Recombinant prefusion RSV F proteins and uses thereof

1. An immunogen comprising a recombinant respiratory syncytial virus (RSV) F protein or a fragment thereof specifically binding to an RSV F prefusion-specific antibody,

wherein the recombinant RSV F protein or the fragment thereof comprises an F1 polypeptide and an F2 polypeptide of any RSV F protein characterized by the following substitutions at amino acid positions corresponding to the following amino acid positions in SEQ ID NO: 1 as a reference sequence:

(i) S155C and S290C substitutions, which form a non-natural disulfide bond;

(ii) a substitution at one or both of positions S190 and V207 by amino acids selected from the group consisting of F, L, W, Y, H, and M; and

(iii) a pair of substitutions forming a non-natural disulfide bond selected from the group consisting of the following substitution pairs: Q98C and Q361C, A149C and Y458C, N183GC and N428C, N88C and N254C, E92C and N254C, and S238C and Q279C;

wherein the recombinant RSV F protein or the fragment thereof does not comprise a pep27 polypeptide; and

wherein (a) the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-103 of SEQ ID NO: 31, and an F1 polypeptide comprising or consisting of amino acids 106-474 of SEQ ID NO: 31; or

(b) the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 32; and

an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 32.

2. The immunogen according to claim 1 , wherein the F1 polypeptide and the F2 polypeptide of the recombinant RSV F protein or of the fragment thereof share at least 80% sequence identity with the F1 polypeptide and the F2 polypeptide, respectively, of a native bovine RSV F protein.

3. The immunogen according to claim 2 , wherein the native bovine RSV F protein comprises an amino acid sequence according to any of SEQ ID NOs: 1-9.

4. The immunogen according to claim 1 , wherein the recombinant RSV F protein, or the fragment thereof, comprises:

(i) an antigenic site Ø that specifically binds to the RSV F prefusion-specific antibody, wherein the antigenic site Ø comprises residues 62-69 and 196-209 of a native bovine RSV F protein sequence set forth in any one of SEQ ID NOs: 1-9;

(ii) an epitope recognized by AM14 antibody, wherein the epitope recognized by AM14 antibody comprises at least residues L160, N183, N426, R429, H514 and H515 of a native bovine RSV F protein sequence set forth in any one of SEQ ID NOs: 1-9; and/or

(iii) an epitope recognized by MPE8 antibody, wherein the epitope recognized by MPE8 antibody comprises at least residues T50, D310, L305, G307, and 1309 of a native bovine RSV F protein sequence set forth in any one of SEQ ID NOs: 1-9.

5. The immunogen according to claim 1 , wherein the F2 polypeptide and the F1 polypeptide are linked by a heterologous peptide linker or are directly linked.

6. The immunogen according to claim 1 , wherein the recombinant RSV F protein is linked to a trimerization domain.

7. The immunogen according to claim 1 , wherein the immunogen comprises or consists of

an F2 polypeptide comprising or consisting of amino acids 26-103 of SEQ ID NO: 31;

an F1 polypeptide comprising or consisting of amino acids 106-474 of SEQ ID NO: 31; and

a foldon domain comprising or consisting of amino acids 475-513 of SEQ ID NO: 31, which is preferably directly linked to the C-terminus of the F1 polypeptide.

8. The immunogen according to claim 1 , wherein the recombinant RSV F protein or the fragment thereof comprises or consists of

an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 32;

an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 32; and

a foldon domain comprising or consisting of amino acids 477-515 of SEQ ID NO: 32, which is preferably directly linked to the C-terminus of the F1 polypeptide.

9. The immunogen according to claim 1 , wherein the immunogen comprises or consists of amino acids 26-474 of SEQ ID NO: 31.

10. The immunogen according to claim 1 , wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 31.

11. The immunogen according to claim 1 , wherein the immunogen comprises or consists of amino acids 26-476 of SEQ ID NO: 32.

12. The immunogen according to claim 1 , wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 32.

13. A virus-like particle comprising the immunogen according to claim 1 .

14. A protein nanoparticle comprising the immunogen according to claim 1 .

15. A nucleic acid molecule comprising a polynucleotide encoding the immunogen of claim 1 , a virus-like particle comprising the immunogen, or a protein nanoparticle comprising the immunogen.

16. A vector comprising the nucleic acid molecule according to claim 15 .

17. An isolated host cell comprising the nucleic acid molecule of claim 15 or a vector comprising the nucleic acid molecule.

18. An immunogenic composition comprising

(i) the immunogen according to claim 1 ;

(ii) a virus-like particle comprising the immunogen according to (i);

(iii) a protein nanoparticle comprising the immunogen according to (i);

(iv) a nucleic acid molecule comprising a polynucleotide encoding the immunogen according to (i), the virus-like particle according to (ii), or the protein nanoparticle according to (iii);

(v) a vector comprising the nucleic acid molecule according to (iv); or

(vi) a host cell comprising the nucleic acid molecule according to (iv) or the vector according to (v);

and a pharmaceutically acceptable carrier.