NZ714594B2 - Stabilized soluble pre-fusion rsv f polypeptides - Google Patents

Abstract

The present invention provides stable pre-fusion respiratory syncitial virus (RSV) F polypeptides, having mutations at positions 161, 173, 182, 486, and 489 of SEQ ID NO: 1, immunogenic compositions comprising said polypeptides and uses thereof for the prevention and/or treatment of RSV infection.

Description

Stabilized soluble pre-fusion RSV F polypeptides The present invention relates to the field of medicine. The invention in particular relates to recombinant pre-fusion RSV F polypeptides and uses thereof, e.g. in genic compositions.

Background of the invention atory syncytial virus (RSV) is an enveloped non-segmented negative- strand RNA virus in the family Paramyxoviridae, genus Pneumovirus. Worldwide, it is ted that 64 million RSV infections occur each year resulting in 160.000 deaths (WHO Acute Respiratory Infections Update September 2009). The most severe disease occurs particularly in ure infants, the elderly and immune-compromised individuals. In children younger than 2 years, RSV is the most common respiratory tract pathogen, accounting for imately 50% of the hospitalizations due to respiratory infections, with a peak of hospitalization occurring at 2-4 months of age. It has been reported that almost all children have been infected by RSV by the age of two. Repeated infection during lifetime is attributed to ineffective natural immunity. The level of RSV disease burden, mortality and morbidity in the elderly are second only to those caused by non-pandemic influenza A infections.

To infect a host cell, RSV, like other enveloped viruses such as influenza virus and HIV, require fusion of the viral membrane with a host cell ne. For RSV the ved fusion protein (RSV F protein) fuses the viral and host cell cellular membranes. In current models, based on paramyxovirus studies, the RSV F protein initially folds into a "pre-fusion" mation. The metastable structure has recently been solved in x with a stabilizing neutralizing antibody Fab fragment (McLellan et al., Science 340(6136):1113-7, 2013). During cell entry, the pre-fusion conformation oes refolding and conformational s to its "post-fusion" conformation (McLellan, J. Virol 85(15):7788-96, 2010; Swanson, PNAS 108(23):9619-24, 2011). Thus, the RSV F protein is a metastable protein that drives membrane fusion by coupling irreversible protein refolding to membrane juxtaposition by initially folding into a metastable form (pre-fusion mation) that subsequently undergoes discrete/stepwise conformational changes to a lower energy conformation (post-fusion conformation).

These observations suggest that pre-fusion and post-fusion RSV F protein are antigenically distinct (Calder, L. J. et al. Virology 271, 122-131 (2000)).

A vaccine against RSV infection is not currently available, but is d. Vaccine candidates based on the RSV F protein have failed due to ms with e.g. stability, purity, reproducibility, and potency. As indicated above, crystal structures have revealed a large conformational change between the pre-fusion and post-fusion states. The magnitude of the ngement suggested that only a n of antibodies directed to the post-fusion conformation of RSV-F will be able to cross react with the native conformation of the pre-fusion spike on the surface of the virus. Accordingly, efforts to e a vaccine against RSV have focused on developing vaccines that contain sion forms of RSV F protein (see, e.g., WO20101149745, WO2010/1149743, WO2009/1079796, WO2012/158613). r, these efforts have not yet yielded stable pre-fusion RSV F polypeptides that could be used as candidates for testing in humans. It is an object of the present invention to go some way towards overcoming these problems and/or to at least provide the public with a useful .

Summary of the invention In a first aspect the present invention es a recombinant pre-fusion respiratory syncytial virus (RSV) Fusion (F) polypeptide, comprising at least one epitope that is specific to the prefusion conformation F protein, wherein the at least one epitope is recognized by a pre-fusion specific onal antibody, comprising a heavy chain CDR1 region of SEQ ID NO: 54, a heavy chain CDR2 region of SEQ ID NO: 55, a heavy chain CDR3 region of SEQ ID NO: 56 and a light chain CDR1 region of SEQ ID NO: 62, a light chain CDR2 region of SEQ ID NO: 63, and a light chain CDR3 region of SEQ ID NO: 64 and/or a pre-fusion specific monoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO: 58, a heavy chain CDR2 region of SEQ ID NO: 59, a heavy chain CDR3 region of SEQ ID NO: 60 and a light chain CDR1 region of SEQ ID NO: 66, a light chain CDR2 region of SEQ ID NO: 67, and a light chain CDR3 region of SEQ ID NO: 68, wherein the polypeptide comprises an F1 domain and an F2 domain, and wherein the ptides comprise at least one mutation, as compared to wildtype F1 and F2 domains, wherein the at least one mutation is selected from the group consisting of: (a) a mutation of the amino acid residue E on on 161 into P, Q or G (E161P, E161Q or E161G); (b) a mutation of the amino acid residue S on position 182 into P (S182P); (c) a mutation of the amino acid residue S, T or N on position 173 into P (S173P); and (d) a on of the amino acid residue D on on 486 into C (D486C) in combination with a mutation of the amino acid residue D on position 489 into C (D489C), and wherein the amino acid positions are given in reference to the sequence of RSV F protein from the A2 strain (SEQ ID NO: 1).

In a second aspect the present invention provides a nucleic acid molecule ng a pre-fusion RSV F polypeptide ing to the first aspect.

In a third aspect the present invention provides a vector comprising a nucleic acid molecule according to the second aspect.

In a fourth aspect the present invention provides a composition comprising a pre-fusion RSV F polypeptide according to the first aspect, a nucleic acid molecule according to the second aspect and/or a vector according to the third aspect.

In a fifth aspect the t invention provides a use of a pre-fusion RSV F polypeptide according to the first aspect, a nucleic acid le according to the second aspect and/or a vector according to the third aspect in the manufacture of a medicament for inducing an immune response against RSV F protein in a patient in need thereof.

In a sixth aspect the present invention provides a use of a pre-fusion RSV F polypeptide according to the first aspect, a nucleic acid molecule ing to the second aspect and/or a vector according to the third aspect in the manufacture of a vaccine.

In a seventh aspect the present invention provides a use of a pre-fusion RSV F ptide according to the first aspect, a c acid molecule according to the second aspect and/or a vector according to the third aspect in the manufacture of a medicament for the prophylaxis and/or treatment of RSV infection in a t in need thereof.

Also described are stable, recombinant, pre-fusion respiratory syncytial virus (RSV) fusion (F) polypeptides, i.e. recombinant RSV F polypeptides that are stabilized in the pre-fusion conformation. The RSV F polypeptides described herein comprise at least one epitope that is specific to the pre-fusion conformation F protein. In certain embodiments, the pre-fusion RSV F polypeptides are soluble. Also described are nucleic acid molecules encoding the pre-fusion RSV F polypeptides described herein and vectors comprising such nucleic acid molecules.

Also described are compositions, preferably immunogenic compositions, comprising a RSV F polypeptide, a nucleic acid molecule and/or a , and to the use thereof in ng an immune response t RSV F n, in particular use thereof as a vaccine. Also described are methods for inducing an anti-respiratory ial virus (RSV) immune response in a subject, comprising stering to the subject an effective amount of a pre-fusion RSV F polypeptide, a nucleic acid molecule encoding said RSV F polypeptide, and/or a vector comprising said nucleic acid molecule. Preferably, the induced immune response is characterized by neutralizing antibodies to RSV and/or protective immunity against RSV. In particular s, bed is a method for inducing neutralizing anti-respiratory ial virus (RSV) F protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic ition comprising a pre-fusion RSV F polypeptide, a nucleic acid molecule encoding said RSV F polypeptide, and/or a vector comprising said nucleic acid molecule.

Brief description of the Figures Reduced and non-reduced SDS-PAGE with RSV sion DM = Double mutant (N67I+S215P = SEQ ID NO:21) and DM+CC = Double mutant + DE486CC = SEQ ID NO:94).

NativePAGE is of supernatants from Lane 2: DM = Double mutant (N67I+S215P = SEQ ID NO 21) and Lane 1: DM+CC = Double mutant + DE486CC = SEQ ID NO: 94).

A) Superdex200 gel filtration chromatogram of the eluate PreF N67I E161P S215P, RSV A2, fibritin (SEQ ID NO: 91) from the ion-exchange column. B) SDS-PAGE analysis of the prefusion F protein containing peak from the SEC chromatogram under reducing conditions. C) NativePAGE is of purified RSV ion F protein (SEQID NO: 91, Lane 2) compared to purified RSV ion F double mutant (SEQ ID NO: 21, Lane 1).

VNA titers of mice at week 6 after a prime boost at week 0 and 4 with immunogens and doses according to Table 14.

VNA titers of cotton rats at week 7 after a prime boost at week 0 and 4 with immunogens and doses according to Table 15.

Lung and nose viral load at 5 days after i.n. RSV challenge.

Detailed description of the invention The fusion protein (F) of the respiratory syncictial virus (RSV) is involved in fusion of the viral membrane with a host cell membrane, which is required for infection.The RSV F mRNA is translated into a 574 amino acid precursor protein ated F0, which contains a signal peptide sequence at the N-terminus (e.g. amino acid residues 1-26 of SEQ ID NO: 1) that is removed by a signal peptidase in the endoplasmic reticulum. F0 is cleaved at two sites (between amino acid residues 109/110 and 136/137) by cellular ses (in particular furin, or furin-like)) removing a short glycosylated intervening sequence (also referred to a p27 region, comprising the amino acid residues 110 to 136, and generating two domains or subunits designated F1 and F2. The F1 domain (amino acid residues 137-574) contains a hydrophobic fusion peptide at its N-terminus and the C-terminus contains the transmembrane (TM) (amino acid residues 530-550) and cytoplasmic region (amino acid es 551-574). The F2 domain (amino acid residues 27-109) is covalently linked to F1 by two disulfide bridges. The F1-F2 heterodimers are assembled as homotrimers in the virion.

A vaccine against RSV infection is not currently available, but is desired. One potential approach to producing a vaccine is a subunit vaccine based on purified RSV F protein. However, for this approach it is desirable that the purified RSV F protein is in a conformation which resembles the mation of the pre-fusion state of RSV F protein, that is stable over time, and can be produced in sufficient quantities. In addition, for a subunit-based vaccine, the RSV F n needs to be truncated by deletion of the transmembrane (TM) and the asmic region to create a soluble secreted F protein (sF). Because the TM region is responsible for membrane anchoring and trimerization, the less e F protein is considerably more labile than the full-length protein and will readily refold into the usion end-state. In order to obtain e F protein in the stable pre-fusion conformation that shows high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized. ization of another paramyxovirus F protein in the pre-fusion conformation has been successfully accomplished for parainfluenza type 5 (PIV5). Yin et al. (Nature 439: 38-44 (2006)) thus stabilized the sion ure of PIV-5 F n by mutation of the furin cleavage site in F0 which blocked processing into F1 and F2. Furthermore, the transmembrane (TM) and cytoplasmic domain were replaced by a well-known helical trimerization domain: GCN4pII.

This domain forms a trimeric helical coiled coil structure and is a modification of the natural dimeric helical coiled coil peptide GCN4 (O’Shea et al., e 243: 2 (1989)). The GCN4-pII peptide, in which the amino acid sequence of the GCN4 Leucine zipper was substituted with cine residues at every a and d position of the heptad, was shown to form a triple stranded el alpha-helical coiled coil (Harbury et al., Science 262: 1401-1407 (1993)).

For the ization of RSV F in the pre-fusion conformation, the same strategy has been tried, i.e. mutation of the furin ge site and fusion of the RSV-F ectodomain to a GCN4pII trimerization domain (as disclosed in e.g.WO2010/149743, WO2010/149745, WO2009/079796, /158613) or to the fibritin trimerization domain (McLellan et al., Nature Struct. Biol.17: 2250 (2010); McLellan et al., Science 340(6136):1113-7 (2013)). This fibritin domain or n’ is derived from T4 fibritin and was described earlier as an artificial natural trimerization domain (Letarov et al., Biochemistry Moscow 64: 817-823 (1993); S-Guthe et al., J. Mol. Biol. 337: 905-915. (2004)). However, these efforts have not resulted in stable pre-fusion RSV-F protein. Moreover, these s have not yet resulted in candidates suitable for testing in humans.

Described herein are recombinant stable pre-fusion RSV F polypeptides, i.e. RSV F ptides that are stabilized in the pre-fusion conformation. In the research that led to the present invention, several modification steps were introduced and/or combined in order to obtain said stable soluble pre-fusion RSV F polypeptides. The stable pre-fusion RSV F polypeptides described herein are in the pre-fusion conformation, i.e. they comprise (display) at least one epitope that is specific to the pre-fusion conformation F protein. An epitope that is specific to the pre-fusion conformation F protein is an epitope that is not presented in the post-fusion conformation. Without wishing to be bound by any ular theory, it is believed that the ion conformation of RSV F protein may contain epitopes that are the same as those on the RSV F protein expressed on natural RSV virions, and therefore may provide advantages for eliciting protective neutralizing antibodies.

The polypeptides described herein comprise at least one epitope that is recognized by a sion specific monoclonal dy, comprising a heavy chain CDR1 region of SEQ ID NO: 54, a heavy chain CDR2 region of SEQ ID NO: 55, a heavy chain CDR3 region of SEQ ID NO: 56 and a light chain CDR1 region of SEQ ID NO: 62, a light chain CDR2 region of SEQ ID NO: 63, and a light chain CDR3 region of SEQ ID NO: 64 (hereafter referred to as CR9501) and/or a pre-fusion specific monoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO: 58, a heavy chain CDR2 region of SEQ ID NO: 59, a heavy chain CDR3 region of SEQ ID NO: 60 and a light chain CDR1 region of SEQ ID NO: 66, a light chain CDR2 region of SEQ ID NO: 67, and a light chain CDR3 region of SEQ ID NO: 68 red to as CR9502).

CR9501 and CR9502 comprise the heavy and light chain variable regions, and thus the binding specificities, of the antibodies 58C5 and 30D8, respectively, which have previously been shown to bind specifically to RSV F protein in its pre-fusion conformation and not to the usion conformation (see WO2012/006596).

In certain embodiments, the recombinant pre-fusion RSV F polypeptides comprise at least one epitope that is recognized by at least one pre-fusion specific monoclonal antibody as described above and are trimeric.

The stable pre-fusion RSV F polypeptides described herein comprise an F1 domain and an F2 , wherein the polypeptides comprise at least one mutation, as compared to pe F1 and F2 domains, ed from the group consisting of: (a) a mutation of the amino acid residue on position 161; (b) a mutation of the amino acid residue on position 182; (c) a on of the amino acid residue on position 173; and (d) a mutation of the amino acid residue D on position 486 into C (D486C) in ation with a mutation of the amino acid residue D on position 489 into C (D489C) or a mutation of the amino acid residue E on position 487 into C (E487C).

In certain embodiments, the stable pre-fusion RSV F polypeptides comprise an F1 domain and a F2 domain, wherein the ptides comprise at least one mutation selected from the group consisting of: (a) a mutation of the amino acid residue E on position 161 into P, Q or G (E161P, E161Q) or E161G); (b) a mutation of the amino acid residue S on position 182 into P ); (c) a mutation of the amino acid residue S, T or N on position 173 into P (S173P); and (d) a mutation of the amino acid residue D on position 486 into C (D486C) in combination with a mutation of the amino acid residue D on position 489 into C (D489C) or a mutation of the amino acid residue E on position 487 into C (E487C).

In certain embodiments, the pre-fusion RSV F polypeptides further comprise a mutation of the amino acid e on on 67 and/or a mutation of the amino acid residue on position 215.

In certain embodiments, the stable pre-fusion RSV F polypeptides thus comprise an F1 domain and a F2 , wherein the polypeptides comprise a mutation of the amino acid residue on position 67 and/or a mutation of the amino acid residue on position 215, and at least one further mutation ed from the group consisting of: (a) a mutation of the amino acid residue on position 161; (b) a on of the amino acid residue on position 182; (c) a mutation of the amino acid residue on position 173; and (d) a mutation of the amino acid residue D on position 486 into C (D486C) in combination with a on of the amino acid residue D on position 489 into C (D489C) or a mutation of the amino acid residue E on position 487 into C (E487C).

In certain embodiments, the stable pre-fusion RSV F polypeptides comprise an F1 domain and a F2 domain, n the polypeptides comprise a on of the amino acid residue N or T on position 67 and/or a mutation of amino acid residue S on position 215, and wherein the polypeptides further comprise at least one further mutation selected from the group consisting of: (a) a mutation of the amino acid residue E on position 161 into P, Q or G (E161P, E161Q) or E161G); (b) a mutation of the amino acid residue S on position 182 into P (S182P); (c) a mutation of the amino acid residue S, T or N on position 173 into P (S173P); and (d) a mutation of the amino acid residue D on position 486 into C (D486C) in combination with a mutation of the amino acid residue D on position 489 into C (D489C) or a mutation of the amino acid residue E on position 487 into C (E487C).

In certain embodiments, the stable pre-fusion RSV F polypeptides se a linking ce sing from 1 to 10 amino acids, linking the F1 domain and F2 domain.

In certain embodiments, the stable pre-fusion RSV F polypeptides described herein thus comprise an F1 domain and an F2 domain, and a linking ce comprising from 1 to 10 amino acid residues, linking said F1 domain to said F2 , n the polypeptides comprise at least one mutation selected from the group consisting of: (a) a mutation of the amino acid residue E on position 161 into P, Q or G (E161P, E161Q) or ; (b) a mutation of the amino acid residue S on on 182 into P (S182P); (c) a mutation of the amino acid residue S, T or N on position 173 into P (S173P), and (d) a mutation of the amino acid residue D on on 486 into C (D486C) in combination with a mutation of the amino acid residue D on position 489 into C (D489C) or a mutation of the amino acid residue E on position 487 into C (E487C) In n embodiments, the stable pre-fusion RSV F polypeptides further comprise a mutation of the amino acid e N or T on position 67 and/or a mutation of amino acid residue S on position 215. In certain ments, the stable pre-fusion RSV F polypeptides further comprise a mutation of the amino acid residue N or T on position 67 (N/T67I) into I and/or a mutation of amino acid residue S on position 215 into P (S215P).

In certain ments, the stable pre-fusion RSV F polypeptides described herein comprise a truncated F1 domain.

In certain embodiments, the stable pre-fusion RSV F polypeptides described herien thus comprise a truncated F1 domain and a F2 domain, and ally a linking sequence comprising from 1 to 10 amino acid residues, linking said truncated F1 domain to said F2 domain, wherein the polypeptides comprise at least one further mutation selected from the group consisting of: (a) a mutation of the amino acid residue E on position 161 into P, Q or G (E161P, E161Q) or E161G); (b) a mutation of the amino acid residue S on position 182 into P (S182P); (c) a mutation of the amino acid residue S, T or N on position 173 into P (S173P); and (d) a mutation of the amino acid residue D on position 486 into C (D486C) in combination with a mutation of the amino acid residue D on position 489 into C (D489C) or a mutation of the amino acid residue E on position 487 into C (E487C) In certain embodiments, the ptides further comprise a mutation of the amino acid residue N or T on position 67 and/or a mutation of amino acid residue S on position 215. In certain embodiments, the stable pre-fusion RSV F polypeptides further comprise a mutation of the amino acid e N or T on position 67 I) into I and/or a mutation of amino acid residue S on position 215 into P (S215P).

According to the present disclosure, the polypeptides described herein thus comprise at least one stabilizing mutation in the F1 and/or F2 domain as compared to the RSV F1 and/or F2 domain in a wild-type RSV F protein. It is known that RSV exists as a single serotype having two antigenic ups: A and B. The amino acid sequences of the mature sed F proteins of the two groups are about 93% identical. As used throughout the present application, the amino acid positions are given in reference to the sequence of RSV F protein from the A2 strain (SEQ ID NO: 1). As used in the present invention, the wording "the amino acid at position "x" of the RSV F n thus means the amino acid corresponding to the amino acid at position "x" in the RSV F protein of the RSV A2 strain of SEQ ID NO: 1. Note that, in the numbering system used throughout this application 1 refers to the N-terminal amino acid of an immature F0 protein (SEQ ID NO: 1) When a RSV strain other than the A2 strain is used, the amino acid positions of the F protein are to be ed with reference to the numbering of the F protein of the A2 strain of SEQ ID NO: 1 by aligning the sequences of the other RSV strain with the F protein of SEQ ID NO: 1 with the ion of gaps as needed. Sequence alignments can be done using methods well known in the art, e.g. by CLUSTALW, t or CLC Workbench.

An amino acid according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as e.g. D-amino acids (the D-enantiomers of amino acids with a chiral center), or any ts that are not naturally found in ns, such as e.g. norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein–protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 17 shows the abbreviations and properties of the standard amino acids.

It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology ures. The mutations bed herien preferably result in increased expression levels and/or increased stabilization of the pre-fusion RSV F polypeptides as compared RSV F polypeptides that do not comprise these mutation(s).

In certain embodiments, the pre-fusion RSV F polypeptides are full .

In certain embodiments, the pre-fusion RSV F polypeptides are soluble.

In certain embodiments, the pre-fusion RSV F polypeptides further comprise a heterologous trimerization domain linked to said truncated F1 . According to the present disclosure, it was shown that by linking a heterologous trimerization domain to the C-terminal amino acid residue of a ted F1 domain, optionally combined with a g sequence linking the F1 and F2 domain and the stabilizing mutation(s), RSV F ptides are described that show high expression and that bind to pre-fusion-specific antibodies, indicating that the ptides are in the pre-fusion mation. In addition, the RSV F polypeptides are stabilized in the pre-fusion conformation, i.e. even after processing of the polypeptides they still bind to the pre-fusion specific antibodies CR9501 and/or CR9502, ting that the sion specific epitope is retained.

In further embodiments, the sion RSV F polypeptides comprise one or more further mutations (as compared to the ype RSV F protein), selected from the group consisting of: (a) a mutation of the amino acid residue on position 46; (b) a mutation of the amino acid residue on position 77; (c) a mutation of the amino acid residue on position 80; (d) a mutation of the amino acid residue on position 92; (e) a mutation of the amino acid residue on position 184; (f) a mutation of the amino acid residue on position 185; (g) a mutation of the amino acid residue on on 201; (h) a mutation of the amino acid residue on position 209; (i) a mutation of the amino acid e on on 421; (j) a mutation of the amino acid residue on position 426; (k) a mutation of the amino acid residue on position 465; (l) a mutation of the amino acid residue on on 486; (m) a mutation of the amino acid residue on position 487; and (n) a mutation of the amino acid residue on position 508.

In preferred embodiments, the one or more further mutations are selected from the group consisting of: (a) a mutation of the amino acid residue S on position 46 into G (S46G); (b) a mutation of the amino acid residue K on position 77 into E (K77E); (c) a mutation of the amino acid residue K on position 80 into E (K80E); (d) a mutation of the amino acid residue E on position 92 into D (E92D); (e) a mutation of the amino acid residue G on position 184 into N (G184N); (f) a mutation of the amino acid residue V on on 185 into N (V185N); (g) a mutation of the amino acid residue K on position 201 into Q (K201Q); (h) a mutation of the amino acid residue K on position 209 into Q (K209Q); (i) a mutation of the amino acid residue K on position 421 into N (K421N); (j) a mutation of the amino acid residue N on position 426 into S (N426S); (k) a mutation of the amino acid residue K on position 465 into E or Q (K465Q); (l) a mutation of the amino acid residue D on position 486 into N (D486N); (m) a mutation of the amino acid residue E on position 487 into Q, N or I /N/I); (n) a mutation of the amino acid residue K on position 508 into E (K508E).

As described above, in certain embodiments, the pre-fusion RSV F polypeptides comprise a on of the amino acid residue D on position 486 into C ) in combination with D489C or E487C. These double mutations to two extra cysteine residues result in an intersubunit disulfide bridge between the F1 proteins to establish a covalent bond between the ers and to stabilize the sion RSV F structure.

It is again noted that for the positions of the amino acid residues reference is made to SEQ ID NO: 1. A skilled person will be able to determine the corresponding amino acid residues in F proteins of other RSV s.

In certain ments, the pre-fusion RSV F polypeptides se at least two mutations (as compared to a wild-type RV F protein).

In certain embodiments, the ptides se at least three mutations.

In n ments, the polypeptides comprise at least four, five or six mutations.

In certain embodiments, the heterologous trimerization domain comprises the amino acid sequence EKKIEAIEKKIEAIEKKIEA (SEQ ID NO: 3). In certain other embodiments, the heterologous ization domain comprises the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 4).

As described above, in certain embodiments the polypeptides described herein comprise a truncated F1 domain. As used herein a "truncated" F1 domain refers to a F1 domain that is not a full length F1 domain, i.e. wherein either N-terminally or inally one or more amino acid residues have been deleted. According to the present disclosure, at least the transmembrane domain and cytoplasmic tail have been deleted to permit expression as a soluble ectodomain.

In certain other embodiments, the F1 domain is truncated after amino acid residue 495 of the RSV F protein (referring to SEQ ID NO: 1), i.e. the C-terminal part of the F1 domain starting from amino acid residue 496 (referring to SEQ ID NO: 1) has been deleted. In certain other embodiments, the F1 domain is truncated after amino acid residue 513 of the RSV F protein. In certain embodiments, the F1 domain is truncated after amino acid residue 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524 or 525.

In certain embodiments, the trimerization domain is linked to amino acid e 495 of the RSV F1 -domain. In certain embodiments, the trimerization domain comprises SEQ ID NO: 4 and is linked to amino acid residue 495 of the RSV F1 domain.

In certain other embodiments, the trimerization domain is linked to amino acid residue 513 of the RSV F1 . In certain embodiments, the trimerization domain comprises SEQ ID NO: 3 and is linked to amino acid residue 513 of the RSV F1 domain.

As described above, in certain embodiments, the F1 domain, which is optionally truncated, and the F2 domain are linked by a linking ce, linking the C-terminal amino acid of the F2 domain to the N-terminal amino acid of the (optionally truncated) F2 domain. In n ments, the linking sequence (or linker) comprises from 1-10 amino acid residues, preferable from 2-9 amino acid residues, preferably from 3-8 amino acid residues, preferably from 4-7 amino acid residues, more preferably the linker comprises 5 or 6 amino acid residues.

Numerous conformationally neutral linkers are known in the art that can be used herein without disrupting the conformation of the pre-fusion RVS F polypeptides. In preferred embodiments, the linker comprises the amino acid sequence GSGSG (SEQ ID NO: 5).

In certain embodiments, the F1 domain and/or the F domain are from an RSV A strain. In certain ments the F1 and/or F2 domain are from the RSV A2 strain of SEQ ID NO: 1.

In certain embodiments, the F1 and/or F2 domain are from the RSV A2 strain of SEQ ID NO: 69.

In certain embodiments, the F1 domain and/or the F domain are from an RSV B strain. In certain ments the F1 and/or F2 domain are from the RSV B strain of SEQ ID NO: 2.

In certain embodiments, the F1 and F2 domain are from the same RSV . In certain embodiments, the pre-fusion RSV F polypeptides are chimeric polypeptides, i.e. comprising F1 and F2 domains that are from different RSV strains.

In certain embodiments, the level of expression of the pre-fusion RSV F polypeptides described herein is increased, as compared to a full length wild-type RSV F polypeptide or a wild-type ectodomain (i.e. without the embrane and cytoplasmic region) without the mutation(s). In certain embodiments the level of expression is increased at least 5-fold, preferably up to 10-fold. In certain embodiments, the level of expression is increased more than -fold.

The pre-fusion RSV F polypeptides described herein are stable, i.e. do not readily change into the usion conformation upon processing of the polypeptides, such as e.g. purification, freeze-thaw cycles, and/or storage etc.

In certain ments, the pre-fusion RSV F polypeptides described herein have an increased stability upon storage a 4ºC as compared to a RSV F polypeptide without the mutation(s). In certain ments, the polypeptides are stable upon storage at 4ºC for at least days, preferably at least 60 days, preferably at least 6 months, even more preferably at least 1 year. With "stable upon storage", it is meant that the polypeptides still display the at least one epitope ic for the a pre-fusion specific antibody (e.g. CR9501) upon storage of the polypeptide in solution (e.g. culture medium) at 4º C for at least 30 days, e.g. as determined using a method as described in Example 8 or 10. In certain ments, the polypeptides display the at least one pre-fusion specific epitope for at least 6 months, preferably for at least 1 year upon storage of the pre-fusion RSV F ptides at 4oC.

In certain embodiments, the pre-fusion RSV F polypeptides described herien have an increased stability when subjected to heat, as compared to RSV F polypeptides without said mutation(s). In certain embodiments, the pre-fusion REV F polypeptides are heat stable for at least 30 minutes at a temperature of 55º C, preferably at 58º C, more ably at 60º C With "heat stable" it is meant that the polypeptides still display the at least one pe-fusion specific epitope after having been subjected for at least 30 minutes to an increased ature (i.e. a temperature of 55ºC or above), e.g. as determined using a method as described in Example 9.

In certain embodiments, the polypeptides display the at least one pre-fusion specific epitope after being subjected to 1 to 6 -thaw cycles in an appropriate formulation buffer.

In certain preferred embodiments, the sion RSV F polypeptide described herien comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 90-94. In certain embodiments, the sion RSV F polypeptide described herien consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 90-94.

As used throughout the present application nucleotide sequences are provided from 5’ to 3’ direction, and amino acid ces from N-terminus to C-terminus, as custom in the art.

In certain embodiments, the encoded polypeptides described herein further comprise a leader sequence, also referred to as signal sequence or signal peptide, corresponding to amino acids 1-26 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 69. This is a short (typically 5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized ns that are destined towards the ory pathway. In certain embodiments, the polypeptides bed herein do not comprise a leader sequence.

In certain embodiments, the ptides comprise a HIS-Tag. A His-Tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least five histidine (H) es, often at the N- or C-terminus of the protein, which is generally used for purification purposes.

In certain ments, the polypeptides do not comprise a HIS-Tag. According to the present disclosure, it has surprisingly been shown that when the HIS-tag is deleted the level of expression and the stability are increased as compared to polypeptides with a HIS-tag.

Also described are nucleic acid molecules encoding the RSV F polypeptides described herein.

In preferred embodiments, the c acid les encoding the polypeptides described herein are codon-optimized for expression in mammalian cells, preferably human cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred. Herein, a nonpreferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon ncy , such as in http://www.kazusa.or.jp/codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons lly leads to higher expression.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acid molecules can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also tood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide ce encoded by the nucleic acid les to reflect the codon usage of any particular host organism in which the polypeptides are to be sed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are rate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine lar biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g.

GeneArt, GenScripts, Invitrogen, Eurofins).

Also described are vectors comprising a c acid molecule as described above. In certain embodiments, a nucleic acid molecule descriebd herein thus is part of a . Such vectors can easily be manipulated by methods well known to the person skilled in the art, and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells. In addition, many s can be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome. The vector used can be any vector that is suitable for cloning DNA and that can be used for transcription of a nucleic acid of interest.

Suitable vectors bed herein are e.g. adenovectors, such as e.g. Ad26 or Ad35, alphavirus, paramyxovirus, ia virus, herpes virus, retroviral vectors etc. The person skilled in the art is capable of choosing suitable expression vectors, and inserting the nucleic acid sequences described herein in a functional manner.

Host cells comprising the c acid les encoding the pre-fusion RSV F polypeptides form also part of the t disclosure. The pre-fusion RSV F polypeptides may be produced h recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain ments, the cells are human cells. In general, the production of a inant ns, such the pre-fusion RSV F polypeptides described herein, in a host cell comprises the introduction of a heterologous nucleic acid le encoding the polypeptide in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the polypeptide in said cell. The nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain sion of a gene in host cells.

Promoters can be constitutive or regulated, and can be obtained from s s, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the pre-fusion RSV F ptides. The suitable medium may or may not contain serum.

A "heterologous nucleic acid molecule" (also referred to herein as ‘transgene’) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into for instance a vector by standard molecular biology techniques. A ene is lly operably linked to expression l ces. This can for instance be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for ing expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt. –735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for e the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g. the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to recombinantly express the protein of interest, or to obtain suitable ers and/or ription terminator sequences, polyA sequences, and the like.

The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the e of a culture vessel or to arriers, as well as suspension culture. Most large-scale suspension es are operated as batch or tch processes because they are the most straightforward to operate and scale up. ys, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for ce in dishes, roller bottles or in ctors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, ic 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 034889-9)).

Also described are compositions comprising a pre-fusion RSV F polypeptide and/or a nucleic acid molecule, and/or a vector, as described above. Also described are compositions comprising a pre-fusion RSV F polypeptide that displays an epitope that is present in a pre- fusion conformation of the RSV F protein but is absent in the post-fusion conformation. Also described are compositions comprising a nucleic acid le and/or a vector, encoding such pre-fusion RSV F polypeptide. Also described are immunogenic compositions sing a prefusion RSV F polypeptide, and/or a nucleic acid molecule, and/or a , as described above.

Also bed is the use of a stabilized pre-fusion RSV F polypeptide, a nucleic acid molecule, and/or a vector, described herein, for inducing an immune response against RSV F protein in a t. Further described are methods for inducing an immune response against RSV F protein in a subject, comprising stering to the subject a pre-fusion RSV F polypeptide, and/or a nucleic acid le, and/or a vector, described herein. Also described are pre-fusion RSV F polypeptides, nucleic acid molecules, and/or vectors, described herein for use in inducing an immune se against RSV F protein in a subject. Further described is the use of the prefusion RSV F polypeptides, and/or nucleic acid molecules, and/or vectors described herein for the manufacture of a ment for use in inducing an immune response against RSV F n in a subject.

The pre-fusion RSV F polypeptides, nucleic acid molecules, or vectors described herein may be used for tion (prophylaxis) and/or treatment of RSV infections. In certain embodiments, the prevention and/or treatment may be targeted at patient groups that are susceptible RSV infection. Such patient groups include, but are not limited to e.g., the elderly (e.g. ≥ 50 years old, ≥ 60 years old, and preferably ≥ 65 years old), the young (e.g. ≤ 5 years old, ≤ 1 year old), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.

The pre-fusion RSV F polypeptides, nucleic acid molecules and/or vectors described herein may be used e.g. in stand-alone treatment and/or prophylaxis of a disease or condition caused by RSV, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.

Also described are methods for preventing and/or treating RSV infection in a subject ing the pre-fusion RSV F polypeptides, nucleic acid molecules and/or vectors described herein. In a specific ment, a method for preventing and/or treating RSV infection in a subject comprises stering to a subject in need thereof an effective amount of a pre-fusion RSV F ptide, nucleic acid molecule and/or a , as bed above. A therapeutically effective amount refers to an amount of a polypeptide, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or ng a disease or condition resulting from infection by RSV. Prevention encompasses inhibiting or reducing the spread of RSV or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by RSV. Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of RSV infection.

For administering to ts, such as humans, the methods described herein may employ pharmaceutical compositions sing a sion RSV F polypeptide, a nucleic acid molecule and/or a vector as described , and a pharmaceutically able carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations ed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see 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 ptides, or c acid molecules, preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The RSV F ptides typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. ally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the RSV F polypeptides may be formulated into an injectable preparation.

In certain ments, a composition described herien further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied nic determinant. The terms "adjuvant" and "immune ant" are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system.

In this context, an adjuvant is used to enhance an immune response to the RSV F polypeptides described herein. es of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water ons, such as MF59 (see e.g. WO 90/14837); n formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, , ); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O- deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or s thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g. antibodies or nts thereof (e.g. directed against the antigen itself or CD1a, CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc), which ate immune se upon interaction with recipient cells. In certain ments the compositions described herein comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations f, in concentrations of 0.05 – 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.

The sion RSV F polypeptides may also be administered in combination with or ated to nanoparticles, such as e.g. polymers, liposomes, virosomes, virus-like particles.

The pre-fusion F polypeptides may be combined with, encapsidated in or conjugated to the nanoparticles with or without adjuvant. Encapsulation within liposomes is described, e.g. in US 4,235,877. Conjugation to macromolecules is disclosed, for example in US 4,372,945 or US 4,474,757.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, described are methods for making a vaccine t respiratory syncytial virus (RSV), comprising providing a composition described herein and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to an agent or composition ning an active component effective to induce a n degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. In the present disclosure, the vaccine comprises an effective amount of a pre-fusion RSV F polypeptide and/or a nucleic acid molecule encoding a pre-fusion RSV F polypeptide, and/or a vector comprising said c acid molecule, which results in an immune response against the F protein of RSV. This provides a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease in frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. The term "vaccine" according to the invention implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises other ents that induce an immune response, e.g. against other proteins of RSV and/or against other infectious agents. The administration of further active components may for instance be done by te administration or by stering combination products of the vaccines described herein and the r active components. itions may be administered to a subject, e.g. a human subject. The total dose of the RSV F polypeptides in a composition for a single administration can for instance be about 0.01 µg to about 10 mg, e.g. 1 µg – 1 mg, e.g. 10 µg – 100 µg. Determining the recommended dose will be carried out by experimentation and is routine for those skilled in the art.

Administration of the compositions described herein can be performed using standard routes of administration. Non-limiting embodiments e parenteral administration, such as intradermal, uscular, subcutaneous, transcutaneous, or mucosal administration, e.g. intranasal, oral, and the like. In one embodiment a composition is administered by intramuscular injection. The skilled person knows the s ilities to ster a composition, e.g. a vaccine in order to induce an immune response to the antigen(s) in the vaccine.

A t as used herein preferably is a mammal, for instance a rodent, e.g. a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject.

The polypeptides, nucleic acid molecules, vectors, and/or compositions may also be administered, either as prime, or as boost, in a homologous or heterologous prime-boost regimen.

If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as ‘priming vaccination’). In certain embodiments, the administration comprises a prime and at least one booster administration.

In addition, the ptides described herein may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the polypeptide described herein. The disclosure thus also relates to an in vitro diagnostic method for ing the ce of an RSV infection in a patient said method comprising the steps of a) contacting a biological sample ed from said patient with a ptide described herein; and b) detecting the presence of antibody-polypeptide xes.

Also described is a method for stabilizing the pre-fusion conformation of an RSV F polypeptide, comprising introducing one or more mutations in a RSV F1 domain, as compared to the wild-type RSV F1 domain, wherein the one or more mutations are selected from the group consisting of: (a) a stabilizing mutation in the HRA region between the secondary structure elements in sion F that are transformed to one large coiled coil in post fusion F; and (b) introduction of two ne residues close to the 3-fold axis at the bottom of the prefusion RSV-F head N-terminal to the pre-fusion stem (residues 493 – 525), N- terminal of HRB) that covalently link the F1 subunits in the trimer.

In certain embodiments, the mutation in the HRA region is at position 161.

In certain ments, the mutation in the HRA region is at on 173.

In n embodiments, the mutation in the HRA region is at position 182.

In certain embodiments, the introduction of two cysteine residues is at position 486 and 489.

In certain embodiments, the introduction of two cysteine es is at position 486 and 487.

Stabilized sion RSV F polypeptides obtainable and/or obtained by such method also form part of the present disclosure, as well as uses thereof as described above.

The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each ent or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

In this specification where reference has been made to patent ications, other external documents, or other sources of information, this is generally for the purpose of providing a t for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current ation. That subject matter should be y identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.

Examples EXAMPLE 1 Preparation of stable pre-fusion RSV F polypeptides – linkers and trimerization domains In the co-pending patent application , stabilized variants of soluble pre-fusion F protein (sF) were ed by stabilizing the two main s that initiate refolding. The first strategy was to arrest the fusion peptide in its on and prevent it from getting released from the head region by fixing and joining the F1-F2 domains by a short loop.

Release of the fusion peptide can be prevented by re-establishing a covalent tion of the N- terminus of F1 to C-terminus of F2. As shown in this example, several different linkers have been tried. The insertion of a 5 amino acid loop between F1 and F2, in ular comprising the amino acid sequence GSGSG (SEQ ID NO: 5), was most successful.

The other unstable region is the second heptad repeat (HRB) region that forms the trimeric helical stem region in pre-fusion F n. Deletion of the transmembrane domain (TM) in the soluble F protein further destabilizes this region, which was compensated by the addition of ent heterologous trimerization domains. The fully processed mature RSV-F ectodomain was fused C-terminally to different trimerization domains and at different ons (i.e. the F1 domain was truncated at different amino acid residues).

Several constructs were made based on either RSV A2 or B1 strains. Different trimerization domains were linked to the RSV F1 , which was truncated at different ons. Trimerization domains that were tested included the Fibritin motif (comprising the amino acid sequence: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 4), and the "Fibritin long" motif, a longer, N-terminal extended Fibritin domain which includes its natural helical regions (comprising the amino acid sequence: SSLQGDVQALQEAGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 6), that were added to the RSV F1 domain in frame (in register) with the presumed heptad repeat of the HRB region.

Further constructs that were made comprised heptad ideal helical trimeric coiled coils, or Isoleucine Zipper domains (IZ) (Suzuki et al., Protein Engineering 11: 1051-1055 (1998)), comprising the amino acid sequence: IEAIEKK (SEQ ID NO: 7). According to the ion different IZ domains were used, referred to as Isoleucine Zipper (L), comprising the amino acid sequence: (I)EKKIEAIEKKIEAIEKKIEAIEAIEKKIEA (SEQ ID NO: 8) and Isoleucine Zipper (S), sing the amino acid sequence EKKIEAIEKKIEAIEKKIEA (SEQ ID NO: 3).

These IZ domains are comparable in structure to GCN4, however, the IZ domains are not natural sequences but designed to be optimal ization domains and therefore more stable.

Further constructs were made with other known ization domains: GCN4II EDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 9) Optimized GCN4II EDKVEELLSKIYHIENRIARIEKLVGEA (SEQ ID NO: 10) Matrilin -1 (long version) CKSIVKFQTKVEELINTLQQKLEAVAKRIEALENKII (SEQ ID NO: 11) Matrillin- 1 short version that only contains zipper domain: EELINTLQQKLEAVAKRIEALENKII (SEQ ID NO: 12) The following constructs were made: Construct F18 comprised the Fibritin trimerization domain (SEQ ID NO: 4) linked to amino acid residue 513 of the F1 domain.

Construct F19 comprised the Fibritin ization domain (SEQ ID NO: 4) linked to amino acid residue 499 of the F1 domain.

Construct F20 sed the Isoleucine Zipper (L) domain (SEQ ID NO: 8) linked to amino acid e 516 of the F1 domain and comprising additional modifications in HRB to optimize the hydrophobic nature of the heptad positions and tate in-frame fusion with the IZ .

Construct F21 also comprised Isoleucine Zipper (L) domain (SEQ ID NO: 8), but linked to amino acid residue 501 of the F1 domain and without additional modifications in the HRB region.

Construct F22 comprised the Isoleucine Zipper (L) domain (SEQ ID NO: 8) linked to amino acid residue 495 of the F1 domain and comprising additional modifications in HRB.

Construct F23 comprised the Isoleucine Zipper (S) domain (SEQ ID NO: 3) linked to amino acid residue 495.

Construct F46 also comprised the Isoleucine Zipper (S) domain (SEQ ID NO: 3) but linked to a longer RSV-F ectodomain, i.e. the F1 domain was truncated after amino acid residue 513.

All constructs comprised a HIS-tag.

The constructs were tested for expression levels, storage stability and antibody binding with the antibody CR9501. The amino acid sequences of the heavy and light chain variable regions, and of the heavy and light chain CDRs of this antibody are given below. CR9501 comprises the binding regions of the antibodies ed to as 58C5 in /006596.

The constructs were synthesized and codon-optimized at Gene Art (Life Technologies, Carlsbad, CA). The constructs were cloned into pCDNA2004 or generated by rd methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. The expression platform used was the 293Freestyle cells (Life Technologies). The cells were transiently transfected using 293Fectin (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37oC and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4oC until use.

Supernatants from day 5 were evaluated for F protein expression by western blot using the monoclonal antibody CR9503, which comprises the heavy and light chain variable regions of the RSV F antibody zumab (referred to as CR9503). The approximate expression levels of the pre-fusion RSV F protein ucts were assessed using CR9503, an anti-human IR-dye conjugated secondary antibody (Li-Cor, Lincoln, NE) or a HRP ated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, PA). The protein ties were then estimated using a on series of purified RSV standard protein, either by eye or using the Odyssey CLx infrared g system. To evaluate construct stability and to identify positive or negative stabilizing s of introduced trimerization motifs, the constructs capable of binding CR9501 were treated at a range of temperatures from 45-65 ºC for 30 minutes to test the stability of the CR9501 epitope. This procedure is described in detail in Example 9. The results are summarized in Table 1.

Table 1. Expression and stability of RSV F constructs with different trimerization motifs Description RSV Trimerization Termination Expression n motif Modifications point (ug/ml) Stability* F18 Fibritin None 513 2 unstable F19 Fibritin None 499 0 ND cine 502 509 516 F20 zipper (L) Ile 516 0 ND Isoleucine F21 zipper (L) None 501 0 ND Isoleucine K483E + F22 zipper (L) E488K 495 0 ND Isoleucine F23 zipper (S) None 495 0.3 1 stable cine Did not F46 zipper (S) None 513 express ND *Stability is defined as described in Example 8; ND: Not determined. 1 Expression level determined by Western Blot as described in e 1.

As can be seen in Table 1, the only ucts that were expressed were the Fibritin variant (F18) and F23. Although F18 was trimeric and showed expression, it was unstable upon storage at 4º C. In st, F23 was stable at 4º C, binds to the pre-fusion–specific antibodies, but ed to be monomeric. Therefore, both variants F18 and F23 were used to optimize for both stability and trimerization.

Next, several constructs were made in which the fusion peptide at the N-terminus of F1 was fixed by fusion to the C-terminus of the F2 domain. All constructs comprised a His-tag. l constructs were made including constructs in which both furin cleavage sites were mutated resulting in a soluble F protein that still contained the p27 peptide (i.e. F12, F15.1, and F17). In other constructs the 27 residue region (P27 loop) that is cleaved from the precursor F0 was replaced by an alternative closed loop: either by replacing the region of RSV-F by the ‘homologous’ region of PIV-5 F, the prefusion F protein that had been produced and crystallized successfully (F25), or by replacing the region by a minimal (GS)n loop that would bridge the termini of F2 and F1 (F24), or by replacing the region by the central ved region of RSV-G (F26). Homology modeling of RSV-F based on PIV-5 and in silico ements resulted in the choice of a minimal loop of 5 amino acid residues between residues 108 and 136. As a linker, Gly (G) and Ser (S) residues were chosen which are flexible and polar and have a high chance to be accommodated (F24). Additionally, F137 was mutated to S because the local modifications caused by the loop could displace the hydrophobic F and cause instabilities. This is shown below. Also the R106 is mutated to Q and 27 residues (109-135) are replaced by GSGSG.

PAANNRARREAPQYMNYTINTTKNLNVSISKKRKRR136FLGFLLGVG AR GSGSGR136SLGFLLGVG As shown in Table 2, all variants showed no or very low expression except for the variant with the short GSGSG loop (F24) which showed a much higher expression (44 µg/ml) compared to wild type RSV F construct, i.e. a similar construct, t said linker (F11). F24 which was trimeric, however, was unstable upon storage like all the other ts with a C-terminal Fibritin trimerization motif. All ts contained a HIS-tag.

Table 2. Expression and stability of RSV F constructs with different F1-F2 linkers Description RSV Trimerization Termination Expr.

Protein Variant motif F1, F2 linker Modifications point (ug/ml) Stability* F11 B1 None None None 513 2.5 stable F18 B1 in None None 513 2 unstable F12 B1 Fibritin p27 Furin site KO 513 0,1 unstable F15.1 B1 None p27 Furin site KO 525 0.5 ND F17 A2 Fibritin p27 Furin site KO 513 0 ND F24 B1 Fibritin Q__GSGSG_S None 513 44 unstable F25 B1 Fibritin PIV None 513 0 ND F26 B1 Fibritin G CR None 513 0 ND *Stability is defined as described in Example 8. Expression level ined as described in Example 1.

Next, the most favorable modifications were ed to find the optimal pre-fusion F polypeptides. Combinations were made of variants with the GSGSG loop, C-terminal truncation of F1, and the addition of either fibritin (SEQ ID NO: 4) or the Isoleucin Zipper (S) motif (SEQ ID NO: 3)(see Table 3).

Table 3. Expression and stability of RSV F constructs with ations of optimizations according to Tables 1 and 2.

Termination Description Stability CR9501 epitope) RSV Protein Variant point Trimerization motif F1, F2 linker (ug/ml) Heat (oC) Storage F11 B1 513 None None 2.5 48 Stable F23 B1 495 Isoleucine zipper (S) None 0.3 ND Stable F24 B1 513 Fibritin Q__GSGSG_S 44 51 Unstable F45 B1 495 Fibritin None 0 ND ND F44 B1 495 in Q__GSGSG_S 0 ND ND F49 B1 495 None None 2 ND Stable F50 A2 495 None None 2 ND Stable F43 B1 495 Isoleucine zipper (S) Q__GSGSG_S 0.4 53 Stable F47 A2 495 Isoleucine zipper (S) Q__GSGSG_S 5 52 Stable F56 B1 513 Isoleucine zipper (S) Q__GSGSG_S 0,4 ND Stable F46 B1 513 Isoleucine zipper (S) None 0 ND unstable F42 B1 513 None Q__GSGSG_S 20 54 Stable F57 A2 513 None Q__GSGSG_S 2-10 54 Stable ND is not determined *Storage stability as ined in e 8. *Heat stability as determined in Example 9.

Expression level as determined by Western blotting (described in Example 1) Addition of the GSGSG-loop always increased the expression of functional ucts as well as the heat stability of the protein. Combination of the loop with the truncated F and isoleucine zipper (S) motif (F43, F47) showed good sion, heat ity and good stability upon storage at 4 ºC. However, these variants were still monomeric. The isoleucine zipper (S) trimerization motif showed higher expression with a F variant that was C-terminally truncated F at on 495 (compare F43 with F56 and F23 with F46). In contrast, for variants with the Fibritin trimerization domain a truncation at position 513 showed high expression compared to truncation at position 495 which showed no expression (compare F24 with F44).

Because the HIS-tag could interfere with the native folding of the trimers, variants were made without the HIS-tag for the Fibritin and the isoleucine zipper (S) variant (Table 4).

Table 4. Expression and stability of RSV F constructs with and without HIS-tag Expressio RSV Trimerization Termination n Trimerization Heat Protein Variant motif F1, F2 linker point ug/ml % (oC) Storage Tags Q__GSGSG_ Trimeric F24 B1 Fibritin S 513 44 (SEC) 51 unstable His-tag SG_ 100% F24- B1 Fibritin S 513 55 (Native) ND unstable None Isoleucine Q__GSGSG_ F47 A2 zipper (S) S 495 5 0% (Odyssey) 52 stable His-tag Isoleucine Q__GSGSG_ 2-5% F47- A2 zipper (S) S 495 10 (Odyssey) 53 stable None Q__GSGSG_ Trimeric A2_F24 A2 Fibritin S 513 5,3 (Native) 48,75 unstable None *Storage stability ined as described in Example 8; Heat stability determined as described in e 9; ND: not determined.

Strikingly, deletion of the HIS-tag increased expression in F47. er, for F47 it increased the trimeric content slightly and for F24 it only sed the expression level moderately.

Next, several alternative trimerization domains and tions were tested in combination with the GSGSG-loop stabilized F variant (F47) (see Table 5). All variants have a GSGSG-loop and contain a HIS-tag.

Table 5. sion and stability of RSV F variants with alternative trimerization domains Description Antibody binding RSV Trimerization Termination sion Trimerization Protein t motif Modifications point ) % CR9501 CR9503 Isoleucine F47 A2 zipper (S) None 495 5 0% + + Isoleucine P1 B1 zipper (S) S502T 502 3.5 0% + + tri and Mat1 A2 Matrillin long None 520 12 hexamers - + Matrillin Mat2 A2 short None 516 0 ND - - Matrillin Mat3 A2 short None 495 1,5 ND - - GCN4II opt GCN A2 optimized None 516 0 ND - - opt GCN4II GCN+L512K A2 optimized L512K 516 1 ND + - Antibody binding is defined as binding on the day of harvest (as bed in Example 8; + indicates binding; - indicates no g.

Expression level is determined as described in Example 1. ND: not determined Only the matrillin 1 domain (Dames-SA et. al., Nat. Struc. Biol., 5(8), 1998) that contains both the N-terminal zipper domain and the C-terminal part with the cysteine residues that can potentially form inter trimeric disulfide bridges was found to enable higher expression levels than F47 (Table 5, Matrillin long). Moreover, the variant with the Matrillin long trimerization motif shows trimeric F proteins. However, the product did not bind to the pre-fusion specific Mab CR9501 and also showed hexameric species which makes the Matrillin 1 ization domain not suitable for production of trimeric native F protein. None of the matrillin-based or the GCN4II based zipper motifs showed increased expression or stability relative to F47 (Table , Matrillin short, GCN4II zed). Again, truncation at 495 results in higher expression levels. Addition of a GCN4 motif which contained an optimized trigger sequence showed no sion.

GCN4 II is a trimerization domain that is used successfully for stabilizing the pre-fusion trimer of parainfluenza type 5 (Yin et al., Nature 439:38-44, 2006) and has also been tried by others to stabilize RSV pre-fusion F (as disclosed in e.g. WO2010/149743, WO2010/14975, WO2009/079796, WO2010/158613). The GCN4II trimerization domain was evaluated and compared with the constructs that n the Isoleucine Zipper (S) domain (SEQ ID NO: 3) or the in (SEQ ID NO: 4) domain (results shown in Table 6). These variants were also compared with annther modifications, i.e. a short linker based on a single Lysine and the L512K on. All variants contained a HIS-tag.

Table 6. Expression and stability of RSV F variants with GCN4II, L512K and p27 ement (single amino acid linker (K) between F1 and F2) Description Stability RSV Trimerization Termination Expr.

Protein Variant motif F1, F2 linker Modifications point (ug/ml) Heat (oC) Storage* F18 B1 Fibritin None None 513 2 ND unstable F24 B1 Fibritin SG_S None 513 44 51 unstable Isoleucine F43 B1 zipper (S) Q__GSGSG_S None 495 0,4 53 stable Isoleucine P1 B1 zipper (S) Q__GSGSG_S S502T 502 3.5 54 ND F42 B1 None Q__GSGSG_S None 513 16.1 54 stable P2 B1 None K None 513 14,3 54 stable P3 B1 GCN4II None L512K 516 0 ND ND P4 B1 GCN4II K L512K 516 0 ND ND P5 B1 GCN4II K L512K 516 0 ND ND P6 A2 I GCN4II K L512K 516 0 ND ND P7 A2 II GCN4II K L512K 516 0 ND ND Storage stability determined as described in Example 8; Expression levels determined as described in Example 1; Heat stability determined as described in Example 9; ND: not determined.

The short linkage between F1 and F2 appears to be comparable to the GSGSG loop.

Addition of the GCN4II motif does not result in any F protein expression in any of the tested constructs (i.e. the RSV A2 F sequence described in WO2010/149743 or /149745, the RSV A2 F sequence used according to the invention, nor the RSV B1 F sequence).

It was shown that the introduction of these two types of cations, i.e. introduction of a linking sequence and the heterologous ization domain, was not enough to enable the expression of stable trimeric pre-fusion F protein. Apart from the two main regions of instability that were stabilized, i.e. the HRB and the fusion e, as bed above, other regions in the pre-fusion F protein also contribute and/or accommodate the dramatic refolding to post-fusion F, and more positions in the sequence can be zed to stop the pre-fusion F protein from refolding. Therefore, different amino acid residues in the HRA and HRB domain and in all s that contact these regions in pre-fusion F were d to increase the pre-fusion structure stability, as described in the following Examples.

EXAMPLE 2 Preparation of stable pre-fusion RSV F polypeptides – stabilizing mutations Because the trimeric content (for construct F47) and storage stability (for construct F24) was not optimal, further variants were made that contained point mutations to increase expression levels, stability and native trimeric structure. The results are shown in Table 7 and 8.

Table 7. Expression and stability of F47- ts Expression RSV Protein (ug/ml) Trimerization % Heat (oC) F47- 10 2-5% 53 F47- + K465E 6 2.4% ND F47- + D479K 5 29% 50,77 F47- + K176M 13 5% ND F47- + K209Q 9 3% 52,9 F47- + S46G 38 11% 59,38 F47- + S215P 8 1-2% 57,21 F47- + N67I 15 2% 59,84 F47- + K465Q 18 2% 54,3 F47- S46G+N67I 31 6% >60 F47- S46G+S215P 38 6% >60 F47- K465Q+K209Q 12 1% 53,3 F47- K465Q+S46G 28 7% 57,7 F47- K465Q+N67I 17 2% 59 F47- K209Q+N67I 15 4% >60 F47- K209Q+S215P 15 2% 56,7 ND: not determined; Expression level determined as described in Example1. Heat stability determined as described in Example 9.

Nomenclature of mutations based on wt sequence (SEQ ID NO: 1).

All constructs are variants of F47- : type A2, Isoleucin Zipper (S) motif (SEQ ID NO: 3), GSGSG linker; termination point 495, no HIS-tag (SEQ ID NO: 16). As shown in Table 7, many mutations increased the expression of F47-, but only the t F47_S46G also showed a higher level of trimers s the high sion.

Table 8 shows the results of the expression and stability of F24 variants. All variants were of RSV type A2, with fibritin motif, GSGSG linker; termination point 513, no HIS-tag.

Table 8. Expression and stability of A2_F24- (SEQ ID NO: 19) variants Expression e RSV Protein (ug/ml) Endpoint Association phase A2_F24 5,3 69 ND A2_F24 K508E 5,3 64 ND A2_F24 K498E 1,7 ND ND A2_F24 E487I 25,0 10 ND A2_F24 E487K 7,1 ND ND A2_F24 E487N 42,4 22 ND A2_F24 E487P 12,8 46 ND A2_F24 E487Q* 14,8 50 ND A2_F24 E487R 8,7 59 ND A2_F24 E487S 6,7 46 ND A2_F24 E487Y 10,5 36 ND A2_F24 D486N 31,2 19 ND A2_F24 D479N 5,2 ND ND A2_F24 D479K 1,5 62 ND A2_F24 E472Q 1,9 ND ND A2_F24 E472K 0,9 ND ND A2_F24 K465E 14,8 76 ND A2_F24 K465Q* 13,6 92 Not stable A2_F24 E463K 3,1 ND ND A2_F24 E463Q 6,0 ND ND A2_F24 G430S 4,8 ND ND A2_F24 N428R 5,2 35 ND A2_F24 N426S 18,6 71 ND A2_F24 K421N 9,2 75 ND A2_F24 E328K 9,5 21 ND A2_F24 T311S 3,5 70 ND A2_F24 I309V 11,3 69 ND A2_F24 D269V 0,0 ND ND A2_F24 S215P* 18,7 99 Stable A2_F24 K209Q 31,4 63 ND A2_F24 V207P 3,3 79 ND A2_F24 I206P 5,4 55 ND A2_F24 L204P 5,9 ND ND A2_F24 L203P 0,8 ND ND A2_F24 Q202P 4,4 ND ND A2_F24 K201Q 21,3 62 ND A2_F24 D194P 1,9 ND ND A2_F24 L193P 6,5 42 ND A2_F24 V192P 0,6 32 ND A2_F24 V185N 50,2 38 ND A2_F24 GV184EG 3,5 ND ND A2_F24 G184N 59,8 37 ND A2_F24 V178P 14,8 23 ND A2_F24 A177P 2,0 ND ND A2_F24 K176M 14,7 58 ND A2_F24 K176E 0,7 ND ND A2_F24 N175P 34,3 55 ND A2_F24 S169P 0,5 ND ND A2_F24 K168P 0,1 ND ND A2_F24 K166P 12,3 45 ND A2_F24 V157P 0,2 ND ND A2_F24 E92D 47,4 94 Not stable A2_F24 K85E 1,1 ND ND A2_F24 K80E 51,9 60 ND A2_F24 K77E 22,4 ND ND A2_F24 N67I* 89,8 101 Stable A2_F24 I57V ND ND A2_F24 VI56IV 16,5 54 ND A2_F24 S46G* 40,7 96 Not stable The d ucts were tested for trimerization and were all found to be trimeric Expression level determined as described in Example 1. Endpoint stability is shown here as the percentage of pre-fusion dy binding (CR9501) after 5 days of storage at 4oC relative to day 1; Association phase stability is determined as described in Example 10.

Many mutations increased the expression of A2_F24-. For most mutations there was an apparent correlation between improved expression in F47- background (Table 7) and - background (Table 8). N67I had more positive impact on F expression in A2_F24- background.

The most significant increase in expression was obtained with the single point mutations: S46G, S215P, N67I, K80E, E92D, D486N, G184N, V185N, E487N, N175P, K209Q, E487I, E487Q, K77E, K201Q, N426S and K465Q. In the initial screening using the nt stability assay (Example 8) the variants with the highest expression showed the best stability upon storage as well (E92D, K465Q, K465E, N426S, S46G, S215P and N67I). To evaluate if these mutations indeed were stabilizing the pre-fusion conformation, culture supernatants were diluted to 5 and 10 µg/ml based on tative western results and these were stored up to 33 days at 4ºC. As single point mutants only N67I and S215P were completely stable over time (see Example 10).

Subsequently, several mutations that showed high expression and good stability of the pre-fusion conformation were combined to evaluate whether the stabilizations were additive or had a possible synergistic effect (Table 9).

Table 9. Expression and stability of variants of A2_F24 with two onal mutations.

Expression RSV Protein (ug/ml) ity* A2_F24 K465Q + S46G 21,8 Not stable A2_F24 K465Q + N67I 122,3 Stable A2_F24 K465Q + E92D 10,5 Stable A2_F24 K465Q + S215P 59,8 Stable A2_F24 S46G + N67I 115,5 Stable A2_F24 S46G + E92D 14,3 Not stable A2_F24 N67I + E92D 134,2 Stable A2_F24 N67I + S215P 152,1 Stable A2_F24 E92D + S215P 49,1 Stable A2_F24 K465Q+S215P 53,3 Stable A2_F24 S46G+S215P 43,8 Stable Storage stability refers to the ation phase analysis illustrated in Example 10.

Expression level was determined as described in e 1.

All variants are ts of F24-: type A2, fibritin motif, GSGSG linker; termination point 513, binding to all Mabs, no HIS-tag (SEQ ID NO: 19).

When the previously fied point mutations were combined very interesting synergistic effects could be observed especially in terms of expression levels with the combinations involving N67I as the most potent. All produced double mutants where either N67I and S215P was included were stable after more than 30 days storage at 4 ºC (Example 10).

Strikingly, the mutation N67I was found to have the strongest effect on expression levels of prefusion F when included in the double mutants. Next, combinations with the S215P mutations resulted in a able expression. Combination of N67I with S215P was selected since it led to a very high expression level, and because both point mutations were stable upon storage.

Additionally it was observed that both N67I and S215P had the ability to ize some of the mutants that as single mutations were unstable indicating that the region where these two mutations are found is central for the conformation changes the protein undergoes during the transition to the post-fusion conformation.

It thus has been shown that at least some mutations ed in increased expression levels and sed stabilization of the pre-fusion RSV protein. It is expected that these phenomena are linked. The mutations described in this Example all resulted in increased tion of pre-fusion F polypeptides. Only a selection of these polypeptides remained stable upon long storage (see Example 10). The stability assay that was used is based on the loss of the pre-fusion specific CR9501 epitope in the top of the pre-fusion F protein in a g assay and it may not be ive enough to measure all contributions to stability of the whole protein. The mutation for which only increased expression is observed are therefore (very likely stabilizing) potential mutations that can be combined with other stabilizing mutations to obtain a pre-fusion F construct with high ity and high expression levels.

Next, it was ed whether the N67I - S215P double mutation, like the single mutations, was able to stabilize point mutations that as single mutants were deemed unstable based on the criteria used. Extra mutations were selected based on the favorable expression levels and ity according to Table 8. Triple mutant RSV-F variants were constructed and tested for expression levels and stability (Table 10).

Table 10. Expression and stability of variants of F24_N67I +S215P with one onal mutation.

Expression RSV Protein (ug/ml) stability* A2_F24 N67I + S215P+K507E 344,6 ++ A2_F24 N67I + S215P+E487I 239,4 +++ A2_F24 N67I + S215P+E487N 285,2 +++ A2_F24 N67I + S215P+E487Q 360,7 +++ A2_F24 N67I + S215P+E487R 130,9 +++ A2_F24 N67I + S215P+D486N 292,6 +++ A2_F24 N67I + D479N 97,1 +++ A2_F24 N67I + S215P+K465Q 283,3 +++ A2_F24 N67I + S215P+N426S 316,3 +++ A2_F24 N67I + S215P+K421N 288,4 +++ A2_F24 N67I + S215P+K209Q 245,0 +++ A2_F24 N67I + S215P+K201Q 231,9 +++ A2_F24 N67I + S215P+V185N 445,1 +++ A2_F24 N67I + S215P+G184N 326,7 +++ A2_F24 N67I + S215P+E92D 308,8 + A2_F24 N67I + S215P+K80E 210,6 + A2_F24 N67I + S215P+S46G 199,4 +++ All variants are variants of A2_F24_N67I +S215P type A2, fibritin motif, GSGSG linker; termination point 513, binding to all Mabs, no HIS-tag (SEQ ID NO: 21). *stability refers to the association phase analysis illustrated in Example 10. + means <10% loss of CR9501 binding after 5 days; ++ means <5% loss of CR9501 binding after 5 days; +++ means 0% loss of CR9501 binding after 5 days.

Again, an ve effect on the expression levels was observed. As expected D479N and E487R triple mutants express at somewhat lower levels e the single mutants were also among the lowest of the selected mutations (Table 8). Because of the stabilizing effect of the N67I+S215P mutation, additional mutations that are unstable as single mutants, ed in stable sion F variants when they were added to the A2_F24 N67I+S215P background. Some very illustrative es are the triple mutants with the additional V185N, G184N or E487N which showed high expression but low stability as single mutants (Table 8) but showed even higher expression and were highly stable when added to the A2_F24 N67I+S215P background. izing mutations also stabilize RSV-F protein from other strains and also in processed F variant.

Several mutations that showed high expression and good stability of the pre-fusion conformation were applied to RSV F proteins of other strains and were applied to a RSV A2 F variant without furin cleavage site mutations (F18: SEQ ID NO 71) to te whether the cations are a universal solution to stabilize RSV prefusion F (Table 11).

Table 11. Expression and stability of variants of A2_F18 with additional mutations and F from strain B1 (SEQ ID NO: 2) and type A CL57-v224 (SEQ ID NO: 69).

Relative* Stability** expression after day 5, RSV protein Seq ID (CR9503) % A2_F18 71 0.018 0.0 A2_F18 N67I 0.449 73.2 A2_F18 S215P 0.129 9.1 A2_F18 E487Q 0.006 NA A2_F18 N67I, S215P 72 0.484 103.4 A2_F18 N67I, E487Q 0.340 92.1 A2_F18 N67I, S215P, E487Q 76 0.355 92.7 A2_F18 N67I, S215P, E92D 78 0.318 96.0 A2_F18 N67I, S215P, D486N 79 0.522 101.3 A2_F18 N67I, S215P, K201N 77 0.643 102.7 A2_F18 N67I, S215P, K66E 0.800 103.0 A2_F18 N67I, S215P, S46G, K66E 0.820 103.5 A2_F18 N67I, S215P, E487Q, K66E 0.704 99.5 A2_F18 N67I, S215P, E92D, K66E 0.905 98.8 A2_F18 N67I, S215P, D486N, K66E 0.863 96.6 A2_F18 N67I, S215P, K201N, K66E 1.021 105.5 A2_F18 N67I, S215P, D486N, K66E, I76V 0.594 95.0 B1_ N67I, S215P 73 0.434 90.9 B1_ N67I, S215P loop 22 0.552 108.2 CL57v224_ N67I, S215P 74 0.698 94.9 CL57v224_ N67I, S215P loop 75 0.615 98.4 n expression (concentration in the supernatant of transiently transfected cells) was measured by Quantitative Octet method.

* Relative expression is normalized to expression of A2_F24_N67I, S215P, E487Q (seq ID #33) ** Stability - is sed as % protein concentration measured after storage at 4C for 5 days, vely to the day of harvest. The concentrations were measured by Quantitative Octet method using CR9501 antibody.NA - data not available: no CR9501 g was detected.

When the previously identified point mutations were introduced in A2_F18 (SEQ ID NO. 71), the stability and expression levels were very similar compared with the single chain F24 (SEQ ID NO. 21) variant that contained a short loop between F1 and F2. Again, synergism was observed showing higher expression and stability when mutations were added to variants that ned the N67I or the double on N67I, S215P. The double point mutation N67I, S215P did not only stabilize the pre-fusion F of the A2 strain but also pre-fusion of of B1 and CL57- v224 strain (Table 11).

Stabilizing ons also stabilize full length RSV-F protein.

Several mutations that showed high expression and good stability of the pre-fusion conformation in the soluble version of RSV-F corresponding to the ectodomain, were applied to the full length RSV-F protein. The ons were introduced in full length RSV-F with or without furin cleavage site mutations. No trimerization domain was fused to these variants (Table 12).

Table 12. sion and stability of variants of full length versions of A2_F18 and A2_F24 with additional mutations.

RSV F nvariant* Attributes SEQ ID No Expression, fold Amino acid substitutions F1, F2 linker increase** Heat-stability*** None (F A2 wildtype, full length) 1 none 1 - N67I none 1.4 N.D.

S215P none 1.4 N.D.

E92D none 1.4 N.D.

N67I, K465Q none 1.4 N.D.

N67I, S46G none 0.2 N.D.

N67I, E92D none 1.4 N.D.

N67I, K80E none 2.3 N.D.

N67I, G184N none 1.5 N.D.

N67I, V185N none 1.4 N.D.

N67I, E487Q none 2.5 N.D.

N67I, S215P,V185N none 2.7 N.D.

N67I, S215P,K508E none 3.0 N.D.

N67I, S215P,K80E none 3.1 N.D.

N67I, S215P,K465Q none 2.9 N.D.

N67I, S215P 80 none 2.4 ++ N67I, S215P, G184N none 7.6 ++ N67I, S215P, E92D 82 none 6.8 N.D.

N67I, S215P, S46G 88 none 6.8 + N67I, S215P, D486N 86 none 5.9 +++ N67I, S215P, E487Q 84 none 6.2 N.D.

N67I, S215P, S46G, K66E none 12.1 +++ N67I, S215P, D486N, K66E none 9.2 +++ N67I, S215P, S46G, E92D, K66E none 11.8 +++ N67I, S215P, S46G, E487Q, K66E none 11.0 +++ N67I, S215P, S46G, D486N, K66E none 10.5 +++ N67I, S215P, D486N, K66E, I76V none 7.2 +++ N67I, S215P, S46G, K66E, I76V none 9.7 +++ N67I, S215P, S46G, K80E none 4.5 N.D.

N67I+S215P+G184N+K80E+E92D+E487Q+S46G none 9.1 N.D.

None Q__GSGSG_S 3.8 - N67I, S215P 81 Q__GSGSG_S 6.2 N.D.

N67I, S215P, G184N Q__GSGSG_S 7.2 ++ N67I, S215P, E92D 83 SG_S 5.9 N.D.

N67I, S215P, S46G 89 Q__GSGSG_S 5.3 ++ N67I, S215P, D486N 87 Q__GSGSG_S 5.2 +++ N67I, S215P, E487Q 85 Q__GSGSG_S 4.6 N.D.

N67I, S215P, S46G, K66E Q__GSGSG_S 11.7 +++ N67I, S215P, D486N, K66E SG_S 13.8 +++ N67I, S215P, D486N, K66E, I76V SG_S 6.8 +++ N67I+S215P+G184N+K80E+E92D+E487Q+S46G Q__GSGSG_S 3.6 N.D. sion level determined using FACS. N.D. – not determined. *all variants are based on RSV A2 F protein sequence. ** comparing to wild type protein, fold increase of MFI on 9503.

Stability was assessed by heat treatment of the HEK293T cells for 5 - 10 minutes at 46, 55.3, 60 oC. *** legend for the stability readout - decrease in binding to prefusion – specific Mab CR9501 binding after 46 oC (e.g. wild type) + slight decrease of CR9501 binding after 46 oC but not to same strong extent as wild type ++ no change in CR9501 binding up to 60 oC, at 60 oC some decrease in CR9501 g +++ no change in CR9501 binding at 60 oC The previously identified izing point ons were also stabilizing in the full length F protein. The increase in expression level was less pronounced but showed the same trend. This may be caused by the different background the mutations were introduced in but may also be caused by the different quantification method (FACS versus Western blot) and a biological maximum of expression due to recycling of surface proteins. Introduction of the linking sequence (or short loop) increased expression and stability and the point mutations did so too. The point mutations were not or hardly synergistic with the short loop (similar as to what we found for the soluble protein (Table 9-11) Because the point mutation at position 67 had such positive effect on expression level and stability, all amino acid substitutions were tested for this position to study whether the most optimal were chosen or whether these positions can be improved. (Table 13) Table 13. Full substitution analysis of expression and stability for position 67 in the A2_F24 background.

Amino acid substitution Relative Expression* Stability** after day 4, % ity** after day 10, % N67A 1.696 0.0 0.0 N67C 1.759 16.7 0.0 N67D 1.702 0.0 0.0 N67E 1.357 0.0 0.0 N67F 2.565 102.2 108.1 N67G 0.853 NA NA N67H 1.509 0.0 0.0 N67I 3.773 98.2 102.7 N67K 0.487 NA NA N67L 3.609 107.5 96.4 N67M 2.579 87.3 78.7 N67P 2.414 14.3 0.0 N67Q 0.955 NA NA N67R 0.523 NA NA N67S 1.277 0.0 0.0 N67T 1.577 0.0 0.0 N67V 2.457 84.2 77.0 N67W 1.794 99.9 104.3 N67Y 1.830 61.3 45.8 * Relative sion - protein concentration was measured by tative Octet method using CR9503 antibody and expressed relative to concentration of A2_F24 (SEQ ID #19) ** Stability - is expressed as % protein concentration ed after storage at 4C for 5 and10 days, relatively to the day of harvest. The concentrations were measured by Quantitative Octet method using CR9501 antibody.NA - data not available: no CR9501 binding was detected.

As shown in Table 13, primarily hydrophobic residues and particularly Ile, Leu and Met at position 67 were able to increase expression and stability. Ile is the residue that increased expression and stability most. Residues Glu and Gln, the smallest residue Gly and the positively charged residues Arg and Lys had the most destabilizing effect at on 67 on the prefusion conformation.

EXAMPLE 3 Preparation of stable pre-fusion RSV F polypeptides according to the present invention In the research that led to the present invention, further stabilized variants of soluble prefusion F protein (sF) were designed by izing the two main regions that initiate refolding.

The first gy was to prevent the refolding of the HRA region into a coiled coil. The second strategy was to uct ide bridges N-terminal to HRB to prevent the relocation of the HRB to form the six helix bundle by docking onto the HRA coiled coil.

The constructs were tested for expression levels, storage stability and antibody binding with the antibody CR9501. The amino acid sequences of the heavy and light chain le regions, and of the heavy and light chain CDRs of this dy are given below. CR9501 comprises the binding regions of the antibodies referred to as 58C5 in WO2012/006596.

The constructs were synthesized and codon-optimized at Gene Art (Life Technologies, ad, CA). The constructs were cloned into pCDNA2004 or ted by standard methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. The expression platform used was the 293Freestyle cells (Life Technologies). The cells were transiently transfected using 293Fectin (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37oC and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4oC until use.

Supernatants from day 5 were ted for F protein expression by western blot using the monoclonal antibody CR9503, which comprises the heavy and light chain variable regions of the RSV F antibody Motavizumab (referred to as CR9503). The approximate expression levels of the pre-fusion RSV F protein constructs were assessed using CR9503, an anti-human IR-dye conjugated secondary antibody (Li-Cor, Lincoln, NE) or a HRP conjugated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, PA). The protein quantities were then estimated using a dilution series of ed RSV standard protein, either by eye or using the Odyssey CLx infrared imaging system. To evaluate construct stability and to identify positive or negative stabilizing effects of introduced trimerization motifs, the constructs were tested for binding to prefusion – ic dies after 5, 14 or 30 days of storage at 4 ºC This procedure is described in detail in Example 10.

Next, the most favorable modifications were combined to find the optimal pre-fusion F polypeptides. Combinations were made of variants with the GSGSG loop, C-terminal truncation of F1, and the on of fibritin (SEQ ID NO: 4). Variants were made that contained point ons to increase sion levels, stability and native trimeric structure. All variants were of RSV type A2, with fibritin motif, GSGSG linker; termination point 513, no HIS-tag.

According to the invention, the amino acid mutations that stabilize the pre-fusion conformation of the RSV F protein can be grouped into different ries that stabilize the mation in different manners.

Amino acid residues 161, 173, 174, 182 and 214 In order to refold from the pre-fusion to the post-fusion mation, the region between residue 160 and 215 has to transform from an assembly of helices, loops and strands to a long continuous helix. This region demonstrates the most dramatic structural transition. Part of this region actually has the highest alpha-helix prediction. The actual helical structures in the prefusion l structure are shown below in grey highlights. This whole region is transformed into one large helix when it refolds to the usion conformation. In the bottom sequence the residues are highlighted in grey with the highest helix prediction based on Agadir (http://agadir.crg.es/). It is clear from this comparison that the C-terminal part that is maintained in a beta-hairpin, a connecting loop and a helix in the pre-fusion conformation (residues 187- 202) has a high tendency to form a alpha-helix. 150 160 170 180 190 200 210 SGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSC hh hhhh sssssss ssssssss hhhhh hhhhh SGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSC Underlined residues have bad angles according to Ramachandran-plot.

The sequence of residues 150 – 212 of RSV-F is shown above. On the second line the secondary structures of the top line are indicated by h (for helix) and s (for s) based on the crystal structure. Helices are ghted with grey shading. The bottom line is the same sequence in which the helices are shaded grey based on the helix propensity of the sequence.

The regions that need zation are the loop regions in between the secondary structural elements (helices and strands) in the labile HRA of pre-fusion RSV-F.

One of the positions in HRA that needs optimization in order to ize the pre-fusion conformation of RSV-F is position 161 in the turn between helices α2 (residues 148-157) and α3 (residues 163-172). There are several reasons why optimization of this on could increase the stability of this region: - The turn positions the negative charge of Glu161 close to the negative charge of Glu163 resulting in destabilizing negative repulsion; - The Ramachandran plot shows that residue 161 has bad/unfavourable dihydral angles; - Residue 161 has a high B-factor which ts high mobility (and suggests instability); - Residues 160 – 172 display high helix propensity.

In this e, residue Glu161 was replaced by Pro to reduce the negative ion and ize the turn and prevent it from refolding, or residue Glu161 was replaced by Gln to reduce the negative charge repulsion, or residue Glu161 was replaced by Gly because it allows a broader range of al angles.

For the region of α2 – turn – α3 (residues 153 – 168), the Brookhaven database was searched for a structurally homologous helix-turn-helix from a stable protein that does not refold in order to find a residue that could replace the unfavourable Glu161. A high structural homology was discovered with a turn in a helix-turn-helix of several proteins that all had a e at the homologous 161 position (PDB codes 2hgs, 3kal, 2o2z, 2zk3, and 2zqp). According to the alignment shown below, the substitution of Glu161 by Pro is a good structural solution to stabilize this turn and t it from refolding.

AVSKVL HL E G EVNKIK RSV-F HRA153 – 168 KVQQELSRP GMLEMLL 2hgs KIQQELAKP GVLERFV 3kal SVLPNLLVP GICEAIK 2o2z avSKVLH-LEGEVNKIK RSV-F HRA 153 – 168 ikTPLVDdLP GAEEAMS 1zk3 AVSKVLH-LEGEVNKIK RSV-F HRA 153 – 168 IMQILVTvVP ALEKLSK 2zqp In certain embodiments, residue Ser173 was replaced by Pro to stabilize the turn and prevent it from refolding. In certain embodiments, residue Thr174 was ed by Pro to stabilize the turn and prevent it from refolding.

The Ramachandran plot shows that the amino acid residue 182 in the turn between ß3 and ß4 also has favourable dihydral angles. Optimization of this position could increase the stability of the turn and stabilize the ß-hairpin.

For the region of ß3 – turn - ß4 (residues 177-189), the Brookhaven database was searched for a structurally homologous ß-hairpin from a stable protein that does not refold in order to find a e that could replace the unfavourable Ser182. A high structural homology was discovered with a turn in a ß-hairpin of a ve electron transfer protein that had a Proline at the homologous 182 position (PDB code 3me8). According to the ent shown below, the substitution of Ser182 by Pro is a good structural solution to ize this turn and prevent it from refolding.

AVVS lSNg V-SVLT VVVLsP ElQiKDYI Cystine bridge formation in the bottom of the head region between residues 486, 487, 489 The negatively charged amino acid residues 486, 487 and 489 are part of a switch mechanism that controls the transition n the pre-fusion and post-fusion RSV-F structure. Mutation of Glu487 to Gln will impair this switch and stabilize contact between the protomers in the trimer.

These same residue positions can also be used to engineer disulfide bridges between the protomers. Mutations of 2 residues by cysteines as described above will reduce the negative charge repulsion and allow disulfide bridges that will further stabilize the ion trimer.

Variants were made that contained point mutations that stabilize the turns between the secondary ural elements in the HRA region of RSV-F pre-fusion protein to increase stability and expression levels of the prefusion conformation. The results are shown in Table 14.

Table 14. Expression and stability of A2_F24- (SEQ ID NO: 19) variants expression Stability relative to A2 protein description F24- day 5-7 day 30 A2 F24- E161P 2,739 75,08 66,24 A2 F24- E161Q 0,410 133,71 N.A.

A2 F24- E161G 0,391 106,42 N.A.

A2 F24- S173P 1,182 85,78 N.A.

A2 F24- I214P 0,288 80,20 N.A.

A2 F24- T174P 0,448 39,82 N.A.

A2 F24- S182P 2,296 87,19 N.A.

A2 F24- N67I S215P E161P 35,766 97,67 100,56 A2 F24- N67I S215P E161Q 9,545 104,40 96,60 A2 F24- N67I S215P E161G 12,035 93,70 81,91 A2 F24- N67I S215P S173P 21,747 103,43 71,89 A2 F24- N67I S215P I214P 8,053 99,47 68,17 A2 F24- N67I S215P T174P 5,431 N.A. N.A.

A2 F24- N67I S215P S182P 14,948 N.A. N.A.

All variants are variants of A2_F24 type A2 that contain a fibritin motif and GSGSG linker between F1 and F2; termination point 513, (SEQ ID NO: 19). ity is expressed as % protein concentration measured by Qoctet (Example 10) after storage at 4C for 5 -30 days, relatively to the day of harvest. The concentrations were measured by Quantitative Octet method using CR9502 antibody. NA: data not available: no CR9502 binding was detected..ND : Not determined Of the single point mutations, substitution of position 173, 182 and especially 161 to e ed in higher expression levels and stability. Removing the charge of residue 161 did stabilize the proteins but did not increase expression levels. The same point mutations had a similar effect in a stabilized pre-fusion F ce that contained the additional stabilizing N67I and S215P mutation. Mutation of residue 182, 173 and especially 161 to Proline showed the highest se in stability and expression levels.

The E161P mutations that showed high expression and good ity of the pre-fusion conformation was also applied to e RSV A2 F ectodomain variants t furin cleavage site mutations (F18: SEQ ID NO 71) to evaluate whether the modifications are a universal solution to stabilize RSV pre-fusion F (Table 15).

Table 15. Expression and stability of variants of A2_F18 (SEQ ID No: 71) with onal mutations stability** relative after 15 RSV protein SEQ ID expression* days (%) A2_F18 71 0,1 0,0 A2_F18 N67I 19,6 29 A2_F18 S215P 8,4 4 A2_F18 E487Q 0,0 ND A2_F18 E161P 4,2 0 A2_F18 N67I, S215P 72 32,1 95 A2_F18 N67I, E161P 34,2 72 A2_F18 N67I, S215P, E161P 56,1 79 A2_F18 N67I, S215P, E161P, E487Q 55,5 91 A2_F18 N67I, S215P, E487Q 76 21,8 95 Protein expression (concentration in the supernatant of ently transfected cells) was measured by Quantitative Octet method.

* Relative expression is normalized to expression of A2_F24_N67I, S215P, E487Q (seq ID #33) ** Stability - is expressed as % protein concentration measured by Qoctet (Example 10) after storage at 4C for 5 days, relatively to the day of harvest. The concentrations were measured by Quantitative Octet method using CR9501 dy. ND : Not determined The E161P mutation also showed a high se in expression levels in the processed RSV-F protein. When combined with stabilizing point mutations at e.g. position 67, 215 and 487, the E161P mutation resulted in prefusion F variants with high expression levels and high stability.

Cystine bridge formation in the bottom of the head region between residues 486, 487, 489 The negatively charged amino acid residues 486, 487 and 489 are part of a switch mechanism that controls the transition between the pre-fusion and post-fusion RSV-F structure.

Mutation of Glu487 to Gln will impair this switch and stabilize contact between the protomers in the trimer (previous patent P00). These same residue positions can also be used to engineer disulfide bridges between the ers. Mutations of 2 residues to cysteines of which one is a negatively charged residue 486, 486 or 489, will reduce the negative charge ion and allow disulfide bridges that will further ize the prefusion trimer. Several of such ts were tested for expression level and stability of the prefusion conformation (Table 16).

Table 16. Expression and stability of A2_F24- (SEQ ID NO: 19) variants Expression Stability relative to A2 protein description F24- day 30 A2 F24 D489C L481C 0 A2 F24 D489C V482C 0 N.D.

A2 F24 D489C D479C 0 N.D.

A2 F24 D489C T374C 0 N.D.

A2 F24 D489C L375C 0 N.D.

A2 F24 D489C P376C 0 N.D.

A2 F24 D489C S377C 0 N.D.

A2 F24 D489C T335C 0 N.D.

A2 F24 D489C D338C 0 N.D.

A2 F24 D489C S398C 0 N.D.

A2 F24 D486C E487C 0,524 N.D.

A2 F24 D489C D486C 0,062 N.D.

A2 F24 N67I S215P D489C D486C 3,875 76,02 A2 F24 N67I S215P D489C S398C 0,003 N.D.

A2 F24 N67I S215P D486C E487C 7,315 79,39 All ts are variants of A2_F24- type A2 that contain a fibritin motif and GSGSG linker between F1 and F2; termination point 513, (SEQ ID NO: 19).

Stability - is expressed as % protein concentration by Qoctet le 10) measured after storage at 4C for 5 -30 days, relatively to the day of harvest. The concentrations were measured by Quantitative Octet method using CR9502 antibody. ND : Not determined In the metastable F24 background (SEQ ID NO: 19), only a disulfide bridge between residues 486 and 487 resulted in a prefusion protein with reasonable sion and stability. Because inter-protomeric disulfides need a t alignment of opposing side-chains, the disulfide tivity may be more sful in a more stable F protein compared to the metastable F24 variant. Therefore, several of the disulfides were also engineered in the F24 variant that contained the 2 stabilizing mutations N67I and S215P. Indeed, in the stabilized background the proteins with engineered disulfides expressed to much higher . Again, the variant with the cysteine mutations at position 486 and 487 expressed to the highest level and expression level was 14 times higher compared with the unstablized variant without the N67I and S215P mutation. Stability of the protein in the supernatant reasonable and still contained 79 % ion conformation. Higher stability may be reached when the protein is ed. Stability may not have reached 100% because not 100% of the cysteines were connected inter-protomeric as shown in example 4 and 5.

EXAMPLE 4 Western blotting Culture atants were run reduced on 4-12% (w/v) Bis-Tris NuPAGE gels (Life logy) and blotted using the iBlot technology (Life Technology). The blots were probed with CR9503 (sequences given below in Table 18) and developed with either a conjugated mouse anti-human IgG (Jackson ImmunoResearch, West Grove, PA) or a IRDye800CW conjugated ty purified anti-human IgG (rabbit) (Rockland Immunochemicals, Gilbertsville, PA). In Figure 1, the expression of DM = Double mutant (N67I+S215P = SEQID 21) and DM+CC = Double mutant + C = SEQID 94) can be seen. Clear difference between the two proteins could be observed when analyzed reduced and non-reduced. Reduced both proteins migrate as a ric species around 55 kDa. Non-reduced the vast majority of the DM is still found as a monomer while the DM+CC predominant species is much higher and predominantly trimeric. This proves that substitution of residue 486 and 487 to cysteine results in a trimer with predominantly inter-protomeric ide bridges.

EXAMPLE 5 NativePAGE For initial determination of the multimeric state of the pre-fusion F polypeptides according to the invention, culture atants from transiently transfected cells were analyzed in a NativePAGE is gel system (Life Technologies). Subsequently the gels were blotted using the iBlot technolog according to the manufacturer’s instructions (Life Technologies). An RSV F protein specific antibody CR9503 (sequences given below in Table 18) was used as primary probe for the detection of pre-fusion RSV F protein and followed by a HRP ated mouse anti-human IgG on ImmunoResearch, West Grove, PA) or a IRDye800CW conjugated affinity purified uman IgG (rabbit) (Rockland Immunochemicals, Gilbertsville, PA). The blots were developed with either standard film (Codak) or using the Odyssey CLx infrared imaging system. Figure 2 shows the NativePAGE analysis of atants from Lane 2: DM = Double mutant (N67I+S215P = SEQID 21) and Lane 1: DM+CC = Double mutant + DE486CC = SEQID 5A) Both the DM and the DM+CC are primarily trimeric on native page showing that the introduction of disulphides may not lead to intertrimeric cross-linking. Since the DM+CC expresses less well than the DM the missing monomer (arrow) may be due to the fact that it is below the limit of detection.

EXAMPLE 6 sion of pre-fusion F protein Expression plasmids encoding the recombinant pre-fusion RSV F protein were generated by standard methods widely known within the art, involving site-directed mutagenesis and PCR.

The expression rm used was the 293Freestyle cells (Life Technologies, Renfreshire, UK).

The cells were transiently transfected using 293Fectin (Life Technologies) according to the manufacturer’s ctions and cultured in a g incubator for 5 days at 37oC and 10% CO2.

The culture supernatant was harvested and spun for 5 min at 300 g to remove cells and cellular debris. The spun supernatant was uently sterile filtered using a 0.22 um vacuum filter and stored at 4oC until use.

EXAMPLE 7 Purification of pre-fusion RSV F n The recombinant polypeptides were purified by a 2-step purification protocol applying a n exchange column for the initial purification and subsequently a superdex200 column for the polishing step to remove residual contaminants. For the initial ion-exchange step the culture supernatant was diluted with 2 volumes of 50 mM NaOAc pH 5.0 and passed over a 5 ml HiTrap Capto S column at 5 ml per minute. Subsequently the column was washed with 10 column volumes (CV) of 20 mM NaOAc, 50mM NaCl, 0.01% (v/v) tween20, pH 5 and eluted 2 CV of mM NaOAc, 1M NaCl, 0.01% (v/v) tween20, pH 5. The eluate was concentrated using a spin concentrator and the protein was further purified using a superdex200 column using 40mM Tris, 500mM NaCl, 0.01% (v/v) tween20, pH 7.4 as running buffer. In Figure 3A the chromatogram from the gel tion column is shown and the dominant peak contains the pre-fusion RSV F protein. The fractions containing this peak were again pooled and the protein concentration was determined using OD280 and stored a 4oC until use. In Figure 3B a reduced SDS-PAGE analysis of the final protein preparation is shown and as can be seen the purity was >95%. The identity of the band was ed using western blotting and n F specific antibodies (not shown).

Next, the purified protein was tested on NativePAGE and compared with a reference stable trimeric prefusion F protein (SEQID NO: 21) (Fig 3C).

Endpoint stability assay The verification of the pre-fusion conformation of the sed ptides according to the invention was done using the octet technology using the pre-fusion specific antibodies CR9501 or CR9502, or the non-conformation specific dy CR9503, which comprises the heavy and light chain variable regions of the commercially available antibody Motavizumab.

The antibodies were biotinylated by rd protocols and immobilized on streptavidin biosensors (ForteBio, Portsmouth, UK). The procedure was as follows. After equilibration of the sensors in kinetic buffer ( ForteBio) for 60s the chips were transferred to PBS with 5 ug/ml of the desired antibody. The loading was carried out for 250s. Subsequently another equilibration step was included for 200s in kinetic buffer. Lastly the chips were transferred to the expression culture supernatant containing the pre-fusion RSV F polypeptides and the total binding signal after 1200s was recorded. This phase is also referred to as the association phase. This was done ately after harvest (day 1) as well as 5 days later (day 5) and the difference in the CR9501 binding was used as a screening tool to identify mutations capable of stabilizing the pre-fusion conformation. A construct was deemed stable if less than 20% loss of binding was observed at day 5 it was deemed stable and if not it was deemed unstable. Stable constructs could then undergo a more stringent stability test if needed. The data analysis was done using the ForteBio Data is 6.4 software (ForteBio).

EXAMPLE 9 Heat stability assay The stabilizing potential of introduced features into the RSV F polypeptides was estimated by heat stress. For that purpose culture supernatant from ently transfected cells or ed protein was heated using a range of temperatures. The samples were uently cooled on ice to prevent further heat induced conformational s and probed using the CR9501 antibody on the octet technology rm as described in Example 11. The responses obtained at end of the association phase at the different temperatures were plotted as a function of the temperature and fitted by non-linear regression using the Prism software. This resulted in an estimation of the temperature where the antibody binding level is 50% of the maximum and this value could be used to compare different constructs in terms of pre-fusion heat stability.

EXAMPLE 10 Association phase stability assay To assess the stability of s point mutations the octet binding assay was developed by using association phase analysis. The CR9501 antibody or CR9502 antibody was used as probes for the prefusion conformation of the RSV-F protein. To reduce potential concentration bias of the endpoint assay, the data points were used from the entire association phase of the experiment. The data were sated for the amount of bound antibody on the chip. The measurements were done at days 1, 5 and 33, and the shapes of the curves from the three days were compared. If identical curves were obtained the construct was deemed stable and if not, unstable.

EXAMPLE 11 Quantitative Octet To e tration of the pre-fusion RSV F protein in cell culture supernatants, quantitative Octet-based method was used. The CR9501 and CR9503 antibodies were biotinylated by standard protocols and immobilized on Streptavidin biosensors (ForteBio, Portsmouth, UK). Afterwards, the coated biosensors were blocked in mock cell culture supernatant. tative experiment was performed as follows: temperature 30C, shaking speed 1000 rpm, time of the assay 300 sec. Concentration of the protein in the cell culture atant was calculated using rd curve. The standard curve was prepared for each coated antibody using the A2_F24_N67I+S215P (SEQ ID# 21) protein, diluted in mock cell culture supernatant.

The measurement was done on the day of supernatant harvest (day1) and after storage of the supernatant at 4C for 5 days or longer. The difference in the concentration determined with the CR9501 or CR9502 was used as a screening tool to identify mutations capable of stabilizing the pre-fusion conformation. A construct was deemed stable if less than 20% decrease of measured tration was observed at day 5.The data analysis was done using the ForteBio Data Analysis 6.4 software (ForteBio).

EXAMPLE 12 Preclinical evaluation of prefusion F immunogenicity To evaluate the genicity of a stabilized pre-fusion RSV F (A2F24,N67I, S215P) (SEQ ID NO: 21) we immunized mice according to Table 19 with 0.5 or 5 ug in a prime – boost regimen at week 0 and week 4. As shown in Figure 4, mice immunized with pre-fusion F showed higher VNA titers than mice immunized with post-fusion RSV F.

Table 19. Immunization scheme Group Preparation Dose Adjuvant N 1 Post-fusion F 0.5 µg - 9 2 Post-fusion F 5 µg - 9 3 Pre-fusion F 0.5 µg - 9 4 Pre-fusion F 5 µg - 9 usion F 0.5 µg Poly(I:C) 9 6 Pre-fusion F 0.5 µg Poly(I:C) 9 8 FI-RSV 1/75 - 8 9 PBS - 3 Next, cotton rats were immunized with two different doses of RSV-F in either the postfusion or the pre-fusion conformation (Table 20). Animals were immunized i.m. at week 0 and week 4. Figure 5 demonstrates high neutralizing antibody titers at the day of nge (week7).

Table 20. Groups, immunogen and dose for immunogenicity evaluation and efficacy in cotton rats Group Preparation Dose nt 1 Post-fusion F 0.5 ug - 2 Post-fusion F 5 ug - 3 Pre-fusion F 0.5 ug - 4 Pre-fusion F 5 ug - 9 Pre-fusion F 0.5 ug Poly IC Pre-fusion F 5 ug Poly IC 11 Pre-fusion F 0.5 ug Adju Phos 12 Pre-fusion F 5 ug Adju Phos 13 Ad26.RSV.FA2 10^8 - 14 PBS - - Five days after challenge the lung and nose viral load was measured (see Figure 6).

As shown, the pre-fusion F polypeptides according to the invention are able to induce a strong tive immune response that reduced viral load in the lung and even in the nose.

Table 17. Standard amino acids, abbreviations and ties Amino Acid 3-Lette r 1-Letter Side chain Side chain charge (pH 7.4) polarity alanine Ala A non-polar Neutral arginine Arg R polar Positive gine Asn N polar Neutral aspartic acid Asp D polar Negative cysteine Cys C non-polar Neutral ic acid Glu E polar Negative glutamine Gln Q polar Neutral glycine Gly G non-polar Neutral histidine His H polar positive(10%) neutral(90%) isoleucine Ile I non-polar Neutral leucine Leu L non-polar Neutral lysine Lys K polar Positive methionine Met M non-polar Neutral phenylalanine Phe F lar Neutral proline Pro P non-polar Neutral serine Ser S polar l threonine Thr T polar Neutral tryptophan Trp W non-polar Neutral tyrosine Tyr Y polar Neutral valine Val V non-polar Neutral Table 18. Amino acid sequences of antibodies CR9501 and CR9502 Ab VH domain VH CDR1 VH CDR2 VH CDR3 Amino acids 1- GASINSDNYYWT HISYTGNTYYTPSLKS CGAYVLISNCGWFDS CR9501 125 of SEQ ID NO: 53 (SEQ ID NO:54) (SEQ ID NO:55) (SEQ ID NO:56) Amino acids 1- GFTFSGHTIA WVSTNNGNTEYAQKI EWLVMGGFAFDH CR9502 121 of SEQ ID QG NO:57 (SEQ ID NO:58) (SEQ ID NO:59) (SEQ ID NO:60) Ab VL domain VL CDR1 VL CDR2 VL CDR3 GASNLET QQYQYLPYT Amino acids 1-107 QASQDISTYLN CR9501 of SEQ ID NO: 61 (SEQ ID NO: 62) (SEQ ID NO:63) (SEQ ID NO:64) GANNIGSQNVH DDRDRPS QVWDSSRDQAVI Amino acids 1-110 CR9502 of SEQ ID NO: 65 (SEQ ID NO:66) (SEQ ID NO:67) (SEQ ID NO:68) The amino acid sequence of several of the pre-fusion RSV F constructs is given below. It is noted that the amino acid numbering in the different constructs described herein is based on the wild-type sequence (SEQ ID NO: 1), which means that all amino acids from position 1 to and including position 108 of the pre-fusion constructs correspond to the amino acid ons 1-108 of the wild-type ce, s the numbering of the amino acids from position 138 to the end is shifted 22 amino acids, i.e. L138 in the wild-type ce (SEQ ID NO: 1) ponds to L116 in all the pre-fusion constructs. This is due to the fact that a deletion has been made in the pre-fusion constructs i.e. the insertion of the GSGSG linker the actual numbering in F1 is not the same between constructs. Thus, the numbering used with respect to the specific mutations according to the invention, e.g. S215P, refers to the position of the amino acid in the wild type sequence.

Sequences RSV F protein A2 full length sequence (SEQ ID NO: 1) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLN NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVV SLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNA GVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV CDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYD PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAV GLLLYCKARSTPVTLSKDQLSGINNIAFSN RSV F protein B1 full length sequence (SEQ ID NO: 2) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTIN TTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVS LSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAG VTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSN RVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTA SNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVF ASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLL YCKAKNTPVTLSKDQLSGINNIAFSK SEQ ID NO: 3 EKKIEAIEKKIEAIEKKIEA SEQ ID NO: 4 GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 5 GSGSG F8: RSV A2, wt ectodomain (SEQ ID NO: 13) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLN NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVV SLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNA GVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYD PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHHHHHHHH F11: RSV B1, wt ectodomain (SEQ ID NO: 14) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTIN TTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVS LSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAG VTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSN RVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTA SNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVF PSDEFDASISQVNEKINQSLAFIRRSDELLHHHHHHHH F47: RSV A2, linker stabilized, IZ(S) (SEQ ID NO: 15) KANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVEKKIEAIEK KIEAIEKKIEAGGIEGRHHHHHHHH F47-: RSV A2, linker ized, IZ(S) (SEQ ID NO: 16) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVEKKIEAIEK KIEAIEKKIEAGG F43: RSV B1, linker stabilized, IZ(S) (SEQ ID NO: 17) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLL GVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLIND MPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTT NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN TDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVEKKIEAIEK KIEAIEKKIEAGGIEGRHHHHHH F24: RSV B1, linker stabilized, fibritin (SEQ ID NO: 18) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLL ASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLIND QKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTT NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN TDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLA FIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGRHHHHHH A2_F24: RSV A2, linker stabilized, fibritin (SEQ ID NO: 19) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR F24-: RSV B1, linker stabilized, in (SEQ ID NO: 20) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLL GVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLIND MPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTT NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN TDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLA FIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P: A2, linker stabilized, in (SEQ ID NO: 21) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR F24-N67I+S215P: RSV B1, linker ized, fibritin (SEQ ID NO: 22) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQL LPIVNQQSCRIPNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPI TNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIK EGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDI FNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGV NTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRR SDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+E92D: RSV A2, linker stabilized, fibritin (SEQ ID NO: 23) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR F24- N67I+E92D RSV B1, linker stabilized, fibritin (SEQ ID NO: 24) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKEIKCNGTDTKVKLIKQELDKYKNAVTDLQLLMQNTPAANNQARGSGSGRSLGFLL GVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLIND MPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTT NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN KYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLA FIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 465Q RSV A2, linker stabilized, fibritin (SEQ ID NO: 25) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR F24- N67I+K465Q RSV B1, linker stabilized, fibritin (SEQ ID NO: 26) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQL LPIVNQQSCRIPNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPI LMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIK LTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDI FNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGV DTVSVGNTLYYVNKLEGQNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRR SDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S46G RSV A2, linker stabilized, fibritin (SEQ ID NO: 27) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR F24- N67I+S46G RSV B1, linker stabilized, fibritin (SEQ ID NO: 28) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFGALRTGWYTSVITIELS NIKEIKCNGTDTKVKLIKQELDKYKNAVTDLQLLMQNTPAANNQARGSGSGRSLGFLL GVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLIND MPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTT NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN TDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN SVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLA FIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 E92D+S215P: A2, linker stabilized, fibritin (SEQ ID NO: 29) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR F24-E92D+S215P: RSV B1, linker stabilized, in (SEQ ID NO: 30) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKETKCNGTDTKVKLIKQELDKYKNAVTDLQLLMQNTPAANNQARGSGSGRSLGFLL GVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRIPNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLIND QKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTT NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN TDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLA FIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K508E: A2, linker stabilized, in (SEQ ID NO: 31) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRESDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+E487I: A2, linker stabilized, fibritin (SEQ ID NO: 32) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDIFDASISQVNEKINQSLAF IRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+E487Q: A2, linker ized, fibritin (SEQ ID NO: 33) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ KQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDQFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+E487N: A2, linker stabilized, fibritin (SEQ ID NO: 34) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDNFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 215P+D486N: A2, linker stabilized, fibritin (SEQ ID NO: 35) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K465E: A2, linker stabilized, fibritin (SEQ ID NO: 36) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGESLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K465Q: A2, linker stabilized, fibritin (SEQ ID NO: 37) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS CNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG SGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+N426S: A2, linker stabilized, fibritin (SEQ ID NO: 38) KANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ KQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASSKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K421N: A2, linker stabilized, fibritin (SEQ ID NO: 39) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTNCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K209Q: A2, linker stabilized, fibritin (SEQ ID NO: 40) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNQQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K201Q: A2, linker stabilized, fibritin (SEQ ID NO: 41) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDQQ KQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN KYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+V185N: A2, linker ized, fibritin (SEQ ID NO: 42) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGNSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+G184N: A2, linker stabilized, in (SEQ ID NO: 43) KANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNNVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+N175P: A2, linker stabilized, fibritin (SEQ ID NO: 44) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTPKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+E92D: A2, linker stabilized, fibritin (SEQ ID NO: 45) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS CNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K80E: A2, linker stabilized, fibritin (SEQ ID NO: 46) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS CNGTDAKIKLIEQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+K77E: A2, linker stabilized, in (SEQ ID NO: 47) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIELIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24 N67I+S215P+S46G: A2, linker ized, fibritin (SEQ ID NO: 48) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR : RSV S46G A2, linker stabilized, fibritin (SEQ ID NO: 49) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24: RSV K465Q A2, linker stabilized, fibritin (SEQ ID NO: 50) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ KQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24: RSV N67I A2, linker stabilized, fibritin (SEQ ID NO: 51) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR A2_F24: RSV E92D A2, linker stabilized, in (SEQ ID NO: 52) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR RSV F protein CL57-v224 full length sequence (SEQ ID NO: 69) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLN NTKNNNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVV SLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNA GVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNVGKSTTNIMITTIIIVIIVILLLLIAV GLFLYCKARSTPVTLSKDQLSGINNIAFSN Ectodomain, RSV CL57-v224 (SEQ ID NO: 70) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLN NTKNNNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVV SVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNA GVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELL PreF, RSV A2, fibritin (SEQ ID NO: 71) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLN NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVV SLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNA GVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYD PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLL STFL PreF N67I S215P, RSV A2, fibritin (SEQ ID NO: 72) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S215P, RSV B1, fibritin (SEQ ID NO: 73) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS CNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTIN TTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVS LSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRIPNIETVIEFQQKNSRLLEINREFSVNAG VTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSN RVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTA SNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVF PSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL RSV N67I S215P, RSV CL57-v224, fibritin (SEQ ID NO: 74) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLN VTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVV SLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNA GVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVV QLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreFL N67I S215P, RSV B1, fibritin, Loop (SEQ ID NO: 22) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELS NIKEIKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNQARGSGSGRSLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQL QSCRIPNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPI TNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIK EGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDI FNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGV DTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRR AIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreFL N67I S215P, RSV CL57-v224, fibritin, Loop (SEQ ID NO: 75) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNQARGSGSGRSLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQL LPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPI TNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNT KEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDI FNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKG VDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR KSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL PreF N67I S215P E487Q, RSV A2, fibritin (SEQ ID NO: 76) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDQFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S215P K201N, RSV A2, fibritin (SEQ ID NO: 77) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDNQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG TYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S215P E92D, RSV A2, fibritin (SEQ ID NO:78) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS VLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S215P D486N, RSV A2, fibritin (SEQ ID NO: 79) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ DTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS Fwt N67I S215P, membrane-bound RSV F, A2, (SEQ ID NO: 80) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK NRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVG LLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P, membrane-bound RSV F, A2, (SEQ ID NO: 81) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN NIAFSN Fwt N67I S215P E92D, membrane-bound RSV F, A2, (SEQ ID NO: 82 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS VLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK NRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVG LLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P E92D, membrane-bound RSV F, A2, (SEQ ID NO: 83) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTDLQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN NIAFSN Fwt N67I S215P E487Q, membrane-bound RSV F, A2, (SEQ ID NO: 84) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDQFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAV GLLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P E487Q, membrane-bound RSV F, A2, (SEQ ID NO: 85) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDQFDASISQVNEKINQSLA FIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN NIAFSN Fwt N67I S215P D486N, membrane-bound RSV F, A2, (SEQ ID NO: 86) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN TLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSNEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVG LLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P D486N, membrane-bound RSV F, A2, (SEQ ID NO: 87) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLA FIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN NIAFSN Fwt N67I S215P S46G, membrane-bound RSV F, A2, (SEQ ID NO: 88) KANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP EFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVG LLLYCKARSTPVTLSKDQLSGINNIAFSN Fsl N67I S215P S46G, membrane-bound RSV F, A2, (SEQ ID NO: 89) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTT NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSN KGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGIN NIAFSN CR9501 heavy chain (SEQ ID NO: 53): QVQLVQSGPGLVKPSQTLALTCNVSGASINSDNYYWTWIRQRPGGGLEWIGHISYTGNT YYTPSLKSRLSMSLETSQSQFSLRLTSVTAADSAVYFCAACGAYVLISNCGWFDSWGQG TQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC CR9501 light chain (SEQ ID NO: 61): EIVMTQSPSSLSASIGDRVTITCQASQDISTYLNWYQQKPGQAPRLLIYGASNLETGVPSR FTGSGYGTDFSVTISSLQPEDIATYYCQQYQYLPYTFAPGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CR9502 heavy chain (SEQ ID NO: 57): EVQLLQSGAELKKPGASVKISCKTSGFTFSGHTIAWVRQAPGQGLEWMGWVSTNNGNT QGRVTMTMDTSTSTVYMELRSLTSDDTAVYFCAREWLVMGGFAFDHWGQGT LLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC CR9502 light chain (SEQ ID NO: 65): QSVLTQASSVSVAPGQTARITCGANNIGSQNVHWYQQKPGQAPVLVVYDDRDRPSGIP DRFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSRDQAVIFGGGTKLTVLGQPKAAPS VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTIAPTECS PreF N67I E161P S215P E487Q, RSV A2, fibritin (SEQ ID NO: 90) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLPGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP QFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I E161P S215P, RSV A2, fibritin (SEQ ID NO: 91) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLPGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ IDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S173P S215P, RSV A2, fibritin (SEQ ID NO: 92) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLPTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK NRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S182P S215P, RSV A2, fibritin (SEQ ID NO: 93) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LPNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS PreF N67I S215P D486C E487C, RSV A2, fibritin (SEQ ID NO: 94) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELS NIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNN AKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ DTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTK CTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSCCFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLS

Claims (24)

Claims

1. A recombinant pre-fusion respiratory syncytial virus (RSV) Fusion (F) polypeptide, comprising at least one epitope that is specific to the pre-fusion conformation F protein, 5 wherein the at least one epitope is recognized by a pre-fusion specific monoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO: 54, a heavy chain CDR2 region of SEQ ID NO: 55, a heavy chain CDR3 region of SEQ ID NO: 56 and a light chain CDR1 region of SEQ ID NO: 62, a light chain CDR2 region of SEQ ID NO: 63, and a light chain CDR3 region of SEQ ID NO: 64 and/or a pre-fusion specific 10 monoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO: 58, a heavy chain CDR2 region of SEQ ID NO: 59, a heavy chain CDR3 region of SEQ ID NO: 60 and a light chain CDR1 region of SEQ ID NO: 66, a light chain CDR2 region of SEQ ID NO: 67, and a light chain CDR3 region of SEQ ID NO: 68, wherein the polypeptide comprises an F1 domain and an F2 domain, and wherein the polypeptides 15 comprise at least one mutation, as compared to wild-type F1 and F2 domains, wherein the at least one mutation is selected from the group consisting of: (a) a mutation of the amino acid residue E on position 161 into P, Q or G (E161P, E161Q or E161G); (b) a mutation of the amino acid residue S on position 182 into P (S182P); 20 (c) a mutation of the amino acid residue S, T or N on position 173 into P (S173P); and (d) a mutation of the amino acid residue D on position 486 into C (D486C) in combination with a mutation of the amino acid residue D on position 489 into C 103 (D489C), and wherein the amino acid positions are given in reference to the sequence of RSV F protein from the A2 strain (SEQ ID NO: 1).

2. Pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide is trimeric. 5

3. Pre-fusion RSV F polypeptide according to claim 1 or 2, wherein the polypeptide further comprises a mutation of the amino acid residue on position 67 and/or a mutation of the amino acid residue on position 215. 10

4. Pre-fusion RSV F polypeptide according to claim 3, wherein the polypeptide comprises a mutation of the amino acid residue N or T on position 67 and/or a mutation of amino acid residue S on position 215.

5. Pre-fusion RSV F polypeptide according to any one of the preceding claims, wherein the 15 polypeptide comprises a linking sequence comprising from 1 to 10 amino acids, linking the F1 domain and F2 domain.

6. Pre-fusion RSV F polypeptide according to any one of the preceding claims, wherein the polypeptide comprises a truncated F1 domain. 20

7. Pre-fusion RSV F polypeptide according to claim 6, wherein the polypeptide comprises a heterologous trimerization domain linked to said truncated F1 domain.

8. Pre-fusion RSV F polypeptide according to claim 7, wherein the heterologous trimerization domain comprises the amino acid sequence

9.GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 4). 5 9. Pre-fusion RSV F polypeptide according to claim 8, wherein the trimerization domain is linked to amino acid residue 513 of the RSV F protein.

10. Pre-fusion RSV F polypeptide according to any one of the preceding claims, wherein the F1 domain and/or the F2 domain are from an RSV A strain. 10

11. Pre-fusion RSV F polypeptide according to any one of the preceding claims 1-10, wherein the F1 domain and/or the F2 domain are from an RSV B strain.

12. Pre-fusion RSV F polypeptide according to any one of the preceding claims, wherein the 15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 90 – SEQ ID NO: 94.

13. Nucleic acid molecule encoding a pre-fusion RSV F polypeptide according to any one of the preceding claims 1-12. 20

14. Nucleic acid molecule according to claim 13, wherein the nucleic acid molecule has been codon-optimized for expression in mammalian cells.

15. Vector comprising a nucleic acid molecule according to claim 13 or 14. 25

16. Composition comprising a pre-fusion RSV F polypeptide according to any one of claims 1-12, a nucleic acid molecule according to claim 13 or 14 and/or a vector according to claim 15. 5

17. A use of a pre-fusion RSV F polypeptide according to any one of claims 1-12, a nucleic acid molecule according to claim 13 or 14 and/or a vector according to claim 15 in the manufacture of a medicament for inducing an immune response against RSV F protein in a patient in need thereof. 10

18. A use of a pre-fusion RSV F polypeptide according to any one of claims 1-12, a nucleic acid molecule according to claim 13 or 14 and/or a vector according to claim 15 in the manufacture of a vaccine.

19. A use of a pre-fusion RSV F polypeptide according to any one of claims 1-12, a nucleic 15 acid molecule according to claim 13 or 14 and/or a vector according to claim 15 in the manufacture of a medicament for the prophylaxis and/or treatment of RSV infection in a patient in need thereof.

20. A pre-fusion RSV F polypeptide as claimed in any one of claims 1-12 substantially as 20 herein described and with reference to any example thereof.

21. A nucleic acid molecule as claimed in claim 13 or 14 substantially as herein described and with reference to any example thereof. 25

22. A vector as claimed in claim 15 substantially as herein described and with reference to any example thereof.

23. A composition as claimed in claim 16 substantially as herein described and with 5 reference to any example thereof.

24. A use as claimed in any one of claims 17-19 substantially as herein described and with reference to any example thereof