RU2811991C2 - Subunit vaccine for treating or preventing respiratory tract infection - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: immunogenic modified F protein of human metapneumovirus (hMPV) or a fragment thereof is described, stabilized in a pre-fusion conformation, containing a recombinant single-chain polypeptide that does not contain a fusion peptide (FP) and contains an F2 domain, a heterologous peptide linker and an F1 domain, with the said linker located between specified F2 and F1 domains and contains a cysteine residue that forms a disulfide bond with a non-natural cysteine residue present in the specified F1 domain. Also the following is presented: an immunogenic composition containing an immunogenic protein or a fragment thereof, additionally containing a pharmaceutically acceptable carrier and/or excipient, wherein the said composition can induce neutralizing antibodies that recognize the native hMPV F protein. Also the following is provided: a method of treating or preventing hMPV infection in a human subject, comprising administering to the human subject an immunogenic composition or vaccine containing an immunogenic protein or fragment thereof.

EFFECT: invention expands the arsenal of means for treating or preventing hMPV infection.

12 cl, 13 dwg, 5 tbl, 6 ex

Description

TECHNICAL FIELD

The present invention relates to modified metapneumovirus (hMPV) F proteins stabilized in a prefusion conformation. The present invention also relates to immunogenic compositions (vaccines) containing these proteins for the prevention and/or treatment of respiratory tract infections in human subjects.

BACKGROUND OF THE ART

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in young children (0–4 years), immunocompromised patients, and the elderly, which can be fatal in these patients (Schildgen et al. 2011. Clinical Microbiology Reviews 24 (4): 734-54). Despite significant efforts, there are currently no licensed vaccines or antiviral drugs to prevent or treat hMPV infection. Among several vaccination strategies that have been studied, the most promising is a subunit vaccine containing a viral protein, in particular the hMPV F protein (Melero & Mas. 2015. Virus Res. 209: 128-35).

hMPV is an enveloped, single-stranded RNA virus of the genus Pneumovirus of the family Paramyxoviridae. The hMPV genome consists of eight genes encoding nine proteins, including three surface glycoproteins F, G and SH. Protection against hMPV is provided primarily by neutralizing antibodies directed against the fusion (F) glycoprotein, which is highly conserved across genotypes and shares similarities with other paramyxoviruses (see van den Hoogen et al. 2004. Emerging Infectious Diseases 10(4): 658- 66; van den Hoogen et al. 2002. Virology 295(1): 119-32).

The paramyxoviral F protein is a type I integral membrane protein that spans the membrane once and contains at its N-terminus a signal peptide that directs the ectodomain to the extracellular membrane. At the C-terminus, a hydrophobic transfer stop domain (TM domain) anchors the protein to the membrane, leaving a short cytoplasmic tail (see Figure 1).

The native F protein is synthesized as an inactive precursor, designated F0, after cleavage of the signal peptide (Yin et al. 2006. Nature 439 (7072): 38-44; Yin et al. 2005. Proc. Nat. Acad. Sci. 102(26 ): 9288-93;Russell et al. 1994. Virology 199(1): 160-8). To become biologically active, F0 is processed by a host protease to form two chains called F1 and F2, which remain covalently linked by disulfide bonds (Schowalter et al. 2006. Journal of Virology 80(22): 10931-41; Biacchesi et al. 2006 Journal of Virology 80(12): 5798-806; Yun et al. 2015. Scientific Reports 5: 15584). The three F1-F2 heterodimers form the mature F protein, which is incorporated into the virion envelope in a metastable conformation before fusion (Battles et al. 2017. Nat. Commun. 8(1): 1528) and mediates fusion of the virion envelope and the host cell plasma membrane. During the fusion process, the F protein undergoes irreversible refolding from a labile conformation before fusion to a stable conformation after fusion (see Figure 2).

Neutralizing antibodies that specifically recognize the prefusion structure of the hMPV F protein have been detected in human sera (Wen et al. 20012. Nat. Struct. Mol. Biol. 19: 461-463; Ngwuta et al. 2015. Science Translational Medicine 7( 309): 309; Rossey et al. 2018. Trends in Microbiology 26(3): 209-19), indicating that the prefusion F protein may be a suitable vaccine candidate (Melero & Mas. 2015. Virus Res. 209: 128-35). One obvious disadvantage of the prefusion conformation of the F protein compared to the postfusion conformation is its instability. Previous attempts to obtain a stabilized F protein before fusion have used careful structural analysis and computer modeling. In particular, one group has described the design of a highly stable prefusion RSV F protein capable of mediating a protective response in rats (see Krarup et al. 2015. Nat. Commun. 6: 8143). Another group disclosed the design of stabilized prefusion forms of hMPV F protein that elicited neutralizing antibodies in mice immunized with these proteins (see WO 2016/103238).

Crystal structures of the F protein in prefusion and postfusion conformations have been determined for hMPV, RSV, and other paramyxoviruses (see, e.g., Battles et al. 2017. Nat. Commun. 8(1): 1528). Despite the overall structural similarity, the prefusion hMPV F protein has been shown to have unique structural features that confer significant functional and immunological differences between the F proteins from hMPV and RSV.

Despite significant progress in understanding the mechanism of action, structure and immunogenic properties of the hMPV F protein, there is no commercially available vaccine based on the F protein. Thus, it is an object of the present invention to provide novel modified candidate hMPV F proteins in a prefusion conformation for the development of a vaccine against respiratory tract infection for use in humans.

SUMMARY OF THE INVENTION

The present disclosure provides recombinant immunogenic human metapneumovirus (hMPV) F proteins and fragments thereof (referred to herein as hMPV F proteins) capable of eliciting neutralizing antibodies and protecting against hMPV infection. The native hMPV F protein coding sequence was modified to a stable conformation before fusion. Such modifications have been developed based on three-dimensional (3D) homology models included as part of the present invention. The present invention further includes methods for producing recombinant immunogenic proteins and methods for using immunogenic proteins to prevent and/or treat hMPV infection in humans.

According to one aspect of the present disclosure, a modified hMPV F protein or fragment thereof is provided, stabilized in a prefusion conformation, comprising a single chain polypeptide consisting of an F2 domain, a heterologous peptide linker, and an F1 domain lacking a fusion peptide (FP), wherein said linker is located between the F2 and F1 domains and contains one or more cysteine residues, each of which forms an unnatural disulfide bond with a cysteine residue present in the F1 domain.

In one embodiment, the single-chain F protein comprises an F2 domain and F1 domains joined such that the C terminus of F2 is proximal to the N terminus of F1. The protease cleavage site between F2 and F1 can be mutated to eliminate cleavage of the precursor protein. In some embodiments, the F1 domain may be a truncated F1 domain, for example, such that it lacks a fusion peptide (FP) spanning amino acid residues at positions 103-118 of the native hMPV F protein sequence SEQ ID NO: 1, corresponding to residues c 1 through 16 of the native F1 domain sequence of SEQ ID NO: 3. Thus, the F1 domain may comprise a fragment corresponding to residues 119-539 of SEQ ID NO: 1 or residues 17-437 of SEQ ID NO: 3. In some embodiments, single-stranded the polypeptide may contain an F1 domain fragment corresponding to residues 119-490 of SEQ ID NO: 1 or residues 17-338 of SEQ ID NO: 3, which does not contain a transmembrane (TM) anchor domain and a cytoplasmic tail at its C-terminus. In addition, the F1 and F2 domains can be connected by a heterologous peptide linker containing, for example, five residues containing at least one cysteine residue, preferably a linker having SEQ ID NO: 4.

In yet another embodiment, the single chain F protein of the present invention has a stable conformation before fusion. On the one hand, the prefusion conformation is stabilized by eliminating protease cleavage between the F1 and F2 domains and the absence of a free N terminus of F1. Another feature that provides stabilization is the presence of at least one additional (including unnatural) disulfide bond that anchors the HRA domain within the cavity formed by trimerization (see Figure 3). In one embodiment, a non-natural disulfide bond may be formed between a cysteine residue of a heterologous peptide linker inserted between F2 and F1 and a cysteine residue located in the F1 domain, preferably in its C-terminal region and located within the cavity. For example, a non-natural disulfide bond may be formed between a cysteine residue in the peptide linker and a non-natural cysteine residue present in the F1 domain at position 338 of the hMPV native F protein sequence SEQ ID NO: 1, corresponding to position 236 of the native F1 domain sequence SEQ ID NO: 3 In such embodiments, the F1 domain present in a single chain F protein (e.g., a truncated F1 domain lacking the fusion peptide of residues 1-16 of SEQ ID NO: 3) may contain a mutation, such as A236C, in the sequence of SEQ ID NO: 3. NO: 3 (corresponding to A338C in SEQ ID NO: 1). The cysteine residue in the peptide linker, for example, may be immediately adjacent to the F2 domain, for example, may be the first residue at the N-terminus of a heterologous linker, and adjacent to the C-terminus of the F2 domain. For example, a cysteine residue in the peptide linker may be present in the recombinant polypeptide at a position equivalent to residue 103 of SEQ ID NO: 1.

In a further embodiment, the single chain F protein may contain one or more additional modifications that compensate for the altered geometry of the trimer containing the single chain F protein. Preferably, said modification(s) are substitution(s) at positions corresponding to provisions 49, 51, 67, 80, 97, 137, 147, 159, 160, 161, 166, 177, 185, 258, 266, 294, 480 and/or 481 native hMPV F protein sequence SEQ ID NO: 1. In particular, the asparagine at position 97 may be replaced by glutamate (N97Q) or the alanine at position 185 may be replaced by proline (A185P). Thus, the recombinant polypeptide may comprise an F2 domain containing one or more substitutions at positions 31, 33, 49, 62 and/or 79 of SEQ ID NO: 2. The recombinant polypeptide may comprise an F1 domain (e.g., a truncated F1 domain in which missing residues 1-16 of SEQ ID NO: 3), containing one or more substitutions at positions 35, 45, 57, 58, 59, 64, 75, 83, 156, 164, 192, 378 and/or 379 of SEQ ID NO: 3.

In addition, some mutations may compensate for insufficient cavity filling. In particular, cavity-filling mutations can be selected from a list containing amino acid substitutions at positions 49, 67, 80, 137, 147, 159, 160, 161, 177 and 258 of the native hMPV F protein sequence SEQ ID NO: 1. According to one In an embodiment, the cavity-filling mutations include a T49M substitution, an I67L substitution, an I137W substitution, an A147V substitution, an A159V substitution, a T160F substitution, an A161M substitution, and/or an I177L substitution in SEQ ID NO: 1. Thus, in some embodiments, the recombinant polypeptide may comprise a domain F2 comprising a T31M or I49L substitution in SEQ ID NO: 2. In other embodiments, the recombinant polypeptide may comprise a (eg, truncated) F1 domain containing one or more substitutions in SEQ ID NO: 3 selected from I35W, A45V, A57V , T58F, A59M, I75L and/or F156I.

In another embodiment, the recombinant single chain F protein may contain one or more substitutions resulting in the formation of unnatural hydrogen bond(s) or salt bridge(s). For example, such substitutions include E80N and S266D in SEQ ID NO: 1. Thus, the recombinant polypeptide may contain an F2 domain containing the E62N substitution in SEQ ID NO: 2. In other embodiments, the recombinant polypeptide may contain a (e.g., truncated) domain F1 containing the replacement of S164D in SEQ ID NO: 3.

In some embodiments, the recombinant single chain F protein may contain additional cysteine substitution(s) to create additional stabilizing disulfide bond(s). For example, the E51C and K166C substitutions can form an unnatural disulfide bond between the cysteine residue at position 51 of the F2 domain β strand and the cysteine at position 166 of the α4 HRA element of the native hMPV F protein sequence SEQ ID NO: 1. This modification disrupts a possible salt bridge between E51 and K138 from the helix in HRA. The S266D mutation introduces an unnatural salt bridge at K138 to compensate for the loss of attachment for this helix. In addition, replacing vicinal residues I480 and L481 of SEQ ID NO: 1 with cysteine allows the introduction of three disulfide bonds among the three protomers to form a covalently linked trimeric protein. Thus, in one embodiment, the recombinant polypeptide may comprise an F2 domain containing the E33C substitution of SEQ ID NO: 2. In other embodiments, the recombinant polypeptide may comprise a (e.g., truncated) F1 domain containing substitution(s) K64C, S164D, I378C and/or L379C in SEQ ID NO: 3.

In another embodiment, the recombinant single chain F protein may contain modification(s) useful for expression of the soluble recombinant protein. For example, substitution of a glycine with a glutamic acid residue may be present at position 294 (G294E) of the native hMPV F protein sequence SEQ ID NO: 1, which may result in higher protein expression yield. Thus, in one embodiment, the recombinant polypeptide may comprise (e.g., a truncated) F1 domain containing the G192E substitution in SEQ ID NO: 3.

In some embodiments, the recombinant single-chain F protein may contain combinations of two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions and/or other modifications.

In some embodiments, the recombinant single-chain F protein may contain an auxiliary trimerization domain, a so-called foldon domain, for example, associated with the C-terminus of a subunit of the recombinant F protein, which enables the formation of a trimer of the protein. The Foldon domain may be derived from bacteriophage T4 fibritin. The recombinant hMPV F protein of the present invention can be produced as a mono- or heterotrimer.

In some embodiments, the recombinant hMPV F protein may comprise or consist of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence of any of SEQ ID NO: 5-9 or 24-28. The recombinant single chain F protein may comprise an F2 domain containing or consisting of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2. The recombinant single chain F protein may comprise an F1 domain containing or consisting of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the recombinant single chain F protein may comprise a truncated F1 domain comprising or consisting of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 17-437 or 17-388 of amino acid sequence SEQ ID NO: 3.

The recombinant hMPV proteins of the present invention are immunogenic and can induce neutralizing antibodies that recognize the native hMPV F protein. The present disclosure also includes immunogenic fragments of recombinant hMPV proteins and immunogenic proteins having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NO: 5-9 or 24-28.

The present disclosure also provides isolated nucleic acid molecules encoding modified hMPV F proteins, vectors containing the isolated nucleic acid molecules, host cells for recombinant expression of modified hMPV F proteins.

The present disclosure also provides immunogenic compositions or vaccines comprising recombinant hMPV F proteins or isolated DNA molecules encoding the hMPV F protein, or vectors of the present invention, further comprising a pharmaceutically acceptable carrier and/or excipient, used with or without an adjuvant. . In particular, the present disclosure provides immunogenic compositions or vaccines for stimulating an immune response in a subject, in particular an immune response that can neutralize hMPV viruses and protect against hMPV infections. The present disclosure also provides immunogenic compositions or vaccines containing additional antigens derived from hMPV, RSV or PIV3 (parainfluenza virus type 3). The immunogenic proteins, isolated DNA molecules, vectors and immunogenic compositions or vaccines disclosed herein are suitable for use as a medicine, in particular for the prophylactic and/or therapeutic treatment of viral respiratory tract infections and associated diseases, in particular infections and diseases caused by hMPV.

Methods for producing recombinant hMPV F proteins or isolated DNA molecules encoding hMPV F protein or immunogenic compositions (vaccines) are included in the present disclosure. Also included are methods of eliciting an immune response in a subject and methods of treating, inhibiting or preventing hMPV infections. According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by one or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains a glutamine residue replacing an asparagine residue at a position corresponding to position 97 of the native hMPV F protein sequence SEQ ID NO: 1 (N97Q). According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by one or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains a glycine residue replaced by a glutamic acid residue at a position corresponding to position 294 of the native hMPV F protein sequence SEQ ID NO: 1 (G294E). According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by one or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains one or more substitutions at positions corresponding to positions 49, 51, 67, 80, 137, 147, 159, 160, 161, 166, 177, 258, 266, 480 and/or 481 sequence of native hMPV F protein SEQ ID NO: 1.

According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by two or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains E51C and K166C substitutions relative to the sequence of the native hMPV F protein SEQ ID NO: 1, and wherein said replaced cysteine residues form a non-native disulfide bond.

According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by one or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains one or more substitutions selected from the group consisting of T49M, E80N, I137W, A147V, A159V, T160F, A161M, I67L, I177L, F258I, S266D, I480C and/or L481C relative to the native one hMPV F protein sequence SEQ ID NO: 1.

According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by three or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains at least substitutions T49M, A161M and I67L or I177L relative to the sequence of the native hMPV F protein SEQ ID NO: 1.

According to a further aspect of the present invention there is provided an immunogenic modified human metapneumovirus (hMPV) F protein or fragment thereof stabilized in a prefusion conformation by three or more amino acid substitutions relative to the sequence of the native hMPV F protein; wherein said modified F protein or fragment thereof contains one of the following combinations of substitutions relative to the sequence of the native hMPV F protein SEQ ID NO: 1:

N97Q, R102G and G294E;

N97Q, R102G, T160F, I177L and G294E;

N97Q, R102G, T49M, I67L, A161M, E80N, F258I and G294E;

N97Q, R102G, T49M, I67L, A161M, E51C, K166C, S266D, G294E, I480C and L481C; or

N97Q, R102G, T49M, A161M, I137W, A159V, A147V, I177L and G294E.

Unless otherwise indicated, all amino acid positions mentioned herein correspond to the amino acid positions of the native hMPV F protein sequence SEQ ID NO: 1. The corresponding positions of such mutations are in the F2 domain having SEQ ID NO: 2 and the F1 domain having SEQ ID NO: 3 can be obtained directly from them. The F2 domain having SEQ ID NO: 2 corresponds to residues 19 to 102 of SEQ ID NO: 1. The F1 domain having SEQ ID NO: 3 corresponds to residues 103 to 539 of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of the native hMPV F protein with the indicated domains and important motifs. F0: precursor protein; F1, F2: F1 and F2 domains; SP: signal peptide; FP: fusion peptide; HRA, HRB: heptad repeat domain A and B, TM: transmembrane domain; CYT: cytoplasmic tail; S-S: disulfide bond.

Figure 2 shows the structural changes in the prefusion and postfusion conformations of the native hMPV F protein. (A) Ribbon diagram of a prefusion F protein trimer in which the C termini of HRB are trimerized with the foldon domain and HRA is folded onto the head domain. (B) Ribbon diagram of an F protein trimer after fusion in which HRA forms a long, parallel three-helix bundle that, together with the displaced helices of HRB, forms a stable six-helix bundle.

Figure 3 shows the three-dimensional structure (ribbon diagram) of the modified hMPV F protein before fusion with the indicated mutations.

Figure 4 shows SE-HPLC analysis of recombinant hMPV F proteins. 1 - sF_A1_K-E294, 2 - sF_A1_K_L7, 3 - L7F_A1_4.2, 4 - L7F_A1_31, 5 - L7F_A1_23, 6 - L7F_A1_33.

Figure 5 shows ELISA data obtained using antibodies specific for recombinant hMPV F proteins pre-fusion or post-fusion: (A) sF_A1_K-E294; (B) sF_A1_K_L7; (C) L7F_A1_4.2; (D) L7F_A1_23; (E) L7F_A1_31; (F) L7F_A1_33; (G) L7F_A1_23.2. In all diagrams except (G), the solid line denotes signals obtained using various dilutions of antibody against the prefusion N113S MPE8 conformation, and the dashed line denotes signals obtained using various dilutions of antibody against the postfusion conformation MF1. In (G): the top line denotes signals obtained using an antibody against the prefusion conformation of MPE8 N113S, and the bottom line denotes signals obtained using an antibody against the postfusion conformation MF1.

Figure 6 shows serum IgG titers in mice immunized with recombinant F proteins in combination with various adjuvants. (A and B) mice immunized with 2 μg of sF_A1_K_L7; (C and D) Mice immunized with 2 μg of sF_A1_MFur.

Figure 7 shows reciprocal dilution titers of serum IgG in mice immunized with recombinant F proteins adjuvanted with Addavax™. The dotted line indicates the detection limit. (A) Titers of fold dilutions of IgG _2a . (B) Titers of fold dilutions of IgG1.

Figure 8 shows titers of fold dilutions of serum IgG in mice immunized with recombinant F proteins adjuvanted IC31 ^® . The dotted line indicates the detection limit. (A) Titers of fold dilutions of IgG _2a . (B) Titers of fold dilutions of IgG1.

Figure 9 shows neutralizing antibody titers (IC ₅₀ , fold dilution titers) in mouse sera that were raised against the sF_A1_K_L7 protein in combination with various adjuvants.

Figure 10 shows neutralizing antibody titers (IC ₅₀ , fold dilution titers) in mouse sera raised against recombinant F proteins adjuvanted with (A) Addavax™ or (B) IC31 ^® .

Figure 11 shows neutralizing antibody titers (IC ₅₀ , fold dilution titers) in mouse sera that were raised against various doses of recombinant F proteins. (A) mice immunized with 6 μg F protein, (B) mice immunized with 2 μg F protein, (C) mice immunized with 0.6 μg F protein, (D) mice immunized with 0.2 μg F-protein protein, and (E) mice immunized with 0.06 μg of F protein.

Figure 12 shows the viral RNA load in the lungs of mice immunized with 2 μg of recombinant F proteins adjuvanted with Addavax and then stimulated with wild-type hMPV (measured by quantitative RT-PCR). A and B represent two independent experiments.

Figure 13 shows protection in mice following immunization with recombinant F proteins adjuvanted with Addavax™ and subsequent challenge with wild-type hMPV (lung colonization assay). (A) mice immunized with 6 μg F protein, (B) mice immunized with 2 μg F protein, (C) mice immunized with 0.6 μg F protein, (D) mice immunized with 0.2 μg F-protein protein, and (E) mice immunized with 0.06 μg of F protein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Adjuvant

By "adjuvant" is meant any substance that is used to specifically or nonspecifically enhance an antigen-specific immune response, possibly through activation of antigen-presenting cells. Examples of adjuvants include oil emulsion (eg, Freund's complete or incomplete adjuvant), Seppic's Montanide incomplete adjuvant such as ISA51, squalene-based oil-in-water emulsion adjuvants such as MF59 ^® (Novartis AG) (Ott G . et al. 1995. Pharm Biotechnol 6: 277-96) or AddaVax™ (InvivoGen), monophosphoryl lipid A (MPL) (Cluff CW. 2010. Adv Exp Med Biol 667:111-23), aluminum salt adjuvant (alum ) (as described in WO 2013/083726), a polycationic polymer, in particular a polycationic peptide, in particular polyarginine, or a peptide containing at least two LysLeuLys motifs, in particular KLKLLLLLLKLK, an immunostimulatory oligodeoxynucleotide (ODN) containing unmethylated dinucleotides cytosine-guanine (CpG), for example CpG 1018 (Dynavax), in a defined base environment (for example, as described in WO 96/02555), or ODN based on inosine and cytidine (for example, as described in WO 01/93903), or a deoxynucleic acid containing deoxyinosine and/or deoxyuridine residues (as described in WO 01/93905 and WO 02/095027), in particular an oligo(dIdC) ₁₃ IC31 ^® (Valneva SE) adjuvant (as described in WO 04/ 084938 and Olafsdottir et al. 2009. Scand J Immunol. 69(3): 194-202), neuroactive compound, in particular human growth hormone (described in WO 01/24822), chemokine (eg defensins 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8 or cytokine (eg, interleukin-1β, -2, -6, -10, or -12; interferon-γ; tumor necrosis factor-α; or granulocyte-monocyte colony-stimulating factor), muramyl dipeptide (MDP) variants, non-toxic bacterial toxin variants, QS-21 (Antigenics Inc.), Quill A, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-glycero-3-(hydroxyphosphoryloxy)]ethylamide (MTP -PE) and others as described in Sarkar et al. 2019. Expert Rev Vaccine: 18(5): 505-521, as well as compositions such as adjuvant systems such as AF03, AS01, AS03 and AS04 (Giudice et al 2018. Seminars in Immunology 39: 14-21) Adjuvants that transduce immunological signals through the TLR3, TLR4, TLR7, TLR8 and TLR9 receptors stimulate immunity with a Th1 bias, while signaling through TLR2/TLR1 , TLR2/TLR6 and TLR5 stimulate immunity with a Th2 bias. For example, adjuvants such as CpG ODN, poly(IC) and MPL preferentially induce Th1 responses, alum is a strong inducer of Th2 responses, while MF59 ^® , Addavax™ and IC31 ^® can induce mixed Th1 and Th2 responses. The adjuvant may be administered with the antigen or may be administered separately, either by the same route as the antigen or by a different route than the antigen. A single adjuvant molecule may have both adjuvant and antigenic properties.

Amino acid substitutions

Amino acid substitution refers to the replacement of one amino acid in a polypeptide with another amino acid or no amino acid (i.e., deletion). As used herein, conservative substitutions are substitutions that do not change the basic structure and function of the protein, for example, the ability of the protein to induce an immune response when administered to a subject.

The following six groups are considered conservative replacements for each other:

1) alanine (A), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (G);

4) arginine (R), lysine (K);

5) leucine (L), isoleucine (I), methionine (M), valine (V); And

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

Non-conservative substitutions are substitutions that reduce the potency of the function of the modified hMPV protein, such as the ability to induce an immune response when administered to a subject.

Antibody

An antibody is a polypeptide or protein that specifically binds and recognizes an antigen, such as the hMPV F protein or an antigenic fragment of the MPV F protein. The term "antibody" is used herein in its broadest sense to cover a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the target antibody. antigen binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain antigen binding affinity. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments obtained either by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (see, for example, Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2 ^nd Ed., Springer Press, 2010).

The following antibodies were used in the present invention: Antibody MPE8 is a monoclonal antibody that specifically binds to an epitope that is present on the surface of the hMPV F protein in the prefusion conformation, but not in the postfusion conformation (see Corti et al. 2013. Nature, 501:439-443). The sequences of the variable regions of the heavy and light chains of the MPE8 antibody have been deposited in GenBank under accession numbers AGU13651.1 and AGU13652.1, respectively. Antibody MF1 recognizes the 6HB domain of the hMPV F protein after fusion, as described in Rodriguez, 2015 (J Virol Methods 224: 1-8). Antibody DS7, described in Williams et al., 2007 (J Virology 81(15): 8315-24), binds to both prefusion and postfusion conformations of the hMPV F protein.

Cavity-filling mutation (or replacement)

A cavity-filling mutation is an amino acid substitution that fills a cavity within the protein core of the hMPV F protein. Cavities are essentially voids within a folded protein in which no amino acids or amino acid side chains are present. In several embodiments, a cavity-filling amino acid substitution is introduced to fill a cavity in the core ectodomain of the hMPV F protein present in the prefusion conformation.

Foldon domain

A foldon domain is an amino acid sequence that naturally forms a trimeric structure and may also be referred to as a trimerization accessory domain. In some examples, a foldon domain may be included in the amino acid sequence of the disclosed recombinant protein such that the antigen will form a trimer. In one example, the foldon domain is a foldon domain derived from bacteriophage T4, comprising the amino acid sequence indicated, for example, GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 10). The foldon domain can be cleaved, for example, from a purified protein, for example, by including a thrombin cleavage site adjacent to the foldon domain.

Glycosylation site

A glycosylation site is an amino acid sequence on the surface of a polypeptide, such as a protein, that mediates the attachment of a glycan. The N-linked glycosylation site is a triplet sequence NX(S/T), in which N is asparagine, X is any residue other than proline, and (S/T) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan can also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or proteoglycan.

Heterologous

The term "heterologous" means coming from a different genetic source. An amino acid sequence that is heterologous to a protein or virus comes from a source other than the protein or virus in which it is present or expressed. In one specific non-limiting example, the heterologous peptide linker present in a recombinant polypeptide between two domains is a peptide sequence that is not naturally present in a wild-type polypeptide between the two domains, e.g., a peptide linker is an artificial sequence connecting two domains in a recombinant polypeptide.

Homologous

Homologous proteins have similar structure and function, for example, proteins from two or more species or viral strains that have similar structure and function in two or more species or viral strains. Homologous proteins have similar protein folding characteristics and can be considered structural homologues. Homologous proteins typically have a high degree of sequence conservation, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence conservation, and a high degree of sequence identity, such as at least 80%, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

hMPV F protein

The hMPV F (fusion) protein is an envelope glycoprotein that promotes fusion of the viral and cellular membranes. In nature, the hMPV F protein is synthesized as a single polypeptide precursor approximately 540 amino acids in length that includes an N-terminal signal peptide (approximately the first 18 residues) that directs localization to the endoplasmic reticulum, where the signal peptide is cleaved. The remaining polypeptide, designated F0, constitutes the F protein monomer (protomer), which is processed at the protease cleavage site between positions 102 and 103 in the native F protein sequence SEQ ID NO: 1, generating two disulfide-linked fragments, F1 and F2. Fragment F2 is derived from the N-terminal portion of the F precursor and includes approximately residues 19-102 of SEQ ID NO: 1. The larger of these fragments, F1, includes the C-terminal portion of the F precursor (approximately residues 103-539 of SEQ ID NO: 1). 1), including the extracellular/luminal region (residues 103-490), the transmembrane domain (residues 491-513) and the cytoplasmic domain (residues 514-539) at the C-terminus. The extracellular portion of the hMPV F protein is the F ectodomain, which includes an F2 domain (approximately positions 19-102 of the hMPV F protein) and an F1 ectodomain (approximately positions 103-490 of the hMPV F protein). The three F2-F1 protomers oligomerize into a trimer of the mature F protein, which adopts a metastable prefusion conformation that undergoes a conformational change to a postfusion conformation triggered upon contact with the target cell membrane. This conformational change exposes a hydrophobic sequence known as a fusion peptide (FP), located at the N-terminus of the F1 domain, which associates with the host cell membrane and stimulates fusion of the viral or infected cell membrane with the target cell membrane. The three ectodomains of hMPV F can form a protein complex of three hMPV F protomers. The present invention relates to a modified hMPV F protein or a fragment thereof, i.e. a recombinant polypeptide containing one or more unnatural amino acid mutations relative to the sequence of the wild type, native or naturally occurring hMPV F protein that stabilize the prefusion conformation.

hMPV F0 polypeptide

The F0 polypeptide is the hMPV F protein precursor remaining after cleavage of the signal peptide, which consists of an F2 domain and an F1 domain, including an extracellular domain, a transmembrane domain, and an F1 cytosolic tail. The native F0 polypeptide is processed at the protease cleavage site separating F1 and F2 (approximately between positions 102 and 103 of SEQ ID NO: 1) to form the F1 and F2 polypeptide fragments (domains).

F1 domain of hMPV

The hMPV F1 domain is part of the amino acid sequence of the hMPV F protein. As used herein, “Fl domain” refers to both native F1 sequences and F1 sequences incorporating modifications (eg, amino acid substitutions, insertions, or deletions). The native F1 domain (SEQ ID NO: 3) comprises approximately residues 103-539 of the native hMPV F protein and includes (N- to C-terminal) the extracellular/luminal region (residues 103-490 of SEQ ID NO: 1), transmembrane domain (residues 491-513 of SEQ ID NO: 1) and a cytosolic domain (residues 514-539 of SEQ ID NO: 1) at the C-terminus. Several embodiments include an F1 domain modified from a native F1 sequence, for example, an F1 domain that lacks a fusion peptide (eg, residues 103-118 of SEQ ID NO: 1). In some embodiments, the F1 domain is an F1 ectodomain, i.e. it lacks a transmembrane and cytosolic domain, for example, the F1 domain corresponds to residues 103 to 490 or 119 to 490 of SEQ ID NO: 1. In further embodiments, the F1 domain includes one or more amino acid substitutions that stabilize the recombinant single chain F protein (containing the F1 domain) in the prefusion conformation.

F2 domain of hMPV

The hMPV F2 domain is part of the amino acid sequence of the hMPV F protein. As used herein, “F2 domain” refers to both native F2 polypeptides and F2 polypeptides incorporating modifications (e.g., amino acid substitutions) relative to the native sequence, e.g., modifications designed to stabilize the recombinant F protein (including the modified F2 polypeptide) in prefusion conformation of the hMPV F protein. The native F2 domain (SEQ ID NO: 2) includes approximately residues 19-102 of SEQ ID NO: 1. In the native mature hMPV F protein, the F2 domain is linked to the F1 domain by two disulfide bonds.

Fusion peptide (FP) of hMPV

The hMPV fusion peptide is part of the amino acid sequence of the hMPV F protein. The fusion peptide may be residues 103-118 of SEQ ID NO: 1, that is, N-terminal residues 1 to 16 of the F1 domain of SEQ ID NO: 3.

Prefusion conformation of hMPV F protein

The prefusion conformation of the hMPV F protein is the structural conformation adopted by the hMPV F protein prior to the initiation of a fusion event that results in the hMPV F protein transitioning to a postfusion conformation and subsequent processing into the mature hMPV F protein in the secretory system. The three-dimensional structure of an exemplary hMPV F protein in a prefusion conformation is discussed herein and, for example, in WO 2016/103238. The prefusion conformation of the hMPV F protein is similar in general structure to the prefusion conformation of the F protein of other paramyxoviruses (such as RSV), although with some significant differences. In several embodiments, the recombinant hMPV F protein, stabilized in the prefusion conformation, specifically binds to an antibody (such as antibody MPE8, see WO 2016/103238) specific for the trimeric form of the hMPV F protein in the prefusion conformation, but not in the postfusion conformation.

hMPV single-chain F protein

The hMPV single chain F protein of the present invention is the ectodomain of a recombinant hMPV F protein (also referred to herein as a “single chain polypeptide”) that is expressed as a single polypeptide chain comprising a (modified) hMPV Fl domain and a (modified) F2 domain hMPV. Single-chain hMPV F protein can typically be trimerized to form a trimeric hMPV F protein subunit, preferably fused to a trimerization auxiliary domain, such as foldon. In some embodiments, the recombinant hMPV single chain F polypeptide does not include a protease cleavage site between the F1 domain and the F2 domain and is not cleaved into separate F1 domain and F2 domain polypeptides when produced in cells. In one embodiment, the F1 domain and the F2 domain are linked to a heterologous peptide linker to produce a single-chain construct.

Immune response

An immune response is the response of an immune system cell, such as a B cell, T cell or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (“antigen-specific response”). In one embodiment, the immune response is a T cell response, such as a CD4 ⁺ response or a CD8 ⁺ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies. “Immune response priming” refers to pre-treatment of a subject with an adjuvant to enhance a target immune response to a subsequently administered immunogenic agent. “Immune response enhancement” refers to the co-administration of an adjuvant and an immunogenic agent, wherein said adjuvant enhances the target immune response to an immunogenic agent relative to administration immunogenic agent to a subject in the absence of an adjuvant.

Immunogen

An immunogen is a compound, composition or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected into the animal or that are absorbed into the animal's body. The immunogen reacts with products of specific humoral or cellular immunity, including products induced by heterologous antigens, such as the disclosed recombinant hMPV F proteins. An immunogen may include one or more epitopes. In some embodiments, the immunogen may be a recombinant hMPV F protein or an immunogenic fragment thereof, a protein nanoparticle, or a virus-like particle comprising a recombinant hMPV F protein or an immunogenic fragment thereof, or a nucleic acid or vector encoding a recombinant hMPV F protein or an immunogenic fragment thereof. a fragment that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen to a subject may result in protective immunity and/or proactive immunity against the pathogen of interest.

Immunogenic composition

An immunogenic composition is a composition containing an immunogen that induces a measurable CTL response against an antigen or induces a measurable B cell response (eg, antibody production) against an antigen included in the immunogen or encoded by a nucleic acid molecule included in the immunogen. In one example, an immunogenic composition is a composition that includes a disclosed recombinant hMPV F protein or an immunogenic fragment thereof that induces a measurable CTL response against the hMPV virus or induces a measurable B cell response (e.g., antibody production) against the hMPV F protein when administered subject. The immunogenic composition may include isolated nucleic acids encoding an immunogenic protein, which can be used to express the immunogenic protein and thereby induce an immune response against the protein. Thus, in another example, an immunogenic composition is a composition that includes a nucleic acid molecule encoding a disclosed recombinant hMPV F protein or an immunogenic fragment thereof that induces a measurable CTL response against the hMPV virus or induces a measurable B cell response (such as antibody production ) against hMPV F polypeptide when administered to a subject. For in vivo use, the immunogenic composition will typically comprise an immunogenic polypeptide or a nucleic acid molecule encoding an immunogenic polypeptide in a pharmaceutically acceptable carrier, and may also include other agents such as an adjuvant. The ability of any particular polypeptide, such as the disclosed recombinant hMPV F protein or the nucleic acid encoding this protein, to induce a CTL or B cell response can be readily tested using assays recognized in the art.

Dedicated

An "isolated" biological component has been substantially separated or purified from other biological components, such as other biological components in which the component is naturally present, such as other chromosomal and extrachromosomal DNA, RNA and proteins. Proteins, peptides and nucleic acids that have been “isolated” include proteins purified using standard purification methods. The term also includes proteins or peptides produced by recombinant expression in a host cell, as well as chemically synthesized proteins, peptides and nucleic acid molecules. The isolated does not require absolute purity and may include protein, peptide or nucleic acid molecules that are at least 50% isolated, for example, at least 75%, 80%, 90%, 95%, 98%, 99% isolated, or even 99.9%. Modified hMPV F proteins disclosed herein that are stabilized in a prefusion conformation are isolated from hMPV F proteins in a postfusion conformation, e.g., at least 80% isolated, at least 90% isolated, 95% isolated, 98%, 99%, or even 99.9% of the hMPV F proteins are in the postfusion conformation.

Linker

A linker is a bifunctional molecule that can be used to join two molecules into one continuous molecule, for example, to join two domains into one polypeptide. Preferably the linker is a peptide linker. The linker can be of any suitable length, for example, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 5 or less amino acid residues. The linker may contain or consist of, for example, alanine, serine, glycine, cysteine and/or valine residues. Preferably, the linker may contain at least one cysteine residue.

Native (or natural) protein, sequence or disulfide bond

A native or natural (used interchangeably herein) polypeptide, sequence, residue, or disulfide bond is one that has not been modified, for example, by selective mutation, to target the antigenicity of the antigen to target epitope or introduce a disulfide bond into the protein that does not occur in the native protein. Native or natural proteins, residues or sequences are also referred to as wild-type proteins, residues or sequences. A non-native or unnatural disulfide bond is a disulfide bond that is not present in a native protein, for example, a disulfide bond that is formed in a protein due to the introduction of one or more cysteine residues into the protein by genetic engineering. Likewise, a non-natural cysteine residue in a domain is a cysteine residue that is not present at that position in the wild-type, native, or natural sequence.

Neutralizing antibody

A neutralizing antibody reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent. In some examples, the infectious agent is a virus. In some examples, an antibody specific for hMPV F protein neutralizes the infectious titer of hMPV. In some embodiments, the neutralizing antibody binds to and inhibits the function of related antigens, such as antigens that have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% antigenic surface identity to the antigen. For an antigen from a pathogen, such as a virus, an antibody may bind to and inhibit the function of an antigen from more than one class and/or subclass of the pathogen. For example, for hMPV, an antibody may bind to and inhibit the function of an antigen, such as the hMPV F protein, from more than one group. In one embodiment, broad spectrum hMPV neutralizing antibodies are distinguished from other hMPV antibodies in that they neutralize a high percentage of many types of hMPV in the bloodstream.

Pharmaceutically acceptable carrier

Pharmaceutically acceptable carriers are used to prepare immunogenic hMPV F protein for clinical administration. In Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens. In general, the nature of the carrier will depend on the particular route of administration employed. For example, parenteral formulations typically contain liquids for injection, which include pharmaceutically and physiologically acceptable liquids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as the carrier. For solid compositions (eg, in the form of a powder, pill, tablet or capsule), conventional non-toxic solid carriers may include, for example, mannitol, lactose, starch or pharmaceutical grade magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions for administration may contain trace amounts of non-toxic excipients such as wetting or emulsifying agents, preservatives, pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In specific embodiments, the carrier suitable for administration to a subject may be sterile and/or suspended, or may otherwise be contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing a targeted immune response against MPV. It may also be accompanied by drugs for its medicinal use. The unit dosage form may be, for example, in a sealed vial that contains sterile contents, or in a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration, or as a solid or controlled-release dosage.

Polypeptide

A polypeptide is any chain of amino acids, regardless of length or post-translational modification (eg, glycosylation or phosphorylation). "Polypeptide" refers to amino acid polymers, including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as those in which one or more amino acid residues is a non-naturally occurring amino acid, such as an artificial chemical mimetic of the corresponding naturally occurring amino acid. "Residue" refers to an amino acid or an amino acid mimetic included in a polypeptide through an amide bond or an amide bond mimetic. The polypeptide has an amino-terminal (N-terminal) end and a carboxy-terminal (C-terminal) end. "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues. In many cases, one or more polypeptides may fold into a specific three-dimensional structure comprising surface-exposed amino acid residues and non-surface-exposed amino acid residues. In some cases, a protein may include several polypeptides that fold together into a functional unit. For example, the mature hMPV F protein at the cell surface contains three F2/F1 heterodimers that trimerize into a multimeric protein. “Surface exposed amino acid residues” are those amino acids that have some degree of exposure on the surface of the protein, for example, such that they can come into contact with a solvent when the protein is in solution. In contrast, non-surface exposed amino acids are those amino acid residues that are not exposed to the surface of the protein such that they do not come into contact with the solution when the protein is in solution. In some examples, amino acid residues not exposed to the surface are part of the core of the protein.

Recombinant polypeptide or protein

A recombinant polypeptide (or protein) is a polypeptide that has a non-naturally occurring sequence or a sequence obtained by artificial combination of two otherwise separated sequence segments. This artificial combination can be achieved by chemical synthesis or, more commonly, by artificial manipulation of selected segments of amino acid residues, such as through genetic engineering techniques. In several embodiments, the recombinant single-stranded polypeptide is encoded by a heterologous (eg, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid may be introduced, for example, into an expression vector having signals capable of expressing a protein encoded by the introduced nucleic acid, or the nucleic acid may be integrated into the chromosome of a host cell.

Sequence identity Sequence identity is often measured as a percentage of identity: the higher the percentage, the more identical the two sequences are. Homologs, orthologs, or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

Methods for aligning sequences for comparison are well known in the art. Various alignment programs and algorithms are described in: Smith & Waterman (Adv. Appl. Math. 2:482, 1981); Needleman & Wunsch (Mol. Biol. 48:443, 1970); Pearson & Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988); Higgins & Sharp (Gene, 73:237-44, 1988); Higgins & Sharp (CABIOS 5:151-3, 1989); Corpet et al. (Nuc. Acids Res. 16:10881-90, 1988); Huang et al. (Computer Appls in the Biosciences 8:155-65, 1992); Pearson et al. (Meth. Mol. Bio. 24:307-31, 1994) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), which provides a detailed discussion of methods for sequence alignment and homology calculations. After alignment, the number of matches is determined by counting the number of positions at which an identical nucleotide or amino acid residue is present in both sequences. Percent sequence identity is determined by dividing the number of matches by either the length of the sequence specified in the identified sequence or by a specified length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence specified in the identified sequence) and then multiplying the resulting value by 100. Percent identity is preferred. sequences is determined along the entire length of the sequence. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13 and 75.14 are rounded to 75.1, while 75.15, 75.16, 75.17, 75.18 and 75.19 are rounded to 75. 2. The length value will always be an integer.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990. Mol. Biol. 215:403) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) , and on the Internet for use in conjunction with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX. A description of how to determine sequence identity using this program is available on the NCBI Internet website. The BLAST and BLAST 2.0 algorithms are also described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1977). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) by default uses word length (W) - 11, alignment (B) - 50, expectation (E) - 10, M=5, N=-4 and comparison of both strands. The BLASTP program (for amino acid sequences) uses a default word length (W) of 3, expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff. 1992. Proc. Natl. Acad. Sci. USA 89:10915- 10919).

Homologues and variants of the polypeptide are typically characterized by being present at least about 75%, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity calculated over at least 50, 100, 150, 250, 500 amino acid residues of the reference sequence, the entire length of the reference sequence, or the entire length of the alignment to the reference amino acid sequence of interest. Proteins with even greater similarity to reference sequences will exhibit increasing percentages of identity when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. To compare nucleic acid sequences, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, subsequence coordinates are indicated if necessary, and the parameters of the sequence comparison program algorithm are specified. The program's default settings are used.

One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the Feng & Doolittle progressive alignment method (Mol. Evol. 35: 351-360, 1987). The method used is similar to the method described by Higgins & Sharp (CABIOS 5:151-153, 1989). With PILEUP, a reference sequence is compared to other test sequences to determine percentage sequence identity using the following parameters: default skip weight (3.00), default skip length weight (0.10), and weighted end skips. PILEUP can be obtained from a GCG sequence analysis software package, for example version 7.0 (Devereaux et al. 1984. Nuc. Acids Res. 12: 387-395).

As used herein, reference to “at least 80% identity” refers to “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93” %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity” to the specified reference sequence, e.g. at least 50, 100, 150, 250, 500 amino acid residues of the reference sequence or the full length of the sequence. As used herein, reference to “at least 90% identity” refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95” %, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity" to the specified reference sequence, for example, at least 50, 100, 150, 250, 500 amino acids residues of the reference sequence or the full length of the sequence.

Therapeutically effective amount

A therapeutically effective amount is an amount of an agent, such as a disclosed immunogen or immunogenic composition, that is sufficient to prevent, treat (including prophylaxis), reduce and/or alleviate the symptoms and/or underlying causes of a disorder or disease, e.g., prevent, inhibit and/or or treatment of hMPV infection. In some embodiments, the therapeutically effective amount is sufficient to reduce or eliminate a symptom of a disease, such as an hMPV infection. For example, it may be the amount necessary to inhibit or prevent viral replication or to measurably change the external symptoms of a viral infection. In general, this amount will be sufficient to measurably inhibit viral replication or infectivity. In one example, the target response is to inhibit, reduce, or prevent hMPV infection. In order for the method to be effective, the infection does not have to be completely eliminated, reduced or prevented. For example, administration of a therapeutically effective amount of agent may reduce infection (measured by cell infection or number or percentage of infected subjects) by a desired amount, e.g., at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% (elimination or prevention of detectable hMPV infection) compared to suitable control.

It is understood that multiple administrations of the immunogenic composition may be required to obtain a protective immune response against the pathogen. Thus, a therapeutically effective amount includes a fractional dose that, in combination with previous or subsequent administrations, promotes the achievement of a protective immune response. For example, a therapeutically effective amount of the agent can be administered in a single dose or in multiple doses, for example, daily, during a course of treatment (such as treatment by priming/vaccine stimulation). However, the therapeutically effective amount may depend on the subject being treated, the severity and type of condition being treated, and the route of administration. The unit dosage form of the agent may be packaged in a therapeutic amount or multiples of a therapeutic amount, for example, in a vial (eg, with a pierceable cap) or in a syringe containing sterile components.

Vaccine

A vaccine is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response to an antigen of a pathogen, such as a viral pathogen, or to a cellular component associated with a pathological condition. The vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell, or one or more cellular components. In one specific non-limiting example, the vaccine reduces the severity of symptoms associated with hMPV infection and/or reduces the viral load compared to a control. In another non-limiting example, the vaccine reduces hMPV infection compared to a control.

Homology modeling

In one aspect, the present disclosure provides a novel modified hMPV F protein stabilized in a prefusion conformation. The present disclosure also provides a method for producing stabilized prefusion F proteins based on crystal structures and homology modeling. A number of structures of available hMPV fusion proteins and homologous fusion proteins were used to analyze the structural basis of the stabilizing modification(s). These included models of the crystal structure of the hMPV F protein ectodomain before fusion (PDB:5WB0) and the hMPV F protein ectodomain after fusion (PDB:5L1X), models of the crystal structure of the RSV F protein ectodomain before fusion (PDB:4DAB) and the F-ectodomain postfusion RSV protein (PDB:3RRR). A homology model of the so-called trimerization auxiliary domain was modeled using the foldon domain and based on the structural data in PDB:2IBL, 1OX3 and 1AVY.

The native mature F protein of hMPV consists of two polypeptides, F2 and F1, covalently linked by two disulfide bonds. The maturation process involves a single cleavage of the F0 precursor by a trypsin-like protease, resulting in the formation of a free N-terminal F1 domain of the FP fusion peptide, which interacts with the target cell membrane and triggers conformational changes. After cleavage, the C-terminal part of F1 moves into the hydrophobic region of the internal trimeric cavity. This may increase the stability of the prefusion metastable state until a trigger event initiates refolding into the postfusion conformer (see Figure 2). In the prefusion conformation, the HRA-containing region is curved and associated with the head domain, while in the postfusion conformation it is part of a long overhanging helix. The transition from the prefusion conformation to the postfusion conformation involves the refolding of the heptad repeat A (HRA) sequences of the F1 subunit into a single long α-helix and the insertion of the fusion peptide (FP), located at the N terminus of the HRA at its end, into the cell membrane. This refolding stimulates the assembly of HRA and HRB sequences into a stable six-helix bundle that drives membrane fusion.

Single chain F protein

Single-chain F protein modeling was performed to obtain a stabilized prefusion conformation while maintaining maximum structural similarity to the native hMPV F protein (see Figure 3). Deletion of the cleavage site between F1 and F2 was proposed to stabilize the conformer prior to fusion. Also, the elimination of one cleavage step is an advantage for the method of producing a recombinant protein. The trypsin-like RQSR motif spans positions 99 to 102 of the native hMPV F protein sequence SEQ ID NO: 1, and cleavage occurs immediately after the second arginine at position 102 (R102). Elimination of cleavage can be achieved by at least one mutation in the cleavage site, preferably an arginine substitution at position 102. In particular, R102 can be replaced with glycine or other suitable amino acid residue. In particular, according to one embodiment of the present invention, the trypsin cleavage site is removed by replacing the arginine at position 102 with glycine (R102G).

To achieve similarity of the three-dimensional structure of the single-chain F ectodomain to the native hMPV F protein, a loop was constructed between the F1 domain and the F2 domain before fusion. In one embodiment, the loop is constructed by insertion of a heterologous peptide linker. In preferred embodiments, the size and composition of the linker are optimized for stability, antibody binding properties, and antigen yield. In one embodiment, the linker length can be from 2 to 10 amino acid residues, preferably from 2 to 5 residues, more preferably from 3 to 5 residues, even more preferably from 4 to 5 residues, most preferably 5 residues. In another embodiment, the linker may be inserted between amino acid residues at positions 95 to 102, preferably at positions 102, most preferably at the glycine residue at position 102 of SEQ ID NO: 1.

In some embodiments, the linker may consist of one or more cysteine, glycine, alanine, phenylalanine, valine, and/or serine residues. The linker may contain one, two or three alanine residues, one or two valine residues and one or two glycine residues. For example, alanine may be at position 1, 2, 3, 4 and/or 5 of the linker; glycine may be at position 2, 3 and/or 4 of the linker; and valine may preferably be at position 3 and/or 5 of the linker. Some non-limiting examples of the linker, along with additional modifications F2 and F1, are presented in Table 1.

In a preferred embodiment, the linker contains at least one (eg, one) cysteine residue. The cysteine may be at any position of the linker, preferably at position 1 or 3, which corresponds to positions 103 and 105 of SEQ ID NO: 1. More preferably, the cysteine is at position 1 of the linker (i.e., at the N-terminus of the linker, preferably adjacent F2 domain), which corresponds to position 103 of the native hMPV F protein sequence SEQ ID NO: 1. Even more preferably, the linker is CGAGA, CGAGV, CGAAV, AGCGA, CAAAV, CAAFV or CGAGA. In a most preferred embodiment, the linker is CGAGA (SEQ ID NO: 4).

Table 1a. Examples of variants of a single-stranded linker connecting the F2 and F1 domains. The insertion/deletion (indel) operation causes deletion of the fusion peptide and results in a net shortening of the ectodomain sequence. Cysteines, marked with appropriate numbers such as C(1) or C(2), form disulfide bonds between the F1 and F2 domains. Cysteines marked with a prime ('C) are located on the adjacent protomer.

In some embodiments, the prefusion conformation of a single-chain F protein can be covalently stabilized by introducing at least one unnatural intra- or interprotomer disulfide bond. For example, a non-natural disulfide bond may be introduced between a cysteine residue of a heterologous peptide linker located between the F2 and F1 domains and a cysteine residue of the F1 domain. The first cysteine residue may be located at any position of the specified linker, for example, at position 1, 2, 3 or 4, preferably at position 1, which corresponds to position 103 of SEQ ID NO: 1. Alternatively, the first cysteine residue can be introduced into the F2 domain at position 96 or 101 or 102 of SEQ ID NO: 1. In a most preferred embodiment, the cysteine at position 103 forms an unnatural disulfide bond with the alanine-substituting cysteine at position 338 of the native F hMPV sequence SEQ ID NO: 1. This The S–S bond may stabilize the prefusion conformation of the single-chain F protein by anchoring the loop between F2 and F1 within the hydrophobic trimeric cavity. This loop fixation mimics the positioning effect of internalized split F1 N termini in the native hMPV protein. In some embodiments, the hMPV single-chain F protein may contain additional cysteine substitution(s) that may introduce non-native interprotomer disulfide bonds, for example, to stabilize the protein trimer by covalently linking it.

In yet another embodiment, the hMPV single chain F protein lacks amino acid residues 1 to 16 at the N terminus of the F1 domain of SEQ ID NO: 3, comprising all or part of the FP fusion peptide sequence. Preferably, the hMPV single-chain F protein lacks amino acid residues at positions 103-118 of the native hMPV F protein sequence SEQ ID NO: 1. The FP deletion further stabilizes the prefusion conformation of the hMPV single-chain F protein.

In some embodiments, the hMPV single-chain F protein may contain one or more additional modifications. On the one hand, additional modification may compensate for the altered geometry of the hMPV single-chain F protein. On the other hand, additional modification may further stabilize the conformation prior to fusion. Additional modifications may include one or more amino acid substitutions, insertions and/or deletions. Among modifications, conservative substitutions may, but are not necessarily, preferred. The following groups of substitutions are considered conservative:

1) alanine (A), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (G);

4) arginine (R), lysine (K);

5) leucine (L), isoleucine (I), methionine (M), valine (V); And

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

In one embodiment, an additional modification that stabilizes the single-chain hMPV F protein is the replacement of an asparagine residue with a glutamine residue at position 97 (N97Q) of the native hMPV F protein sequence SEQ ID NO: 1.

In another embodiment, an additional modification of the single-stranded F ectodomain may contain one or more cavity-filling substitutions, including, but not limited to, substitutions at positions 49, 67, 137, 159, 147, 160, 161, and/or 177 relative to the native F sequence -hMPV protein SEQ ID NO: 1. In particular, the cavity-filling substitution may be selected from, but is not limited to, the T49M substitution, the I67L substitution, the I137W substitution, the A147V substitution, the A159V substitution, the T160F substitution, the A161M substitution, or the I177L substitution. In addition, combinations of two or more cavity-filling replacements are possible. In one particular embodiment, the combination comprises substitutions T160F and I177L. In another specific embodiment, the combination comprises substitutions T49M, I67L, and A161M. In yet another specific embodiment, the combination comprises substitutions T49M, A161M, I137W, A147V, A159V, and I177L.

Strengthening α3 HRA by cavity filling. In native and cleaved F protein, the N-terminal part of F1 carries an HRA-containing domain, a long extended helix in the thermodynamically more stable postfusion F protein, but stacked with several individual small helices, even bearing a beta hairpin element, in the prefusion conformation ( "tensioned spring") Additional contacts of this element may help stabilize the protein in the prefusion conformation. To fill the cavity beneath the α3 HRA, two small residues were replaced by a space-filling aliphatic methionine residue at positions T49M and A161M to produce a complementary pair that packs together and enhance the aliphatic anchorage of this surface-located helix. Battles et al., 2017 reported mutations at position 161 for the prefusion F ectodomain of hMPV, resulting in either unexpressed F protein subunits (A161F) or subunits that react poorly with the MPE8 antibody (A161L) (see Battles et al. 2017 Nat Commun 8(1): 1528.

Covalent attachment of α4 HRA via disulfide bond. The α4 HRA extends to the end of the F protein and is located at the C terminus of the beta hairpin element after the α3 HRA in the prefusion structure. All of these elements are involved in conversion to a long alpha-helical element in the postfusion conformation, which involves movement from the head domain to the host cell membrane during viral fusion. In this case, a disulfide bridge is introduced between the α4 helical element of HRA (K166C) and the long beta chain provided by the F2 moiety (E51C). This change modifies two charged residues that participate in the polar network, including contacts with the α3 HRA. An additional modification, S266D, was introduced to partially restore the disruption in the salt bridge network caused by this disulfide bond-forming mutation.

Strengthening α2-α3 HRA by tryptophan administration. The α2-α3 HRA forms a curved substructure on the hMPV F protein before fusion (while being part of a long helix in the postfusion conformation). Strengthening this bend may prevent conversion to the postfusion conformation. For this bend, similar folded substructures can be observed, in which 1137 hMPV F protein presents tryptophan in a similar position and provides a more densely packed substructure, for example in the crystal structure of the N-terminal part of the split heparin-bound protein C inhibitor (PDB:3DY0 , W271 circuit A). Based on homology modeling and molecular modeling, two additional mutations A147V and A159V were introduced to provide filling of excess space in this substructure to force the tryptophan side chain to adopt the orientation observed in the protein C inhibitor structure, allowing further stabilization of the tryptophan side chain amide with a polar contact with S149 (asparagine in protein C inhibitor). In addition, positions similar to A147 and A159 provide residues with space-filling side chains.

In some embodiments, the hMPV single-chain F protein may contain one or more additional stabilizing substitutions, such as substitutions that result in the formation of unnatural hydrogen bond(s), variant core packing, or salt bridge(s). In particular, the modified hMPV single-chain F protein may contain E80N, F258I and/or G294E substitutions. The E80N substitution may establish an interprotomer H-bond with D224' (the prime indicates an adjacent protomer) and reduce the repulsion with D209. On the other hand, the E80N substitution enhances recombinant expression of hMPV single-chain F protein. Another modification that helps increase the yield of the recombinant protein is the G294E substitution.

In some embodiments, replacing the vicinal residues I480 and L481 with cysteine residues allows for the introduction of three disulfide bonds among the three protomers in the form of a covalent ring. It is assumed that the covalently bound trimer is more stable than the foldon trimerized particle. For a functional ring to form, all three disulfide bonds or, in the case of multiple rings, at least one disulfide bond between each protomer must be formed. The distance from the ring to the foldon domain is short, and the foldon attachment position is optimized for a more rigid geometry.

In a preferred embodiment, the substitution combinations are:

N97Q, R102G and G294E (L7F A123) (for example, those present in SEQ ID NO: 5)

N97Q, R102G, T160F, I177L and G294E (sF_A1_K_L7) (eg, those present in SEQ ID NO: 6);

N97Q, R102G, T49M, I67L, A161M, E80N, F258I and G294E (L7F_A1_31) (for example, those present in SEQ ID NO: 7);

N97Q, R102G, T49M, I67L, A161M, E51C, K166C, S266D, G294E, I480C and L481C (L7F_A1_33) (for example, those present in SEQ ID NO: 8) and

N97Q, R102G, T49M, A161M, I137W, A159V, A147V I177L and G294E (L7F A14.2) (for example, those present in SEQ ID NO: 9).

Protein trimer

In some embodiments, the modified single-chain hMPV F proteins of the present invention differ from the native hMPV F protein in that they do not possess a transmembrane domain and a cytoplasmic tail. However, in some embodiments, the modified hMPV single-chain F proteins can form mono- or heterotrimers. To form a trimer, an auxiliary trimerization domain, the so-called "foldon", can be inserted into the C-terminal part of the F ectodomain. The addition of an auxiliary trimerization domain, which maintains a soluble state, to the C-terminus of the subunit ectodomain supports the formation of a stable trimeric and soluble trimer of the protein. In one embodiment, the foldon domain may be derived from bacteriophage T4 fibritin and comprises the sequence SEQ ID NO: 10.

In another embodiment, the fibritin foldon can be modified by the insertion of one or more N-glycosylation sites (NxT/S motif, where "x" is any amino acid residue other than proline), which can help hide the non-specific epitope(s) of hMPV. The following are some non-limiting examples of modified foldon domain sequences:

Alternatively, the foldon domain may contain structural elements from the GCN4 leucine zipper (Harbury et al. 1993. Science 262:1401) or self-assembled nanoparticle monomers allowing attachments around the C3 axis (eg, ferritin and lumazine synthase).

In another embodiment, a foldon domain is attached to the C-terminus of the F protein, replacing its transmembrane and cytosolic domains. The glycine residue at the N-terminus of the foldon can be attached to the F1 domain directly or through a peptide linker of varying length, which can include at least one protease site. Longer linkers allow the movement of the foldon domain to be decoupled, but are less effective at keeping the HRB (stem) region helices in position. The helices can move with transverse displacement and bending of the auxiliary trimerization domains at an angle off the axis of the main c3 axis of the particle. Shorter linkers provide a more rigid attachment of the foldon domain with a stronger locking effect on the three helices of the stem domain.

In particular, the foldon domain can be attached by an alanine residue inserted after S482 of the native hMPV F protein sequence SEQ ID NO: 1. This allows S482 to be retained as the C-terminal cap of the helix and reproduces the local geometry of the foldon contact surface. Such a geometry can be achieved by foldon attachment via a short linker, for example a short linker called "VSL" or "VSA", consisting of the sequence ILSA (SEQ ID NO: 34) and CCS A (SEQ ID NO: 35), respectively . In this linker, alanine (A483) is in a similar position to the alanine at position n-2 relative to tyrosine in the PDBTAVY crystal structure (corresponding to position 2 in SEQ ID NO: 10), and shows similar contacts with the tyrosine side chain in the structure models. In addition, a short linker "VSL" or "VSA" can be used in combination with other mutations, such as amino acid substitutions in close proximity to the linker. For example, the combination of a “VSA” linker with substitutions C480 and C481 allows three protomers to be covalently linked by forming a disulfide ring across the three protomers. In this geometry, the cysteine residues of the disulfide ring are held in spatial proximity and hence the formation of fully closed rings is supported, increasing the overall stability of the protomer trimer. An example of a less stringent foldon addition retains residues 483-485 of SEQ ID NO: 1 (ILSSAE or CCSSAE with a disulfide ring). Some examples of modified foldon linkers that form more than one cysteine ring are shown below:

Other non-limiting examples of short linkers are: GG, SG, GS, GGG, GGA, GGS, SGG, SSG, SGS, SGA, GGA, SSA and SGGS. Such linkers can be used in combination with cleavage sites introduced, for example, by substitution A496. The cleavage site is preferably a thrombin cleavage site, a TEV (tobacco etch virus protease) cleavage site, or an Xa (factor Xa) cleavage site disclosed in Table 2.

In some embodiments, to facilitate purification of the recombinant protein, the single chain polypeptide may contain any purification tag sequences known in the art. Examples of polypeptides that facilitate purification include, but are not limited to, His tag, myc tag, S-peptide tag, MBP tag, GST tag, FLAG tag, thioredoxin tag, GFP tag, BCCP tag, calmodulin tag , streptavidin tag, HSV epitope tag, V5 epitope tag and CBP tag. The proteins of the present invention preferably contain His and/or streptavidin tags having the sequences SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

Non-limiting examples of combinations that can be used are shown in Table 3, which can produce a parallel bundle of three helices with two disulfide rings. Trimerization can occur with a region of the sequence containing residues 480-495, but can be facilitated by the presence of a foldon domain. The presence of cysteine rings ensures the formation of disulfide bonds that form a covalent bond between the three protomers. The function of the trimerization helper domain is then unnecessary and the packaging helper can be cleaved off, with the advantage that the immunogenic side effects of the heterologous sequence (and eg non-hMPV) can be avoided.

In some embodiments, the recombinant hMPV F protein may comprise or consist of an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any of SEQ ID NO: 5-9 or 24-28, for example, wherein the specified percentage of sequence identity is determined over the entire length of the reference sequence. The recombinant hMPV single chain F protein may comprise an F2 domain containing or consisting of an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2. Recombinant single chain F The α-protein may comprise an F1 domain containing or consisting of an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 3, preferably with respect to at least residues 17-437 or 17-388 from SEQ ID NO: 3.

Coding nucleic acids and vectors

The present application provides isolated nucleic acid molecules encoding recombinant hMPV proteins according to the present invention. The nucleic acid may encode, for example, a polypeptide containing, for example, a) a (modified) F1 domain of the hMPV F protein; b) (modified) F2 domain of hMPV F protein; c) a heterologous peptide linker located between the F1 and F2 domains; d) trimerization auxiliary domain; and optionally e) a purification tag, wherein said F1 and F2 domains are covalently linked by at least one non-natural disulfide bond introduced between a cysteine in the linker and a cysteine in the F1 domain. Nucleic acids encoding proteins of the present invention may contain or consist of the sequences of SEQ ID NOs 19-23. The present application also includes isolated nucleic acid molecules encoding proteins having at least 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequence of any of SEQ ID NOs 5-9 or 24-28. The present application also includes isolated nucleic acid molecules having at least 85%, 90%, 95%, 98% or 99% sequence identity with the sequence of SEQ ID NOs 19-23, for example, wherein said percentage sequence identity is determined over the entire length of the reference sequences.

The present application also provides vectors containing isolated nucleic acid molecules for the expression of recombinant proteins according to the present invention. The present invention also provides expression systems designed to facilitate the expression and provision of isolated polypeptides. The present application also provides host cells for the expression of recombinant hMPV F proteins according to the present invention. The host cell may be a prokaryote. The prokaryote may be, for example, E. coli. The host cell may be a eukaryotic cell.

Immunogenic compositions and formulations

The recombinant single chain hMPV F proteins of the present invention are immunogenic and can induce neutralizing antibodies that recognize the native hMPV F protein. The present disclosure also includes immunogenic fragments of recombinant hMPV proteins and immunogenic proteins having at least 85% sequence identity with proteins having SEQ ID NOs 5-9 or 24-28.

The present disclosure also provides immunogenic compositions or vaccines comprising recombinant hMPV F proteins or isolated DNA molecules encoding the hMPV F protein, or vectors of the present invention, containing a suitable carrier and/or excipient or stabilizers known in the art (See generally Remington, 2005. The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins). An immunogenic composition is any composition of material that elicits an immune response in a mammalian host when the immunogenic composition is administered by injection or otherwise. The immune response can be humoral, cellular, or both. A booster effect refers to the enhancement of the immune response to an immunogenic composition upon subsequent exposure of a mammalian host to the same immunogenic composition. The humoral response results in the production of antibodies by the mammalian host upon exposure to the immunogenic composition. Immunogenic compositions or vaccines may additionally contain an adjuvant. The adjuvant may be selected depending on the route of administration and may include mineral oil adjuvants such as Freund's complete and incomplete adjuvant, Seppic's Montanide incomplete adjuvant such as ISA, oil-in-water emulsion adjuvants such as such as the Ribi adjuvant system, oil-in-water emulsion adjuvants such as MF59 ^® (Novartis AG) or Addavax™ (InvivoGen) (Ott G. et al. 1995. Pharm Biotechnol 6: 277-96), monophosphoryl lipid A (MPL) (Chuff CW. 2010. Adv Exp Med Biol 667:111-23), aluminum salt (alum) adjuvant (e.g. as described in WO 2013/083726), adjuvant formulation from Syntex containing muramyl and peptide (MDP), a polycationic polymer, in particular a polycationic peptide, in particular polyarginine or a peptide containing at least two LysLeuLys motifs, in particular KLKLLLLLKLK, an immunostimulatory oligodeoxynucleotide (ODN) containing unmethylated cytosine-guanine dinucleotides (CpG), for example, CpG 1018 (Dynavax), in a defined base environment (for example, as described in WO 96/02555), or ODN based on inosine and cytidine (for example, as described in WO 01/93903), or deoxynucleic acid containing deoxyinosine residues and/or deoxyuridine (as described in WO 01/93905 and WO 02/095027), in particular oligo(dIdC) ₁₃ (as described in WO 01/93903 and WO 01/93905), IC31 ^® (Valneva SE), as described in WO 04/084938 and Olafsdottir et al. 2009 (Scand JImmunol. 69(3): 194-202), a neuroactive compound, in particular human growth hormone (described in WO 01/24822) and others described in Sarkar I. et al. 2019 (Expert Rev Vaccine: 18(5): 505-521), or combinations thereof such as AF03, AS01, AS03 and AS04 described in Giudice GD et al. 2018 (Seminars in Immunology 39: 14-21). Some combinations correspond to those described for example in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857, WO 02/095027 and WO 03/047602. In one preferred embodiment, the adjuvant is aluminum hydroxide or an aluminum salt that induces a strong antibody response and a Th2-biased immune response. In another preferred embodiment, the adjuvant is an adjuvant or composition of adjuvants that induce mixed Th1/Th2 responses, such as ^MF59® or Addavax™, MPL/alum and ^IC31® .

The present disclosure also provides pharmaceutical compositions comprising the recombinant hMPV F proteins of the present invention, further comprising a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition may further contain pharmaceutically acceptable carriers and/or excipients. Pharmaceutically acceptable carriers and/or excipients may include buffers, stabilizers, diluents, preservatives and solubilizers (see Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975).

Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations administered. The carriers, excipients or stabilizers may additionally contain buffers. Examples of excipients include, but are not limited to, carbohydrates (such as monosaccharide and disaccharide), sugars (such as sucrose, mannitol and sorbitol), phosphate, citrate, antioxidants (such as ascorbic acid and methionine), preservatives (such as phenol, butanol, benzanol; alkylparabens, catechol, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, resorcinol, cyclohexanol, 3-pentanol, benzalkonium chloride, benzethonium chloride and m-cresol), low molecular weight polypeptides, proteins (such as serum albumin or immunoglobulins), hydrophilic polymers, amino acids , chelating agents (such as EDTA), salt-forming counterions, metal complexes (such as Zn-protein complexes) and nonionic surfactants (such as TWEEN™ and polyethylene glycol). The immunogenic compositions of the present invention elicit an immune response in a mammalian host, including a human. The immune response can be either a cell-dependent response, an antibody-dependent response, or both. These immunogenic compositions can be used as vaccines and can provide a protective response against hMPV infection.

The present disclosure further provides immunogenic compositions or vaccines containing one or more additional antigens derived from at least one other infectious virus, in particular a virus that causes respiratory tract infection such as hMPV, RSV (respiratory syncytial virus), PIV3 (parainfluenza virus type 3), influenza virus or coronavirus (for example, SARS-CoV, SARS-CoV-2, MERS or similar). Preferably, the additional antigen is RSV F protein, PIV3 F protein, influenza virus hemagglutinin or coronavirus S protein.

The immunogenic recombinant proteins, isolated DNA or RNA molecules, vectors and immunogenic compositions or vaccines disclosed herein are suitable for use as a medicinal product, in particular for the prophylactic and/or therapeutic treatment of viral respiratory tract infections and associated diseases, in particular , infections and diseases caused by hMPV. Methods for producing recombinant hMPV F proteins or isolated nucleic acid molecules (DNA or RNA) encoding the hMPV F protein or immunogenic compositions (vaccines) are included in the present disclosure. Also included are methods of eliciting an immune response in a subject and methods of treating, inhibiting or preventing respiratory tract infections, particularly those caused by hMPV. The present invention will now be described by way of example only with reference to the following non-limiting embodiments.

EXAMPLES

EXAMPLE 1: Design of Modified F Proteins

Structural models

The design of mutated hMPV F proteins in a stabilized prefusion conformation was performed based on homology models obtained from the available crystal structures of the RSV prefusion F proteins (PDB: 4JHW and 4MMV, and 4MMU, 4MMS), PIV-5 prefusion F proteins (PDB: 5GIP), F hMPV before fusion (PDB:5WB0) and F hMPV after fusion (PDB:5L1X), representing models of different transition phases of the fusion process. The Foldon domain was adapted from the PDB:2IBL model. Model construction and structural analysis were performed using the open source version of the PyMol structure editor software package (Schrodinger LLC, https://github.com/schrodinger/pymol-open-source). The models were refined using the NAMD modeling package (Phillips et al. 2005. J Comput Chem. 26(16): 1781-802) and the charmm36 force field (McKerell et al. 1998. J Phys Chem B. 102(18): 3586-3616) or Gromacs (Berendsen et al. 1995. Al Phys. Comm. 91:43-56; Hess et al. 2008, J. Chem Theory Comput. 4, 435-447; www.gromacs.org)/ OPLS-AA (Jorgensen WL, Yale University). Candidate models were typically refined in a protocol applied after in vacuo relaxation at NVT, NPT sequence simulations for a total of 13 ns using three cycles of symmetric annealing (adapted from Anishkin et al. 2010. Proteins. 78(4): 932 -949) followed by free sampling and energy minimization.

EXAMPLE 2: Production of recombinant F proteins

Strains

The native hMPV F protein can be selected from any hMPV strain and any serotype represented by SEQ ID NOs 1, 13-18 or variants thereof. In certain exemplary embodiments, the hMPV F protein is derived from strain NL/1/00, serotype A1, represented by SEQ ID NO: 1, and strain CAN97-83, serotype A2, represented by SEQ ID NO: 14.

Expression vectors

Plasmid pVVS 1371 used for cloning contains:

- HS4 insulator sequence from the chicken β-globin locus,

- two CMV promoters,

- two chimeric introns located in the 3' direction from the CMV promoters, consisting of a 5-donor site from the first intron of the human β-globin gene and a branch point and 3' acceptor sites from the intron of the immunoglobulin heavy chain variable region gene. The sequences of the donor and acceptor sites, as well as the branch point site, were adapted to match the splicing consensus sequences. The intron is located in the 5' direction of the cDNA insert to prevent the use of possible hidden 5' donor splice sites in the cDNA sequence,

- bovine growth hormone polyadenylation signal sequence (bGH A),

- neomycin phosphotransferase gene from Tn5, regulated by the enhancer and early promoter region of SV40,

- the HSV TK polyadenylation signal from the herpes virus thymidine kinase gene is located in the 3' direction from the neomycin phosphotransferase gene,

- a kanamycin resistance gene regulated by a bacterial promoter, and

- origin of replication pUC.

The wild-type F protein coding sequence was isolated from hMPV strain NL/1/00, subline A1, and codon optimized for expression in CHO cells. The coding sequences of wild-type and modified F proteins were cloned into plasmid pVVS1371 for transient or stable expression of the protein in CHO cells.

In general terms, coding sequences were cloned between the chimeric intron and the bGH A polyadenylation site of vector pVVS1371 using SalI and PacI restriction sites. The vector and synthesized coding sequence (synthesis was performed by GeneArt) were digested with SalI and PacI before purification on an agarose gel. The fragments were ligated with T4 DNA ligase, and the ligation product was used to transform Max efficiency DH5α competent cells. Selected clones were tested to identify constructed mutations using sequence analysis.

Expression in CHO cells

Protein expression is based on transient transfection of CHO cells using the MaxCyte ^® STX Scalable Transfection System device according to the supplier's experimental guidelines. In general, prior to electroporation, CHO cells are pelleted, suspended in ^MaxCyte® electroporation buffer and mixed with the appropriate expression plasmid DNA. The cell-DNA mixture is transferred to a cassette processing unit and loaded into the MaxCyte ^® STX Scalable Transfection System. Cells are electroporated using the CHO protocol, preloaded into the device, immediately transferred to culture flasks and incubated for 30-40 minutes at 37°C with 8% CO ₂ . After a recovery period, cells are resuspended at high density in EX-CELL ACF CHO medium (Sigma Aldrich). Cell cultivation after electroporation is carried out at 37°C with 8% CO ₂ and orbital shaking.

Product kinetics consist of lowering the culture temperature to 32°C and feeding transfected cells daily using a fed-batch medium designed for transient protein expression in CHO cells (CHO CD EfficientFeed™ A (ThermoFischer Scientific) supplemented with yeast extract, glucose and glutaMax). After approximately 7-14 days of culture, cell viability is checked and conditioned media is collected after cell clearing, corresponding to two cycles of centrifugation at maximum speed for 10 minutes. The purified product is filtered through a sterile 0.22 µm membrane and stored at -80°C before protein purification.

Detection of proteins using intracellular immunostaining

On day 7 posttransfection, cells are washed once in phosphate-buffered saline (PBS) and fixed for 10 minutes in 4% paraformaldehyde (PFA) at room temperature. Fixed cells are permeabilized in BD Perm for 15 minutes at room temperature and incubated with primary antibody diluted in BD Perm for 1 hour at 4°C. Finally, a secondary antibody coupled to the fluorescent marker was added for 1 hour at 4°C and stored in PBS at 4°C until analysis by flow cytometry (MacsQuant Analyzer, Miltenyi Biotec). The primary antibody used was the MPE8 N113S antibody (PRO-2015-026-01), which specifically recognizes the prefusion conformation of the hMPV F protein, or the DS7 IgG1 antibody (PRO-2016-003), which recognizes the hMPV F protein both before fusion and and after the merger. The fluorescent FITC-conjugated secondary antibody was goat anti-mouse Ig+IgM (JIR 115-096-068).

Protein purification

The frozen supernatant is brought to room temperature and dialyzed using a standard grade Spectra/Por ^® 1-7 CR (HOMM: 3.5 kDa) regenerated cellulose dialysis membrane (Spectrum) against PBS. It is then equilibrated with 50 mM Na ₂ HPO ₄ buffer at pH 8.0, 300 mM NaCl, and protein purification is performed using immobilized metal ion affinity chromatography (IMAC) followed by gel filtration chromatography.

For IMAC, agarose resin containing Ni ²⁺ (His GraviTrap) is packaged into chromatography columns by the manufacturer (GE Healthcare). The resin is washed with two volumes of deionized water and equilibrated with three volumes of equilibration and wash buffer (20 mM sodium phosphate, pH 7.4, with 0.5 M sodium chloride and 20 mM imidazole) as specified by the manufacturer. After loading the sample, the column is washed with 10 ml of wash buffer. The His-tagged protein is eluted from the column using 3-10 column volumes of elution buffer as specified by the manufacturer (50 mM sodium phosphate, pH 8.0, with 0.5 M sodium chloride and 500 mM imidazole). The eluate is then filtered on a 0.22 µm filter and dialyzed twice in Slide-A-lyzer™ dialysis cassettes against storage buffer (50 mM Na ₂ HPO ₄ , 300 mM NaCl, 5 mM EDTA, pH 8.0) before aliquoting and storing at -20°C.

Analysis of the purity, size and aggregation of recombinant proteins is performed using size exclusion chromatography (SE-HPLC), and SDS-PAGE with SE-HPLC (Shimadzu) is performed on a SUPERDEX200 column (GE Healthcare). For SDS-PAGE, proteins are diluted in sample buffer (0.08 M Tris-HCl pH 8.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue), boil for five minutes in the presence of beta -mercaptoethanol (or DTT) and perform electrophoretic separation on 4-12% bis-Tris-glycine-polyacrylamide Criterion XT (BioRad) gels (SDS-PAGE). Gels are stained in a solution of Coomassie blue (Instant blue, Sigma Aldrich). Excess dye was removed with water and bands were visualized using an Imager 600 (Amersham). Figure 4 shows SE-HPLC analysis of purified recombinant F proteins. Approximate yields of recombinant F proteins are shown in Table 4.

EXAMPLE 3: Conformation of recombinant hMPV F proteins

Determination of conformational profile using sandwich ELISA

Moderate binding plates (Greiner) were coated with human DS7 IgG1 capture antibody (Williams et al., 2007) at 200 ng/well and incubated overnight at 4°C. Plates are saturated for 2 hours at 37°C using PBS + 0.05% Tween 20 and 5% skim milk powder with stirring (saturation buffer). The wells are drained of liquid and the plates are incubated for 1 hour at 37°C with 2.5 ng/well of purified proteins of interest diluted in saturation buffer. After washing, 5-fold serial dilution in saturation buffer with mouse antibody MPE8 N113S (Corti et al., 2013) directed against the prefusion hMPV F protein or mouse antibody MF1 (Melero, personal communication) directed against the hMPV F protein after fusion, incubate for 1 hour at 37°C. Immune complexes are then detected by incubation for one hour at 37°C with horseradish peroxidase (HRP)-conjugated secondary species-specific goat anti-mouse IgG α-Ig antibody (Covalab, catalog no. lab0252) followed by 50 μl of peroxidase substrate (TMB) , Sigma). The colorimetric reaction is stopped by adding 3 N. H ₂ SO ₄ and the absorbance of each well was measured at 490 nm using a spectrophotometer (MultiSkan). Figure 5 shows the results of an ELISA performed on recombinant F proteins with antibodies specific for prefusion conformation or postfusion conformation. All candidates tested exhibit an abundant prefusion profile, with the exception of one candidate L7F A14.2, which exists in both a prefusion and a postfusion conformation. Recombinant protein L7F A123.3 differs from L7F A123 in only one amino acid residue in the heterologous linker, that is, it contains a valine at position 5 of the linker (CGAGV), which corresponds to the valine located at position 118 of SEQ ID NO: 1.

EXAMPLE 4: Immunogenicity Study

Immunogenicity in mice

Groups of five to ten BALB/c mice are immunized subcutaneously three times at three-week intervals (eg, days 0, 14, or 21, and 28 or 42) with recombinant F proteins (used in various experiments in amounts ranging from 0.06 μg to 6.0 μg per mouse) with or without various adjuvant, in particular, alum, alum +MPL, IC31 ^® , Addavax™ (InvivoGen). Serums are collected by drawing blood from the retroorbital sinus. One to four weeks after the last vaccination, blood is drawn and serum is prepared. Assessment of the Th ₁ /Th ₂ immune response is performed by determining the IgG ₁ /IgG _2a subtypes in sera using an indirect ELISA, as described below.

IgG subclass ELISA

Recombinant F protein is diluted in carbonate/bicarbonate buffer at pH 9.6, and 50 ng of protein per well is added to a 96-well high-binding plate (50 μl/well, Greiner). The plates are incubated overnight at 4°C. Wells are saturated for 30 minutes at room temperature with 150 μl of PBS with 0.05% Tween 20 and 5% skim milk powder (saturation buffer). The wells are drained of liquid and the plates are incubated for 1 hour at room temperature with 50 μl/well of sera from immunized mice at various dilutions (5-fold serial dilution) in saturation buffer. After washing three times with PBS and 0.05% Tween 20, immune complexes are detected by incubating for one hour at room temperature with 50 μl of horseradish peroxidase-conjugated secondary antibody specific for mouse IgG ₁ or IgG _2a , followed by 50 μl of peroxidase substrate. (TMB, Sigma). The colorimetric reaction is stopped by adding orthophosphoric acid, and the absorbance of each well is measured at 490 nm using a spectrophotometer (MultiSkan).

Figure 6 shows the results of the IgG ELISA obtained by testing sera from mice immunized with 2 μg per mouse of recombinant protein sF_A1_K_L7 (SEQ ID NO: 6) or sF_A1_MFur (SEQ ID NO: 53), administered in combination with various adjuvants. Induction of high IgG ₁ /IgG _2a titers was demonstrated for both F proteins, regardless of the adjuvant used.

Figure 7 shows the results for IgG ₁ and IgG _2a titers that were measured in mice immunized with one of the recombinant F proteins (2 μg) with Addavax™ adjuvant mixed at 1:1 (v/v).

Figure 8 also shows serum IgG ₁ and IgG _2a titers measured in mice immunized with one of the recombinant F proteins (2 μg) adjuvanted with IC31 ^® . Despite some differences, the data clearly show that all vaccine candidates are highly immunogenic and capable of inducing IgG ₁ /IgG _2a antibodies.

EXAMPLE 5: Induction of Neutralizing Antibodies

Neutralization Analysis

In general terms, the plaque neutralization test (PRNT) is used to determine the titer of serum/antibody from an immunized subject required to reduce hMPV plaque burden by 50% (PRNT50) compared to control serum/antibody. PRNT50 is performed using cell monolayers that can be infected with hMPV. Subject sera are diluted and incubated with live hMPV virus. Plaques formed on cell monolayers are counted and compared with the number of plaques formed by the virus in the absence of serum or control antibody. A threshold of neutralizing antibodies at a serum dilution of 1:10 in PRNT50 is generally considered evidence of protection (Hombach et. al. 2005. Vaccine 23: 5205-5211).

PRNT protocol

LLC-MK2 cells are seeded into a 24-well plate, 1.2×10 ⁵ cells per well, under 1 ml of EMEM buffer (Lonza), 2 mM L-Gln (Ozyme), 5% FBS and 1% NeAA (Ozyme), and incubated for 24 hours at 37°C and 5% CO ₂ . hMPV Al virus (Valneva bank MVB 1611-009) is diluted to 50 PFU/62.5 μl (per well) or 800 PFU/ml. Equal volumes of virus and serum dilutions (2 x 62.5 μl) were pooled and incubated at 37°C with 5% CO ₂ for approximately 1 hour. After washing the plate with PBS, 125 μl of the virus/serum mixture is added to each well, and the plate is incubated for 2 hours at 37°C with shaking (100 rpm). 225 μl/well of overlay solution (EMEM, 2 mM Gln, 0.75% methylcellulose) is then added and the plate is incubated for 5 days at 37°C with 5% CO ₂ . To fix the cells, add 225 μl/well of 4% PFA/PBS, incubate for 10 minutes at room temperature and then wash three times with PBS. To permeabilize the cells, add 0.5 ml of PBS, 0.5% Tween, 0.1% BSA to each well and incubate for 30 minutes at 4°C. The primary antibody (human DS7-PRO-2016-003) (0.91 μg/ml) is diluted in PBS to a final concentration of 5 μl/ml, 150 μl of the primary antibody dilution per well is added and incubated for 45 minutes at 37°C with shaking. After removing the primary antibody and washing the plate three times with blocking buffer, 150 μl/well of diluted anti-human HRP-conjugated secondary antibody (UP 783493) is added and the plate is incubated for a further 45 minutes at 37°C with shaking. The secondary antibody is then removed and the plate is washed three times with blocking buffer. For staining, add 150 µl of DAB 1x substrate per well. After 1 hour of incubation at room temperature, cells are counted manually under a microscope.

Figure 9 shows the neutralization assay results obtained with sera collected on day 57 from mice immunized with recombinant sF_A1_K_L7 protein (2 μg) with various adjuvants. Typically, neutralizing antibodies against the F protein tested are present in mouse sera regardless of the adjuvant used.

In experiments in which all candidate proteins were tested simultaneously, IC ₅₀ values were highest for candidate L7F_A1_23 with Addavax™ adjuvant and for candidate L7F_A1_23 with ^IC31® adjuvant (see Figure 10 and Table 5). The lowest IC ₅₀ values were calculated for candidate L7F_A1_31 regardless of the adjuvant used.

Figure 11 shows a dose response study performed to evaluate the immunogenicity of recombinant F proteins when inoculated into mice in combination with Addavax™. In contrast to the placebo group (data not shown), neutralizing antibodies were detected in the serum of mice immunized with 0.06-6.0 μg of recombinant F protein. No significant differences in IC ₅₀ between constructs were observed.

EXAMPLE 6: Protection in mice

Immunostaining of viral plaques (foci)

An assay to quantify hMPV foci was developed based on methods published in Williams et al, 2005. J Virology 79(17): 10944-51; Williams et al. 2007. J Virology 81(15):8315-24; and Cox et al, 2012. J Virology 86(22):12148–60. In general, confluent cultures of Vero cells or LLC-MK2 cells in 24-well plates were infected with 50 μl/well of hMPV virus preincubated for 30 minutes at room temperature in the presence or absence of mouse sera diluted in medium. To detect hMPV, the mouse monoclonal antibody IgG _2a DS7 is used. After a virus adsorption period of two hours at 37°C, a top layer of 1.5% methylcellulose containing EMEM medium supplemented with 2 mM L-Gln and 20 to 50 μg/ml trypsin is added. On day 6 postinfection, the supernatant was removed and the cells were washed twice with PBS. Cell monolayers are fixed and stained with human IgG ₁ DS7 antibody. Foci are counted and plaque reduction titers of 50% are calculated, taking into account appropriate negative and positive controls. Images of cells are recorded using a Zeiss microscope with a 2.5× or 10× objective. Immunostaining results are expressed in plaque forming units per milliliter or PFU/mL.

Stimulation protocol

Animal stimulation experiments use hMPV A1 and A2 isolates grown on LLC-MK2 cells. BALB/c mice are immunized three times at two-week intervals with recombinant F-protein adjuvant as described previously, and on day 42 postimmunization they are challenged intranasally with approximately 1 x 10 ⁶ PFU of hMPV. After four to five days, the animals are sacrificed, individual serum samples are collected and frozen. Lung tissue samples are collected, weighed and homogenized to determine the viral titer. The viral load in lung tissue is determined by immunostaining of viral foci, as described above. Alternatively or additionally, quantitative RT-PCR (RT-qPCR) is used to determine the viral load in collected tissues.

Figure 12 shows the viral RNA load (GCE) in the lungs of mice immunized with recombinant F-protein adjuvant after stimulation with wild-type hMPV, assessed by RT-qPCR. The highest hMPV RNA load was observed in the placebo groups, while a strong reduction in viral load was observed in the lungs of immunized mice, demonstrating protection by the vaccine candidates. The protective effect is even more evident when immunostaining of viral plaques (foci) is used. Strong reductions (up to 4 log) in viral load, measured in PFU/mL, were observed in mice immunized with varying doses of protein (from 0.06 to 6.0 μg per mouse) compared with the placebo group for all candidate F proteins tested hMPV as shown in Figure 13. It will be understood that the precise details of the described methods or compositions may be changed or modified without departing from the spirit of the described embodiments. The claims include all such modifications and variations that fall within the scope and spirit of the following claims. All publications described in this application are incorporated herein by reference.

SEQUENCES

SEQ ID NO: 1

Sequence of native F protein of hMPV strain NL/1/00, serotype genotype A1 (GenBank: AAK62968.2)

SEQ ID NO: 2

Sequence of the native F2 domain of hMPV strain NL/1/00, serotype A1

SEQ ID NO: 3

Sequence of the native Fl domain of hMPV strain NL/1/00, serotype A1

SEQ ID NO: 4

Heterologous peptide linker

SEQ ID NO: 5

Protein sequence L7F_A1_23

SEQ ID NO: 6

Protein sequence sF_A1_K_L7

SEQ ID NO: 7

Protein sequence L7F_A1_31

SEQ ID NO: 8

Protein sequence L7F_A1_33

SEQ ID NO: 9

Protein sequence L7F_A1_4.2

SEQ ID NO: 10

Auxiliary trimerization domain (foldon) from bacteriophage T4 fibritin

SEQ ID NO: 11

His tag sequence with leader GS as linker

SEQ ID NO: 12

Streptavidin tag sequence

SEQ ID NO: 13

Sequence of the native F protein of hMPV strain NL/17/00, serotype A2 (GenBank: AY304360.1)

SEQ ID NO: 14

Sequence of the native F protein of hMPV strain CAN97-83, serotype A2 (Uniprot Q6WB98)

SEQ ID NO: 15

Sequence of native F protein of hMPV strain NCL174, serotype A2 (Uniprot G0ZRI7)

SEQ ID NO: 16

Sequence of the native F protein of hMPV strain NL/1/99, serotype B1 (GenBank: AY304361.1)

SEQ ID NO: 17

Sequence of native F protein of hMPV strain NDL00-1, serotype Bl (GenBank: AAK62968.2)

SEQ ID NO: 18

Sequence of native F protein of hMPV strain CAN98-75, serotype B2 (Uniprot: 6WBA7)

SEQ ID NO: 19

Codon-optimized coding nucleotide sequence sF_A1_K_L7

SEQ ID NO: 20

Codon optimized coding nucleotide sequence L7F_A1_23

SEQ ID NO: 21

Codon optimized coding nucleotide sequence L7F_A1_31

SEQ ID NO: 22

Codon optimized coding nucleotide sequence L7F_A1_33

SEQ ID NO: 23

Codon-optimized coding nucleotide sequence L7F_A1_4.2

SEQ ID NO: 24

Sequence of mature protein sF_A1_K_L7 without tags for purification

SEQ ID NO: 25

Sequence of mature protein L7F_A1_23 without tags for purification

SEQ ID NO: 26

Mature protein sequence L7F_A1_31 without purification tags

SEQ ID NO: 27

Sequence of mature protein L7F_A1_33 without tags for purification

SEQ ID NO: 28

Sequence of mature protein L7F_A1_4.2 without tags for purification

SEQ ID NO: 29 Foldon-glyc-1

SEQ ID NO: 30 Foldon-glyc-2

SEQ ID NO: 31 Foldon-glyc-3

SEQ ID NO: 32 Foldon-glyc-4

SEQ ID NO: 33 Foldon-glyc-5

SEQ ID NO: 34

Trimerization auxiliary domain VSL motif

SEQ ID NO: 35

Trimerization auxiliary domain VSA motif

SEQ ID NO: 36

SEQ ID NO: 37

SEQ ID NO: 38

SEQ ID NO: 39

SEQ ID NO: 40

SEQ ID NO: 41

SEQ ID NO: 42

Thrombin cleavage site

SEQ ID NO: 43

TEV cleavage site

SEQ ID NO: 44

Factor Xa cleavage site

SEQ ID NO: 45

SEQ ID NO: 46

SEQ ID NO: 47

SEQ ID NO: 48

SEQ ID NO: 49

SEQ ID NO: 50

SEQ ID NO: 51

Protein sequence sF_A1_K-E294 with substitutions A113C, A339C, T160F, I177L and an auxiliary KLL trimerization domain

SEQ ID NO: 52

Codon-optimized coding nucleotide sequence sF_A1_K-E294

SEQ ID NO: 53

Protein sequence sF_A1_MFur with deletion of amino acids in positions 103 to 111, replacement of R102 by the furin site KKRKRR and substitution G294E, stabilized in the post-fusion conformation

SEQ ID NO: 54

Codon-optimized coding nucleotide sequence sF_A1_MFur

--->

List of sequences

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respiratory tract

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His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

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Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

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Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

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Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

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Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

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Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

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Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala

275 280 285

Ala Pro Ser Cys Ser Gly Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile

465 470 475 480

Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile

485 490 495

Leu Ile Ala Val Leu Gly Ser Thr Met Ile Leu Val Ser Val Phe Ile

500 505 510

Ile Ile Lys Lys Thr Lys Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser

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Gly Val Thr Asn Asn Gly Phe Ile Pro His Asn

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Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu

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Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu

35 40 45

Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg

50 55 60

Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Asn Pro

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Arg Gln Ser Arg

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Phe Val Leu Gly Ala Ile Ala Leu Gly Val Ala Thr Ala Ala Ala Val

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Thr Ala Gly Val Ala Ile Ala Lys Thr Ile Arg Leu Glu Ser Glu Val

20 25 30

Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr Asn Glu Ala Val Ser Thr

35 40 45

Leu Gly Asn Gly Val Arg Val Leu Ala Thr Ala Val Arg Glu Leu Lys

50 55 60

Asp Phe Val Ser Lys Asn Leu Thr Arg Ala Ile Asn Lys Asn Lys Cys

65 70 75 80

Asp Ile Ala Asp Leu Lys Met Ala Val Ser Phe Ser Gln Phe Asn Arg

85 90 95

Arg Phe Leu Asn Val Val Arg Gln Phe Ser Asp Asn Ala Gly Ile Thr

100 105 110

Pro Ala Ile Ser Leu Asp Leu Met Thr Asp Ala Glu Leu Ala Arg Ala

115 120 125

Val Ser Asn Met Pro Thr Ser Ala Gly Gln Ile Lys Leu Met Leu Glu

130 135 140

Asn Arg Ala Met Val Arg Arg Lys Gly Phe Gly Phe Leu Ile Gly Val

145 150 155 160

Tyr Gly Ser Ser Val Ile Tyr Met Val Gln Leu Pro Ile Phe Gly Val

165 170 175

Ile Asp Thr Pro Cys Trp Ile Val Lys Ala Ala Pro Ser Cys Ser Gly

180 185 190

Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg Glu Asp Gln Gly Trp Tyr

195 200 205

Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr Pro Asn Glu Lys Asp Cys

210 215 220

Glu Thr Arg Gly Asp His Val Phe Cys Asp Thr Ala Ala Gly Ile Asn

225 230 235 240

Val Ala Glu Gln Ser Lys Glu Cys Asn Ile Asn Ile Ser Thr Thr Asn

245 250 255

Tyr Pro Cys Lys Val Ser Thr Gly Arg His Pro Ile Ser Met Val Ala

260 265 270

Leu Ser Pro Leu Gly Ala Leu Val Ala Cys Tyr Lys Gly Val Ser Cys

275 280 285

Ser Ile Gly Ser Asn Arg Val Gly Ile Ile Lys Gln Leu Asn Lys Gly

290 295 300

Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp Thr Val Thr Ile Asp Asn

305 310 315 320

Thr Val Tyr Gln Leu Ser Lys Val Glu Gly Glu Gln His Val Ile Lys

325 330 335

Gly Arg Pro Val Ser Ser Ser Phe Asp Pro Val Lys Phe Pro Glu Asp

340 345 350

Gln Phe Asn Val Ala Leu Asp Gln Val Phe Glu Ser Ile Glu Asn Ser

355 360 365

Gln Ala Leu Val Asp Gln Ser Asn Arg Ile Leu Ser Ser Ala Glu Lys

370 375 380

Gly Asn Thr Gly Phe Ile Ile Val Ile Ile Leu Ile Ala Val Leu Gly

385 390 395 400

Ser Thr Met Ile Leu Val Ser Val Phe Ile Ile Ile Lys Lys Thr Lys

405 410 415

Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser Gly Val Thr Asn Asn Gly

420 425 430

Phe Ile Pro His Asn

435

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Cys Gly Ala Gly Ala

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Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Gln Pro Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala

100 105 110

Ile Ala Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn

115 120 125

Ala Leu Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val

130 135 140

Arg Val Leu Ala Thr Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys

145 150 155 160

Asn Leu Thr Arg Ala Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu

165 170 175

Lys Met Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val

180 185 190

Val Arg Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu

195 200 205

Asp Leu Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro

210 215 220

Thr Ser Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val

225 230 235 240

Arg Arg Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val

245 250 255

Ile Tyr Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys

260 265 270

Trp Ile Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr

275 280 285

Ala Cys Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly

290 295 300

Ser Thr Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp

305 310 315 320

His Val Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser

325 330 335

Lys Glu Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val

340 345 350

Ser Thr Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly

355 360 365

Ala Leu Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn

370 375 380

Arg Val Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr

385 390 395 400

Asn Gln Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu

405 410 415

Ser Lys Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser

420 425 430

Ser Ser Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala

435 440 445

Leu Asp Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp

450 455 460

Gln Ser Asn Arg Ile Leu Ser Ala Gly Tyr Ile Pro Glu Ala Pro Arg

465 470 475 480

Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser

485 490 495

Thr Phe Leu Gly Gly Leu Val Pro Arg Gly Ser His His His His

500 505 510

His Ser Ala Trp Ser His Pro Gln Phe Glu Lys

515 520

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Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

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His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Gln Pro Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala

100 105 110

Ile Ala Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn

115 120 125

Ala Leu Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val

130 135 140

Arg Val Leu Ala Phe Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys

145 150 155 160

Asn Leu Thr Arg Ala Leu Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu

165 170 175

Lys Met Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val

180 185 190

Val Arg Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu

195 200 205

Asp Leu Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro

210 215 220

Thr Ser Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val

225 230 235 240

Arg Arg Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val

245 250 255

Ile Tyr Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys

260 265 270

Trp Ile Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr

275 280 285

Ala Cys Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly

290 295 300

Ser Thr Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp

305 310 315 320

His Val Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser

325 330 335

Lys Glu Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val

340 345 350

Ser Thr Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly

355 360 365

Ala Leu Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn

370 375 380

Arg Val Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr

385 390 395 400

Asn Gln Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu

405 410 415

Ser Lys Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser

420 425 430

Ser Ser Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala

435 440 445

Leu Asp Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp

450 455 460

Gln Ser Asn Arg Ile Leu Ser Ser Ala Glu Ser Ala Ile Gly Gly Tyr

465 470 475 480

Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly

485 490 495

Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu Val Pro Arg Gly

500 505 510

Ser His His His His His Ser Ala Trp Ser His Pro Gln Phe Glu

515 520 525

Lys

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Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Met Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Leu Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Asn

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Gln Pro Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala

100 105 110

Ile Ala Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn

115 120 125

Ala Leu Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val

130 135 140

Arg Val Leu Ala Thr Met Val Arg Glu Leu Lys Asp Phe Val Ser Lys

145 150 155 160

Asn Leu Thr Arg Ala Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu

165 170 175

Lys Met Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val

180 185 190

Val Arg Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu

195 200 205

Asp Leu Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro

210 215 220

Thr Ser Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val

225 230 235 240

Arg Arg Lys Gly Phe Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val

245 250 255

Ile Tyr Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys

260 265 270

Trp Ile Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr

275 280 285

Ala Cys Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly

290 295 300

Ser Thr Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp

305 310 315 320

His Val Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser

325 330 335

Lys Glu Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val

340 345 350

Ser Thr Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly

355 360 365

Ala Leu Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn

370 375 380

Arg Val Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr

385 390 395 400

Asn Gln Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu

405 410 415

Ser Lys Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser

420 425 430

Ser Ser Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala

435 440 445

Leu Asp Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp

450 455 460

Gln Ser Asn Arg Ile Leu Ser Ala Gly Tyr Ile Pro Glu Ala Pro Arg

465 470 475 480

Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser

485 490 495

Thr Phe Leu Gly Gly Leu Val Pro Arg Gly Ser His His His His

500 505 510

His Ser Ala Trp Ser His Pro Gln Phe Glu Lys

515 520

<210> 8

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<212> PROTEIN

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Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Met Leu Cys Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Leu Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Gln Pro Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala

100 105 110

Ile Ala Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn

115 120 125

Ala Leu Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val

130 135 140

Arg Val Leu Ala Thr Met Val Arg Glu Leu Cys Asp Phe Val Ser Lys

145 150 155 160

Asn Leu Thr Arg Ala Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu

165 170 175

Lys Met Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val

180 185 190

Val Arg Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu

195 200 205

Asp Leu Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro

210 215 220

Thr Ser Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val

225 230 235 240

Arg Arg Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Asp Val

245 250 255

Ile Tyr Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys

260 265 270

Trp Ile Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr

275 280 285

Ala Cys Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly

290 295 300

Ser Thr Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp

305 310 315 320

His Val Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser

325 330 335

Lys Glu Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val

340 345 350

Ser Thr Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly

355 360 365

Ala Leu Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn

370 375 380

Arg Val Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr

385 390 395 400

Asn Gln Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu

405 410 415

Ser Lys Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser

420 425 430

Ser Ser Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala

435 440 445

Leu Asp Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp

450 455 460

Gln Ser Asn Arg Cys Cys Ser Ala Gly Tyr Ile Pro Glu Ala Pro Arg

465 470 475 480

Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser

485 490 495

Thr Phe Leu Gly Gly Leu Val Pro Arg Gly Ser His His His His

500 505 510

His Ser Ala Trp Ser His Pro Gln Phe Glu Lys

515 520

<210> 9

<211> 529

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 9

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Met Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Gln Pro Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala

100 105 110

Ile Ala Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Trp Lys Asn

115 120 125

Ala Leu Lys Lys Thr Asn Glu Val Val Ser Thr Leu Gly Asn Gly Val

130 135 140

Arg Val Leu Val Thr Met Val Arg Glu Leu Lys Asp Phe Val Ser Lys

145 150 155 160

Asn Leu Thr Arg Ala Leu Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu

165 170 175

Lys Met Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val

180 185 190

Val Arg Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu

195 200 205

Asp Leu Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro

210 215 220

Thr Ser Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val

225 230 235 240

Arg Arg Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val

245 250 255

Ile Tyr Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys

260 265 270

Trp Ile Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr

275 280 285

Ala Cys Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly

290 295 300

Ser Thr Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp

305 310 315 320

His Val Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser

325 330 335

Lys Glu Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val

340 345 350

Ser Thr Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly

355 360 365

Ala Leu Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn

370 375 380

Arg Val Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr

385 390 395 400

Asn Gln Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu

405 410 415

Ser Lys Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser

420 425 430

Ser Ser Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala

435 440 445

Leu Asp Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp

450 455 460

Gln Ser Asn Arg Ile Leu Ser Ser Ala Glu Ser Ala Ile Gly Gly Tyr

465 470 475 480

Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly

485 490 495

Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu Val Pro Arg Gly

500 505 510

Ser His His His His His Ser Ala Trp Ser His Pro Gln Phe Glu

515 520 525

Lys

<210> 10

<211> 27

<212> PROTEIN

<213> Bacteriophage T4

<400> 10

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 11

<211> 8

<212> PROTEIN

<213> Artificial

<400> 11

Gly Ser His His His His His

15

<210> 12

<211> 10

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 12

Ser Ala Trp Ser His Pro Gln Phe Glu Lys

1 5 10

<210> 13

<211> 539

<212> PROTEIN

<213> Human metapneumovirus

<400> 13

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ser Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Thr Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Asp Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Thr Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala

275 280 285

Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Asn Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile

465 470 475 480

Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile

485 490 495

Leu Ile Ala Val Leu Gly Ser Ser Met Ile Leu Val Ser Ile Phe Ile

500 505 510

Ile Ile Lys Lys Thr Lys Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser

515 520 525

Gly Val Thr Asn Asn Gly Phe Ile Pro His Ser

530 535

<210> 14

<211> 539

<212> PROTEIN

<213> Human metapneumovirus

<400> 14

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ser Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Thr Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Asp Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala

275 280 285

Ala Pro Ser Cys Ser Gly Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Asn Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile

465 470 475 480

Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile

485 490 495

Leu Ile Ala Val Leu Gly Ser Ser Met Ile Leu Val Ser Ile Phe Ile

500 505 510

Ile Ile Lys Lys Thr Lys Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser

515 520 525

Gly Val Thr Asn Asn Gly Phe Ile Pro His Ser

530 535

<210> 15

<211> 539

<212> PROTEIN

<213> Human metapneumovirus

<400> 15

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Ser Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Lys Pro Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Asp Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ala Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala

275 280 285

Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Asn Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile

465 470 475 480

Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile

485 490 495

Leu Ile Ala Val Leu Gly Ser Ser Met Ile Leu Val Ser Val Phe Ile

500 505 510

Ile Ile Lys Lys Thr Arg Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser

515 520 525

Gly Val Thr Asn Asn Gly Phe Ile Pro His Ser

530 535

<210> 16

<211> 539

<212> PROTEIN

<213> Human metapneumovirus

<400> 16

Met Ser Trp Lys Val Met Ile Ile Ile Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Thr Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Ile Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Asn Ala Ile Lys Gly Ala Leu Lys Gln Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Glu Phe Val Ser Lys Asn Leu Thr Ser Ala

165 170 175

Ile Asn Arg Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Tyr Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Ile Lys Ala

275 280 285

Ala Pro Ser Cys Ser Glu Lys Asn Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Lys Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Arg Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Trp Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Pro Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Lys Ile

465 470 475 480

Leu Asn Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Val Ile

485 490 495

Leu Val Ala Val Leu Gly Leu Thr Met Ile Ser Val Ser Ile Ile Ile

500 505 510

Ile Ile Lys Lys Thr Arg Lys Pro Thr Gly Ala Pro Pro Glu Leu Asn

515 520 525

Gly Val Thr Asn Gly Gly Phe Ile Pro His Ser

530 535

<210> 17

<211> 539

<212> PROTEIN

<213> Human metapneumovirus

<400> 17

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala

275 280 285

Ala Pro Ser Cys Ser Gly Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile

465 470 475 480

Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile

485 490 495

Leu Ile Ala Val Leu Gly Ser Thr Met Ile Leu Val Ser Val Phe Ile

500 505 510

Ile Ile Lys Lys Thr Lys Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser

515 520 525

Gly Val Thr Asn Asn Gly Phe Ile Pro His Asn

530 535

<210> 18

<211> 539

<212> PROTEIN

<213> Human metapneumovirus

<400> 18

Met Ser Trp Lys Val Met Ile Ile Ile Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Thr Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Ala Thr Ala Ala Ala Val Thr Ala Gly Ile Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Asn Ala Ile Lys Gly Ala Leu Lys Thr Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr

145 150 155 160

Ala Val Arg Glu Leu Lys Glu Phe Val Ser Lys Asn Leu Thr Ser Ala

165 170 175

Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Tyr Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Ile Lys Ala

275 280 285

Ala Pro Ser Cys Ser Glu Lys Asp Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Lys Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Lys Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Arg Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Pro Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Lys Ile

465 470 475 480

Leu Asn Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile

485 490 495

Leu Ile Ala Val Leu Gly Leu Thr Met Ile Ser Val Ser Ile Ile Ile

500 505 510

Ile Ile Lys Lys Thr Arg Lys Pro Thr Gly Ala Pro Pro Glu Leu Asn

515 520 525

Gly Val Thr Asn Gly Gly Phe Ile Pro His Ser

530 535

<210> 19

<211> 1590

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 19

atgtcttgga aggtggtcat catcttctcc ctgctgatca cccctcagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgaga 120

accggctggt acaccaacgt gttcaccctg gaagtgggcg acgtggaaaa cctgacctgt 180

gctgatggcc ccagcctgat caagaccgag ctggacctga ccaagtctgc cctgagagaa 240

ctgaggaccg tgtctgccga tcagctggcc agagaggaac agatcgagca gcctagacag 300

tccggatgtg gtgctggtgc tacagctggc gtggccattg ccaagaccat ccggctggaa 360

tctgaagtga ccgccatcaa gaacgccctg aaaaagacca acgaggccgt gtctaccctc 420

ggcaatggcg ttagagtgct ggcctttgct gtgcgcgagc tgaaggactt cgtgtccaag 480

aacctgacca gggctctgaa caagaacaag tgtgatatcg ccgacctgaa gatggccgtg 540

tcctttagcc agttcaaccg gcggttcctg aacgtcgtgc ggcagttctc tgataacgcc 600

ggcatcaccc ctgccatcag cctggatctg atgaccgatg ccgagctggc tagagccgtg 660

tccaacatgc ctacctctgc cggccagatc aagctgatgc tggaaaacag agccatggtc 720

cgacggaaag gcttcggctt tctgatcggc gtgtacggct cctccgtgat ctacatggtg 780

cagctgccta tcttcggcgt gatcgacacc ccttgctgga tcgtgaaggc cgctcctagc 840

tgctctgaga agaagggcaa ctacgcctgc ctgctgagag aggaccaagg ctggtactgt 900

cagaacgccg gctccaccgt gtactacccc aacgagaagg actgcgagac aagaggcgac 960

cacgtgttct gcgatacctg cgctggcatc aatgtggccg agcagtccaa agagtgcaac 1020

atcaacatct ccaccaccaa ctatccctgc aaggtgtcca ccggcaggca ccctatttcc 1080

atggtggctc tgtctccact gggcgccctg gtggcttgtt ataagggcgt gtcctgctcc 1140

atcggctcca acaggtggg catcatcaag cagctgaaca agggctgcag ctacatcacc 1200

aaccaggacg ccgataccgt gaccatcgac aataccgtgt atcagctgtc caaggtggaa 1260

ggcgagcagc acgtgatcaa gggcagacct gtgtcctcca gcttcgaccc cgtgaagttc 1320

cctgaggatc agttcaacgt ggccctggac caggtgttcg agtccatcga gaactctcag 1380

gctctggtgg accagtccaa ccggatcctg tcctctgccg agtctgctat cggcggctat 1440

atccccgagg ctcctagaga tggccaggcc tatgttcgga aggatggcga atgggtgctg 1500

ctgtctacct tcctcggagg cctggtgcct agaggctctc accaccatca tcaccactcc 1560

gcttggtccc atccacagtt cgagaagtga 1590

<210> 20

<211> 1572

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 20

atgtcttgga aggtggtcat catcttctcc ctgctgatca cccctcagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgaga 120

accggctggt acaccaacgt gttcaccctg gaagtgggcg acgtggaaaa cctgacctgt 180

gctgatggcc ccagcctgat caagaccgag ctggacctga ccaagtctgc cctgagagaa 240

ctgaggaccg tgtctgccga tcagctggcc agagaggaac agatcgagca gcctagacag 300

tccggatgtg gtgctggtgc tacagctggc gtggccattg ccaagaccat ccggctggaa 360

tctgaagtga ccgccatcaa gaacgccctg aaaaagacca acgaggccgt gtctaccctc 420

ggcaatggcg ttagagtgct ggccacagcc gtgcgcgagc tgaaggattt cgtgtccaag 480

aacctgacca gggccatcaa caagaacaag tgtgatatcg ccgacctgaa gatggccgtg 540

tccttcagcc agttcaaccg gcggttcctg aatgtcgtgc ggcagttctc tgacaacgcc 600

ggcatcaccc ctgccatcag cctggatctg atgaccgatg ccgagctggc tagagccgtg 660

tccaacatgc ctacctctgc cggccagatc aagctgatgc tggaaaacag agccatggtc 720

cgacggaaag gcttcggctt tctgatcggc gtgtacggct cctccgtgat ctacatggtg 780

cagctgccta tcttcggcgt gatcgacacc ccttgctgga tcgtgaaggc cgctcctagc 840

tgctctgaga agaagggcaa ctacgcctgc ctgctgagag aggaccaagg ctggtactgt 900

cagaacgccg gctccaccgt gtactacccc aacgagaagg actgcgagac aagaggcgac 960

cacgtgttct gcgatacctg cgctggcatc aatgtggccg agcagtccaa agagtgcaac 1020

atcaacatct ccaccaccaa ctatccctgc aaggtgtcca ccggcaggca ccctatttcc 1080

atggtggctc tgtctccact gggcgccctg gtggcttgtt ataagggcgt gtcctgctcc 1140

atcggctcca acaggtggg catcatcaag cagctgaaca agggctgcag ctacatcacc 1200

aaccaggacg ccgataccgt gaccatcgac aataccgtgt atcagctgtc caaggtggaa 1260

ggcgagcagc acgtgatcaa gggcagacct gtgtcctcca gcttcgaccc cgtgaagttc 1320

cctgaggatc agttcaacgt ggccctggac caggtgttcg agtccatcga gaactctcag 1380

gctctggtgg accagtccaa ccggattctg tctgccggct acatccccga ggctcctaga 1440

gatggacagg cctacgtcag aaaggacggc gaatgggtgc tgctgtctac ctttctcgga 1500

ggcctggtgc ctagaggctc tcaccaccat catcaccact ccgcttggtc ccatccacag 1560

ttcgagaagtga 1572

<210> 21

<211> 1572

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 21

atgtcttgga aggtggtcat catcttctcc ctgctgatca cccctcagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgaga 120

accggctggt acaccaacgt gttcatgctg gaagtgggcg acgtggaaaa cctgacctgt 180

gctgatggcc ccagcctgct gaaaacagag ctggacctga ccaagagcgc cctgagaaat 240

ctgaggaccg tgtctgccga tcagctggcc agagaggaac agatcgagca gcctagacag 300

tccggatgtg gtgctggtgc tacagctggc gtggccattg ccaagaccat ccggctggaa 360

tctgaagtga ccgccatcaa gaatgccctg aaaaagacca acgaggccgt gtctaccctc 420

ggcaatggcg ttagagtgct ggccacaatg gtccgagagc tgaaggactt cgtgtccaag 480

aacctgacca gggccatcaa caagaacaag tgtgatatcg ccgacctgaa gatggccgtg 540

tcctttagcc agttcaaccg gcggttcctg aacgtcgtgc ggcagttctc tgataacgcc 600

ggcatcaccc ctgccatcag cctggatctg atgaccgatg ccgagctggc tagagccgtg 660

tccaacatgc ctacctctgc cggccagatc aagctgatgc tcgagaacag agctatggtc 720

cgacggaaag gcttcggcat cctgatcggc gtgtacggct cctccgtgat ctacatggtg 780

cagctgccta tcttcggcgt gatcgacacc ccttgctgga tcgtgaaggc cgctcctagc 840

tgctctgaga agaagggcaa ctacgcctgc ctgctgagag aggaccaagg ctggtactgt 900

cagaacgccg gctccaccgt gtactacccc aacgagaagg actgcgagac aagaggcgac 960

cacgtgttct gcgatacctg cgctggcatc aatgtggccg agcagtccaa agagtgcaac 1020

atcaacatct ccaccaccaa ctatccctgc aaggtgtcca ccggcaggca ccctatttcc 1080

atggtggctc tgtctccact gggcgccctg gtggcttgtt ataagggcgt gtcctgctcc 1140

atcggctcca acaggtggg catcatcaag cagctgaaca agggctgcag ctacatcacc 1200

aaccaggacg ccgataccgt gaccatcgac aataccgtgt atcagctgtc caaggtggaa 1260

ggcgagcagc acgtgatcaa gggcagacct gtgtcctcca gcttcgaccc cgtgaagttc 1320

cctgaggatc agttcaacgt ggccctggac caggtgttcg agtccatcga gaactctcag 1380

gctctggtgg accagtccaa ccggattctg tctgccggct acatccccga ggctcctaga 1440

gatggacagg cctacgtcag aaaggacggc gaatgggtgc tgctgtctac ctttctcgga 1500

ggcctggtgc ctagaggctc tcaccaccat catcaccact ccgcttggtc ccatcctcag 1560

ttcgagaagtga 1572

<210> 22

<211> 1572

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 22

atgtcttgga aggtggtcat catcttctcc ctgctgatca cccctcagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgaga 120

accggctggt acaccaacgt gttcatgctg tgtgtgggcg acgtggaaaa cctgacctgt 180

gctgatggcc ccagcctgct gaaaacagag ctggacctga ccaagagcgc cctgagagaa 240

ctgaggaccg tgtctgcaga tcagctggcc agagaggaac agatcgagca gcctagacag 300

tccggatgtg gtgctggtgc tacagctggc gtggccattg ccaagaccat ccggctggaa 360

tctgaagtga ccgccatcaa gaatgccctg aaaaagacca acgaggccgt gtctaccctc 420

ggcaatggcg ttagagtgct ggccacaatg gtccgagagc tgtgcgactt cgtgtccaag 480

aatctgaccc gggccatcaa caagaacaag tgtgatatcg ccgacctgaa gatggccgtg 540

tccttcagcc agttcaaccg gcggttcctg aatgtcgtgc ggcagttctc tgacaacgcc 600

ggcatcaccc ctgccatcag cctggatctg atgaccgatg ccgagctggc tagagccgtg 660

tccaacatgc ctacctctgc cggccagatc aagctgatgc tcgagaacag agctatggtc 720

cgacggaaag gcttcggctt cctgatcggc gtgtacggct ctgacgtgat ctacatggtg 780

cagctgccta tcttcggcgt gatcgacacc ccttgctgga tcgtgaaggc cgctcctagc 840

tgctctgaga agaagggcaa ctacgcctgc ctgctgagag aggaccaagg ctggtactgt 900

cagaacgccg gctccaccgt gtactacccc aacgagaagg actgcgagac aagaggcgac 960

cacgtgttct gcgatacctg cgctggcatc aatgtggccg agcagtccaa agagtgcaac 1020

atcaacatct ccaccaccaa ctatccctgc aaggtgtcca ccggcagaca ccccatttcc 1080

atggtggctc tgtctccact gggtgccctg gtggcttgtt ataagggcgt gtcctgctcc 1140

atcggctcca acaggtggg catcatcaag cagctgaaca agggctgcag ctacatcacc 1200

aaccaggacg ccgataccgt gaccatcgac aataccgtgt atcagctgtc caaggtggaa 1260

ggcgagcagc acgtgatcaa gggcagacct gtgtcctcca gcttcgaccc cgtgaagttc 1320

cctgaggatc agttcaacgt ggccctggac caggtgttcg agtccatcga gaactctcag 1380

gctctggtgg accagtccaa cagatgctgt tccgccggct acatccccga ggctcctaga 1440

gatggacagg cctacgtcag aaaggacggc gaatgggtgc tgctgtctac ctttctcgga 1500

ggcctggtgc ctagaggctc tcaccaccat catcaccact ccgcttggtc ccatccacag 1560

ttcgagaagtga 1572

<210> 23

<211> 1590

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 23

atgtcttgga aggtggtcat catcttctcc ctgctgatca cccctcagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgaga 120

accggctggt acaccaacgt gttcatgctg gaagtgggcg acgtggaaaa cctgacctgt 180

gctgatggcc ccagcctgat caagaccgag ctggacctga ccaagtctgc cctgagagaa 240

ctgaggaccg tgtctgccga tcagctggcc agagaggaac agatcgagca gcctagacag 300

tccggatgtg gtgctggtgc tacagctggc gtggccattg ccaagaccat ccggctggaa 360

tctgaagtga ccgcctggaa gaacgccctg aaaaagacca acgaggtggt gtctaccctc 420

ggcaacggcg tcagagtgct ggtcacaatg gtccgagagc tgaaggactt cgtgtccaag 480

aacctgacca gggctctgaa caagaacaag tgtgatatcg ccgacctgaa gatggccgtg 540

tctttcagcc agttcaaccg gcggttcctg aacgtcgtgc ggcagttctc tgataacgcc 600

ggcatcaccc ctgccatcag cctggatctg atgaccgatg ccgagctggc tagagccgtg 660

tccaacatgc ctacctctgc cggccagatc aagctgatgc tggaaaacag agccatggtc 720

cgacggaaag gcttcggctt tctgatcggc gtgtacggct cctccgtgat ctacatggtg 780

cagctgccta tcttcggcgt gatcgacacc ccttgctgga tcgtgaaggc cgctcctagc 840

tgctctgaga agaagggcaa ctacgcctgc ctgctgagag aggaccaagg ctggtactgt 900

cagaacgccg gctccaccgt gtactacccc aacgagaagg actgcgagac aagaggcgac 960

cacgtgttct gcgatacctg cgctggcatc aatgtggccg agcagtccaa agagtgcaac 1020

atcaacatct ccaccaccaa ctatccctgc aaggtgtcca ccggcaggca ccctatttcc 1080

atggtggctc tgtctccact gggcgccctg gtggcttgtt ataagggcgt gtcctgctcc 1140

atcggctcca acaggtggg catcatcaag cagctgaaca agggctgcag ctacatcacc 1200

aaccaggacg ccgataccgt gaccatcgac aataccgtgt atcagctgtc caaggtggaa 1260

ggcgagcagc acgtgatcaa gggcagacct gtgtcctcca gcttcgaccc cgtgaagttc 1320

cctgaggatc agttcaacgt ggccctggac caggtgttcg agtccatcga gaactctcag 1380

gctctggtgg accagtccaa ccggatcctg tcctctgccg agtctgctat cggcggctat 1440

atccccgagg ctcctagaga tggccaggcc tatgttcgga aggatggcga atgggtgctg 1500

ctgtctacct tcctcggagg cctggtgcct agaggctctc accaccatca tcaccactcc 1560

gcttggtccc atccacagtt cgagaagtga 1590

<210> 24

<211> 487

<212> PROTEIN

<213> artificial

<220>

<223> Artificial

<400> 24

Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly

1 5 10 15

Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu

20 25 30

Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu

35 40 45

Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg

50 55 60

Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Gln Pro

65 70 75 80

Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala Ile Ala

85 90 95

Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu

100 105 110

Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val

115 120 125

Leu Ala Phe Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu

130 135 140

Thr Arg Ala Leu Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met

145 150 155 160

Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg

165 170 175

Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu

180 185 190

Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser

195 200 205

Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg

210 215 220

Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr

225 230 235 240

Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile

245 250 255

Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys

260 265 270

Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr

275 280 285

Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val

290 295 300

Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu

305 310 315 320

Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr

325 330 335

Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu

340 345 350

Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val

355 360 365

Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln

370 375 380

Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys

385 390 395 400

Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser

405 410 415

Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp

420 425 430

Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser

435 440 445

Asn Arg Ile Leu Ser Ser Ala Glu Ser Ala Ile Gly Gly Tyr Ile Pro

450 455 460

Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp

465 470 475 480

Val Leu Leu Ser Thr Phe Leu

485

<210> 25

<211> 481

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 25

Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly

1 5 10 15

Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu

20 25 30

Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu

35 40 45

Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg

50 55 60

Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Gln Pro

65 70 75 80

Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala Ile Ala

85 90 95

Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu

100 105 110

Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val

115 120 125

Leu Ala Thr Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu

130 135 140

Thr Arg Ala Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met

145 150 155 160

Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg

165 170 175

Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu

180 185 190

Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser

195 200 205

Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg

210 215 220

Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr

225 230 235 240

Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile

245 250 255

Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys

260 265 270

Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr

275 280 285

Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val

290 295 300

Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu

305 310 315 320

Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr

325 330 335

Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu

340 345 350

Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val

355 360 365

Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln

370 375 380

Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys

385 390 395 400

Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser

405 410 415

Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp

420 425 430

Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser

435 440 445

Asn Arg Ile Leu Ser Ala Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly

450 455 460

Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe

465 470 475 480

Leu

<210> 26

<211> 481

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 26

Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly

1 5 10 15

Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Met Leu

20 25 30

Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu

35 40 45

Leu Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Asn Leu Arg

50 55 60

Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Gln Pro

65 70 75 80

Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala Ile Ala

85 90 95

Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu

100 105 110

Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val

115 120 125

Leu Ala Thr Met Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu

130 135 140

Thr Arg Ala Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met

145 150 155 160

Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg

165 170 175

Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu

180 185 190

Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser

195 200 205

Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg

210 215 220

Lys Gly Phe Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr

225 230 235 240

Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile

245 250 255

Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys

260 265 270

Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr

275 280 285

Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val

290 295 300

Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu

305 310 315 320

Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr

325 330 335

Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu

340 345 350

Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val

355 360 365

Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln

370 375 380

Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys

385 390 395 400

Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser

405 410 415

Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp

420 425 430

Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser

435 440 445

Asn Arg Ile Leu Ser Ala Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly

450 455 460

Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe

465 470 475 480

Leu

<210> 27

<211> 481

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 27

Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly

1 5 10 15

Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Met Leu

20 25 30

Cys Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu

35 40 45

Leu Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg

50 55 60

Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Gln Pro

65 70 75 80

Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala Ile Ala

85 90 95

Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu

100 105 110

Lys Lys Thr Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val

115 120 125

Leu Ala Thr Met Val Arg Glu Leu Cys Asp Phe Val Ser Lys Asn Leu

130 135 140

Thr Arg Ala Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met

145 150 155 160

Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg

165 170 175

Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu

180 185 190

Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser

195 200 205

Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg

210 215 220

Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Asp Val Ile Tyr

225 230 235 240

Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile

245 250 255

Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys

260 265 270

Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr

275 280 285

Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val

290 295 300

Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu

305 310 315 320

Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr

325 330 335

Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu

340 345 350

Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val

355 360 365

Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln

370 375 380

Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys

385 390 395 400

Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser

405 410 415

Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp

420 425 430

Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser

435 440 445

Asn Arg Cys Cys Ser Ala Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly

450 455 460

Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe

465 470 475 480

Leu

<210> 28

<211> 487

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 28

Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly

1 5 10 15

Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Met Leu

20 25 30

Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu

35 40 45

Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg

50 55 60

Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Gln Pro

65 70 75 80

Arg Gln Ser Gly Cys Gly Ala Gly Ala Thr Ala Gly Val Ala Ile Ala

85 90 95

Lys Thr Ile Arg Leu Glu Ser Glu Val Thr Ala Trp Lys Asn Ala Leu

100 105 110

Lys Lys Thr Asn Glu Val Val Ser Thr Leu Gly Asn Gly Val Arg Val

115 120 125

Leu Val Thr Met Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu

130 135 140

Thr Arg Ala Leu Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met

145 150 155 160

Ala Val Ser Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg

165 170 175

Gln Phe Ser Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu

180 185 190

Met Thr Asp Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser

195 200 205

Ala Gly Gln Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg

210 215 220

Lys Gly Phe Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr

225 230 235 240

Met Val Gln Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile

245 250 255

Val Lys Ala Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys

260 265 270

Leu Leu Arg Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr

275 280 285

Val Tyr Tyr Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val

290 295 300

Phe Cys Asp Thr Cys Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu

305 310 315 320

Cys Asn Ile Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr

325 330 335

Gly Arg His Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu

340 345 350

Val Ala Cys Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val

355 360 365

Gly Ile Ile Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln

370 375 380

Asp Ala Asp Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys

385 390 395 400

Val Glu Gly Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser

405 410 415

Phe Asp Pro Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp

420 425 430

Gln Val Phe Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser

435 440 445

Asn Arg Ile Leu Ser Ser Ala Glu Ser Ala Ile Gly Gly Tyr Ile Pro

450 455 460

Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp

465 470 475 480

Val Leu Leu Ser Thr Phe Leu

485

<210> 29

<211> 27

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 29

Gly Tyr Ile Pro Glu Ala Pro Arg Asn Gly Thr Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 30

<211> 27

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 30

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asn Gly Thr Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 31

<211> 27

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 31

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Asn Trp Thr Leu Leu Ser Thr Phe Leu

20 25

<210> 32

<211> 27

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 32

Gly Tyr Ile Pro Glu Ala Pro Arg Asn Gly Thr Ala Tyr Val Arg Lys

1 5 10 15

Asn Gly Thr Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 33

<211> 27

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 33

Gly Tyr Ile Pro Glu Ala Pro Arg Asn Gly Thr Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Asn Trp Thr Leu Leu Ser Thr Phe Leu

20 25

<210> 34

<211> 4

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 34

Ile Leu Ser Ala

1

<210> 35

<211> 4

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 35

Cys Cys Ser Ala

1

<210> 36

<211> 18

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 36

Cys Cys Lys Gln Thr Asn Glu Cys Cys Lys Asn Leu Glu Arg Ala Val

1 5 10 15

Ser Ala

<210> 37

<211> 18

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 37

Cys Cys Arg Glu Leu Lys Glu Cys Cys Lys Asn Leu Glu Asn Ala Val

1 5 10 15

Ser Ala

<210> 38

<211> 18

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 38

Cys Cys Arg Glu Leu Lys Asp Cys Cys Lys Asn Leu Glu Asn Ala Val

1 5 10 15

Ser Ala

<210> 39

<211> 18

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 39

Cys Cys Arg Glu Leu Lys Asp Cys Cys Lys Asn Leu Glu Arg Ala Val

1 5 10 15

Ser Ala

<210> 40

<211> 18

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 40

Cys Cys Arg Glu Leu Lys Asp Cys Cys Lys Gln Leu Asn Lys Ala Val

1 5 10 15

Ser Ala

<210> 41

<211> 18

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 41

Cys Cys Arg Glu Leu Lys Glu Cys Cys Lys Gln Leu Asn Lys Ala Val

1 5 10 15

Ser Ala

<210> 42

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 42

Leu Val Pro Arg Gly Ser

15

<210> 43

<211> 7

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 43

Glu Asn Leu Tyr Phe Gln Gly

15

<210> 44

<211> 4

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 44

Ile Glu Gly Arg

1

<210> 45

<211> 17

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 45

Cys Cys Lys Gln Thr Asn Glu Cys Cys Lys Asn Leu Glu Arg Ala Val

1 5 10 15

Ser

<210> 46

<211> 17

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 46

Cys Cys Lys Gln Thr Asn Glu Cys Cys Lys Asn Leu Glu Arg Ala Val

1 5 10 15

Ser

<210> 47

<211> 17

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 47

Cys Cys Lys Gln Thr Asn Glu Cys Cys Lys Asn Leu Glu Arg Ala Val

1 5 10 15

Ser

<210> 48

<211> 17

<212> PROTEIN

<213> Artificial

<400> 48

Cys Cys Arg Glu Leu Lys Glu Cys Cys Lys Asn Leu Glu Asn Ala Val

1 5 10 15

Ser

<210> 49

<211> 17

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 49

Cys Cys Arg Glu Leu Lys Glu Cys Cys Lys Asn Leu Glu Asn Ala Val

1 5 10 15

Ser

<210> 50

<211> 17

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 50

Cys Cys Arg Glu Leu Lys Glu Cys Cys Lys Asn Leu Glu Asn Ala Val

1 5 10 15

Ser

<210> 51

<211> 540

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 51

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val

100 105 110

Cys Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile

115 120 125

Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr

130 135 140

Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Phe

145 150 155 160

Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala

165 170 175

Leu Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser

180 185 190

Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser

195 200 205

Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp

210 215 220

Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln

225 230 235 240

Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe

245 250 255

Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln

260 265 270

Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala

275 280 285

Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg

290 295 300

Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr

305 310 315 320

Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp

325 330 335

Thr Ala Cys Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile

340 345 350

Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His

355 360 365

Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys

370 375 380

Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile

385 390 395 400

Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp

405 410 415

Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly

420 425 430

Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro

435 440 445

Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe

450 455 460

Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile

465 470 475 480

Leu Ser Ser Ala Glu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro

485 490 495

Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu

500 505 510

Ser Thr Phe Leu Gly Gly Leu Val Pro Arg Gly Ser His His His His

515 520 525

His His Ser Ala Trp Ser His Pro Gln Phe Glu Lys

530 535 540

<210> 52

<211> 1623

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 52

atgtcttgga aggtggtcat catcttctcc ctgctgatca cccctcagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgaga 120

accggctggt acaccaacgt gttcaccctg gaagtgggcg acgtggaaaa cctgacctgt 180

gctgatggcc ccagcctgat caagaccgag ctggacctga ccaagtctgc cctgagagaa 240

ctgaggaccg tgtctgccga tcagctggcc agagaggaac agatcgagaa ccctcggcag 300

tccagattcg tgctgggagc tattgctctg ggcgtgtgta cagccgctgc tgtgacagct 360

ggtgtcgcta tcgccaagac catccggctg gaatctgaag tgaccgccat caagaacgcc 420

ctgaaaaaga ccaacgaggc cgtgtccaca ctcggcaatg gcgttagagt gctggccttt 480

gctgtgcgcg agctgaagga cttcgtgtcc aagaacctga ccagggctct gaacaagaac 540

aagtgtgata tcgccgacct gaagatggcc gtgtctttca gccagttcaa ccggcggttc 600

ctgaacgtcg tgcggcagtt ctctgataac gccggcatca cccctgccat cagcctggat 660

ctgatgaccg atgccgagct ggctagagcc gtgtctaaca tgcctacctc tgccggccag 720

atcaagctga tgctggaaaa cagagccatg gtccgacgga aaggcttcgg ctttctgatc 780

ggcgtgtacg gctcctccgt gatctacatg gtgcagctgc ctatcttcgg cgtgatcgac 840

accccttgct ggatcgtgaa ggccgctcct agctgctctg agaagaaggg caactacgcc 900

tgcctgctga gagaggacca aggctggtac tgtcagaacg ccggctccac cgtgtactac 960

cccaacgaga aggactgcga gacaagaggc gaccacgtgt tctgcgatac cgcctgtggc 1020

atcaatgtgg ccgagcagtc caaagagtgc aacatcaaca tctccaccac caactatccc 1080

tgcaaggtgt ccaccggcag gcaccctatt tccatggtgg ctctgtctcc actgggcgcc 1140

ctggtggctt gttataaggg cgtgtcctgc tccatcggct ccaacagagt gggcatcatc 1200

aagcagctga acaagggctg cagctacatc accaaccagg acgccgatac cgtgaccatc 1260

gacaataccg tgtatcagct gtccaaggtg gaaggcgagc agcacgtgat caagggcaga 1320

cctgtgtcct ccagcttcga ccccgtgaag ttccctgagg atcagttcaa cgtggccctg 1380

gaccaggtgt tcgagtccat cgagaactct caggctctgg tggaccagtc caaccggatc 1440

ctgtcctctg ccgagtctgc tatcggcggc tatatccccg aggctcctag agatggccag 1500

gcctatgttc ggaaggatgg cgaatgggtg ctgctgtcta ccttcctcgg aggcctggtg 1560

cctagaggct ctcaccacca tcatcaccac tccgcttggt cccatccaca gttcgagaag 1620

tga 1623

<210> 53

<211> 538

<212> PROTEIN

<213> Artificial

<220>

<223> Artificial

<400> 53

Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln

1 5 10 15

His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr

20 25 30

Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe

35 40 45

Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro

50 55 60

Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu

65 70 75 80

Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu

85 90 95

Asn Pro Arg Gln Ser Lys Lys Arg Lys Arg Arg Val Ala Thr Ala Ala

100 105 110

Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile Arg Leu Glu Ser

115 120 125

Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr Asn Glu Ala Val

130 135 140

Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr Ala Val Arg Glu

145 150 155 160

Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala Ile Asn Lys Asn

165 170 175

Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser Phe Ser Gln Phe

180 185 190

Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser Asp Asn Ala Gly

195 200 205

Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp Ala Glu Leu Ala

210 215 220

Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln Ile Lys Leu Met

225 230 235 240

Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe Gly Phe Leu Ile

245 250 255

Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln Leu Pro Ile Phe

260 265 270

Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala Ala Pro Ser Cys

275 280 285

Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg Glu Asp Gln Gly

290 295 300

Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr Pro Asn Glu Lys

305 310 315 320

Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp Thr Ala Ala Gly

325 330 335

Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile Asn Ile Ser Thr

340 345 350

Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His Pro Ile Ser Met

355 360 365

Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys Tyr Lys Gly Val

370 375 380

Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile Lys Gln Leu Asn

385 390 395 400

Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp Thr Val Thr Ile

405 410 415

Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly Glu Gln His Val

420 425 430

Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro Val Lys Phe Pro

435 440 445

Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe Glu Ser Ile Glu

450 455 460

Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile Leu Ser Ser Ala

465 470 475 480

Glu Lys Gly Asn Thr Ser Gly Arg Glu Asn Leu Tyr Phe Gln Gly Gly

485 490 495

Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

500 505 510

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

515 520 525

Ile Glu Gly Arg His His His His His

530 535

<210> 54

<211> 1617

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 54

atgtcctgga aggtcgtgat catcttctcc ctgctgatca ccccccagca cggcctgaaa 60

gagtcctacc tggaagagag ctgctccacc atcaccgagg gctacctgtc tgtgctgcgg 120

accggctggt acaccaacgt gttcaccctg gaagtgggcg acgtggaaaa cctgacctgc 180

gccgatggcc ccagcctgat caagaccgag ctggacctga ccaagtccgc cctgcgggaa 240

ctgagaaccg tgtctgccga tcagctggcc agagaggaac agatcgagaa cccccggcag 300

tccaagaaac ggaagcggag agtggccacc gccgctgctg tgacagctgg cgtggccatt 360

gccaagacca tccggctgga atccgaagtg accgccatca agaacgccct gaaaaagacc 420

aacgaggccg tgtctaccct gggcaatggc gtgcgagtgc tggctacagc tgtgcgcgag 480

ctgaaggact tcgtgtccaa gaacctgacc cgggccatca acaagaacaa gtgtgatatc 540

gccgacctga agatggccgt gtcctttagc cagttcaacc ggcggttcct gaacgtcgtg 600

cggcagttct ctgacaacgc cggcatcacc cctgccatct ccctggatct gatgaccgac 660

gccgagctgg ctagagccgt gtccaacatg cctacctctg ccggccagat caagctgatg 720

ctggaaaacc gggccatggt gcgacggaag ggcttcggct ttctgatcgg cgtgtacggc 780

tcctccgtga tctacatggt gcagctgcct atcttcggcg tgatcgacac cccctgctgg 840

atcgtgaagg ccgctcctag ctgctccgag aagaagggca actacgcctg cctgctgaga 900

gaggaccagg gctggtactg tcagaacgcc ggctccaccg tgtactaccc caacgagaag 960

gactgcgaga cacggggcga ccacgtgttc tgtgataccg ctgctggcat caacgtggcc 1020

gagcagtcca aagagtgcaa catcaacatc tccaccacca actacccctg caaggtgtcc 1080

accggcaggc accccatctc tatggtggcc ctgtctcctc tgggcgccct ggtggcttgt 1140

tacaagggcg tgtcctgctc catcggctcc aacagagtgg gcatcatcaa gcagctgaac 1200

aagggctgca gctacatcac caaccaggac gccgacaccg tgaccatcga caataccgtg 1260

tatcagctgt ccaaggtgga aggcgagcag cacgtgatca agggcagacc cgtgtcctcc 1320

agcttcgacc ccgtgaagtt ccccgaggat cagttcaatg tggccctgga ccaggtgttc 1380

gagtccatcg agaactccca ggctctggtg gaccagtcca accggatcct gtcctctgcc 1440

gagaagggaa acacctccgg cagagagaac ctgtattttc aaggcggcgg aggctccggc 1500

tacatccctg aggctcctag agatggccag gcctacgtgc ggaaggatgg cgaatgggtg 1560

ctgctgtcca ccttcctggg cggcatcgag ggcagacacc accatcatca ccactga 1617

<---

Claims (22)

1. An immunogenic modified F protein of human metapneumovirus (hMPV) or a fragment thereof, stabilized in a pre-fusion conformation, containing a recombinant single-chain polypeptide that does not contain a fusion peptide (FP) and containing an F2 domain, a heterologous peptide linker and an F1 domain, wherein said linker is located between said F2 and F1 domains and contains a cysteine residue which forms a disulfide bond with a non-natural cysteine residue present in said F1 domain. 2. A protein or a fragment thereof according to claim 1, characterized in that the length of said linker is from 1 to 10 amino acid residues. 3. A protein or fragment thereof according to claim 1 or 2, characterized in that said F2 domain contains or consists of an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99 % sequence identity to SEQ ID NO: 2. 4. Protein or its fragment according to any one of paragraphs. 1-3, characterized in that said F1 domain contains or consists of an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 3 or its residues 17-338. 5. Protein or its fragment according to any of the preceding paragraphs, characterized in that: (a) said F1 domain contains a cysteine residue substituted at position 236 of SEQ ID NO: 3; (b) said recombinant single chain polypeptide contains a cysteine residue substituted at position 338 relative to the amino acid positions of the native hMPV F protein sequence SEQ ID NO: 1; (c) said linker contains a cysteine residue that forms a disulfide bond with a non-natural cysteine residue substituted at position 236 in said F1 domain of SEQ ID NO: 3; and/or (d) said linker contains a cysteine residue that forms a disulfide bond with a non-natural cysteine residue substituted at position 338 relative to the amino acid positions of the native hMPV F protein sequence SEQ ID NO: 1. 6. A protein or fragment thereof according to any of the preceding paragraphs, characterized in that the length of said linker is from 2 to 10 amino acid residues or said linker consists of 1-5 amino acids and is preferably selected from the group consisting of the sequences CGAGA, CGAGV, CGAAV, AGCGA, CAAAV and CAAFV. 7. A protein or fragment thereof according to any of the preceding claims, characterized in that said single-chain polypeptide contains one of the following combinations of substitutions: N97Q, R102G and G294E; N97Q, R102G, T160F, I177L and G294E; T49M, I67L, E80N, N97Q, R102G, A161M, F258I and G294E; T49M, E51C, I67L, N97Q, R102G, A161M, K166C, S266D, G294E, I480C and L481C; or T49M, N97Q, R102G, I137W, A147V, A159V, A161M, I177L and G294E, wherein said positions are relative to the amino acid positions of the native hMPV F protein SEQ ID NO: 1. 8. A protein or fragment thereof according to any one of the preceding claims, characterized in that said single chain polypeptide contains an auxiliary trimerization domain (foldon) consisting of the amino acid sequence of SEQ ID NO: 10 or any sequence from SEQ ID NO: 29-33, and optionally additionally containing the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35 before the specified foldon domain. 9. A protein or fragment thereof according to any one of the preceding claims, characterized in that said single chain polypeptide contains or consists of an amino acid sequence having any of SEQ ID NO: 5-9 or 24-28. 10. An immunogenic composition containing an immunogenic protein or a fragment thereof according to any of the preceding paragraphs, additionally containing a pharmaceutically acceptable carrier and/or excipient, and the said composition can induce neutralizing antibodies that recognize the native hMPV F protein. 11. Immunogenic composition according to claim 10, additionally containing an adjuvant. 12. A method of treating or preventing hMPV infection in a human subject, comprising administering to the human subject an immunogenic composition or vaccine containing an immunogenic protein or fragment thereof according to any one of claims. 1-9.