitution with K;

[0602] Substitution at position 315 with I or V; optionally substitution with I;

[0603] Substitution at position 346 with Q, D, H, K, N, R, S or W; optionally Q, D, H, K, N, R or S; optionally substitution with Q;

[0604] Optionally, substitution at position 445 with D;

[0605] Substitution with V or I at position 455; optionally substitution with V;

[0606] And / or, optionally and;

[0607] Substitution with M at position 459.

[0608] 49. The RSV-F protein according to any one of the preceding embodiments, further comprising, relative to SEQ ID NO:1:

[0609] Substitution with K, R, Q, N or A at position 228; optionally substitution with K, R, Q or N; optionally substitution with K, R or Q; optionally substitution with K or R; optionally substitution with K;

[0610] Substitution with I or V at position 315; optionally substitution with I;

[0611] Substitution with N at position 241

[0612] Substitution with V or I at position 455; optionally substitution with V;

[0613] And / or, optionally and;

[0614] Substitution with M at position 459.

[0615] 50. The RSV-F protein according to any one of the preceding embodiments, further comprising, relative to SEQ ID NO:1:

[0616] Substitution with K, R, Q, N or A at position 228; optionally substitution with K, R, Q or N; optionally substitution with K, R or Q; optionally substitution with K or R; optionally substitution with K;

[0617] Substitution with I or V at position 315; optionally substitution with I;

[0618] Substitution with V or I at position 455; optionally substitution with V;

[0619] And / or, optionally and;

[0620] Substitution with M at position 459.

[0621] 51. The RSV-F protein according to any one of the preceding embodiments, comprising a substitution with K, R, Q, N or A at position 228 of SEQ ID NO:1; optionally substitution with K, R, Q or N; optionally substitution with K, R or Q; optionally substitution with K or R.

[0622] 52. The RSV-F protein according to embodiment 51, comprising a substitution with K at position 228 of SEQ ID NO:1.

[0623] 53. The RSV-F protein according to any one of the preceding embodiments, which comprises a substitution of R at position 152 of SEQ ID NO:1, and / or a substitution of Q at position 346 of SEQ ID NO:1.

[0624] 54. The RSV-F protein according to any one of the preceding embodiments, which comprises a substitution of N at position 211 of SEQ ID NO:1, and / or a substitution of D at position 445 of SEQ ID NO:1, optionally comprising both substitutions.

[0625] 55. The RSV-F protein according to any one of the preceding embodiments, wherein the RSV-F protein is subtype A.

[0626] 56. The RSV-F protein according to any one of embodiments 1-54, wherein the RSV-F protein is subtype B.

[0627] 57. The RSV-F protein according to any one of the preceding embodiments, which is specifically bound by a prefusion mAb, as measured by SPR, with a K D less than 10 nM; optionally 1 pM - 10 nM.

[0628] 58. The RSV-F protein according to any one of the preceding embodiments, which is specifically bound by a prefusion mAb comprising an LC and an HC according to SEQ ID NO:2 and 3 respectively, as measured by SPR, with a K D less than 1000, 900, 800, 700, 650, 600, 550, 100, 90, 80, 70, 60, 50 or 35 pM; wherein the RSV-F protein is in trimeric form.

[0629] 59. The RSV-F protein according to any one of the preceding embodiments, which is specifically bound by a prefusion mAb comprising an LC and an HC according to SEQ ID NO:4 and 5 respectively, as measured by SPR, with a K D less than 200, 180, 160, 140, 130, 100, 95, 90, 85, 80 or 70 pM.

[0630] 60. The RSV-F protein according to any one of the preceding embodiments, which is specifically bound by a prefusion mAb comprising an LC and an HC according to SEQ ID NO:8 and 9 respectively, as measured by SPR, with a K D less than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM.

[0631] 61. The RSV-F protein according to any of the foregoing embodiments, which is specifically bound by a prefusion mAb comprising LC and HC according to SEQ ID NO: 8 and 9 respectively, and as measured by SPR, its K D is less than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM.

[0632] 62. The RSV-F protein according to any of the foregoing embodiments, which is specifically bound by a mAb comprising LC and HC according to SEQ ID NO: 6 and 7 respectively, and as measured by SPR, its K D is less than 200, 180, 160, 140, 120, 110, 100, 95, 80, 70, 60, 55, 50, 45 or 40 pM.

[0633] 63. A recombinant RSV-F protein in a prefusion conformation, which comprises at least one mutation relative to the wild-type RSV-F according to SEQ ID NO: 1, wherein the at least one mutation does not introduce an artificial disulfide bond or a P residue into the wild-type protein.

[0634] 64. The RSV-F protein according to embodiment 63, which comprises the features of any one of embodiments 1-62, provided that a P residue is not introduced into the protein by the at least one mutation.

[0635] 65. A trimer, which comprises three RSV-F proteins according to any of the foregoing embodiments.

[0636] 66. A nucleic acid, which encodes an RSV-F protein according to any one of embodiments 1-64.

[0637] 67. The nucleic acid according to embodiment 66, wherein the nucleic acid is a viral vector, or is contained within a viral vector; optionally wherein the viral vector is an adenovirus vector.

[0638] 68. The nucleic acid according to embodiment 66, wherein the nucleic acid is DNA; optionally wherein the DNA is a DNA plasmid.

[0639] 69. The nucleic acid according to embodiment 66, wherein the nucleic acid is RNA.

[0640] 70. The RNA according to embodiment 69, which is a non-self-replicating RNA.

[0641] 71. The RNA according to embodiment 69, which is a self-replicating RNA.

[0642] 72. The RNA according to any one of embodiments 69 - 71, which comprises, in the 5' to 3' direction: a 5' cap, a 5' UTR, an open reading frame encoding at least one RSV - F protein according to any one of embodiments 1 - 64, a 3' UTR, and a 3' poly - A tail.

[0643] 73. The RNA according to embodiment 72, wherein the 5' cap comprises 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleoside by a triphosphate bridge, and wherein the first 5' ribonucleoside comprises 2'-O-methylated ribose (2'-O-Me).

[0644] 74. The RNA according to embodiment 72 or 73, wherein the 3' poly - A tail comprises a continuous stretch of 100 - 500 A ribonucleotides.

[0645] 75. The RNA according to embodiment 72 or 73, wherein the 3' poly - A tail comprises at least two non - continuous stretches of A ribonucleotides; optionally having lengths of 25 - 35 and 65 - 90 ribonucleotides respectively; optionally oriented in the 5' to 3' direction.

[0646] 76. The RNA according to any one of embodiments 69 - 75, which comprises modified ribonucleotides.

[0647] 77. The RNA according to embodiment 76, wherein the modified ribonucleotide is 1mΨ.

[0648] 78. The RNA according to embodiment 77, wherein the RNA comprises 1mΨ and does not comprise standard U ribonucleotides or other modified U ribonucleotides; optionally wherein the RNA comprises 1mΨ and does not comprise standard U ribonucleotides or other modified ribonucleotides.

[0649] 79. The RNA according to any one of embodiments 69 - 78, which has a GC content of 55 - 70%.

[0650] 80. The RNA according to any one of embodiments 69 - 78, which has a GC content of 40 - 60%.

[0651] 81. A vector, which comprises a nucleic acid according to any one of embodiments 66, 68 or 69 - 80.

[0652] 82. The vector according to embodiment 81, which is a lipid nanoparticle.

[0653] 83. The lipid nanoparticle according to embodiment 82, which comprises a mixture of a cationic lipid, a neutral lipid, a sterol, and a polymer - conjugated lipid.

[0654] 84. The lipid nanoparticle according to embodiment 83, wherein the pKa of the cationic lipid is 5.0 - 8.0; optionally 5.0 - 7.6.

[0655] 85. The lipid nanoparticle according to embodiment 83 or 84, wherein the cationic lipid comprises a tertiary amine group.

[0656] 86. The lipid nanoparticle according to any one of embodiments 83 - 85, wherein the polymer-conjugated lipid is a PEGylated lipid; optionally wherein the average molecular weight of the PEG is 1 - 3 kDa.

[0657] 87. The lipid nanoparticle according to embodiment 86, wherein the weight-average molecular weight of the PEG is 1 - 3 kDa.

[0658] 88. The lipid nanoparticle according to any one of embodiments 83 - 87, wherein the sterol is cholesterol or a cholesterol-based lipid.

[0659] 89. The lipid nanoparticle according to any one of embodiments 83 - 88, which comprises (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.

[0660] 90. A pharmaceutical composition comprising the RSV-F protein according to any one of embodiments 1 - 64, the trimer according to embodiment 65, the nucleic acid according to any one of embodiments 66 - 80, or the carrier according to any one of embodiments 81 - 89; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant.

[0661] 91. A vaccine composition comprising the RSV-F protein according to any one of embodiments 1 - 64, the trimer according to embodiment 65, the nucleic acid according to any one of embodiments 66 - 80, or the carrier according to any one of embodiments 81 - 89; optionally comprising a pharmaceutically acceptable excipient; optionally further comprising an adjuvant.

[0662] 92. The pharmaceutical composition according to embodiment 90, which is for medical use.

[0663] 93. The pharmaceutical composition for the use according to embodiment 92, which is for a method of enhancing the immune response of a subject, optionally the protective immune response of the subject.

[0664] A pharmaceutical composition for use according to embodiment 92 or 93, for use in the treatment or prevention of RSV.

[0665] 95. A pharmaceutical composition for use according to embodiment 94, for use in a method of vaccinating a subject against RSV; optionally wherein the vaccination is prophylactic.

[0666] 96. A pharmaceutical composition for use according to any one of embodiments 93-95, wherein the subject is a human infant; optionally 2-6 months old.

[0667] 97. A pharmaceutical composition for use according to any one of embodiments 93-95, wherein the subject is an elderly human; optionally ≥60 years old.

[0668] 98. A pharmaceutical composition for use according to any one of embodiments 93-95, wherein the subject is a pregnant human female; optionally pregnant ≥28 weeks.

[0669] 99. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of the RSV-F protein according to any one of embodiments 1-64, the trimer according to embodiment 65, the nucleic acid according to any one of embodiments 66-80, the vector according to any one of embodiments 81-89, the pharmaceutical composition according to embodiment 90 or the vaccine composition according to embodiment 91.

[0670] 100. Use of the RSV-F protein according to any one of embodiments 1-64, the trimer according to embodiment 65, the nucleic acid according to any one of embodiments 66-80, the vector according to any one of embodiments 81-89 in the manufacture of a medicament.

[0671] 101. Use according to embodiment 84, wherein the medicament is for the prevention or treatment of RSV.

[0672] 102. Use according to embodiment 85, wherein the medicament is a vaccine; optionally a prophylactic vaccine.

[0673] 103. A kit comprising the RSV-F protein according to any one of embodiments 1-64, the trimer according to embodiment 65, the nucleic acid according to any one of embodiments 66-80, the vector according to any one of embodiments 81-89, the pharmaceutical composition according to embodiment 90 or the vaccine composition according to embodiment 91, and instructions for use.

[0674] 104. A respiratory syncytial virus fusion (RSV-F) protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1 and comprises the following (a), (b) and (c):

[0675] (a) (ai) At least one mutation relative to the wild-type in the 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

[0676] (aii) At least one mutation relative to the wild-type in the 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 a residue selected from M, F, I and V into the region by substitution or insertion;

[0677] (b) At least one mutation relative to the wild-type in the region corresponding to positions 208 - 216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208 - 216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion; and

[0678] (c) At least one mutation relative to the wild-type in the region corresponding to positions 345 - 352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion.

[0679] 105. The RSV-F protein according to embodiment 104, wherein:

[0680] (a) The regions corresponding to positions 38 - 60 and 296 - 318 of SEQ ID NO:1 comprise beta-sheets;

[0681] (b) The region corresponding to positions 208 - 216 of SEQ ID NO:1 comprises a loop;

[0682] And / or, optionally and,

[0683] (c) The region corresponding to positions 345 - 352 of SEQ ID NO:1 comprises a beta-sheet and a loop.

[0684] 106. The RSV-F protein according to embodiment 104 or 105, wherein:

[0685] (a) The region corresponding to positions 38 - 60 of SEQ ID NO:1 has at least 90% or 95% sequence identity with positions 38 - 60 of SEQ ID NO:1; and / or, optionally, the region corresponding to positions 296 - 318 of SEQ ID NO:1 has at least 90% or 95% sequence identity with positions 296 - 318 of SEQ ID NO:1;

[0686] (b) The region corresponding to positions 38 - 60 of SEQ ID NO:1 has at least 75% or 85% sequence identity with positions 208 - 216 of SEQ ID NO:1;

[0687] and / or, optionally,

[0688] (c) The region corresponding to positions 345 - 352 of SEQ ID NO:1 has at least 75% or 85% sequence identity with positions 345 - 352 of SEQ ID NO:1.

[0689] 107. The RSV - F protein according to any one of embodiments 104 - 106, comprising:

[0690] (a) A substitution at position 55 of SEQ ID NO:1 with T, C, V, I or F; optionally a substitution with T, C or V; optionally a substitution with T or V;

[0691] (b) A substitution at position 215 of SEQ ID NO:1 with A, P, V, I or F; optionally a substitution with A or P;

[0692] and / or, optionally,

[0693] (c) A substitution at position 348 of SEQ ID NO: with T or N.

[0694] 108. The RSV - F protein according to embodiment 107, comprising:

[0695] (a) A substitution at position 55 of SEQ ID NO:1 with T;

[0696] (b) A substitution at position 215 of SEQ ID NO:1 with A;

[0697] and / or, optionally,

[0698] (c) A substitution at position 348 of SEQ ID NO:1 with N.

[0699] 109. The RSV - F protein according to embodiment 107 or 108, ...

Claims

1. A respiratory syncytial virus fusion (RSV-F) protein in a pre-fusion conformation, which is mutated relative to SEQ ID NO:1 and comprises the following (a) and (b): (a) (ai) a substitution at position 55 of SEQ ID NO:1 with T, C, V, I or F; and / or (aii) a substitution at position 301 of SEQ ID NO:1 with M, F or I, and / or a substitution at position 303 of SEQ ID NO:1 with V, M, F or I; and (b) at least one mutation in the region corresponding to positions 208-216 of SEQ ID NO:1 relative to SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion.

2. The RSV-F protein according to any one of the preceding claims, which comprises: (a) a substitution at position 55 of SEQ ID NO:1 with T, C, V, I or F; optionally T, C or V; optionally T or V; and (b) a substitution at position 215 of SEQ ID NO:1 with A, P, V, I or F; optionally A or P.

3. The RSV-F protein according to claim 2, which comprises: (a) a substitution at position 55 of SEQ ID NO:1 with T; and (b) a substitution at position 215 of SEQ ID NO:1 with A.

4. The RSV-F protein according to any one of the preceding claims, which comprises a substitution at position 228 of SEQ ID NO:1 with K, R or Q; optionally K or R; optionally K.

5. The RSV-F protein according to any one of the preceding claims, which comprises at least 2 or at least 3 substitutions selected from the following, or comprises the following substitutions: a substitution at position 228 of SEQ ID NO:1 with K, R or Q; a substitution at position 315 of SEQ ID NO:1 with I or V; a substitution at position 455 of SEQ ID NO:1 with V or I; and a substitution at position 459 of SEQ ID NO:1 with M.

6. The RSV-F protein according to claim 5, which comprises at least 2 or at least 3 substitutions selected from the following, or comprises the following substitutions: a substitution at position 228 of SEQ ID NO:1 with K; a substitution at position 315 of SEQ ID NO:1 with I; a substitution at position 455 of SEQ ID NO:1 with V; and a substitution at position 459 of SEQ ID NO:1 with M.

7. The RSV-F protein according to claim 6, which comprises the following substitutions: a substitution at position 55 of SEQ ID NO:1 with T; a substitution at position 215 of SEQ ID NO:1 with A; optionally a substitution at position 228 of SEQ ID NO:1 with K; A substitution of I at position 315 of SEQ ID NO:1; A substitution of V at position 455 of SEQ ID NO:1; and A substitution of M at position 459 of SEQ ID NO:

1.

8. The RSV-F protein according to any one of the preceding claims, which comprises at least 4 or at least 5 substitutions selected from the following, or comprises the following substitutions: A substitution of R, L or W at position 152 of SEQ ID NO:1; Optionally, a substitution of N at position 211 of SEQ ID NO:1; A substitution of K, R or Q at position 228 of SEQ ID NO:1; A substitution of I or V at position 315 of SEQ ID NO:1; A substitution of Q, D, H, K, N, R, S or W at position 346 of SEQ ID NO:1; Optionally, a substitution of D at position 445 of SEQ ID NO:1; A substitution of V or I at position 455 of SEQ ID NO:1, and A substitution of M at position 459 of SEQ ID NO:

1.

9. The RSV-F protein according to claim 8, which comprises at least 4 or at least 5 substitutions selected from the following, or comprises the following substitutions: A substitution of R at position 152 of SEQ ID NO:1; Optionally, a substitution of N at position 211 of SEQ ID NO:1; A substitution of K at position 228 of SEQ ID NO:1; A substitution of I at position 315 of SEQ ID NO:1; A substitution of Q at position 346 of SEQ ID NO:1; Optionally, a substitution of D at position 445 of SEQ ID NO:1; A substitution of V at position 455 of SEQ ID NO:1; and A substitution of M at position 459 of SEQ ID NO:

1.

10. The RSV-F protein according to claim 9, which comprises the following substitutions: A substitution of T at position 55 of SEQ ID NO:1; A substitution of R at position 152 of SEQ ID NO:1; Optionally, a substitution of N at position 211 of SEQ ID NO:1; A substitution of A at position 215 of SEQ ID NO:1; Optionally, a substitution of K at position 228 of SEQ ID NO:1; A substitution of I at position 315 of SEQ ID NO:1; A substitution of Q at position 346 of SEQ ID NO:1; Optionally, a substitution of D at position 445 of SEQ ID NO:1; A substitution of V at position 455 of SEQ ID NO:1; and A substitution of M at position 459 of SEQ ID NO:

1.

11. The RSV-F protein according to any one of the preceding claims, which comprises an F2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with positions 26-108 or 26-109 of SEQ ID NO:

1.

12. The RSV-F protein according to any one of the preceding claims, which comprises an F1 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:

1.

13. The RSV-F protein according to any one of the preceding claims, which comprises a heterotrimerization domain at its C-terminus and / or at the C-terminus of the F1 domain; optionally wherein the heterotrimerization domain is the T4 minor fiber protein foldon domain.

14. The RSV-F protein according to any one of claims 1-12, which comprises a transmembrane domain at its C-terminus and / or at the C-terminus of the F1 domain; and optionally a cytoplasmic domain at the C-terminus of the transmembrane domain.

15. The RSV-F protein according to any one of claims 1-12 or 14, wherein the RSV-F protein comprises a cytoplasmic domain; wherein 2-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein relative to the cytoplasmic domain according to SEQ ID NO:109 or 110.

16. The RSV-F protein according to claim 15, wherein 16-20, 17-20, 18-20, 19-20 or 20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

17. The RSV-F protein according to any one of the preceding claims, wherein the signal peptide is absent from the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO:

1.

18. The RSV-F protein according to any one of the preceding claims, wherein the p27 peptide is absent from the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO:

1.

19. The RSV-F protein according to any one of the preceding claims, wherein the RSV-F protein comprises the E residue at position 66 and the P residue at position 101 of SEQ ID NO:

1.

20. A nucleic acid encoding the RSV-F protein according to any one of claims 1-19.

21. The nucleic acid according to claim 20, wherein the nucleic acid is RNA.

22. A lipid nanoparticle comprising the nucleic acid according to claim 20 or 21.

23. A pharmaceutical composition comprising an RSV-F protein according to any one of claims 1-19, a nucleic acid according to claim 20 or 21, or a lipid nanoparticle according to claim 22; optionally for medical use.

24. A pharmaceutical composition for use according to claim 23, for a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; an elderly human, optionally ≥50 or ≥60 years old; or a pregnant human female, optionally pregnant ≥28 weeks.

25. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of an RSV-F protein according to any one of claims 1-19, a nucleic acid according to claim 20 or 21, a lipid nanoparticle according to claim 22, or a pharmaceutical composition according to claim 23.

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