Stabilized trimeric RSV fusion proteins lacking a heterotrimerization domain

Abstract

The present invention relates to a respiratory syncytial virus (RSV) F protein comprising a head region and a stem region, wherein the stem region comprises at least one amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein, wherein the at least one mutation in the stem region comprises a mutation of the amino acid residue at position 509 to F, I, or L, wherein the amino acid positions are numbered according to the numbering of the amino acid residues in SEQ ID NO: 1. The present invention also relates to a respiratory syncytial virus F protein comprising a head region and a stem region, wherein the stem region comprises at least one amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein, wherein the amino acid residue at position 509 is numbered according to the numbering of the amino acid residues in SEQ ID NO: 1. The present invention also relates to a respiratory syncytial virus F protein comprising at least one amino acid mutation in the stem region of a wild-type RSV F protein, wherein the amino acid residue at position 509 is numbered according to the numbering of

Description

[Technical Field]

[0001] The present invention relates to the field of medicine. In particular, the present invention relates to recombinant pre-fusion RSV F proteins, nucleic acid molecules encoding RSV F proteins, and their use in, for example, vaccines. [Background technology]

[0002] Respiratory syncytial virus (RSV) is associated with upper and lower respiratory tract infections in humans. Worldwide, an estimated 64 million RSV infections occur annually, resulting in 160,000 deaths (WHO Acute Respiratory Infections Update September 2009). The most severe disease occurs particularly in premature infants, the elderly, and immunocompromised individuals. In children under 2 years of age, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of hospitalizations due to respiratory infections, with peak hospitalizations occurring between 2 and 4 months of age. Nearly all children have been reported to have been infected with RSV by the age of 2 years. Lifelong recurrent infections are due to ineffective innate immunity. In older adults, the disease burden caused by RSV is similar to that caused by nonpandemic influenza A infections. Older adults are at particularly high risk for severe RSV-mediated illness, with an estimated 177,000 hospitalizations and 14,000 deaths due to RSV in adults aged 65 and older in the United States alone. (Centers for Disease Control and Prevention. Respiratory Syncytial Virus Infection (RSV). Trends and Surveillance. https: / / www.cdc.gov / rsv / research / us-surveillance.html (2020). Accessed March 28, 2022).

[0003] RSV is a paramyxovirus belonging to the Pneumoviridae subfamily. Its genome encodes various proteins, including the RSV glycoprotein (G) and RSV fusion protein (F) membrane proteins, the latter of which is the primary antigenic target for neutralizing antibodies. Antibodies against the F protein can prevent viral entry into cells, thereby exerting a neutralizing effect.

[0004] RSV F fuses viral and host cell membranes through irreversible protein refolding from an unstable pre-fusion conformation to a stable post-fusion conformation. The structures of both conformations of RSV F (McLellan JS et al. (2010, 2013, 2013), Swanson KA et al. (2011)) and fusion proteins from related paramyxoviruses have been determined, providing insight into the complex process this fusion protein undergoes. Like other class I fusion proteins, the inactive precursor, RSV F0, requires cleavage by a furin-like protease during intracellular maturation. Cleavage of RSV F at two furin cleavage sites yields three proteins: F2, p27, and F1. The p27 fragment is not part of the mature F protein; F2 and F1 are linked by two disulfide bridges, and the F1 domain contains a hydrophobic fusion peptide (FP) at its N-terminus. To refold from the pre-fusion conformation to the post-fusion conformation, refolding region 1 (RR1) between residues 137 and 216, which contains FP and heptad repeat A (HRA, also referred to as "HR1"), must transform from an assembly of helices, loops, and strands into a long, continuous helix. Then, FP, located in the N-terminal segment of RR1, extends away from the viral membrane and can insert into the proximal membrane of the target cell. Next, refolding region 2 (RR2), which forms the C-terminal stem in the pre-fusion F protein and contains heptad repeat B (HRB, also referred to as "HR2"), relocates to the other side of the RSV F head, and binds the HRA coiled-coil trimer with the HRB domain to form a six-helix bundle. The formation of the RR1 coiled coil and the relocation of RR2 to complete the six-helix bundle are the most dramatic structural changes that occur during the refolding process.

[0005] Soluble subunit-based vaccines require truncation of the viral fusion protein at the C-terminus by deleting the transmembrane (TM) and cytoplasmic regions. The remaining ectodomain of the fusion protein is significantly less stable due to the removal of the membrane anchor and either does not form as a trimeric protein or more readily refolds to the post-fusion terminal state. Therefore, soluble viral fusion proteins generally contain a C-terminal heterotrimerization domain (e.g., Foldon or GCN4) to restore trimer formation (Welch et al. (2012) Proc Natl Acad Sci USA 109:16672-16677; McLellan et al. (2013) Science 342:592-598; Walls et al. (2016) Nat Struct Mol Biol 23:899-905). However, the addition of a non-native trimerization domain may induce unrelated antibodies to this domain that do not cross-react with the virus, which may interfere with immunogenicity if the vaccine is used repeatedly and in other vaccines where the domain increases its immunodominance.

[0006] A vaccine to prevent RSV infection is not currently available, but it is sought after due to the high disease burden. The RSV fusion glycoprotein F is an attractive vaccine antigen because it is the primary target of neutralizing antibodies in human serum. Most neutralizing antibodies in human serum are directed against the pre-fusion conformation, but due to its instability, the pre-fusion conformation tends to prematurely refold to the post-fusion conformation. As described above, the crystal structure demonstrated a large conformational change between the pre-fusion and post-fusion states. The magnitude of the rearrangement suggests that only a portion of antibodies directed against the post-fusion conformation of RSV F can cross-react with the native conformation of the pre-fusion spike on the surface of the virus. Therefore, efforts to produce vaccines against RSV have focused on developing vaccines containing or expressing the pre-fusion form of the RSV F protein.

[0007] There remains a need for an effective vaccine against RSV, particularly a vaccine based on or comprising a pre-fusion conformation RSV F protein. The present invention aims to provide such a stable pre-fusion RSV F protein for use in vaccination against RSV. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Welch et al. (2012), Proc Natl Acad Sci USA, 109:16672-16677 [Non-patent document 2] McLellan et al. (2013), Science, 342:592-598 [Non-patent document 3] Walls et al. (2016) Nat Struct Mol Biol 23:899-905 Summary of the Invention

[0009] Provided herein are vaccine components that can be used to induce an immune response against RSV.

[0010] In a first aspect, the present invention provides a respiratory syncytial virus (RSV) F protein comprising a head region and a stem region, wherein the protein comprises at least one amino acid substitution in the stem region relative to the amino acid sequence of a wild-type RSV F protein, wherein the at least one substitution in the stem region comprises a substitution of the amino acid residue at position 509 with F, I, or L, wherein the amino acid positions are numbered according to the numbering of the amino acid residues in SEQ ID NO: 1.

[0011] The present invention further provides nucleic acids encoding proteins according to the invention, as well as vectors comprising such nucleic acids.

[0012] Compositions comprising the proteins, nucleic acids and / or vectors according to the invention, and uses thereof, are also provided.

[0013] The present invention also provides an isolated host cell comprising a nucleic acid according to the present invention.

[0014] The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the accompanying drawings. It is to be understood that the invention is not limited to the precise embodiments set forth in the examples. [Brief explanation of the drawings]

[0015] [Figure 1] RSV F forms monomers in the absence of a heterotrimerization domain. Detection of (A) RSV-A and (B) RSV-B F trimers (T) and monomers (M) in cell culture supernatants using analytical size exclusion chromatography (SEC). [Figure 2] The S509 substitution in HR2 results in trimeric RSV-A F in the absence of a heterotrimerization domain. Detection of RSV-A F trimers (T) and monomers (M) in cell culture supernatants with mutations at position 509 using analytical SEC. [Figure 3] The F505W + S509F substitution in HR2 improves trimer yield in RSV-A and RSV-B F in the absence of a heterotrimerization domain. (A) Detection of RSV-A F trimers in cell culture supernatants using analytical SEC. (B) Detection of RSV-B F trimers (T) and monomers (M) in cell culture supernatants using analytical SEC. [Figure 4] The F505W + S509F substitution in HR2 confers temperature stability to trimeric RSV-A F in the absence of a heterotrimerization domain. Detection of RSV-A F trimers in cell culture supernatants incubated for 15 minutes at 4, 60, 65, and 70°C (see caption) using analytical SEC. [Figure 5]Head domain substitution improves trimer yield in RSV-A and RSV-B F in the absence of a heterotrimerization domain. (A) Detection of RSV-A F trimers in cell culture supernatants using analytical SEC. (B) Detection of RSV-B F trimers (T) and monomers (M) in cell culture supernatants using analytical SEC. [Figure 6] RSV-B F trimer stability is increased by combined HR2 and head domain stabilization in the absence of the heterotrimerization domain. (A) Detection of RSV-B F trimers (T) and monomers (M) in cell culture supernatants on the day of harvest using analytical SEC. (B) Detection of RSV-B F trimers (T) and monomers (M) in cell culture supernatants after 16 days of storage at 4°C using analytical SEC. [Figure 7] HR2 extension improves trimer yield and stability in RSV-A and RSV-B F in the absence of a heterotrimerization domain. (A) Detection of RSV-A F trimers of short (left) and long (right) HR2 mutants in cell culture supernatants using analytical SEC. (B) Detection of RSV-A F trimers in cell culture supernatants on the day of harvest (day 0) and after 2 days of storage at 4°C (day 2) using analytical SEC. (C) Detection of RSV-B F trimers (T) and monomers (M) in cell culture supernatants using analytical SEC. [Figure 8] The G519V + T523I substitution in the extended HR2 improves trimer expression in RSV-A F in the absence of a heterotrimerization domain. RSV-A F trimer detection in cell culture supernatants using analytical SEC. [Figure 9]Purification and characterization of tag-free stabilized RSV-A protein without the heterotrimerization domain. (A) Analytical SEC-MALS of purified RSV-A F trimers. (B) Melting temperature (Tm50) of purified RSV-A F trimers determined by differential scanning fluorimetry (DSF). N=3 replicates were performed, and individual and average values ​​are reported as gray and black solid lines, respectively. (C) Trimer yield of purified RSV-A F trimers. (D) Pre-fusion, post-fusion, and pan-specific RSV-A F antibody binding of purified RSV-A F trimers using quantitative Octet analysis. Initial binding rates are plotted. [Figure 10] Purification and characterization of stabilized RSV-A proteins in the presence and absence of heterotrimerization domains. (A) Overview of amino acid substitutions in purified RSV-A F mutants and the presence (+) or absence (-) of the Foldon trimerization domain. (B) Analytical SEC-MALS of purified RSV-A F trimers. (C) Melting temperatures (Tm50) of purified RSV-A F trimers determined by differential scanning fluorimetry (DSF). N=3 replicates were performed, and individual and average values ​​are reported as gray and black solid lines, respectively. (D) Pre-fusion, post-fusion, and pan-specific RSV-A F antibody binding of purified RSV-A F trimers using quantitative Octet analysis. Initial binding rates are plotted. (E) Analytical SEC-MALS of purified RSV-A F trimers immediately after purification (t=0) and after 19 weeks of storage at 4°C (t=19 weeks). [Figure 11] Immunogenicity of stabilized RSV-A proteins in the presence and absence of heterotrimerization domains. (A) Virus neutralizing antibody titers (VNT) measured by a firefly luciferase (FFL) reporter-based assay for RSV-A CL57. (B) Foldon antibody binding titers measured by ELISA. (C) Pre-fusion RSV-F antibody binding titers measured by ELISA. (D) Post-fusion RSV-F antibody binding titers measured by ELISA. (E) Ratio of pre-fusion / post-fusion RSV-F antibody binding titers from (C) and (D). [Figure 12]Purification, characterization, and immunogenicity of stabilized foldon-less and tagless RSV-A and RSV-B pre-fusion F proteins. (A) Analytical SEC-MALS of purified RSV F trimers. (B) Melting temperature (Tm50) of purified RSV F trimers determined by differential scanning fluorimetry (DSF). N = 3 replicates were performed, and individual and average values ​​are reported as gray and black solid lines, respectively. (C) Pre-fusion (CR9501, RSV90, RSD5-GL), post-fusion (ADI-15644), and pan-specific (CR9506) RSV F antibody binding of purified RSV F trimers using quantitative Octet analysis. Initial binding rates are plotted. (D) Analytical SEC-MALS of purified RSV F trimers immediately after purification (t = 0) and after storage at either 4 or 37 °C for 1, 3, and 6 months. DETAILED DESCRIPTION OF THE INVENTION

[0016] As mentioned above, respiratory syncytial virus (RSV) is a respiratory virus that infects the lungs and respiratory tract. RSV is a leading cause of severe viral lower respiratory tract disease in infants worldwide and an important cause of respiratory disease in the elderly.

[0017] Human RSV (HRSV) is divided into two major subtypes, HRSV-A and HRSV-B, which are generally distinguished based on differences in the sequence of the G protein. The F proteins of A and B strains show a high degree of sequence identity (approximately 95% in the mature ectodomain).

[0018] The human RSV F glycoprotein is initially translated from mRNA as a single 574 amino acid polypeptide precursor (termed "F0" or "F0 precursor") containing a signal peptide sequence (amino acids 1-25) at its N-terminus. During translation, the signal peptide is removed by signal peptidases in the endoplasmic reticulum. The remaining portion of the F0 precursor (i.e., residues 26-574) is further cleaved by cellular proteases (specifically furin) at two polybasic sites (amino acids 109 / 110 and 136 / 137), removing a 27-amino acid intervening sequence (amino acids 110-136) called p27, generating two linked fragments termed F1 (C-terminal, amino acids 137-574) and F2 (N-terminal, amino acids 26-109). F1 contains a hydrophobic fusion peptide at its N-terminus and two heptad repeat regions (HR1 and HR2). HR1 is near the fusion peptide, and HR2 is near the TM domain. The F1 and F2 fragments are linked to each other via two disulfide bonds. Either the uncleaved F0 protein without the signal peptide sequence or the F1-F2 heterodimer can form the RSV F promoter. Three such promoters assemble to form the final RSV F protein complex, which is a homotrimer of three promoters.

[0019] As mentioned above, no vaccine against RSV infection is currently available. One possible approach to producing a vaccine is to provide a subunit vaccine based on a soluble purified RSV F protein. However, in this approach, it is desirable that the purified RSV F protein has a conformation similar to the pre-fusion state conformation of the RSV F protein, is stable over time, i.e., remains in a pre-fusion conformation as determined, for example, by specific binding of the RSV F protein to an antibody specific for the pre-fusion conformation of the RSV F protein, and can be produced in sufficient quantities. Furthermore, in the case of a soluble subunit-based protein vaccine, the RSV F protein must be truncated by deletion of the transmembrane (TM) and cytoplasmic regions to produce a soluble secreted F protein (sF protein). Because the TM region is involved in membrane anchoring and increases stability, the anchor-free soluble F protein is significantly less stable than the full-length protein and more easily refolds to the post-fusion terminal state.

[0020] Therefore, to obtain an F protein in a stable pre-fusion conformation that exhibits high expression levels and high stability, it is necessary to stabilize the pre-fusion conformation. Because the full-length (membrane-bound) RSV F protein is also metastable, for example, any live attenuated genetic immunization or vector-based vaccine approach requires stabilization of the pre-fusion conformation of the full-length RSV F protein, i.e., including the TM and cytoplasmic regions.

[0021] The present invention provides a respiratory syncytial virus (RSV) F protein comprising a head region and a stem region, wherein the protein comprises at least one amino acid substitution in the stem region relative to the amino acid sequence of a wild-type RSV F protein, wherein the at least one substitution in the stem region comprises a substitution of an amino acid residue at position 509 with F, I, or L, and the amino acid positions are numbered according to the numbering of the amino acid residues in SEQ ID NO: 1. According to the present invention, it has been shown that RSV F proteins comprising at least one substitution at position 509 have reduced fusogenicity (i.e., reduced fusogenicity) compared to RSV F proteins not comprising a substitution at position 509, and may result in reduced reactogenicity when used as a vaccine component.

[0022] Furthermore, it has been shown that a stable trimeric RSV F ectodomain can be obtained when the amino acid residue at position 509 is substituted with F, I, or L.

[0023] As used herein, the head domain (including amino acids 27-490) is defined as the RSV F ectodomain without the signal peptide and without the HR2 region. The stem domain is defined herein as the HR2 region (i.e., including amino acids 491-524).

[0024] Thus, the present invention provides a trimeric recombinant pre-fusion RSV F protein with reduced fusogenicity.

[0025] Additionally or alternatively, the present invention provides a trimeric RSV F protein stabilized in a pre-fusion conformation. In the research leading to the present invention, several modifications, such as amino acid mutations (substitutions), were introduced compared to the amino acid sequence of the wild-type RSV F protein, particularly the amino acid sequence of SEQ ID NO: 1, to obtain the stable trimeric pre-fusion RSV F protein. The stable pre-fusion RSV F proteins of the present invention are pre-fusion conformations, i.e., they contain (display) at least one epitope specific to the pre-fusion conformation F protein. The epitope specific to the pre-fusion conformation F protein is an epitope that is not present in the post-fusion conformation. Without wishing to be bound by any particular theory, it is believed that the pre-fusion conformation of the RSV F protein may contain the same epitope as the RSV F protein expressed on native RSV virions, and therefore may provide an advantage for inducing protective neutralizing antibodies. In certain embodiments, the proteins of the present invention comprise at least one epitope recognized by a pre-fusion specific anti-RSV monoclonal antibody. An example of such a pre-fusion specific RSV F antibody is RSV90 (Mousa et al. (2017), Nat Microbiol, 2:16271).

[0026] In a particularly preferred embodiment, the recombinant pre-fusion RSV F protein comprises at least one epitope that is recognized by at least one pre-fusion-specific monoclonal antibody as described above and is a trimer.

[0027] In a specific embodiment, the protein comprises at least one amino acid substitution in the head region and at least one amino acid substitution in the stem region relative to the amino acid sequence of the wild-type RSV F protein, wherein at least one mutation in the stem region comprises a substitution of the amino acid residue at position 509 with F, I, or L.

[0028] In certain embodiments, at least one amino acid mutation in the head region is a P substitution of the amino acid at residue 215 or a N substitution of the amino acid residue at position 486. In accordance with the present invention, these mutations have been shown to stabilize the pre-fusion conformation and increase trimer yield and / or trimer stability.

[0029] In certain embodiments, the protein comprises at least one additional substitution in the head region, where one or more specific mutations in the head region increase trimer yield and / or trimer stability and / or pre-fusion conformation stability.

[0030] In a further embodiment, the protein comprises at least two amino acid substitutions in the head region and at least one amino acid substitution in the stem region relative to the amino acid sequence of the wild-type RSV F protein, wherein at least one substitution in the stem region comprises a substitution of amino acid residue 509 to F, I, or L.

[0031] In certain embodiments, the at least two amino acid mutations in the head region comprise a P substitution of the amino acid at residue 215 and a N substitution of the amino acid residue at position 486.

[0032] In a specific embodiment, the protein comprises at least three amino acid substitutions in the head region and at least one amino acid substitution in the stem region relative to the amino acid sequence of the wild-type RSV F protein, wherein at least one substitution in the stem region comprises a substitution of amino acid residue 509 with F, I, or L. According to the present invention, proteins comprising at least three substitutions in the head region in combination with a substitution of amino acid residue 509 have been shown to have increased trimer yield and / or trimer stability and / or pre-fusion conformation stability.

[0033] According to the present invention, the RSV F protein may be a RSV A subtype F protein or a RSV B subtype F protein. Thus, according to the present invention, substitutions can be introduced into a RSV A subtype F protein, such as a RSV F protein comprising the amino acid sequence of SEQ ID NO: 1. In an alternative embodiment, substitutions can be introduced into a RSV B subtype F protein, such as a RSV F protein comprising the amino acid sequence of SEQ ID NO: 40.

[0034] In certain embodiments, particularly when the RSV F protein is derived from a RSV A subtype, at least one additional mutation in the head region is selected from the group consisting of a mutation of the amino acid residue at position 328 to P, a mutation of the amino acid residue at position 354 to L, a mutation of the amino acid residue at position 487 to L, a mutation of the amino acid residue at position 489 to Y, a mutation of the amino acid residue at position 494 to I, a mutation of the amino acid residue at position 519 to V, and a mutation of the amino acid residue at position 523 to I.

[0035] Additionally or alternatively, the amino acid residue at position 101 is not P. Preferably, the amino acid residue at position 101 is Q, S, T, A or G.

[0036] In a further embodiment, particularly when the RSV F protein is derived from a RSV B subtype, at least one additional mutation in the head region is selected from the group consisting of a mutation of the amino acid residue at position 152 to M, a mutation of the amino acid residue at position 203 to I, a mutation of the amino acid residue at position 354 to L, a mutation of the amino acid residue at position 487 to L, and a mutation of the amino acid residue at position 489 to Y.

[0037] Additionally or alternatively, the amino acid residue at position 101 is not P. Preferably, the amino acid residue at position 101 is Q, S, T, A or G.

[0038] In a preferred embodiment, at least one mutation of the amino acid residue at position 509 in the stem region is a mutation to F. The presence of F at position 509 has been shown to increase trimer yield and / or trimer stability.

[0039] In certain embodiments, the protein further comprises a second mutation in the stem region, wherein said second mutation is a mutation of amino acid residue 505 to W. The presence of the second mutation at position 505 has been shown to increase trimer yield and / or trimer stability.

[0040] In a particularly preferred embodiment, the protein comprises a substitution of the amino acid residue at position 509 to F, I or L, preferably F, and a mutation of the amino acid residue at position 505 to W.

[0041] In a specific embodiment, the protein comprises at least two amino acid substitutions in the head region relative to the amino acid sequence of the wild-type RSV F protein, particularly the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 40, and a substitution of the amino acid residue at position 509 with F, I, or L, preferably F, and a substitution of the amino acid residue at position 505 with W.

[0042] According to the present invention, trimer expression (trimer yield) is shown to be increased compared to the RSV F protein not containing the substitutions of the present invention.

[0043] Additionally, or alternatively, trimer stability is increased compared to RSV F proteins not containing the substitutions of the present invention.

[0044] In a specific embodiment, the RSV F protein is a trimer after incubation at 65°C for 15 minutes.

[0045] Additionally or alternatively, the protein is a trimer after storage at 4°C for at least 2 days, in particular the protein is a trimer after storage at 4°C for at least 16 days, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 weeks at 4°C.

[0046] In addition, or alternatively, the RSV F protein has a higher melting temperature compared to the RSV F protein not containing the substitutions of the present invention.

[0047] As used herein, a mutation refers to a change (modification or substitution) of one amino acid in a protein to a different amino acid. The terms amino acid mutation and substitution are used interchangeably throughout this application. As used throughout this application, the position of an amino acid residue (or amino acid position) is indicated with reference to the sequence of the RSV F protein of SEQ ID NO: 1. Thus, as used herein, the phrase "amino acid residue, for example, at position 509 of the RSV F protein" refers to the amino acid corresponding to amino acid position 509 of the RSV F protein of SEQ ID NO: 1. Note that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature F0 protein (SEQ ID NO: 1), i.e., including the signal peptide. When using an RSV strain other than the RSV-A strain of SEQ ID NO: 1, such as the RSV B strain, the amino acid position of the F protein can be numbered with reference to the numbering of the F protein of SEQ ID NO: 1 by aligning the sequence with the F protein of SEQ ID NO: 1, inserting gaps as necessary. Sequence alignment can be performed using methods well known in the art, for example, by CLUSTALW, Bioedit, or CLC Workbench.

[0048] Amino acids according to the present invention can be any of the 20 naturally occurring (or "standard" amino acids) or variants thereof, such as D-amino acids (D-enantiomers of amino acids with a chiral center), or any variant not naturally found in proteins, such as norleucine. Standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional group. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine, which can form covalent disulfide bonds (or disulfide bridges) with other cysteine ​​residues, proline, which induces or stabilizes turns in the protein backbone, and glycine, which is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids. Those skilled in the art will understand that mutations can be made in proteins using routine molecular biology procedures.

[0049] In a specific embodiment, the stable pre-fusion RSV F protein of the present invention is a soluble protein, i.e., the RSV F protein ectodomain. Thus, in a specific embodiment, the RSV F protein (particularly the F1 domain) is truncated (i.e., the transmembrane and cytoplasmic regions are (partially) deleted). In a specific embodiment, the RSV F protein is truncated after amino acid residue 513.

[0050] In a preferred embodiment, the RSV F protein is truncated after amino acid residue 524. The present invention has shown that a longer HR2 can increase the stability of the RSV F protein. Thus, the present invention has shown that a longer HR2 results in an increased melting temperature.

[0051] In a particularly preferred embodiment, the RSV F protein is truncated after amino acid 524 and contains at least three amino acid mutations in the head region and at least one amino acid mutation in the stem region relative to the amino acid sequence of the wild-type RSV F protein, wherein at least one mutation in the stem region includes a mutation of the amino acid residue at position 509 to F, I, or L, preferably, the amino acid residue at position 509 is F.

[0052] In certain other preferred embodiments, the RSV F protein is a RSV F protein from the RSV A subtype, truncated after amino acid 524, and includes at least three amino acid mutations in the head region and at least one amino acid mutation in the stem region relative to the amino acid sequence of the wild-type RSV F protein, wherein at least one mutation in the stem region includes a mutation of the amino acid residue at position 509 to F, I, or L, preferably, the amino acid residue at position 509 is F.

[0053] In a specific embodiment, the protein is derived from an RSV B subtype, truncated after amino acid 524, and contains at least three amino acid substitutions in the head region relative to the amino acid sequence of the wild-type RSV F protein, and a substitution of the amino acid residue at position 509 with F, I, or L, preferably F, and a substitution of the amino acid residue at position 505 with W.

[0054] As used herein, a "truncated" protein refers to an RSV F protein that is not a full-length protein, i.e., a protein lacking one or more amino acid residues at the C-terminus. In certain embodiments, at least the transmembrane and cytoplasmic domains are deleted to allow expression as a soluble ectodomain. Because the TM region is responsible for membrane anchoring and trimerization, an "anchorless" (i.e., TM and cytoplasmic domain-free) soluble F protein is a monomer and exhibits low expression. Therefore, to obtain a soluble trimeric F protein in a stable pre-fusion conformation, it is necessary to stabilize the pre-fusion conformation. To promote trimerization, it is known to replace the TM / CT region with a heterotrimerization domain, such as a Foldon domain.

[0055] In a preferred embodiment, the truncated RSV F protein does not contain a heterotrimerization domain. According to the present invention, a soluble trimeric RSV pre-fusion F protein can be provided without a heterotrimerization domain. According to the present invention, it has surprisingly been found that the presence of one or more stabilizing mutations in HR2 (or the stem domain) increases trimer content compared to an RSV F protein that does not have one or more stabilizing mutations in the HR2 domain, even in the absence of a heterotrimerization domain.

[0056] In certain embodiments, the protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 17-29, 34, 38-39, and 43-44, or an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence identity, or a fragment thereof. In certain preferred embodiments, the protein consists of SEQ ID NO: 43 or a fragment thereof, or comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 43. In certain preferred embodiments, the protein consists of SEQ ID NO: 44 or a fragment thereof, or comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 44.

[0057] Fragments of the RSV F protein described herein are also encompassed by the present invention. Fragments can result from either or both amino-terminal (e.g., by cleaving the signal sequence) and carboxy-terminal deletions (e.g., by (partially) deleting the transmembrane domain and / or cytoplasmic tail). Fragments can be selected to contain immunologically active fragments of the F protein, i.e., portions that generate an immune response in a subject. This can be readily determined using computer, in vitro, and / or in vivo methods, all of which are routine for those skilled in the art.

[0058] In certain embodiments, proteins according to the invention do not contain a signal sequence, also called a leader sequence or signal peptide, corresponding to amino acids 1-26 of SEQ ID NO: 1. Signal sequences are short (e.g., 5-30 amino acids in length) amino acid sequences typically present at the N-terminus of most newly synthesized proteins destined for the secretory pathway, and are typically cleaved by a signal peptidase to generate the free signal peptide and mature protein.

[0059] In certain embodiments, amino acids 110-136 (i.e., the p27 peptide) are not present in the RSV F protein. Furin is typically cleaved between amino acids 109 and 110, but in certain embodiments, the C-terminal arginine residue of the F2 domain has been shown to be cleaved in the mature protein. Thus, in certain embodiments, amino acids 109-136 or 108-136 are not present in the RSV F protein.

[0060] In certain embodiments, the protein comprises a His tag, a Strep tag, or a C tag. A His tag or polyhistidine tag is an amino acid motif in a protein consisting of at least five histidine (H) residues, a Strep tag is an amino acid sequence consisting of eight residues (WSHPQFEK (SEQ ID NO: 41)), and a C tag is an amino acid motif consisting of four residues (EPEA, SEQ ID NO: 42). Tags are often located at the N- or C-terminus of a protein and are generally used for purification purposes. In a preferred embodiment, the protein does not comprise such an N- or C-terminal tag.

[0061] The present invention further provides nucleic acids encoding the proteins described herein. Such nucleic acids can be used, for example, in recombinant protein expression systems to produce the proteins according to the present invention. Furthermore, nucleic acid vaccines represent another vaccine approach that uses synthetic sequences to express antigenic peptides or proteins in vivo. Genetic immunization can promote superior adaptive immunity by activating both humoral and cell-mediated responses, and has manufacturing advantages over conventional vaccines.

[0062] In a preferred embodiment, the nucleic acid molecule encoding the protein according to the present invention is codon-optimized for expression in mammalian cells, preferably human cells. Codon optimization methods are known and have been previously described (e.g., WO 96 / 09378). A sequence is considered codon-optimized if, compared to the wild-type sequence, at least one non-preferred codon is replaced with a more preferred codon. Here, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The codon usage frequency of a particular organism can be found in a codon frequency table, such as http: / / www.kazusa.or.jp / codon. Preferably, two or more non-preferred codons, preferably most or all non-preferred codons, are replaced with more preferred codons. Preferably, the codon most frequently used in the organism is used in the codon-optimized sequence. Replacement with a preferred codon generally results in higher expression.

[0063] Those skilled in the art will understand that many different polynucleotides and nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. Those skilled in the art will also understand that, using routine techniques, nucleotide substitutions that do not affect the protein sequence encoded by nucleic acid molecules can be made to reflect the codon usage of any specific host organism in which the protein is expressed. Therefore, unless otherwise specified, "nucleotide sequences or nucleic acid molecules encoding amino acid sequences" include all nucleotide sequences or nucleic acid molecules that are degenerate versions of each other and encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNAs may or may not contain introns.

[0064] In certain embodiments, a nucleic acid molecule according to the invention encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 17-29, 34, 38-39 and 43-44, or a fragment thereof.

[0065] Nucleic acid sequences can be cloned using routine molecular biology techniques or generated de novo by DNA synthesis, which can be carried out using routine procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScripts, Invitrogen, Eurofins).

[0066] Nucleic acids according to the present invention can be DNA or RNA.

[0067] In certain embodiments, the RNA is mRNA, modified mRNA, self-replicating RNA, or circular mRNA. The RNA vaccines provided herein may have the excellent properties of generating much larger antibody titers and generating responses faster than antiviral therapeutic treatments. Without wishing to be bound by theory, it is believed that because RNA vaccines utilize natural cellular mechanisms, RNA vaccines are better designed to generate appropriate protein conformations during translation. Unlike traditional vaccines that are produced outside the body and may induce undesirable cellular responses, the RNA vaccines provided herein are introduced into cell systems in a more natural way.

[0068] Thus, the present invention also provides a RSV vaccine comprising (i) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one RSV F protein or immunogenic fragment thereof (e.g., an immunogenic fragment capable of eliciting an immune response against RSV) described herein, and (ii) a pharmaceutically acceptable carrier. In a specific embodiment, the nucleic acid molecule is an RNA polynucleotide having an open reading frame encoding the RSV F protein of the present invention, formulated in a cationic lipid nanoparticle.

[0069] The present invention also provides a vector comprising the above-described nucleic acid molecule. Thus, in certain embodiments, the nucleic acid molecule according to the present invention is part of a vector. In certain embodiments of the present invention, the vector is an adenovirus vector.

[0070] Host cells containing nucleic acid molecules encoding pre-fusion RSV F proteins also form part of the present invention. Pre-fusion RSV F proteins can be produced by recombinant DNA technology, including expression of molecules in host cells, such as Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells and PER.C6 cells, or yeast, fungi, insect cells, or transgenic animals or plants. In certain embodiments, the cells are derived from multicellular organisms, and in certain embodiments, they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. Generally, production of a recombinant protein in a host cell, such as a pre-fusion RSV F protein of the present invention, involves introducing a heterologous nucleic acid molecule encoding the protein in an expressible form into the host cell, culturing the cell under conditions conducive to expression of the nucleic acid molecule, and allowing expression of the protein in the cell. The nucleic acid molecule encoding the protein in an expressible form may be in the form of an expression cassette and typically requires sequences capable of effecting expression of the nucleic acid, such as an enhancer, promoter, polyadenylation signal, etc. Those skilled in the art will recognize that a variety of promoters can be used to obtain expression of a gene in a host cell. Promoters can be constitutive or regulated, and can be obtained from a variety of sources, including viral, prokaryotic or eukaryotic sources, or can be artificially designed.

[0071] Cell culture media are available from various suppliers, and suitable media can be routinely selected for host cells expressing the protein of interest, in this case the pre-fusion RSV F protein. Suitable media may or may not contain serum.

[0072] A "heterologous nucleic acid molecule" (also referred to herein as a "transgene") is a nucleic acid molecule that does not naturally occur in a host cell. It is introduced into a vector, for example, by standard molecular biology techniques. The transgene is generally operably linked to an expression control sequence. This can be done, for example, by placing the nucleic acid encoding the transgene under the control of a promoter. Additional regulatory sequences may be added. Many promoters can be used to express the transgene and are known to those skilled in the art. These may include, for example, viral, mammalian, and synthetic promoters. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is the CMV promoter (U.S. Pat. No. 5,385,839), e.g., the CMV immediate-early promoter comprising nt. -735 to +95 from the CMV immediate-early gene enhancer / promoter. A polyadenylation signal, e.g., the bovine growth hormone polyA signal (U.S. Pat. No. 5,122,458), may be present after the transgene. Alternatively, several widely used expression vectors are available in the art and from commercial sources, such as Invitrogen's pcDNA and pEF vector series, BD Sciences' pMSCV and pTK-Hyg, Stratagene's pCMV-Script, etc., which can be used to recombinantly express a protein of interest or to provide an appropriate promoter and / or transcription termination sequence, polyA sequence, etc.

[0073] Cell culture can be any type of cell culture, including adherent cell cultures, for example, of cells attached to the surface of a culture vessel or to microcarriers, as well as suspension cultures. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are easiest to operate and scale up. Today, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to those skilled in the art and can generally be obtained in large quantities from commercial sources or custom-made according to standard protocols. Culturing can be carried out, for example, in dishes, roller bottles, or bioreactors using batch, fed-batch, or continuous systems. Suitable conditions for culturing cells are known (e.g., Tissue Culture, Academic Press, Kruse and Paterson, editors (1973) and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0074] The present invention further provides pharmaceutical compositions comprising a pre-fusion RSV F protein and / or a fragment thereof, and / or a nucleic acid molecule, and / or a vector as described herein. Thus, the present invention provides compositions comprising a pre-fusion RSV F protein or a fragment thereof that displays an epitope present in the pre-fusion conformation of the RSV F protein but not in the post-fusion conformation. The present invention also provides compositions comprising nucleic acid molecules and / or vectors encoding such pre-fusion RSV F proteins or fragments. The present invention particularly provides pharmaceutical compositions, e.g., vaccine compositions, comprising the above-described pre-fusion RSV F protein, RSV F protein fragment, and / or nucleic acid molecule, and / or vector, and one or more pharmaceutically acceptable excipients.

[0075] The present invention also provides for the use of stabilized pre-fusion RSV F proteins (fragments), nucleic acid molecules, and / or vectors according to the present invention to vaccinate a subject against RSV.

[0076] The present invention also provides the use of stabilized pre-fusion RSV F proteins (fragments), nucleic acid molecules and / or vectors according to the present invention to induce an immune response against the RSV F protein in a subject. Further provided is a method for inducing an immune response against the RSV F protein in a subject, comprising administering to the subject a pre-fusion RSV F protein (fragment), and / or nucleic acid molecule, and / or vector according to the present invention. Further provided is the use of pre-fusion RSV F proteins (fragments), and / or nucleic acid molecules, and / or vectors according to the present invention to manufacture a medicament for use in inducing an immune response against the RSV F protein in a subject. The immune response is typically characterized by an increase in antibodies against the RSV F protein.

[0077] The present invention particularly provides pre-fusion RSV F proteins (fragments) and / or nucleic acid molecules and / or vectors of the present invention for use as vaccines.

[0078] The pre-fusion RSV F protein (fragment), nucleic acid molecule, or vector of the present invention can be used to prevent (prevent) and / or treat RSV infection. In certain embodiments, prevention and / or treatment can be targeted to patient groups susceptible to RSV infection. Such patient groups include, but are not limited to, elderly people (e.g., 50 years or older, 60 years or older, preferably 65 years or older), young people (e.g., 5 years or younger, 1 year or younger), pregnant women (for maternal-fetal immunization), as well as hospitalized patients and patients who have been treated with antiviral compounds but have shown an inadequate antiviral response.

[0079] The pre-fusion RSV F proteins, fragments, nucleic acid molecules and / or vectors of the present invention can be used to treat and / or prevent diseases or conditions caused by RSV, or in combination with other preventative and / or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and / or monoclonal antibodies.

[0080] The present invention further provides a method for preventing and / or treating RSV infection in a subject using a pre-fusion RSV F protein or fragment thereof, a nucleic acid molecule, and / or a vector according to the present invention. In a specific embodiment, the method for preventing and / or treating RSV infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion RSV F protein (fragment), a nucleic acid molecule, and / or a vector as described above. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule, or vector that is effective for preventing, ameliorating, and / or treating a disease or condition caused by infection with RSV. Prevention includes inhibiting or reducing the spread of RSV, or inhibiting or reducing the onset, development, or progression of one or more symptoms associated with RSV infection. As used herein, improvement may refer to a reduction in visible or perceptible disease symptoms, viremia, or any other measurable symptoms of RSV infection.

[0081] For administration to a subject, such as a human, the present invention can use pharmaceutical compositions comprising the pre-fusion RSV F protein (fragment), nucleic acid molecule, and / or vector described herein, and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that the carrier or excipient does not cause any undesirable or harmful effects in the subject to which it is administered at the dosage and concentration used. Such pharmaceutically acceptable carriers and excipients are well known in the art (Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., Mack Publishing Company

[1990] ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis

[2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press

[2000] ). The RSV F protein or nucleic acid molecule is preferably formulated and administered as a sterile solution, although lyophilized preparations may also be utilized. Sterile solutions are prepared by sterile filtration or other methods known in the art. The solution is then lyophilized or filled into pharmaceutical administration containers. The pH of the solution generally ranges from pH 3.0 to 9.5, for example, pH 5.0 to 7.5. The RSV F protein is typically in a solution with an appropriate pharmaceutically acceptable buffer, and the composition may also contain salts. A stabilizer such as albumin may be present. In certain embodiments, a surfactant is added. In certain embodiments, the RSV F protein may be formulated into an injectable formulation.

[0082] In certain embodiments, the composition according to the present invention further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to the applied antigenic determinant. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that stimulate the immune system. In this context, adjuvants are used to enhance the immune response to the RSV F protein of the present invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and / or aluminum phosphate, squalene-water emulsions, oil emulsion compositions (or oil-in-water compositions) including, for example, MF59 (see, for example, WO 90 / 14837), saponin formulations such as QS21 and immunostimulating complexes (ISCOMS) (see, for example, U.S. Pat. No. 5,057,540, WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620), examples of which include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG motif-containing oligonucleotides, ADP-ribosyl Examples of adjuvants include bacterial or microbial derivatives of oxidized bacterial toxins or variants thereof, such as E. coli heat-labile enterotoxin LT and cholera toxin CT; eukaryotic proteins (e.g., antibodies or fragments thereof (e.g., against antigens themselves or CD1a, CD3, CD7, CD80)); and ligands for receptors that stimulate an immune response upon interaction with recipient cells (e.g., CD40L, GMCSF, GCSF, etc.). In certain embodiments, the compositions of the invention comprise aluminum as an adjuvant, for example, in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, or combinations thereof, at a concentration of 0.05 to 5 mg, e.g., 0.075 to 1.0 mg, of aluminum per dose.

[0083] In other embodiments, the composition does not include an adjuvant.

[0084] In a specific embodiment, the present invention provides a method for producing a vaccine against respiratory syncytial virus (RSV), comprising providing an RSV F protein (fragment), nucleic acid, or vector according to the present invention and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to a drug or composition containing an active ingredient effective to induce a degree of immunity in a subject against a particular pathogen or disease, resulting in at least a reduction (up to a complete absence) in the severity, duration, or other symptoms associated with infection by the pathogen or disease. In the present invention, the vaccine comprises an effective amount of a pre-fusion RSV F protein (fragment) and / or a nucleic acid molecule encoding a pre-fusion RSV F protein and / or a vector containing the nucleic acid molecule, which induces an effective immune response against RSV. This provides a method for preventing severe lower respiratory tract diseases that lead to hospitalization and reducing the frequency of complications, such as pneumonia and bronchiolitis, caused by RSV infection and replication in subjects. The term "vaccine" according to the present invention means that it is a pharmaceutical composition and therefore typically contains a pharmaceutically acceptable diluent, carrier, or excipient. It may or may not contain additional active ingredients. In certain embodiments, the vaccine may be a combination vaccine that further comprises, for example, other proteins of RSV and / or other components that induce an immune response against other infectious agents, such as HMPV, PIV, and / or influenza. The administration of the additional active ingredient can be carried out, for example, by separate administration or by administering a product that combines the vaccine of the present invention with the additional active ingredient.

[0085] Furthermore, the proteins of the present invention can be used as diagnostic tools to test the immune status of an individual, for example, by determining whether antibodies capable of binding to the proteins of the present invention are present in the serum of such an individual. Thus, the present invention also relates to an in vitro diagnostic method for detecting the presence of RSV infection in a patient, the method comprising: a) contacting a biological sample obtained from the patient with a protein of the present invention; and b) detecting the presence of an antibody-protein complex.

[0086] The present invention is further described in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.

[0087] [Example] [Example 1] Instability of soluble RSV F ectodomain protein.

[0088] Plasmids encoding recombinant RSV-A and RSV-B F protein ectodomains truncated after amino acid 513 according to SEQ ID NO: 1 were synthesized and codon-optimized in Genscript. The constructs were cloned into pCDNA2004 and sequenced using standard methods well known in the art, including site-directed mutagenesis and PCR. The RSV-A and RSV-B F ectodomains were designed with and without the Foldon trimerization domain. Additionally, RSV-A F had N67I, S215P, and D486N substitutions, while RSV-B F had P101Q, I152M, L203I, S215P, D486N, and D489Y substitutions. The proteins were expressed in Expi293F cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37 ° C and 10% CO for 3 days. To assess RSV F trimer expression, sterile-filtered crude cell culture supernatant was loaded at 0.35 mL / min onto a Unix-C SEC-300, 15 cm column (Sepax Technologies) equipped with a corresponding guard column (Sepax Technologies) equilibrated with running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0). Analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package. OD280 values ​​were displayed after subtraction of the OD280 signal from mock-transfected cells.

[0089] RSV-A and RSV-B F trimers containing the Foldon trimerization domain eluted at a retention time of approximately 4.4 minutes (RSV150042 and RSV200125, respectively). Trimers were not detected upon expression of the RSV-A and RSV-B F ectodomains without the Foldon trimerization domain (RSV220961 and RSV220982, respectively) (Figure 1). Removal of the trimerization domain instead resulted in a monomer peak eluting at a retention time of approximately 4.8 minutes, indicating the instability of both the RSV-A and RSV-B F ectodomain trimers.

[0090] Example 2: Stabilizing mutations in HR2 improve trimerization of RSV-A and RSV-B F in the absence of a heterotrimerization domain.

[0091] To stabilize the HR2 region of RSV F (amino acids 491-524) and enable RSV F ectodomain trimer expression in the absence of a heterotrimerization domain, amino acid residue S509 was substituted with a hydrophobic amino acid in the stem region of RSV-A F (RSV220963-RSV220968).

[0092] The recombinant RSV-A F protein ectodomain did not have a heterotrimerization domain, was truncated after amino acid 524, and further contained N67I, S215P, and D486N substitutions. Trimer expression was evaluated 3 days after transfection in Expi293F cells as described in Example 1.

[0093] RSV220962, which lacks the trimerization domain and has N67I, S215P, D486N, and wild-type S509, eluted as a monomer peak at a retention time of approximately 4.8 minutes, as did the S509Y and S509V substitutions (RSV220964, RSV220967) (Figure 2). A minimal trimer peak eluting at a retention time of approximately 4.4 minutes was detected for S509M (RSV220968), and more prominent trimer peaks were observed with the substitutions S509F, S509I, and S509L (RSV220963, RSV220965, RSV220966) (Figure 2).

[0094] The S509F substitution was then combined with another amino acid substitution in the HR2 region of RSV F at position F505, and their individual and combined effects were evaluated in RSV-A and RSV-B F ectodomain mutants without heterotrimerization domains (FIG. 3). For this purpose, a plasmid encoding a recombinant RSV-A F protein ectodomain truncated after amino acid 513 and further containing a C-tag and P101Q, S215P, Q354L, D486N, E487L, and D489Y substitutions was used. For RSV-B F, a protein ectodomain truncated after amino acid 513 and further containing P101Q, I152M, L203I, S215P, D486N, and D489Y substitutions was used. Trimer expression was evaluated 3 days after transfection in Expi293F cells as described in Example 1.

[0095] The RSV-A F backbone RSV211965 without HR2 substitution and without the heterotrimerization domain eluted as a trimer peak with a retention time of approximately 4.6 minutes (Fig. 3A). The trimer RSV-A F ectodomain RSV211965 eluted later than the RSV-A F mutants described in Fig. 1 due to the shorter ectodomain used in this mutant. Introduction of the single substitutions F505W (RSV211964) and S509F (RSV211963) had minimal effect on trimer yield, while introduction of the combination of F505W + S509F (RSV210789) demonstrated a more significant increase in RSV-A F trimer yield with a shift to a longer retention time, indicating a more closed conformation of the stabilized mutant (Fig. 3A). Furthermore, introduction of S509F, particularly the combination of F505W + S509F, increased trimer stability, as shown by minimal loss of the RSV-A F trimer peak after 15 minutes of incubation at 65°C (Fig. 4).

[0096] The RSV-B F backbone RSV220982, which does not have an HR2 substitution and does not have a heterotrimerization domain, eluted as a monomer peak with a retention time of approximately 4.8 minutes (Fig. 3B). Substitution with either the single mutation F505W (RSV220983) or S509F (RSV220984) resulted in similar expression of monomers and no trimer expression. However, introduction of the combination of F505W + S509F reduced the monomer content and resulted in detectable trimer expression (RSV220985, Fig. 3B).

[0097] In summary, HR2 stabilization by F505W and S509F substitutions improved RSV F ectodomain trimer expression and stability.

[0098] Example 3: Stabilizing head domain mutations improve trimerization of RSV-A and RSV-B F in the absence of a heterotrimerization domain.

[0099] To stabilize the RSV F head domain (amino acids 27-490) and enable RSV F ectodomain trimer expression in the absence of a heterotrimerization domain, various amino acid substitutions were evaluated for improved trimer expression. Plasmids encoding recombinant RSV-A and RSV-B F protein ectodomains truncated after amino acid 513 without the heterotrimerization domain were generated. The RSV-A F mutant had a C tag and P101Q, D486N, F505W, and S509F substitutions (RSV210776). The RSV-B F mutant had P101Q, I152M, L203I, S215P, D486N, and D489Y (RSV220982). Trimer expression was evaluated 3 days after transfection in Expi293F cells as described in Example 1.

[0100] RSV-A F trimer expression was increased by the introduction of E328P (RSV210845), Q354L (RSV210843), Q494I (RSV210844), and especially E487L (RSV210842) (Fig. 5A). Introduction of Q354L (RSV221265) and E487L (RSV221269) into RSV-B F both resulted in detectable trimer expression (Fig. 5B). The combined substitution of Q354L + E487L (RSV221273) significantly increased RSV-B F trimer yield and reduced monomer expression (Fig. 5B), demonstrating that head domain stabilization improved RSV F ectodomain trimer expression.

[0101] The stability of RSV-B F ectodomain trimers was assessed by analytical SEC of crude cell culture supernatants after 16 days of storage at 4°C. These data demonstrated that for RSV221273, which has the head domain substitutions Q354L+E487L, significant trimer expression was initially detected (Figures 5B and 6A), but these trimers were significantly reduced after 16 days of storage at 4°C (Figure 6B). Similarly, the HR2-stabilized F505W+S509F in RSV220985 was sufficient to detect RSV-B F trimer expression on the day of harvest (Figures 3B and 6A), but did not result in stable trimer expression after 16 days of storage at 4°C (Figure 6B). However, combined stabilization of the RSV-B F head domain and HR2 region resulted in stable trimer expression (Fig. 6B, RSV221276), indicating that stabilization of both regions contributes to overall trimer ectodomain stability in the absence of a heterotrimerization domain. These results are consistent with the improved thermostability observed with RSV-A F with stabilization of both the head domain and HR2 region (RSV210789, Fig. 4).

[0102] [Example 4] Effect of HR2 length and stabilization on trimerization of RSV-A and RSV-B F in the absence of a heterotrimerization domain.

[0103] The effect of the cleavage position (i.e., HR2 length) on RSV-A and RSV-B F trimer ectodomain expression was evaluated in various backbones. HR2 extension involved adding 11 amino acids (HNVNAGKSTTN) from the full-length RSV-A and RSV-B F sequences to a frequently used variant truncated at position 513. Mutants with a "short" HR2 (truncated at position 513) were compared with a "long" HR2 variant (truncated at position 524) by assessing trimer expression in cell culture supernatants, as described in Example 1.

[0104] Improved trimer expression due to S509 substitutions in the RSV-A F ectodomain was measured for long HR2 mutants with substitutions N67I, S215P, and D486N (RSV220963, RSV220965, RSV220966, RSV220968) but was not detectable in the expression of equivalent mutants with shorter HR2 regions (RSV221289, RSV221292, RSV221291, RSV221293) (Fig. 7A ). Furthermore, the stability of RSV-A F trimer expression was enhanced in a mutant with a long HR2 (RSV220775) containing P101Q, S215P, D486N, D489Y, F505W, and S509F compared to the equivalent mutant with a short HR2 (RSV220940), and RSV-A F trimers were undetectable after 2 days of storage at 4°C (Figure 7B). Similarly, HR2 extension positively affected RSV-B F trimer ectodomain expression in a mutant with P101Q, I152M, L203I, S215P, D486N, D489Y, F505W, and S509F (RSV221277). Compared to the same mutant with a short HR2 (RSV220985), trimer expression was significantly enhanced and monomer expression was reduced (Figure 7C). As expected, due to the longer HR2 (i.e., the addition of 11 amino acids), the trimer of RSV221277 eluted slightly earlier than the shorter HR2 variant (Fig. 7C ).

[0105] The extended HR2 region was further stabilized by the introduction of G519V and T523I substitutions. In a stabilized long HR2 RSV-A F ectodomain mutant lacking the heterotrimerization domain and containing P101Q, S215P, Q354L, D486N, E487L, D489Y, F505W, and S509F (RSV220974), the introduction of G519V + T523I increased trimer expression (RSV220975) (Figure 8A).

[0106] Stabilized RSV-A F mutants with either a short HR2 (RSV211956), a long HR2 (RSV220974), or a long stabilized HR2 domain (RSV220975) were purified. None of the mutants contained the Foldon trimerization domain or the C-tag purification tag. All contained the stabilizing mutations P101Q, S215P, Q354L, D486N, E487L, D489Y, F505W, and S509F. Expi293F cells were transiently transfected with the plasmids using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37°C and 10% CO2 for 5 days. The culture supernatant was harvested and spun at 600 g for 10 minutes to remove cells and debris, then sterile filtered using a 0.22 μm vacuum filter. RSV-A F protein was purified using a two-step purification protocol, including ion exchange (cation) purification at pH 5.0 and polishing by size-exclusion chromatography using a Superdex 200 16 / 600 pg column. Portions of the trimer were pooled and further characterized by analytical SEC-MALS using an ultra-high-performance liquid chromatography system (Vanquish, Thermo Scientific) coupled to an Optilab μT-rEX Refractive Index Detector (Wyatt) and a μDAWN TREOS instrument (Wyatt) in combination with an in-line Nanostar DLS reader (Wyatt). Protein was loaded onto a Unix-C SEC-300, 15 cm column (Sepax Technologies) with a corresponding guard column (Sepax Technologies), equilibrated with running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL / min. Analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package, and the conformation, hydrodynamic radius, and molecular weight of the F trimer were calculated by Astra software and compared with the calculated weight to confirm the trimer conformation (Figure 9A).

[0107] Melting temperature (Tm) of purified RSV-A F trimer 50 ) was determined by differential scanning fluorimetry (DSF). For this purpose, the fluorescence emission of Sypro Orange Dye (ThermoFisher Scientific) added to the RSV-A F protein in solution was measured. Measurements were performed at a starting temperature of 25 °C and an ending temperature of 95 °C (increasing 54 °C per hour). Melting curves were measured using a ViiA7 real-time PCR instrument (Applied Biosystems), and Tm50 values ​​were obtained from the negative first derivative as previously described (Rutten et al. (2020) Cell Rep 30:4540-4550). RSV220974, which has a long HR2, had a higher melting temperature of 74.0 °C compared to the melting temperature of 68.7 °C for RSV211956, which has a short HR2, confirming the improved stability of extending the HR2 region of the RSV-A F ectodomain (Figure 9B). The substitutions G519V+T523I in RSV220975 did not affect the melting temperature compared to RSV220975 (74.2°C and 74.0°C, respectively), but increased the trimer yield (Figure 9C).

[0108] The pre-fusion conformation of the purified RSV-A F protein was confirmed by quantitative Octet biolayer interferometry (BLI) measurements using the RSV-F A pre-fusion-specific monoclonal antibody RSV90 (Mousa et al., (2017) Nat Microbiol, 2:16271), the post-fusion-specific monoclonal antibody ADI-15644 (Gilman et al., (2016) Sci Immunol, 1:1aaj 1879), and the pan-specific monoclonal antibody CR9506 (containing the heavy and light chain variable regions disclosed in WO 20 / 099383). The antibodies were immobilized on an anti-human IgG sensor at a concentration of 5 μg / ml, and the initial binding rate of RSV-F A at 20 μg / ml during 300 seconds of association was plotted (Figure 9D). Post-fusion RSV-A F protein (RSV150043) was included as an assay control. Pan-specific CR9506 binding was observed for all four RSV-A F proteins, but post-fusion F-specific binding was only detected for the post-fusion F protein. Conversely, pre-fusion F-specific binding was only detected for the stabilized RSV-A F mutant (Fig. 9D).

[0109] Example 5: Purification, characterization and immunogenicity of stabilized RSV-A F mutants with and without heterotrimerization domains.

[0110] RSV-A F mutants with (RSV150042) and without (RSV210789 and RSV211957) the Foldon trimerization domain and stabilizing mutations listed in Figure 10A were transiently transfected into Expi293F cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37 °C and 10% CO for 5 days. The culture supernatant was collected and spun at 600 g for 10 minutes to remove cells and cell debris, then sterile filtered using a 0.22 μm vacuum filter. The RSV-A F protein with the Foldon trimerization domain (RSV150042) was purified using a two-step purification protocol, including ion exchange (cation) purification at pH 5.0 and size exclusion chromatography using a Superdex 200 16 / 600 pg column. RSV-A F proteins (RSV210789 and RSV211957) lacking the Foldon trimerization domain and carrying a C-tag purification tag were purified using a two-step purification protocol, including a CaptureSelect™ C-tag affinity column and size-exclusion chromatography using a Superdex 200 16 / 600 pg column. Portions of the trimers were pooled and further characterized by analytical SEC-MALS using an ultra-high-performance liquid chromatography system (Vanquish, Thermo Scientific) coupled to an Optilab μT-rEX Refractive Index Detector (Wyatt) and a μDAWN TREOS instrument (Wyatt) in conjunction with an in-line Nanostar DLS reader (Wyatt). A Unix-C SEC-300, 15 cm column (Sepax Technologies) with a corresponding guard column (Sepax Technologies) equilibrated with running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) was filled with protein at 0.35 mL / min.The analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package, and the conformation, hydrodynamic radius, and molecular weight of the F trimer were calculated by Astra software and compared with the calculated weight, confirming the trimer conformation (Figure 10B).

[0111] The melting temperature (Tm50) of purified RSV-A F trimers was determined by differential scanning fluorimetry (DSF). To this end, the fluorescence emission of Sypro Orange Dye (ThermoFisher Scientific) added to RSV-A F protein in solution was measured. Measurements were performed at a starting temperature of 25°C and an ending temperature of 95°C (increasing 54°C per hour). Melting curves were measured using a ViiA7 real-time PCR instrument (Applied Biosystems), and Tm50 values ​​were obtained from the negative first derivative as previously described (Rutten et al. (2020) Cell Rep 30:4540-4550). The melting temperature of RSV150042, which has a Foldon trimerization domain, was lower at 64.7 ° C. compared to RSV210789 and RSV211957, which do not have a trimerization domain and have stabilizing amino acid substitutions according to the present invention (70.4 ° C. and 69.9 ° C., respectively) (FIG. 10C).

[0112] The prefusion conformation of the purified RSV-A F protein was confirmed by quantitative Octet biolayer interferometry (BLI) measurements using the RSV-F A prefusion-specific monoclonal antibody RSV90 (see above), the postfusion-specific monoclonal antibody ADI-15644 (see above), and the pan-specific monoclonal antibody CR9506 (see above). The antibodies were immobilized on an anti-human IgG sensor at a concentration of 5 μg / ml, and the initial binding rate of RSV-F A at 20 μg / ml during 300 seconds of association was plotted (Figure 10D). The postfusion RSV-A F protein (RSV150043) was included as an assay control. Binding to the pan-specific CR9506 was confirmed for all four RSV-A F proteins, while postfusion F-specific binding was only detected for the postfusion F protein. Conversely, pre-fusion F-specific binding was only detected for stabilized RSV-A F mutants, regardless of the presence or absence of the Foldon trimerization domain (Fig. 10D).

[0113] Storage stability at 4°C was evaluated for purified RSV-A F mutants lacking the heterotrimerization domain. RSV210789 remained stable after 19 weeks of storage at 4°C, whereas some aggregates were detected in RSV211957, which eluted between 3 and 3.5 minutes (Figure 10E). The presence of aggregates in RSV211957 indicates that S215P removal is not optimal for RSV-A F protein stability. Therefore, without the S215P mutation, the protein can still transition to the post-fusion conformation.

[0114] The immunogenicity of purified RSV-A F proteins RSV150042, RSV210789, and RSV211957 was evaluated by intramuscularly immunizing naive female Balb / c mice with 15, 5, or 1.5 μg of the RSV-A F protein variants (n=5 / dose). As a control, mice were intramuscularly injected with formulation buffer (n=3).

[0115] Serum samples were isolated two weeks after the second immunization (week 6) and used to measure virus neutralizing antibody titers (VNT) for RSV A CL57, RSV-A pre-fusion F by a firefly luciferase (FFL) reporter-based assay, and post-fusion F-binding and Foldon-binding antibody titers by ELISA.

[0116] RSV-A pre-fusion F and post-fusion F binding antibody titers (FIGS. 11C, 11D) and RSV CL57 VNT (FIG. 11A) were induced by RSV150042 and by RSV210789 and RSV211957 (two stabilized trimeric pre-fusion RSV-A F proteins without heterotrimerization domains according to the present invention), and there was no apparent difference in the level of induction by the pre-fusion F protein variants across administrations. For samples with at least one titer above the LLOD, the pre-fusion F / post-fusion F binding antibody titer ratio was calculated (FIG. 11E). RSV150042 and RSV210789 showed similar pre-fusion F / post-fusion F binding antibody ratios, while RSV211957 showed a pre-fusion F / post-fusion F binding antibody ratio that was more biased towards post-fusion F. Two stable trimeric pre-fusion RSV-A F proteins that do not contain the Foldon trimerization domain did not induce Foldon-binding antibody titers, and RSV150042 was confirmed to induce Foldon-binding antibody titers (FIG. 11B).

[0117] In summary, these data demonstrate that a stable trimeric pre-fusion RSV-A F protein can be produced that does not contain a heterotrimerization domain and that this protein is immunogenic in mice.

[0118] Example 6: Purification, characterization, and immunogenicity of stabilized Foldon-less tagless RSV-A and RSV-B pre-fusion F proteins. Next, we generated stabilized Foldon-less tagless RSV-A and RSV-B pre-fusion F proteins based on the sequences of currently circulating strains. The complete 524 amino acid ectodomain sequence was used, containing head substitutions P101S, I152M, S215P, Q354L, D486N, E487L, and D489Y, and HR2-stem substitutions F505W, S509F, G519V, and T523I. Stabilized RSV-A (RSV23316) and RSV-B (RSV23321) F trimers were expressed in Expi293F cells, purified (IEX followed by SEC polishing), characterized, and compared to a benchmark RSV-A pre-fusion F protein ("PRPM") containing a Foldon trimerization domain (Krarup et al., 2015). Analytical SEC revealed that the proteins eluted at the expected retention time (Rt), with RSV23316 and RSV23321 eluting at a slightly longer Rt than PRPM due to the absence of Foldon (Figure 12A). The melting temperature of the Foldon-less RSV-A F protein RSV23316 was 75°C, an increase of more than 10°C compared to the benchmark Foldon-containing RSV-A F mutant PRPM (Figure 1B). The Foldon-less RSV-B protein RSV23321 had an intermediate melting temperature of 70°C (Figure 12B). The antigenic profiles of all three proteins were typical of the pre-fusion RSV F conformation, demonstrating binding of the pre-fusion specific CR9501, RSV90, and RSD5-GL antibodies in the absence of binding of the post-fusion specific ADI-15644 antibody (Figure 12C). As expected, RSV90 does not bind to the RSV-B F protein due to mutations in the RSV90 epitope (Mousa et al., 2017). Both foldon-free pre-fusion F proteins exhibited excellent stability profiles, retaining a trimeric conformation during long-term storage at 4°C and 37°C for up to 6 months (Figure 12D).

[0119] [Table 1]

[0120] array SEQ ID NO: 1 - RSV-A full length (consensus) [ka]

[0121] SEQ ID NO:2-RSV150042 [ka]

[0122] SEQ ID NO:3-RSV220961 [ka]

[0123] SEQ ID NO:4-RSV220962 [ka]

[0124] SEQ ID NO:5-RSV220964 [ka]

[0125] SEQ ID NO:6-RSV220967 [ka]

[0126] SEQ ID NO:7-RSV220968 [ka]

[0127] SEQ ID NO:8-RSV221293 [ka]

[0128] SEQ ID NO:9-RSV220963 [ka]

[0129] SEQ ID NO: 10-RSV221289 [ka]

[0130] SEQ ID NO: 11-RSV220965 [ka]

[0131] SEQ ID NO: 12-RSV221292 [ka]

[0132] SEQ ID NO: 13-RSV220966 [ka]

[0133] SEQ ID NO: 14 - RSV221291 [ka]

[0134] SEQ ID NO: 15-RSV211965 [ka]

[0135] SEQ ID NO: 16-RSV211964 [ka]

[0136] SEQ ID NO: 17-RSV211963 [ka]

[0137] SEQ ID NO: 18-RSV210789 [ka]

[0138] SEQ ID NO: 19-RSV210776 [ka]

[0139] SEQ ID NO:20-RSV210845 [ka]

[0140] SEQ ID NO:21-RSV210843 [ka]

[0141] SEQ ID NO:22-RSV210844 [ka]

[0142] SEQ ID NO:23-RSV210842 [ka]

[0143] SEQ ID NO:24-RSV220775 [ka]

[0144] SEQ ID NO:25-RSV220940 [ka]

[0145] SEQ ID NO: 26-RSV211956 [ka]

[0146] SEQ ID NO:27-RSV220974 [ka]

[0147] SEQ ID NO: 28-RSV220975 [ka]

[0148] SEQ ID NO: 29-RSV211957 [ka]

[0149] SEQ ID NO: 30-RSV200125 [ka]

[0150] SEQ ID NO:31-RSV220982 [ka]

[0151] SEQ ID NO:32-RSV220983 [ka]

[0152] SEQ ID NO: 33 - RSV220984 [ka]

[0153] SEQ ID NO: 34 - RSV220985 [ka]

[0154] SEQ ID NO: 35 - RSV221265 [ka]

[0155] SEQ ID NO: 36 - RSV221269 [ka]

[0156] SEQ ID NO: 37 - RSV221273 [ka]

[0157] SEQ ID NO: 38 - RSV221276 [ka]

[0158] SEQ ID NO: 39 - RSV221277 [ka]

[0159] SEQ ID NO: 40 - RSV-B full length (consensus) [ka]

[0160] SEQ ID NO: 41 - Strep tag WSHPQFEK

[0161] SEQ ID NO: 42-C tag EPEA

[0162] SEQ ID NO: 43 - RSV23316 [ka]

[0163] SEQ ID NO: 44 - RSV23321 [ka]

Claims

1. 1. A respiratory syncytial virus (RSV) F protein comprising a head region and a stem region, the protein comprising at least one amino acid mutation in the stem region relative to the amino acid sequence of a wild-type RSV F protein, the at least one mutation in the stem region comprising a mutation of amino acid residue at position 509 to F, I, or L, wherein the amino acid positions are numbered according to the numbering of the amino acid residues in SEQ ID NO:

1.

2. 2. The protein of claim 1, wherein the protein comprises at least one amino acid mutation in the head region and at least one amino acid mutation in the stem region relative to the amino acid sequence of a wild-type RSV F protein, wherein the at least one mutation in the stem region comprises a mutation of amino acid residue 509 to F, I, or L.

3. 3. The protein of claim 2, wherein the at least one amino acid mutation in the head region is a mutation of amino acid at residue 215 to P or a mutation of amino acid residue at position 486 to N.

4. The protein of claims 2 and 3, wherein the protein comprises at least one additional mutation in the head region.

5. 5. The protein of claim 4, wherein the protein comprises at least two amino acid mutations in the head region and at least one amino acid mutation in the stem region relative to the amino acid sequence of a wild-type RSV F protein, wherein the at least one mutation in the stem region comprises a mutation of amino acid residue 509 to F, I, or L.

6. 5. The protein of claim 4, comprising at least three amino acid mutations in the head region and at least one amino acid mutation in the stem region relative to the amino acid sequence of a wild-type RSV F protein, wherein the at least one mutation in the stem region comprises a mutation of amino acid residue 509 to F, I, or L.

7. The protein of any one of claims 1 to 6, wherein the RSV F protein is an F protein of an RSV A subtype.

8. The protein of any one of claims 1 to 6, wherein the RSV F protein is an F protein of an RSV B subtype.

9. The protein according to any one of claims 4 to 8, wherein the amino acid at position 101 is not P.

10. 10. The protein of any one of claims 1 to 7 or 9, wherein the at least one further mutation in the head region is selected from the group consisting of a mutation of the amino acid residue at position 101 to Q, S, T, A or G, a mutation of the amino acid residue at position 328 to P, a mutation of the amino acid residue at position 354 to L, a mutation of the amino acid residue at position 487 to L, a mutation of the amino acid residue at position 489 to Y, a mutation of the amino acid residue at position 494 to I, a mutation of the amino acid residue at position 519 to V, and a mutation of the amino acid residue at position 523 to I.

11. 10. The protein of any one of claims 1 to 6, 8 or 9, wherein the at least one further mutation in the head region is selected from the group consisting of: a mutation of the amino acid residue at position 101 to Q, S, T, A or G, a mutation of the amino acid residue at position 152 to M, a mutation of the amino acid residue at position 203 to I, a mutation of the amino acid residue at position 354 to L, a mutation of the amino acid residue at position 487 to L, and a mutation of the amino acid residue at position 489 to Y.

12. The protein according to any one of claims 1 to 11, wherein the at least one mutation at amino acid residue 509 in the stem region is a mutation to F.

13. The protein of any one of claims 1 to 12, further comprising a second mutation in the stem region, wherein the second mutation is a mutation of the amino acid residue at position 505 to W.

14. The protein of any one of claims 5 to 13, wherein the protein is truncated after amino acid residue 524.

15. The protein of any one of claims 6 to 13, wherein the protein is truncated at the C-terminus after amino acid residue 513.

16. The protein of claim 14 or 15, wherein the protein does not contain a heterotrimerization domain.

17. The protein of any one of claims 1 to 16, wherein the protein is a trimer.

18. The protein according to any one of claims 1 to 17, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 17-29, 34, 38-39, and 43-44, or an amino acid sequence having at least 90% amino acid sequence identity thereto, or a fragment thereof.

19. A fragment of the protein described in any one of claims 1 to 18.

20. A nucleic acid encoding a protein according to any one of claims 1 to 18 or a fragment according to claim 19.

21. 21. The nucleic acid of claim 20, wherein the nucleic acid is DNA or RNA.

22. 22. The nucleic acid of claim 21, wherein the RNA is mRNA, a modified mRNA, a self-replicating RNA, or a circular mRNA.

23. 23. The nucleic acid of claim 20, 21 or 22, encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 17-29, 34, 38-39, and 43-44, or an amino acid sequence having at least 90% amino acid sequence identity, or a fragment thereof.

24. A vector comprising the nucleic acid according to any one of claims 20 to 23.

25. A composition comprising a protein according to any one of claims 1 to 18, a fragment according to claim 19, a nucleic acid according to any one of claims 20 to 23 and / or a vector according to claim 24.

26. 26. A method of vaccinating a subject against RSV, comprising administering to said subject the composition of claim 25.

27. 26. A method of preventing RSV infection and / or replication in a subject, comprising administering to said subject the composition of claim 25.

28. An isolated host cell comprising a nucleic acid according to any one of claims 20 to 23.

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