WO2023110618A1 - Stabilized pre-fusion hmpv fusion proteins - Google Patents

Abstract

The present invention relates to stabilized pre-fusion human pneumovirus (HMPV) F proteins, to nucleic acid molecules encoding said HMPV F proteins, as well as to the use thererof.

Description

STABILIZED PRE-FUSION HMPV FUSION PROTEINS

The present invention relates to the field of medicine. The invention in particular relates to recombinant pre-fusion HMPV F proteins and to fragments thereof and to nucleic acid molecules encoding the HMPV F proteins and fragments thereof, and to uses thereof, e.g. in vaccines.

BACKGROUND OF THE INVENTION

Human metapneumovirus (HMPV) belongs to the family Orthopneumovirinae which also includes hRSV. Genetic analysis of HMPV isolates have revealed two major groups A and B and four minor groups (Al, A2, Bl and B2) mainly based on the diversity of the attachment protein (G) and the fusion protein (F) (van Hoogen et al., Emerg. Infec.t Dis. 10(4): 658-666, 2004). Recently Noa et al. (Microorganisms. 2020 Aug 21 ;8(9): 1280.) described the subdivision of A2 into A2a and A2b, with the latter currently circulating.

To infect a host cell, HMPV, like other enveloped viruses such as influenza virus, RSV and HIV, requires fusion of the viral membrane with a host cell membrane. For HMPV the conserved fusion protein (HMPV F protein) fuses the viral and host cell cellular membranes. The HMPV F protein initially folds into a "pre-fusion" conformation. This metastable structure has recently been solved (Battles et al., Nat Commun. Nov 16;8(1): 1528, 2017.) During cell entry, the prefusion conformation undergoes refolding and conformational changes to its "post-fusion" conformation (McLellan, J. Virol. 85(15): 7788-7796, 2010; Swanson, PNAS 108(23): 9619- 9624, 2011). Thus, the HMPV F protein is a metastable protein that drives membrane fusion by coupling irreversible protein refolding to membrane juxtaposition by initially folding into a metastable form (pre-fusion conformation) that subsequently undergoes discrete/stepwise conformational changes to a lower energy conformation (post-fusion conformation). HMPV was first identified in 2001 in clinical samples from pediatric patients who had disease resembling that of human Respiratory Syncytial Virus (RSV) but in samples from whom RSV could not be identified (van den Hoogen et al., Nat. Med. 7(6): 719-724, 2001). Subsequent studies showed that HMPV is a major cause of both upper and lower respiratory tract infections in infants, young children, the elderly and among immunocompromised persons or those with underlying chronic medical conditions. The clinical manifestation of HMPV infections is similar to that caused by RSV, ranging from mild respiratory illness to bronchiolitis and pneumonia. HPMV infections appear to be ubiquitous since virtually all children are seropositive by the age of 5 years. Previous epidemiological studies have suggested that HMPV infections cause lower respiratory tract infection in 5-15% of otherwise healthy infants (Falsey et al., J. Infect. Dis. 187: 785-790, 2003). Among children younger than 5 years who were hospitalized with acute respiratory infection or fever, HMPV was detected in 4.9% of the children, with a population similar to that of influenza and higher than that of parainfluenza virus type 3 (PIV-3) (Williams et al., J Infect Dis. 201(12): 1890-1898, 2010). Incidence of disease is the highest for children younger than 0-6 months. Recurrent infection with HMPV also has been described.

To date, most of the infection data are derived from studies in children, however evidence accumulates that HMPV can cause serious illness in adults as well. As for influenza and RSV, infection in adults is most severe in the elderly and patients with chronic underlying medical conditions. The incidence of symptomatic infection in the adult population is typically less than 5% in most studies (Falsey, Pediatr. Infect. Dis. J. 27: S80- 83, 2008). It has been quantified by Gaunt et al. that influenza A, influenza B and RSV are the leading viral cause of respiratory disease in older adults (>65 years), followed by HMPV, which attributed to 2.1 disability-adjusted life years (DALYs) per 1000 hospitalized population (Gaunt et al., J. Clin. Virol. 52(3): 215-221, 2011). There are currently no approved therapeutic or prophylactic treatments for the management of HMPV infections. Since HPMV, next to RSV, represents a major cause of acute viral respiratory tract infection, there is a need for effective therapies or vaccines against HMPV.

SUMMARY OF THE INVENTION

The present invention provides recombinant stabilized pre-fusion human pneumovirus (HMPV) fusion (F) proteins, i.e. recombinant HMPV F proteins that are stabilized in the prefusion conformation. The HMPV F proteins of the invention comprise at least one epitope that is specific to the pre-fusion conformation of the F protein. In certain embodiments, the prefusion hMPV F proteins are soluble proteins (i.e. are not membrane-bound, and lack the transmembrane and cytoplasmic regions). The invention also provides nucleic acid molecules encoding the pre-fusion HMPV F proteins according to the invention and vectors comprising such nucleic acid molecules. The invention also relates to pharmaceutical compositions, preferably vaccine compositions, comprising one or more HMPV F proteins, nucleic acid molecules and/or vectors according to the invention, and to the use thereof in inducing an immune response against HMPV F protein, in particular to the use thereof as a vaccine. The invention also relates to methods for inducing an anti-human pneumovirus (HMPV) immune response in a subject, comprising administering to the subject an effective amount of a prefusion HMPV F protein, a nucleic acid molecule encoding said HMPV F proteins, and/or a vector comprising said nucleic acid molecule. Preferably, the induced immune response is characterized by neutralizing antibodies to HMPV, T cells and/or protective immunity against HMPV. In particular aspects, the invention relates to a method for inducing neutralizing antihuman pneumovirus (HMPV) F protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a pre-fusion HMPV F protein, a nucleic acid molecule encoding said HMPV F protein, and/or a vector comprising said nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

FIG. 1: Simplified schematic drawing of soluble versions of protein monomers of the invention. The residue position is numbered as in the full-length wild type protein including signal peptide. Processed protein designs (a and b) and single chain protein design (c) are depicted. Fl and F2 domains are indicated, as well as F2 C-terminal amino acid after processing, fusion peptide (FP), fibritin trimerization domain (foldon), and the linker (GSGSGR) in single chain proteins (c) between F2 and Fl. The depicted proteins are examples.

FIG. 2: Expi-HEK expressed proteins corresponding to the ectodomain of HMPV F of strain TN/00/3-14 with furin cleavage site between F2 and Fl. In crude supernatant at the day of harvest (a) the proteins are evaluated by binding to pre-fusion specific antibody ADI- 14448 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on the Octet (Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation), (b) Analytical SEC analysis of monomeric HMPV F content in the crude supernatant at day of harvest.

FIG. 3: Expi-HEK expressed proteins corresponding to the ectodomain of HMPV F with furin cleavage site between F2 and Fl and C-terminal foldon trimerization domain. HMPV without stabilizing mutations (MPV190845) is compared to designs with stabilizing mutations, (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The average per design is reported with error bar indicating the standard deviation, (b) Additionally, expression levels and trimer content are analyzed in analytical SEC. (c) One of the hMPV preF monomers of the trimer x-ray structure is depicted. The refolding region 2 (RR2) is highlighted in dark gray. The location of P360C/A459C is indicated with spheres on the cartoon representation of a hMPV preF monomer. C459, located in P-sheet 23, is in the refolding region 2 (RR2; dark gray) and once the disulfide bridge is formed to C360, located in the loop between a8 and P 14, the RR2 is locked in preF conformation, (d) One preF monomer of the trimeric x-ray structure with RR2 in dark gray and the monomer+DS7 with missing RR2 are shown as cartoons. The location of the disulfide bridge is not in the footprint of DS7.

FIG. 4: Expi-HEK expressed HMPV F proteins with furin cleavage site between F2 and Fl and foldon trimerization domain. Designs with different linker between Fl and foldon and designs with different stabilizing mutations are compared, (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) Expression levels and trimer content are analyzed in analytical SEC. A 0.05min shift in retention time between the proteins is indicated with vertical dashed lines.

FIG. 5: Expi-HEK expressed HMPV F proteins with furin cleavage site between F2 and Fl, foldon trimerization domain and SGGG linker. Mutations optimizing the dimer interface in the apex of preF are evaluated in a backbone with D185P and P360C/A459C. (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) Expression levels and trimer content are analyzed in analytical SEC. The shoulder before the trimer peak for MPV201028 is indicated.

FIG. 6: Expi-HEK expressed proteins with furin cleavage site between F2 and Fl, foldon trimerization domain and SAIG or SGGG linker. Non-stabilized designs are compared to designs with stabilizing mutations, (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) The trimer content and retention time of the trimer are analyzed in analytical SEC. The crude supernatant and supernatant incubated for 30 min at 50, 60 and 70 °C are analyzed. Trimer elutes between 4.5 and 4.6 min. The protein designs with stabilizing mutations show increased expression and resilient to heat stress.

FIG. 7: Purification and characterization of selected preF hMPV proteins with furin cleavage site between F2 and Fl, foldon trimerization domain and SAIG or SGGG linker. PostF was produced as control protein, (a) After harvest the proteins are purified first via affinity chromatography C-tagXL 5ml column using the C-terminal C-tag (data not shown) followed by size exclusion chromatography on a Superose 6 (10/300) column. A representative example of MPV190444 is depicted. Fractions between the dashed lines corresponding to HMPV F trimer were pooled, (b) SDS-PAGE analysis under reduced conditions for selected preF proteins (Coomassie stained). Cleaved (Fl) and uncleaved material (F0) is observed for preF hMPV proteins, (c) Trimer content of purified hMPV proteins analyzed by SEC-MALS. Samples were analyzed after purification and after storage at 4°C for 6 months or longer. Aggregates (A), Trimer (T) and smaller molecular species (monomers; M) are indicated, (d) In vitro antigenicity of purified HMPV-F proteins. Binding to pre-fusion specific antibodies ADI-14448 and ADI-15614, pre and post- binding antibody ADI-18992 and non-preF specific antibody DS7 are measured using biolayer interferometry on the Octet, (e) Melting temperature of the HMPV-F proteins measured by DSF. For each sample the derivative is plotted, and the assigned melting points are indicated in degree Celsius. HMPV-F proteins can have multiple melting temperatures, (f) Trimer content and stability after freezing evaluated by SEC-MALS in a buffer without (left) and with (right) sucrose. Reference sample storage at 4°C (light gray line) compared to one-time (dashed line), 5 times (dotted line) and 10 times (dash-dotted line) snap freezing, (g) Stability and trimer content after 2 (g) and 14 (h) weeks at 37°C evaluated by SEC-MALS. Reference sample stored at 4°C (dark gray) is compared to samples incubated at 37°C (light gray). Aggregates (A) and Trimer (T) are indicated.

FIG. 8: Expi-HEK expression of stabilized single chain HMPV F proteins with a linker between F2 and Fl, foldon trimerization domain and an SAIG linker between Fl and foldon. (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) The trimer content and retention time of the trimer are analyzed in analytical SEC. Trimer elutes between 4.2 and 4.35 min. (c) Images obtained by nsEM of the purified HMPV-F proteins with wild-type L73 (left) and with L73W (right).

FIG. 9: Expi-HEK expression of stabilized single chain HMPV F proteins with a linker between F2 and Fl, foldon trimerization domain and a SAIG or SGGG liker between Fl and foldon. Non-stabilized designs are compared to designs with stabilizing mutations, (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) The trimer content and retention time of the trimer are analyzed in analytical SEC. FIG. 10: Expi-HEK expression of stabilized single chain HMPV F proteins with a linker between F2 and Fl, foldon trimerization domain and a SGGG liker between Fl and foldon.

Combination of various stabilizing mutations are compared, (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) The trimer content and retention time of the trimer are analyzed in analytical SEC. The crude supernatant and supernatant incubated for 30 min at 50, 60 and 70 °C are analyzed. Trimer elutes between 4.4 and 4.6 min. The reduced retention time and trimer reduction is an indication for instability.

FIG. 11: Expi-HEK expression of stabilized HMPV F proteins without cleavage site between F2 and Fl. Amino acids of F2 are joined to Fl to create single chain proteins comprising different linkers, (a) In crude supernatant at the day of harvest proteins are evaluated by antibody binding (as Figure 2). Transfections are performed with n=2. The Average per design is reported with error bar indicating the standard deviation, (b) The retention times of the trimer are analyzed in analytical SEC (b). Trimer elutes between 4.16 and 4.25 min. A reduced retention time is an indication for opening up of the trimer. (c) The trimer content and retention time of the trimer are analyzed in analytical SEC for selected designs.

FIG. 12: Purification and characterization of selected preF hMPV single chain proteins with foldon trimerization domain and SAIG or SGGG linker between Fl and foldon. PostF was added as control protein, (a) After harvest the proteins are purified first via affinity chromatography C-tagXL 5ml column using the C-terminal C-tag (data not shown) followed by size exclusion chromatography on a Superose 6 (10/300) column. A representative example is depicted, (b) SDS-PAGE analysis under reduced conditions (Coomassie stained), (c) Trimer content of purified hMPV proteins analyzed by SEC-MALS. Samples were analyzed after purification and after storage at 4°C for 6 months or longer. Aggregates (A), Trimer (T) and smaller molecular species (monomers; M) are indicated, (d) In vitro antigenicity of purified HMPV-F proteins (as Figure 7). (e) Melting temperature of the HMPV-F proteins measured by DSF. For each sample the derivative is plotted, and the assigned melting points are indicated in degree Celsius. HMPV-F proteins can have multiple melting temperatures, (f) Trimer content and stability after freezing evaluated by SEC-MALS in a buffer without (left) and with (right) sucrose. Reference sample storage at 4°C (light gray line) compared to one-time (dashed line), 5 times (dotted line) and 10 times (dash-dotted line) snap freezing. Stability and trimer content after 2 (g) and 14 (h) weeks at 37°C evaluated by SEC-MALS. Reference sample stored at 4°C (dark gray) is compared to samples incubated at 37°C (light gray). Aggregates (A) and Trimer (T) are indicated.

FIG. 13: A. Expi-HEK expression of stabilized HMPV F proteins without cleavage site between F2 and Fl. Amino acids of F2 are joined to Fl to create single chain proteins comprising different linkers, (a) The trimer content and retention time of the trimer are analyzed in analytical SEC in crude supernatant at the day of harvest.

B. In crude supernatant at the day of harvest the F proteins are evaluated by antibody binding using Octet and binding was measured as nm shift after 300 seconds.

DETAILED DESCRIPTION OF THE INVENTION

The fusion protein (F) of the human pneumovirus (HMPV or hMPV) is involved in fusion of the viral membrane with a host cell membrane, which is required for infection. The HPMV F mRNA is translated into a 539 amino acid precursor protein designated F0, which contains a signal peptide sequence at the N-terminus (i.e. amino acid residues 1-18 of SEQ ID NO: 1) (Ulbrandt et al, Journal of General Virology (2008), 89, 3113-3118) which is removed by a signal peptidase in the endoplasmic reticulum. F0 is cleaved by cellular proteases generating two domains or subunits designated Fl and F2. The Fl domain (corresponding to amino acid residues 103-539 of SEQ ID NO: 1) contains a 23 hydrophobic fusion peptide at its N-terminus (corresponding to amino acids 103-126 of SEQ ID NO: 1), the refolding region 2 (RR2) (corresponding to amino acids 426-491 of SEQ ID NO: 1) and the C-terminus contains the transmembrane region (TM) (corresponding to amino acid residues 492-513 of SEQ ID NO: 1) and the cytoplasmic region (corresponding to amino acid residues 514 - 539) (Ulbrandt et al, Journal of General Virology (2008), 89, 3113-3118). The F2 domain (corresponding to amino acid residues 19-102 of SEQ ID NO: 1) is covalently linked to Fl by two disulfide bridges (Ulbrandt et al, Journal of General Virology (2008), 89, 3113-3118). The F1-F2 heterodimers are assembled as homotrimers in the virion.

A vaccine against HMPV infection is not currently available but is desired. One potential approach to producing a vaccine is a subunit vaccine based on purified HMPV F protein. However, for this approach it is desirable that the purified HMPV F protein is in a conformation which resembles the conformation of the pre-fusion state of HMPV F protein, and which is stable over time, and can be produced in sufficient quantities. In addition, for a subunit-based vaccine, the HMPV F protein needs to be truncated by deletion of the transmembrane (TM) and the cytoplasmic region to create a soluble secreted F protein (F or sF). Because the TM region is responsible for membrane anchoring and trimerization, the anchorless soluble F protein is considerably more labile than the full-length protein and will readily refold into the post-fusion end-state. In order to obtain soluble F protein in the stable pre-fusion conformation that shows high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized.

The present invention now provides recombinant stable pre-fusion HMPV F proteins, i.e. HMPV F proteins that are stabilized in the pre-fusion conformation. In the research that led to the present invention, several modifications, such as mutations, deletions, insertions, and fusions of amino acids as compared to the amino acid sequence of a wild-type HMPV F protein, were introduced in order to obtain said stable pre-fusion HMPV F proteins. The stable pre-fusion HMPV F proteins of the invention are in the pre-fusion conformation, i.e. they comprise (display) at least one epitope that is specific to the pre-fusion conformation F protein. An epitope that is specific to the pre-fusion conformation F protein is an epitope that is not present in the post-fusion conformation. Without wishing to be bound by any particular theory, it is believed that the pre-fusion conformation of HMPV F protein may contain epitopes that are the same as those present on the HMPV F protein expressed on natural HMPV virions, and therefore may provide advantages for eliciting protective neutralizing antibodies. In certain embodiments, the proteins of the invention comprise at least one epitope that is recognized by a pre-fusion specific anti-HMPV monoclonal antibody. Examples of such pre-fusion HMPV antibodies are MPE8 (Corti et. al., Nature 50(7467): 439-443, 2013) and ADI-14448 (Gilman et al, Sci Immunol. 2016 Dec 16;l(6):eaaj l879. doi: 10.1126/SciImmunol.aaj l879. Epub 2016 Dec 9). In certain embodiments, the recombinant pre-fusion HMPV F proteins comprise at least one epitope that is recognized by at least one pre-fusion specific monoclonal antibody as described above and are trimeric. In certain embodiments, the stable pre-fusion HMPV F proteins according to the invention are soluble and thus comprise a truncated Fl domain (i.e. the transmembrane and cytoplasmic region have been (partially) deleted).

The present invention in particular provides stabilized pre-fusion human pneumovirus (HMPV) F proteins, comprising an Fl and an F2 domain, comprising an amino acid sequence wherein the amino acid residue at position 69 is Y, and/or the amino acid residue at position 73 is W, and/or the amino acid residue at position 191 is I, and/or the amino acid residue at position 116 is H, and/or the amino acid residue at position 342 is P, and/or the amino acid residue at position 453 is Q, wherein the numbering of the amino acid positions is according to the numbering is amino acid residues in SEQ ID NO: 1.

As used throughout the present application, the position of the amino acid residues (or amino acid positions) are given in reference to the sequence of the HMPV F protein of SEQ ID NO: 1. As used in the present invention, the wording “the amino acid residue at position “x” of the HMPV F protein thus means the amino acid corresponding to the amino acid at position “x” in the HMPV F protein of SEQ ID NO: 1. It is noted that, in the numbering system used throughout this application 1 refers to the N-terminal amino acid of an immature F0 protein (SEQ ID NO: 1), i.e. including the signal peptide. When a HMPV strain other than the strain CAN97-83 of SEQ ID NO: 1 is used, the amino acid positions of the F protein are to be numbered with reference to the numbering of the F protein of the strain of SEQ ID NO: 1, by aligning the sequences of the other HMPV strain with the F protein of SEQ ID NO: 1 with the insertion of gaps as needed. Sequence alignments can be done using methods well known in the art, e.g. by CLUSTALW, Bioedit or CLC Workbench.

An amino acid according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as e.g. D-amino acids (the D- enantiomers of amino acids with a chiral center), or any variants that are not naturally found in proteins, such as e.g. norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the protein backbone, and glycine that is more flexible than other amino acids. Table 4 shows the abbreviations and properties of the standard amino acids. It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology procedures.

According to the present invention novel trimeric HMPV proteins in the pre-fusion conformation have been generated. The modifications according to the invention preferably result in increased expression levels and/or increased stabilization of the pre-fusion HMPV F trimers as compared to HMPV F proteins that do not comprise these modification(s). In addition, or alternatively, the modifications according to the invention result in increased trimer content and trimer stability after storage at 4°C as compared to HMPV F proteins that do not comprise these modification(s). In particular, the modifications according to the invention result in increased trimer content and trimer stability after storage at 4°C for at least 6 months as compared to HMPV F proteins that do not comprise these modification(s).

In certain embodiments, the proteins further comprise one or more non-native intra- or inter-protomer disulfide bonds that further stabilize the proteins in the pre-fusion conformation. In particular embodiments, the one or more disulfide bonds are selected from an intraprotomeric disulfide bond between the amino acid residues 140 and 147 and/or an intraprotomeric disulfide bond between the amino acid residues 141 or 161, and/or an intraprotomeric disulfide bond between the amino acid residues 360 and 459.

According to the invention it has been demonstrated that the presence of specific amino acids and/or one or more disulfide bridges at the indicated positions increase the stability of the proteins in the pre-fusion conformation. According to the invention, the specific amino acids or disulfide bridges are introduced by substitution (mutation) of the amino acid at that position into a specific amino acid according to the invention. According to the invention, the proteins thus comprise one or more mutations in their amino acid sequence, i.e. the naturally occurring amino acids at the indicated positions have been substituted with another amino acid. In further embodiments, the amino acid residue at position 185 is P.

In further embodiments, the amino acid residue at position 294 is E, and/or the amino acid residue at position 368 is N.

In certain embodiments, the proteins comprise a non-native cleavage site between the Fl and F2 domain. In a preferred embodiment, the non-native cleavage site is a furin cleavage site, e.g. RRRR or RRAR, in order to improve the processing of the protein in the cells that are used to produce the proteins.

In certain embodiments, The F2 and Fl domains are directly linked through a linking sequence, in order create a single chain polypeptide. In certain instances, part of the Fl (at the N-terminus) and/or F2 domain (at the C-terminus) have been deleted and replaced by a linking sequence. In particular embodiments, at least the amino acids 97-106 have been deleted and replaced by a linking sequence of 1-10 amino acids. In a preferred embodiment, the amino acids 91-110 have been deleted and replaced by a linking sequence of 1-10 amino acids

In certain embodiments, the pre-fusion HMPV F proteins are soluble proteins, i.e. not membrane bound. Thus, in certain embodiments, the proteins comprise a truncated Fl domain. In particular embodiments, the truncated Fl domain does not comprise the transmembrane and cytoplasmic regions. The Fl domain may be truncated after the amino acid at position 481, 482, 483, 484, 485, 486, 487, 488 or 489. In certain preferred embodiments, the truncated Fl domain comprises the amino acids 103-481, 107-481, 111- 481, 103-482, 107-482, 111-482, 103-489, 107-489, or 111-489 of the HMPV F protein.

In certain embodiments, a heterologous trimerization domain is linked to the truncated Fl domain, optionally through a linking sequence. By linking a heterologous trimerization domain to the C-terminal amino acid residue of a truncated Fl domain, either directly or through a linking sequence, combined with the stabilizing mutation(s), HMPV F proteins are provided that show high expression and that bind to pre-fusion-specific antibodies, indicating that the proteins are in the pre-fusion conformation. In addition, the HMPV F proteins remain stabilized in the pre-fusion conformation, i.e. even after processing of the proteins (i.e. after cleaving into Fl and F2) the proteins still bind to the pre-fusion specific antibodies, indicating that the pre-fusion specific epitope is retained after processing of the proteins.

In certain embodiments, a fibritin - based trimerization domain is fused to the C- terminus of the soluble HMPV-F. This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was described earlier as an artificial natural trimerization domain (Letarov et al., Biochemistry Moscow 64: 817-823 (1993); S-Guthe et al., J. Mol. Biol. 337: 905-915. (2004)). In certain embodiments, the Fl domain is truncated at position 481, 482 or 489 and fused C terminally to the heterologous trimerization domain using an amino acid linker, e.g. SAIG or SGGG. In certain embodiments, the heterologous trimerization domain is the fibritin domain comprising the amino acid sequence: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 2).

In certain embodiments, the pre-fusion HMPV F proteins thus comprise one or more mutations (as compared to the wild-type HMPV F protein, in particular the HMPV F protein of SEQ ID NO: 1) selected from the group consisting of: a) a mutation of the amino acid residue T at position 69; b) a mutation of the amino acid residue L at position 73; c) a mutation of the amino acid residue A at position 116; d) a mutation of the amino acid residue A at position 140; e) a mutation of the amino acid residue L at position 141; f) a mutation of the amino acid residue A at position 147; g) a mutation of the amino acid residue A at position 161; h) a mutation of the amino acid residue D at position 185; i) a mutation of the amino acid residue V at position 191; j) a mutation of the amino acid residue N at position 342; k) a mutation of the amino acid residue P at position 360; l) a mutation of the amino acid residue H at position 368; m) a mutation of the amino acid residue E at position 453; and n) a mutation of the amino acid residue A at position 459, wherein the numbering is according to the numbering of amino acids of SEQ ID NO: 1.

In certain embodiments, the pre-fusion HMPV F proteins comprise one or more further mutations (as compared to the wild-type HMPV F protein, in particular the HMPV F protein of SEQ ID NO: 1) selected from the group consisting of: a) a mutation of the amino acid residue T at position 69 into Y; b) a mutation of the amino acid residue L at position 73 into W; c) a mutation of the amino acid residue A at position 116 into H; d) a mutation of the amino acid residue A at position 140 into C; e) a mutation of the amino acid residue L at position 141 into C; f) a mutation of the amino acid residue A at position 147 into C; g) a mutation of the amino acid residue A at position 161 into C h) a mutation of the amino acid residue D at position 185 into P; i) a mutation of the amino acid residue V at position 191 into I; j) a mutation of the amino acid residue N at position 342 into P; k) a mutation of the amino acid residue P at position 360 into C; l) a mutation of the amino acid residue H at position 368 into N; m) a mutation of the amino acid residue E at position 453 into Q; and n) a mutation of the amino acid residue A at position 459 into C. It is again noted that for the positions of the amino acid residues reference is made to SEQ ID NO: 1. A skilled person will be able to determine the corresponding amino acid residues in F proteins of other HMPV strains. In certain embodiments, the pre-fusion HMPV F proteins comprise at least two mutations (as compared to a wild-type HMPV F protein). In certain embodiments, the proteins comprise at least three mutations. In certain embodiments, the proteins comprise at least four, five or six mutations.

As described above, in certain embodiments (e.g. in the case of soluble F proteins), the proteins of the invention comprise a truncated Fl domain. As used herein a “truncated” Fl domain refers to a Fl domain that is not a full length Fl domain, i.e. wherein either N- terminally or C-terminally one or more amino acid residues have been deleted. In certain embodiments, at least the transmembrane domain and cytoplasmic domain have been deleted to permit expression as a soluble ectodomain. In certain embodiments, the Fl domain is truncated after amino acid residue 481, 482, 483, 484, 485, 486, 487, 488 or 489.

In a preferred embodiment, the HMPV F protein comprises an amino acid sequence wherein the amino acid at position 73 is W, the amino acid at position 116 is H, the amino acid at position 185 is P, the amino acid at position 432 is P, and the amino acid at position 368 is N, the amino acid at position 453 is Q, and comprises an intraprotomeric disulfide bond between the amino acid residues 140 and 147.

In another preferred embodiment, the HMPV F protein comprises a truncated Fl domain consisting of the amino acids 103-481 and an F2 domain, and an amino acid sequence wherein the amino acid at position 73 is W, the amino acid at position 116 is H, the amino acid at position 185 is P, the amino acid at position 432 is P, and the amino acid at position 368 is N, the amino acid at position 453 is Q, and comprises an intraprotomeric disulfide bond between the amino acid residues 140 and 147. In certain embodiments, the proteins according to the invention comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8-16, 18-20, 22-24, 28-31,

33-34, 37-64, or 66, or fragments thereof.

In certain preferred embodiments, the protein according to the invention comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 66.

In certain embodiments, the proteins do not comprise a signal sequence.

As used throughout the present application nucleotide sequences are provided from 5’ to 3’ direction, and amino acid sequences from N-terminus to C-terminus, as custom in the art. It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology procedures. The modifications according to the invention preferably result in increased expression levels and/or increased stabilization of the prefusion HMPV F proteins as compared to HMPV F proteins that do not comprise these modifcation(s).

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

The nucleic acid molecule may be DNA or RNA. In certain embodiments, the RNA is mRNA, modified mRNA, self-replicating (or self-amplifying) RNA, or circular mRNA. The present invention thus also encompasses RNA molecules, e.g. self-amplifying RNA molecules (or replicons) encoding a protein as described herein.

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

In certain embodiment, the nucleic acid molecules according to the invention encode a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-16, 18-20, 22-24, 28-31, 33-34, 37-64, and 66, or fragments thereof.

Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScripts, Invitrogen, Eurofins). The invention also provides vectors comprising a nucleic acid molecule as described above. In certain embodiments, a nucleic acid molecule according to the invention thus is part of a vector.

In certain embodiments of the invention, the vector is an adenovirus vector. An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), a canine adenovirus (e.g., CAdV2), a porcine adenovirus (e.g., PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd). In the invention, a human adenovirus is meant if referred to as Ad without indication of species, e.g., the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.

Most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, a recombinant adenovirus according to the invention is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials. Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g., US6083716; WO 2005/071093; WO 2010/086189; WO 2010/085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine 24: 849-62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P. In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see, e.g., WO 2012/172277), or ChAdOx 2 (see, e.g., WO 2018/215766). In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see, e.g., WO 2019/086466). In certain embodiments, the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see, e.g., WO 2019/086456), or BZ1 (see, e.g., WO 2019/086466).

In a preferred embodiment of the invention, the adenoviral vectors comprise capsid proteins from rare serotypes, e.g. including Ad26. In the typical embodiment, the vector is an rAd26 virus. An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein a “capsid protein” for a particular adenovirus, such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber are of Ad26.

One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of a first serotype with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like. See for example WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also e.g. WO 2019/086461 for chimeric adenoviruses Ad26HVRPtrl, Ad26HVRPtrl2, and Ad26HVRPtrl3, that include an Ad26 virus backbone having partial capsid proteins of Ptrl, Ptrl2, and Ptrl3, respectively)

In certain preferred embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26). In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome. For adenoviruses being derived from non-group C adenovirus, such as Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the El genes of Ad5, such as for example 293 cells, PER.C6 cells, and the like (see, e.g., Havenga, et al., 2006, J Gen Virol 87: 2135-43; WO 03/104467). However, such adenoviruses will not be capable of replicating in non-complementing cells that do not express the El genes of Ad5. The preparation of recombinant adenoviral vectors is well known in the art.

Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Examples of vectors useful for the invention for instance include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.

Typically, a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.

The adenovirus vectors useful in the invention are typically replication deficient. In these embodiments, the virus is rendered replication deficient by deletion or inactivation of regions critical to replication of the virus, such as the El region. The regions can be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding the stabilized pre-fusion HMPV F protein (usually linked to a promoter), or a gene encoding the pre-fusion HMPV F protein fragment (usually linked to a promoter) within the region. In some embodiments, the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions. For E2- and/or E4-mutated adenoviruses, generally E2- and/or E4-complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication. A packaging cell line is typically used to produce sufficient amounts of adenovirus vectors for use in the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenoviruses with a deletion in the El region include, for example, PER.C6, 911, 293, and El A549.

In a preferred embodiment of the invention, the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the El region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p. 4654-63, which is incorporated herein by reference. Typically, the nucleic acid sequence encoding the pre-fusion HMPV F protein is cloned into the El and/or the E3 region of the adenoviral genome.

Host cells comprising the nucleic acid molecules encoding the pre-fusion HMPV F proteins form also part of the invention. The pre-fusion HMPV F proteins may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In general, the production of a recombinant proteins, such the pre-fusion HMPV F proteins of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in said cell. The nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.

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

A “heterologous nucleic acid molecule” (also referred to herein as ‘transgene’) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into for instance a vector by standard molecular biology techniques. A transgene is generally operably linked to expression control sequences. This can for instance be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for example the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g. the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like. The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley -Liss Inc., 2000, ISBN 0-471-34889-9)).

The invention further provides pharmaceutical compositions comprising a pre-fusion HMPV F protein, and/or fragment thereof, and/or a nucleic acid molecule, and/or a vector, as described herein. The invention thus provides compositions comprising a pre-fusion HMPV F protein, or fragment thereof, that displays an epitope that is present in a pre-fusion conformation of the HMPV F protein but is absent in the post-fusion conformation. The invention also provides compositions comprising a nucleic acid molecule and/or a vector, encoding such pre-fusion HMPV F protein or fragment. The invention in particular provides pharmaceutical compositions, e.g. vaccine compositions, comprising a pre-fusion HMPV F protein, a HMPV F protein fragment, and/or a nucleic acid molecule, and/or a vector, as described above and one or more pharmaceutically acceptable excipients.

The invention also provides the use of a stabilized pre-fusion HMPV F protein (fragment), a nucleic acid molecule, and/or a vector, according to the invention, for vaccinating a subject against HMPV. The invention also provides the use of a stabilized pre-fusion HMPV F protein (fragment), a nucleic acid molecule, and/or a vector, according to the invention inducing an immune response against HMPV F protein in a subject. Further provided are methods for inducing an immune response against HMPV F protein in a subject, comprising administering to the subject a pre-fusion HMPV F protein (fragment), and/or a nucleic acid molecule, and/or a vector, according to the invention. Further provided is the use of the prefusion HMPV F protein (fragments), and/or nucleic acid molecules, and/or vectors according to the invention for the manufacture of a medicament for use in inducing an immune response against HMPV F protein in a subject. The invention in particular provides pre-fusion HMPV F protein (fragments), and/or nucleic acid molecules, and/or vectors according to the invention for use as a vaccine.

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

The pre-fusion HMPV F proteins, fragments, nucleic acid molecules and/or vectors according to the invention may be used in stand-alone treatment and/or prophylaxis of a disease or condition caused by HMPV, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies. The invention further provides methods for preventing and/or treating HMPV infection in a subject utilizing the pre-fusion HMPV F proteins or fragments thereof, nucleic acid molecules and/or vectors according to the invention. In a specific embodiment, a method for preventing and/or treating HMPV infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion HMPV F protein (fragment), nucleic acid molecule and/or a vector, as described above. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by HMPV. Prevention encompasses inhibiting or reducing the spread of HMPV or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by HMPV. Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of HMPV infection.

For administering to subjects, such as humans, the invention may employ pharmaceutical compositions comprising a pre-fusion HMPV F protein (fragment), a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The HMPV F proteins, or nucleic acid molecules, preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The HMPV F proteins typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the HMPV F proteins may be formulated into an injectable preparation.

In certain embodiments, a composition according to the invention further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant. The terms “adjuvant” and "immune stimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the HMPV F proteins of the invention. Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g. antibodies or fragments thereof (e.g. directed against the antigen itself or CDla, CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc), which stimulate immune response upon interaction with recipient cells. In certain embodiments the compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose. In certain embodiments, the compositions comprise a combination of adjuvants, e.g. alum and CpG.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, the invention provides methods for making a vaccine against respiratory syncytial virus (HMPV), comprising providing an HMPV F protein (fragment), nucleic acid or vector according to the invention and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. In the present invention, the vaccine comprises an effective amount of a pre-fusion HMPV F protein (fragment) and/or a nucleic acid molecule encoding a prefusion HMPV F protein, and/or a vector comprising said nucleic acid molecule, which results in an effective immune response against HMPV. This provides a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease in frequency of complications such as pneumonia and bronchiolitis due to HMPV infection and replication in a subject. The term “vaccine” according to the invention implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises other components that induce an immune response, e.g. against other proteins of HMPV and/or against other infectious agents, e.g. against RSV, HMPV and/or influenza. The administration of further active components may for instance be done by separate administration or by administering combination products of the vaccines of the invention and the further active components.

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

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

The proteins, fragments, nucleic acid molecules, vectors, and/or compositions may also be administered, either as prime, or as boost, in a homologous or heterologous primeboost regimen. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as ‘priming vaccination’). In certain embodiments, the administration comprises a prime and at least one booster administration.

The invention further provides methods for making a vaccine against HMPV, comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a pre-fusion HMPV F protein or fragment thereof as described herein, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition. In certain embodiments, provided herein are methods of producing an adenoviral particle comprising a nucleic acid molecule encoding a HMPV F protein or fragment thereof (transgene). The methods comprise (a) contacting a host cell of the invention with an adenoviral vector of the invention and (b) growing the host cell under conditions wherein the adenoviral particle comprising the transgene is produced. Recombinant adenovirus can be prepared and propagated in host cells, according to well- known methods, which entail cell culture of the host cells that are infected with the adenovirus. The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.

Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable (see, e.g., WO 2010/060719, and WO 2011/098592, both incorporated by reference herein, which describe suitable methods for obtaining and purifying large amounts of recombinant adenoviruses).

The invention further provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a HMPV F protein or fragment thereof, as described herein.

In addition, the proteins of the invention may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the protein of the invention. The invention thus also relates to an in vitro diagnostic method for detecting the presence of an HMPV infection in a patient said method comprising the steps of a) contacting a biological sample obtained from said patient with a protein according to the invention; and b) detecting the presence of antibody-protein complexes.

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

EXAMPLES

EXAMPLE 1 : Design of a soluble proteins

Several pre-fusion HMPV F protein variants were produced (Figure 1). The soluble candidates were truncated at amino acid position 481 or 482 or 489 (Figure la) (for numbering see SEQ ID NO: 1). With a four amino acid linker (SAIG or SGGG) a fibritin trimerization domain (foldon) (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 2) was fused to the Fl domain (Figure lb).

To allow processing of the protein by furin, the cleavage site between F2 and Fl was changed to a polybasic cleavage site, e.g. RRRR, for proteins based on Figure la and lb (e.g. SEQ ID NO: 4). Alternative single chain designs, that are not processed (i.e. not cleaved between Fl and F2) (Figure 1c) were also designed. An example with the linker sequence GSGSGR to replace the amino acids 97-106 (e.g. SEQ ID NO: 36) is depicted in Fig. 1C.

The designs may further have a linker and C-tag, C-terminal to the foldon sequence to allow affinity purification (e.g. SEQ ID NO: 4).

To stabilize the pre-fusion conformation of the proteins, several point mutations and combinations of point mutations were introduced, e.g one or more of the following mutations T69Y, L73W, A116H, A140C/A147C, L141C/A161C, D185P, V191I, N342P, P360C/A459C, H368N and E453Q (wherein the numbering is according to the numbering of amino acid positions in SEQ ID NO: 1).

EXAMPLE 2: Expression of non-stabilized HMPV proteins without trimerization domain

Based on the F protein of the wild type HMPV strain TN/00/3-14 (SEQ ID NO: 1), the DNA fragments encoding for proteins MPV201042 (SEQ ID NO: 4) and MPV201043 (SEQ ID NO: 5) with furin cleavage site and truncation after position 482 or 489 were synthesized (Genscript, Piscataway, NJ) and cloned in the pcDNA2004 expression vector (modified pcDNA3 plasmid with an enhanced CMV promotor). The expression platform used is the Expi293F expression system (Thermo Fisher Scientific, Waltham, USA) in 96- well format. Designs were co-transfected with furin in a 1 :5 furin: HMPV F DNA ratio. The constructs were transfected in duplicate. Three days after transfection the levels of the prefusion HMPV-F proteins in crude cell supernatant were assessed using biolayer interferometry on the Octet (ForteBio, Portsmouth, UK) and using the monoclonal antibodies ADI-14448 (Gilman et al., Sci Immunol. 2016 Dec 16;l(6):eaaj 1879. doi: 10.1126/sciimmunol.aaj l879. Epub 2016 Dec 9.) and DS7 (Wen et al., Nat Struct Mol Biol . 2012 Mar 4;19(4):461-3. doi: 10.1038/nsmb.2250.)). (Figure 2a). ADI-14448 has been described as cross neutralizing antibody to RSV and HMPV, it is pre-fusion specific and binds to the antigenic site III of the RSV preF. DS7 has been reported as neutralizing antibody to HMPV and the complex between HMPV fusion protein and DS7 is solved by x- ray crystallography. The complex of HMPV F + DS7 depicts that the heptad repeat 2 (HR2), which is part of a region that we defined as the refolding region 2 is in non-preF conformation. For a preferred HPMV F protein according to the invention, DS7 binding is therefore classified as unwanted. For the Octet assay the antibodies are immobilized on anti-human Fc biosensors

(ForteBio, Portsmouth, UK). The procedure is as follows. After equilibration of the sensors in kinetic buffer (ForteBio, Portsmouth, UK) for 600s the sensors are transferred to kinetic buffer with 5 ug/ml of the desired antibody. Subsequently another equilibration step is included in mock cell medium. Lastly the sensors are transferred to a solution of supernatant that contains the pre-fusion HMPV F proteins. The initial slope (also referred to as the association phase) and binding after 300 seconds in nm are reported. The data analysis is done using the ForteBio Data Analysis 8.2 software (ForteBio, Portsmouth, UK). Bar plots were plotted in GraphPad Prism (version 9.0.0, GraphPad Software).

Three days after transfection the crude cell supernatant is analyzed for trimer content on analytical SEC. The content of the expressed proteins in the Expi293F (Thermo Fisher Scientific, Waltham, USA) cell culture harvests was assessed by analytical Size Exclusion Chromatography (SEC) in an Ultra High-Performance Liquid Chromatography (UHPLC) system using a Vanquish system (ThermoFisher Scientific, Waltham, USA) with a Sepax Unix-C SEC-300 4.6X150mm 1.8 pm column (Sepax (231300-4615), injection volume 20pL, flow 0.35mL/min). The constructs transfected in duplo were pooled for SEC analysis. The elution was monitored by a UV detector. The SEC profiles were analyzed by the Chromeleon software version (version 7.2.7, Thermo Fisher Scientific). Chromatograms were plotted in GraphPad Prism (version 9.0.0, GraphPad Software) and are shown in Figure 2b.

Results and conclusion

Non-stabilized HMPV proteins MPV201042 (SEQ ID NO: 4) and MPV201043 (SEQ ID NO: 5) with furin cleavage site and truncation after position 481 and 489, respectively, were expressed (Figure 2a). In octet both proteins showed both anti pre-F and anti non-pre-F binding indicating that the protein is not fully in the prefusion trimeric conformation. The retention time (about 4.8-4.9 min) in analytical SEC showed that the proteins are monomeric (Figure 2b). Further design strategies were applied to obtain trimeric pre-F proteins, in a backbone which is C-terminally truncated at position 481.

EXAMPLE 3 : Trimerization of the proteins and stabilization of pre-F conformation

To the non-stabilized backbone with furin cleavage site, a foldon trimerization domain was added via a 4 residue SAIG linker (MPV190845; SEQ ID NO: 6). Subsequently, one or more of stabilizing mutations D185P (MPV190842; SEQ ID NO: 7), E453Q (MPV190843; SEQ ID NO: 8), Al 16H (MPV190997 SEQ ID NO: 9), H368N (MPV190856; SEQ ID NO: 10), N342P (MPV190991; SEQ ID NO: 11), L73W (MPV19993; SEQ ID NO: 12), A140C/A147C (MPV191012; SEQ ID NO: 13) and P360C/A459C (MPV191013; SEQ ID NO: 16) were introduced (see Figure 3a for the mutations) Furthermore, additional combinations of the stabilizing mutations were evaluated (MPV191386 SEQ ID NO: 14 (with L73W, Al 16H, A140C/A147C, D185P, N342P, H368N and E453Q) and MPV191388 SEQ ID NO: 15 (with L73W, Al 16H, L141/A161C, D185P, N342P, H368N and E453Q)). Expression and analysis of the HMPV F proteins were performed as described in Example 2.

For the structural analysis one monomer of the trimeric HMPV pre-F structure with the pdb code 5WB0 (Battles et al. Nat Commun. 2017 Nov 16;8(1): 1528. doi: 10.1038/s41467-017-01708-9.)) and HMPV monomer in complex with DS7 with the pdb code 4DAG were compared. Pictures were prepared with pymol (version 1.8.6.2 Schrodinger, LLC).

Results and conclusion

With the introduction of the stabilizing mutations and a C-terminal foldon, the signal for pre-F binding in Octet increased (initial slope of ADI-4448 in Figure 3a). All proteins were expressed as trimers according to the peak at 4.4 - 4.5 minutes in analytical SEC (Figure 3b). Although the increase in pre-F trimer based on the initial slope in Figure 3a did not completely correspond to the AUC in analytical SEC, the trend was similar and all proteins improved on trimer content after introduction of one or more stabilizing mutations (peak at 4.45 in Figure 3b).

Combining stabilizing mutations D185P, E453Q, A116H, H368N, N342P, L73W and A140C/A147C (MPV191386; SEQ ID NO: 14) or mutations D185P, E453Q, Al 16H, H368N, N342P, L73W and L141C/A161C (MPV191388; SEQ ID NO: 15) resulted in increased trimer content compared to the other proteins tested in this example (Figure 3b).

The level of unfavorable binding (Binding after 300sec of DS7 in Figure 3a) was reduced somewhat by introduction of L73W (MPV19993; SEQ ID NO: 12) and strongly by P360C/A459C (MPV191013; SEQ ID NO: 16).

In the HMPV preF structure (PDB 5WB0 Battles et. al., 2017, supra) (Figure 3c) the refolding region 2 is well refined enabling the connection of a disulfide bridge between position 360 in Fl and position 459 in RR2 (Figure 3d). In the complex of HMPV monomer+DS7 (PDB 4DAG, Wen et al., 2012, supra) the RR2 is not in pre-F conformation. The superposition of both structures would show a clash between RR2 and DS7. According to the prefusion structure (PDB 5WB0) the position of the P360C/A459C disulfide bridge is not in the footprint of DS7. It is likely that the RR2 is fixed in pre-F conformation by the disulfide bridge P360C/A459C making the binding of DS7 impossible.

EXAMPLE 4: Comparing different linkers between Fl and foldon and additional combinations of stabilizing mutations.

In a stabilized backbone with furin cleavage site, foldon and one or more stabilizing mutations, two different linkers between the Fl domain and the foldon domain, i.e. SAIG (MPV190843; SEQ ID NO: 8 or MPV191386; SEQ ID NO: 14) and SGGG (MPV190894; SEQ ID NO: 17 or MPV191757; SEQ ID NO: 18) were evaluated. Expression and analysis of HMPV F constructs with the different linkers and additional combinations of stabilizing mutations with disulfide P360C/A459C were explored (MPV200641; SEQ ID NO: 19 and MPV1200718; SEQ ID NO: 20) as described in example 2.

Results and conclusion

Both linker variants expressed as trimers. The SGGG-variants (MPV190894; SEQ ID NO: 17 and MPV191757; SEQ ID NO: 18) resulted in an increased pre-F signal in Octet (ADI-4448 initial slope in Figure 4a) and trimer content in analytical SEC (Figure 4b) compared to variants with SAIG linker (MPV190843; SEQ ID NO: 8 and MPV191386; SEQ ID NO: 14). The retention time for the SGGG variant was shifted by 0.05 min to a longer retention time indicating a more compact structure. Adding disulfide bridge P360C/A459C to the combination of L73W, A140C/A147C, D185P, N342P, H368N and E453Q (MPV200641; SEQ ID NO: 19) or L73W, D185P, N342P, H368N and E453Q (MPV190718; SEQ ID NO: 20) reduced the pre-F signal in Octet (ADI-4448 initial slope in Figure 4a) and trimer content in analytical SEC (Figure 4b) compared to MPV191757 (SEQ ID NO: 18). However, the non-preF binding in Octet (DS7 Binding rate in Figure 4a) was strongly decreased with the introduction of the disulfide bridge, likely due to the fixation of RR2 (see Example 3, Figure 3c).

In conclusion the P360C/A459C disulfide bridge had some impact on the expression levels but based on the ratio of preF-binding and non-preF binding, the quality of the preF trimer was improved.

EXAMPLE 5 : Stabilizing the trimer apex HMPV F protein with furin cleavage site, foldon, SGGG linker between Fl and foldon and with stabilizing mutation D185P and disulfide A360P/A489C (MPV201028; SEQ ID NO: 21) was further modified by introduction of mutations in the protomer interface in the apex: MPV201031 (SEQ ID NO: 22) with T69Y, MPV201032 (SEQ ID NO: 23) with T69Y and L73W mutations and MPV201033 (SEQ ID NO: 24) with T69Y, L73W, and V191I mutations. Expression and analysis of HMPV F proteins were performed as described in example 2.

Results and conclusion

The introduction of the dimer interface mutations showed a reduction of the non-preF antibody binding according to the endpoint binding in Octet (DS7 Binding after 300s in Figure 5a). Introduction of T69Y resulted in a more uniform trimer peak according to analytical SEC without the shoulder at 4.35 minutes (Figure 5b). The peak at 4.35 minutes is likely a more open trimer species which has a shorter retention time (see also example 8). T69Y is improving the closed trimer quality. Further improvement by L73W and L191I was not detected in the protein analysis of the supernatant. In conclusion the T69Y mutation at position 69 resulted in a more compact trimer with a higher ratio of preF -binding and non- preF binding and therefore improved the quality of the trimer.

EXAMPLE 6: Combinations of stabilizing mutations

Different combinations of stabilizing mutations were introduced in the non-stabilized protein MPV101285 (SEQ ID NO: 25), comprising a furin cleavage site and a SGGG linker between the Fl domain and the foldon: (i) MPV201027 with disulfide P360C/A459C (SEQ ID NO: 26); MPV201028 with disulfide P360C/A459C and D185P (SEQ ID NO: 21); (ii) to MPV201028, the disulfide A140C/A147C (MPV201029; SEQ ID NO: 27) or E453Q (MPV201030; SEQ ID NO: 28) or T69Y (MPV201031; SEQ ID NO: 22) were added; (iii) to MPV201031 A140C/A147C alone or in combination with E453Q were added (MPV201216; SEQ ID NO: 30 or MPV201038; SEQ ID NO: 29).

Additionally, a non-stabilized and stabilized variant were evaluated with a SAIG- linker between the Fl domain and foldon (MP VI 90845; SEQ ID NO: 6 and MPV201286; SEQ ID NO: 31). Expression and analysis of HMPV F constructs were performed as described in Example 2. For the analytical SEC analysis, the crude supernatant was additionally incubated for 30 minutes at 50, 60 and 70 °C.

Results and conclusion

To stabilize the HMPV protein in its pre-F conformation mutations were subsequently introduced. The disulfide bridge between residues 360 and 459 (MPV201027, SEQ ID NO: 26) resulted in reduced binding to the non-preF antibody DS7 (Figure 6a). Furthermore, the disulfide between residues 360 and 459 increased protein stability compared to the nonstabilized protein (MPV201285 SEQ ID NO: 25) after 30 min heat stress at 50 °C (Figure 6b). The addition of D185P mutation increased expression and heat stability of HMPV preF as measured by analytical SEC (MPV201028; SEQ ID NO: 21).

Addition of A140C/A147C (MPV201029; SEQ ID NO: 27) showed increased HMPV preF trimer expression. Addition of T69Y decreased unfavorable antibody binding of DS7 (MPV201031 (SEQ ID NO: 22) in Figure 6a) and reduced the shoulder before the trimer peak in analytical SEC (Figure 6b). Although the addition of T69Y reduced expression according to analytical SEC (Figure 6b), this can be compensated by the addition A140C/A147C (MPV201216; SEQ ID NO: 30). Further addition of E453Q (MPV201038; SEQ ID NO: 29) showed no further improvements. Comparing linker SAIG (non-stabilized: MPV190845; SEQ ID NO: 6 and stabilized: MPV201286; SEQ ID NO: 31) to linker SGGG (non-stabilized: MPV201285 SEQ ID NO: 25 and stabilized MPV201216 SEQ ID NO: 30) only minor differences were detected.

Example 7: Production, purification and characterization of selected proteins

Production and purification

Several HMPV proteins based on a processed design with furin cleavage site and foldon (Figure lb) were produced and purified. Designs were explored of F proteins derived from several HMPV strains, i.e. strain TN/00/3-14 (SEQ ID NO: 1), Yokohama. JPN/P8356/2016 (SEQ ID NO: 35) and Yokohama.JPN/P8674/2016 (SEQ ID NO: 65). The linker between Fl and foldon was SAIG or SGGG. Several stabilizing mutations were stepwise introduced and explored. MPV190444 (SEQ ID NO: 32) has an additional strep-tag before the C-tag (see Table 1 for detailed list of mutation included per design).

On 70ml-scale the cells were transiently transfected using ExpiFectamine 293 (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions and cultured in a shaking incubator for 5 days at 37°C and 10% CO2. For MPV201031 (SEQ ID NO: 22) and MPV201216 (SEQ ID NO: 30) more protein was produced in 400ml-scale transfection in HEK-E cells and 6 days of production. The culture supernatants were harvested, centrifuged for 10 min at 600rpm and filtered over a 0.22pm PVDF filter to remove cells and cellular debris.

The proteins were purified by means of a two-step protocol. First, the harvested and clarified culture supernatant was loaded on a pre-packed C-tagXL 5- or 6-ml column

(Thermo Fisher Scientific, cat# 494307205, Waltham, USA). This column was pre-packed with an affinity resin (Capture Select) that consists of a C-tag specific single domain antibody, immobilized on an Agarose based bead. This resin is highly specific for binding proteins with the C-tag. Elution of the C-tagged proteins was performed using a TRIS buffer containing 2M MgC12. Based on the UV signal (A280) the eluted fractions were pooled and concentrated using Amicon Ultracel 50kDa MWCO centrifugal filter devices (Merck Millipore, cat# UFC805024, Darmstadt, Germany). Subsequently, the concentrated collected elution peak was applied to a Superose 6 Increase 10/300 column (GE Healthcare, cat# 17- 5172-01, Chicago, USA) equilibrated in running buffer (20mM Tris, 150mM NaCl, pH7.4) for polishing purpose, i.e. remove the minimal amount of multimeric and monomeric protein. Based on the UV signal (A280) the trimer fractions were pooled (Figure 7a).

In addition, a postF hMPV protein (SEQ ID NO: 3) as described by Mas et. al., PLoS Pathog. 2016 Sep 9;12(9):el005859. doi: 10.1371/joumal.ppat.1005859. eCollection 2016 Sep.) was produced and purified. Expression plasmid encoding the recombinant post-fusion hMPV F protein are prepared as in in Example 2. On 300ml-scale the cells are transiently transfected and subsequently purified by means of a two-step protocol (see details above). Subsequently, TEV cleavage was performed to remove the foldon and c-tag. For 15 pg of protein 1 pL of TEV (10 000 Units/mL) was used. The protein-TEV mixture was incubated overnight at 4°C. The TEV-His protease was removed from the protein sample by a Ni Sepharose excel beads (GE Healthcare, 17-3712-03) pull down. Ni Sepharose excel beads were added to the protein-TEV mixture and incubated for 2 hours at room temperature. Flow through was collected via a micro bio-spin column (Bio Rad, 7326204). The cleaved protein sample was heat-shocked for 30 minutes at 45°C. Subsequently, the protein sample was applied to a Superose 6 Increase 10/300 column (GE Healthcare, Chicago, USA) equilibrated in running buffer (20mM Tris, 150mM NaCl, pH7.4) for polishing purpose, i.e. remove the minimal amount of multimeric and monomeric protein. Proteins were subsequently analyzed on Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis (SDS-PAGE) (Figure 7b) under reducing conditions. Proteins were visualized on the gel upon staining with Instant Blue.

Results and conclusion With the preparative SEC aggregates could efficiently be separated from trimer

(Figure 7a) proteins. Yields of the different variants are shown in Table 1. In a processed backbone (i.e. with a furin cleavage site) the addition of H368N and E453Q (MPV190856 SEQ ID NO: 10) showed no improved yield compared to MPV190444 (SEQ ID NO: 32) with D185P only. Additional stabilizing mutations L73W, A116H, A141C/A147C and N342P (MPV191386; SEQ ID NO: 14) resulted in more than 10-fold increase in yield.

Variants with a SGGG linker (e.g. MPV191757 SEQ ID NO: 18) had an additional 1.75-fold increase in expression. These stabilizing mutations also resulted in high trimer yields for the type A2 and B2 Yokohama strains. Designs with disulfide bridge P360C/A459C showed reduced yields. Table 1.: Purified proteins of Example 7 and expression yields

*Yokohama.JPN/P8356/2016; ~ Yokohama.JPN/P8674/2016 Main bands on the reduced SDS-PAGE are corresponding to Fl domains (Figure 7b).

MPV191757 shows minimal unprocessed material indicated with F0.

Trimer content and trimer stability after storage at 4°C

The purified proteins were assessed by analytical Size Exclusion Chromatography (SEC) to study trimer content after purification and trimer content and stability after storage at 4°C. SEC was performed with an Ultra High-Performance Liquid Chromatography (UHPLC) using a Vanquish system (ThermoFisher Scientific) with a Sepax Unix-C SEC-300 4.6X150mm 1.8 pm column (Sepax (231300-4615), injection volume 20pL, flow 0.3mL/min.). The elution was monitored by a UV detector (Thermo Fisher Scientific), a pDawn Light Scatter (LS) detector (Wyatt Technologies), a pT-rEx Refractive Index (RI) detector (Wyatt Technologies) and a Nanostar Dynamic Light Scattering (DLS) detector (Wyatt Technologies). The SEC profiles were analyzed by the Astra 7.3.2.19 software package (Wyatt Technology). Due to the long-time span between the sample measurements the samples were run on column with different lot numbers. A small shift in retention time for the standard was observed and used to correct the retention times of the proteins, allowing superposition of the chromatograms. Chromatograms were plotted in GraphPad Prism (version 9.0.0, GraphPad Software)

Results and conclusion

All purified proteins are trimeric after purification (Figure 7c). The proteins remain trimeric for 6 months, only slight differences in the trimer content could be observed. The design with SGGG linker (MPV191757) showed less changes in the retention time after 6 months storage compared to the design with SAIG linker (MPV191386), which is an indication for a more closed timer (see example 8). Grafting the set of stabilizing mutations into a recent A2 strain (MPV191769; SEQ ID NO 33) and recent B2 strain (MPV191806; SEQ ID NO: 34) strain resulted also in stable trimers at 6 monhs.

In conclusion, proteins of the invention show a high trimer content after purification and the best storage stability was observed for MPV191757 with mutations L73W, Al 16H, A140C/A147C, D185P, N342P, H368N and E453Q.

In vitro antigenicity

In vitro antigenicity of selected purified proteins was tested using biolayer interferometry technology with octet. In addition to the antibodies described in Example 2, also preF specific Antibody ADI-15614 described to bind to site III (Gilman et al, Sci Immunol. 2016 Dec 16;l(6):eaaj 1879. doi: 10.1126/sciimmunol.aaj l879. Epub 2016 Dec 9.) and antibody ADI-18992 recognizing the preF and postF conformation described to bind to site IV (Gilman et al Sci Immunol. 2016 Dec 16;l(6):eaaj 1879. doi: 10.1126/sciimmunol.aaj l879. Epub 2016 Dec 9.) were used. The antibodies were immobilized as described in Example 2. After equilibration of the sensors in kinetic buffer (ForteBio) for 600s the sensors were transferred to kinetic buffer with 5 ug/ml of the desired antibody. Subsequently another equilibration step was included in kinetic buffer. Lastly the sensors were transferred to a solution of the proteins (20 pg/mL in IxKB). Analysis was performed as described in Example 2.

Results and conclusion

The binding of the purified proteins is summarized in Figure 7d. The reference postF protein was binding only minimally to the pre-fusion specific antibodies in the initial slope but the binding after 300 seconds indicated there may be minimal amounts of protein in preF conformation in the sample. All pre-fusion HMPV-F proteins showed binding to the preF-specific antibodies ADI- 18444 and ADI-15614. The preF proteins showed reduced non-preF binding (DS7 binding) compared to the postF reference according to the initial slope signal. However, the binding after 300 seconds showed a signal for non-preF, indicating that a subpopulation may be in non-preF trimeric conformation. Also, the variant that showed full processing after purification (MPV190444, Figure 7b) showed non-preF binding.

With the introduction of H368N and E453Q DS7 binding was reduced. Furthermore, the introduction of the disulfide bridge P360C/A459C reduced the non-preF binding rate completely. Proteins based on the F protein of the recent HMPV A2 strain

Yokohama. JPN/P8356/2016 (SEQ ID NO: 35) (MPV191769 SEQ ID NO: 33) and recent HMPV B2 strain Yokohama.JPN/P8674/2016 (SEQ ID NO: 65) (MPV191806 SEQ ID NO: 34) were comparable to the constructs based on strain TN/00/3-14 (SEQ ID NO: 1). ADI- 15614 did not bind to the recent B strain. Stabilizing mutations are therefore independent of hMPV subtypes.

Thermostability of proteins

Thermo-stability of the purified pre-fusion HMPV F proteins was determined by Differential Scanning Fluorimetry (DSF) by monitoring the fluorescent emission of Sypro Orange Dye (ThermoFisher Scientific) in a 96 well optical qPCR plate. 15 pl of a 66.67pg/ml protein solution was used per well (buffer as described in Example 2; for MPV190856 5% sucrose was added to the buffer). To each well, 5 pl of 20x Sypro orange solution was added. Upon gradual increase of the temperature, from 25°C to 95°C (0.015°C/s), the proteins unfold and the fluorescent dye binds to the exposed hydrophobic residues leading to a characteristic change in emission. The melting curves were measured using a ViiA7 real time PCR machine (Applied BioSystems). The 1st derivative of the fluorescent signal (a.u.) versus the temperature (°C) of three individual samples (technical triplicate), as well as the averaged melting curve, were plotted with Graphpad Prism software (Dan Diego, CA, US). From the averaged melting curve, the Tm50 was deducted (lowest point on the curve). The Tm50 values represent the temperature at which 50% of the protein is unfolded and thus are a measure for the temperature stability of the proteins.

Results and conclusion

With the addition of stabilizing mutations, most HMPV F variants showed two melting temperatures (Figure 7E) which could suggest that there are 2 different species in the sample or that the protein has two separate melting events. The only construct with one melting temperature is MPV190444 (SEQ ID NO: 32). Since this purified F protein was fully processed (Figure 7B) on SDS-PAGE, it could be that the double melting points reflect cleaved and uncleaved species. Introduction of a disulfide bridge at amino acid positionl41C/161C (MPV191388; SEQ ID NO: 15) resulted in a major melting event at 86.9°C reflecting increased stability. Constructs based on the F protein of the recent A2 strain Yokohama.JPN/P8356/2016

(SEQ ID NO: 35) (MPV191769 SEQ ID NO: 33) and recent B2 strain Yokohama.JPN/P8674/2016 (SEQ ID NO: 65) (MPV191806 SEQ ID NO: 34) showed a similar melting temperature compared to designs in reference strain (TN/00/3-14, SEQ ID NO: 1), showing that the mutations are transferable to other strains.

In MPV201031 (SEQ ID NO: 22) and MPV201216 (SEQ ID NO: 30) the disulfide bridge in position P360C/A459C was introduced. A melting temperature for MPV201031 (SEQ ID NO: 22) was not detected likely due to the low concentration of the sample. For MPV201216 (SEQ ID NO: 30) two melting temperatures were detected. The first melting event was increased compared to designs without P360C/A459C indicating that the disulfide resulted in increased thermo-stability.

Freezing stability

The purified proteins were snap frozen in liquid nitrogen once, 5 times and 10 times and assessed by analytical Size Exclusion Chromatography (SEC) (as described above for Figure 7A). As control a sample stored at 4°C was measure.

Results and conclusion

HMPV F protein (Figure 7F) with D185P, H368N and E453Q (MPV190856 SEQ ID NO: 10) was least stable after 10-time snap freezing. Addition of stabilizing mutations L73W, Al 16H, A140C/A147C and N342P (MPV191386 SEQ ID NO: 14) improved the stability after 10-time snap freezing. A construct with an alternative disulfide bridge (MPV191388 SEQ ID NO: 15) showed similar results. With the addition of sucrose to the buffer all designs showed increased freezing stability. For the designs with disulfide bridge quality was maintained best after 10-times snap freezing.

37°C storage stability

The purified proteins were assessed for preF trimer stability by analytical Size Exclusion Chromatography (SEC) after storage at 37°C for 2 or 14 weeks (as described above for Figure 7a).

Results and conclusion

High trimer content was observed for all designs after 2 (Figure 7G) and 14 (Figure 7H) weeks. For MPV191388 some aggregates were detected after 2 weeks. For all tested constructs aggregations were detected after 14 weeks (Figure 7G). MPV191386 (SEQ ID NO: 14) showed best trimer stability (Figure 7G).

EXAMPLE 8: Single chain design to avoid incomplete processing & evaluation of trimer opening

To avoid incomplete processing of the F protein, the stabilizing mutations were transferred into a single chain, i.e. a non-processed polypeptide (Figure 1C) (as described in WO 2014/174018; Krarup et. al., 2015 (Nat Commun. 2015 Sep 3;6:8143. doi: 10.1038/ncomms9143.)). The designs all had a C-terminal foldon domain and a SAIG linker between Fl and foldon. Single chain protein non-stabilized (MPV190862; SEQ ID NO: 36) and constructs stabilized with D185P and E453Q (MPV190860 SEQ ID NO: 37) and D185P, E453Q and L73W (MPV191031 SEQ ID NO: 38) were compared. Expression and analysis of HMPV F constructs were performed as described in Example 2. Purification was performed as in Example 7. The purified proteins were analyzed by negative stain Transmission Electron Microscopy (nsTEM).

Continuous carbon grids (copper, EMS) were glow discharged for 30 seconds in a easiglow plasma cleaner. Four microliters of the sample diluted (20mM Tris, 150mM NaCl, pH7.4) to concentrations ranging from 5 to 25 pg/ml were applied to glow-discharged grids and incubated for Imin. The sample solution was partially absorbed by gentle side blotting, and the grid was immediately stained with by depositing it on top of a 40 pl drop of a 2% (w/v) uranyl acetate solution for a total of 1 min. After staining, the grid was blotted dry and stored at room temperature prior to imaging.

The prepared grids were imaged in a Talos L120C TEM (Thermo Fisher Scientific) equipped with a Ceta camera.

Resulting pixel ranged from 2.4 to 2.8 ang per pixel depending on imaging conditions.

The parameters of the Contrast Transfer Function (CTF) were estimated on each micrograph using CTFFIND4 and the rest of the processing (picking, 2D classification, 3d model generation) was done in RELION (version 3 or 4).

Results and conclusion

All single chain HMPV F constructs bound to the preF-specific antibody ADI-4448 in Octet (Figure 8A) in supernatant. With subsequent introduction of stabilizing mutations D185P and E453Q (MPV190860 SEQ ID NO: 37) or D185P, E453Q and L73W (MPV191031 SEQ ID NO: 38) the un-favorable binding to DS7 was slightly reduced (Figure 8A) but the retention time in analytical SEC of crude supernatant was significantly higher (4.2 minutes to 4.35 minutes (Figure 8B) indicating a more compact trimer structure for the variant with the L73W mutation. The more closed conformation of the variant with the L73W substitution was confirmed by nsEM followed 2D averaging and 3D reconstruction of the purified proteins (Figure 8C). The antigenicity, longer retention time in SEC and the EM structures clearly showed that a stabilizing substitution like L73W can stabilize an HMPV preF trimer with a more closed Apex.

EXAMPLE 9: Further stabilization of the preF single chain proteins

Compared to a non-stabilized single chain HMPV protein (MPV190862; SEQ ID NO: 36) stabilizing mutations L73W, Al 16H, H368N and E453Q (MPV191394; SEQ ID NO: 40) were tested. In addition, further stabilizing mutations A140C/A147C (MPV191395; SEQ ID NO: 41) or A140C/A147C and D185P (MPV191392; SEQ ID NO: 39) were introduced. The linker between Fl and foldon was evaluated in constructs comprising the same set of stabilizing mutations (SAIG: MPV191392; SEQ ID NO: 39 and SGGG: MPV191746; SEQ ID NO: 42). Further, MPV191703 (SEQ ID NO: 43) with T69Y, L73W, D185P, H368N and E453Q and MPV191708 (SEQ ID NO: 44) with L73W, D185P, V191I, H368N and E453Q were tested to evaluate apex mutations (example 5) in amino acid position 69 and 191.

Expression and analysis of HMPV F constructs were performed as described in Example 2.

Results and conclusion

All single chain HMPV F designs bound to the preF-specifc antibody ADI-4448 in Octet (Figure 9A) and were trimeric (analytical SEC in Figure 9B). Improved pre-F binding and trimer content, compared to a design with L73W, Al 16H, H368N and E453Q (MPV191394; SEQ ID NO: 40), was observed when introducing mutations A140C/A147C (MPV191395; SEQ ID NO: 41) or A140C/A147C and D185P (MPV191392; SEQ ID NO: 39). Additionally, a subsequent increase in trimer content and shift to later retention times (MPV190862<MPV191394<MPV191395<MPV191392) were observed indicating higher expression and a more closed trimer (see Example 8).

A SGGG linker (MPV191746; SEQ ID NO: 42) increased pre-F binding and trimer content compared to a SAIG linker (MPV191392; SEQ ID NO: 39), however the retention time was minimally reduced (Figure 9B). Single chain designs with additional apex mutations in amino acid position 69 (MPV191703: SEQ ID NO: 43) and 191 (MPV191708; SEQ ID NO: 44) and a SGGG linker had longer retention times (MPV191708<MPV191703). Additionally, a slight reduction in un-favorable binding to DS7 (Figure 9A) was observed.

In conclusion, the combination of a SGGG linker between Fl and foldon and the apex mutation T69Y is preferred due to the lowest non-preF signal in binding and longer retention time measured.

EXAMPLE 10: Further stabilization of the preF single chain proteins with disulfide bridge P360C/A459C

A single chain HMPV F protein was stabilized by the introduction of T69Y, L73W, A140C/A147C, D185P, H368H and E453Q (MPV200620; SEQ ID NO: 45; numbering of the positions is based on SEQ ID NO: 1). Subsequently, disulfide P360C/A459C (MPV200622; SEQ ID NO: 46), mutation V191I (MPV200631; SEQ ID NO: 47) and mutation N342P (MPV200632: SEQ ID NO: 48) were added. Finally, back mutation to wildtype amino acids were tested based on MPV200631 (SEQ ID NO: 47) to evaluate the contributions of N368H, E453Q and A140C/A147C (MPV200633: SEQ ID NO: 49 and MPV201022 (SEQ ID NO: 50 and MPV201222 (SEQ ID NO: 51). Expression and analysis of HMPV F constructs were performed as described in Example 2. For the analytical SEC analysis, the crude supernatant was additionally incubated for 30 minutes at 50, 60 and 70 °C.

Results and conclusion

All single chain HMPV F constructs bound to the preF-specific antibody ADI-4448 in Octet (Figure 10A) and were trimeric (analytical SEC in Figure 10B). With subsequent introduction of stabilizing mutations P360C/A459C (MPV200622; SEQ ID NO: 46) and V191I (MPV200631; SEQ ID NO: 47) the un-favorable binding to DS7 was reduced (Figure 10A) and the resilient to heat stress was increased (Figure 10B). Addition of N342P (MPV200632; SEQ ID NO: 48) showed an increase in expression and a slight increase of unfavorable binding to DS7 ( Figure 10A).

Back mutation to the original wild-type residue at position 368, 453 and 140/147 compared to MPV20631 (SEQ ID NO: 47) showed that A140C/A147C has a clear contribution to expression and quality of the single chain HMPV F trimer (Figure 10B). Whereas the point mutations H368N (MPV201222 SEQ ID NO: 51) showed a benefit, the contribution of E453Q (MPV200633; SEQ ID NO: 49) was less clear in heat stress.

EXAMPLE 11 : Single chain variants

Additional single chain constructs to the one described in Example 1 (Figure 1C) were made (Table 2). Expression and analysis of HMPV F constructs were performed as described in Example 2. Table 2. Overview of single chain designs

Results and Discussion

The single chain backbone MPV191746 (SEQ ID NO: 42) was compared to other single chain designs (Figure 11 A). Level of non-preF trimer binding was comparable for the different constructs. As described in Example 8 and 9, a later retention time indicates a closed trimer (Figure 11B). MPV191748 (SEQ ID NO: 53) and MPV191756 (SEQ ID NO: 61) had similar retention times as the original design (MPV191746), indicating a correctly closed trimer. However, MPV191748 showed reduced preF -binding and MPV191748 and MPV191756 both showed reduced expression levels compared to MPV191746 (Figure

11C) Several single chain designs are possible but the design according MPV191746, having a deletion of amino acids 97 to 106 and a linker with the sequence GSGSGR is most preferred.

EXAMPLE 12: Production, purification and characterization of selected single chain proteins

Production and purification

Proteins were produced and purified as described in Example 7. (MPV201222 was produced on an increased 400ml transfection scale in HEK-E cells for 6 days).

Results and Discussion

One elution peak was observed in preparative SEC for the trimer (Figure 12A) proteins. In general, the yield (Table 3) was higher compared to the corresponding processed designs of Example 7 (Table 1). Comparing MPV191391 (SEQ ID NO: 62) and MPV101392 (SEQ ID NO: 39) or MPV191703 (SEQ ID NO: 43) and MPV200620 (SEQ ID NO: 45), disulfide bridge A140C/A147C improved expression by 1.5-fold. Additionally, 2- fold improvement in expression was observed with the SGGG linker between Fl and the foldon (comparing MPV1913192 (SEQ ID NO: 39) with MPV191746 (SEQ ID NO: 42)).

The stabilizing mutations increased expression and stabilized preF of several diverse HMPV strains (MPV191746, MPV191768, MPV191807). Designs with disulfide bridge P360C/A459C show reduced yields. Table 3.: Purified proteins of example 12 and yields

*Yokohama.JPN/P835b, 2<J16, Yokohama.JPN/P8674/2016; wild type variant A185

Analysis on SDS page (Figure 11B) shows all designs as F0, the unprocessed form of the proteins as desired for a single chain design. Trimer content and trimer stability after storage at 4°C

The purified proteins were assessed by analytical Size Exclusion Chromatography (SEC) to study trimer content after purification and trimer content and stability after storage at 4°C (Figure 12C) as described in Example 7.

Results and Discussion All purified proteins were trimeric after purification (Figure 12C) and showed trimer stability up to 28 weeks.

The SGGG linker variant (MPV191746; SEQ ID NO: 42) of MPV191392 (SEQ ID NO: 39) showed a smaller shift in retention time after 6 months, indicative of a more compact trimer conformation (see Example 8). Introducing the set of stabilizing mutations into a recent A2 (MPV191768; SEQ ID

NO: 63) and recent B2 (MPV191807; SEQ ID NO: 64, containing also N342P) strain also resulted in stable trimers at week 13. In conclusion, all proteins showed high trimer content after purification and can be stored at 4°C up to 6 months.

In vitro antigenicity

In vitro antigenicity of selected produced and purified proteins was performed as described in Example 7.

Results and conclusion

The binding of the purified proteins is summarized in Figure 12D. The binding of the reference postF protein is described in Example 7. All pre-fusion HMPV-F proteins showed binding to the preF-specific antibodies ADI-18444 and ADI-15614 (design based on B strain was not binding). MPV191394 (SEQ ID NO: 40) with L37W, Al 16H and H368N showed reduced preF binding compared to design with D185P and/or A140C/A147C (MPV191391; SEQ ID NO: 62, MPV191392; SEQ ID NO: 39 and MPV191395; SEQ ID NO: 41). After 300 seconds a strong signal for non-preF was observed, indicating that with longer incubation times, a non-preF epitope is are exposed. With the introduction of the disulfide bridge P360C/A459C the non-preF binding was strongly reduced.

Designs based on the recent A2 strain Yokohama. JPN/P8356/2016 (SEQ ID NO: 35) (MPV191768 SEQ ID NO: 63) and recent B2 strain Yokohama.JPN/P8674/2016 (SEQ ID NO: 65) (MPV191807 SEQ ID NO: 64 containing also N342P) were comparable to the designs based on strain TN/00/3-14 (SEQ ID NO: 1). Only exception is, that ADI-15614 seems not to bind to the recent B strain. Stabilizing mutations are therefore independent of hMPV subtypes. Thermostability of proteins

Thermo-stability of several the purified pre-fusion HMPV F proteins were determined by Differential Scanning Fluorimetry (DSF) as described in Example 7. Melting temperatures of MPV191703, MPV200620, MPV200622 and MPV200632 were measured after 6 months storage of the purified proteins at 4°C.

Results and conclusion

In contrast to the processed designs of Example 7 most of the designs showed one single meting event (Figure 12E).

Designs based on the recent A2 strain Yokohama. JPN/P8356/2016 (SEQ ID NO: 35) (MPV191768 SEQ ID NO: 63) and recent B2 strain Yokohama.JPN/P8674/2016 (SEQ ID NO 65) (MPV191807 SEQ ID NO: 64; containing also N342P) showed a slightly increased melting temperature compared to designs in reference stain (TN/00/3-14 SEQ ID NO: 1).

The addition of the apex mutation T69Y in a backbone without (MPV191703; SEQ ID NO: 43) or with (MPV200620; SEQ ID NO: 45) A140C/A147C showed no increased melting temperature.

In MPV200622 (SEQ ID NO: 46), MPV200632 (SEQ ID NO: 48) and MPV201222 (SEQ ID NO: 51) the disulfide bridge in position P360C/A459C was introduced resulting in an increased melting temperature of 64.7, 65.5 and 68.4°C, respectively, which is an increase of about 10°C compared to designs without disulfide bridge.

Best improvement in temperature stability is due to P360C/A459C. Freezing stability

The purified proteins were tested for freezing stability as described in example 7.

Results and conclusion

All tested proteins can resist one-time freezing without trimer loss (Figure 12F). With the addition of sucrose to the buffer all designs showed increased freezing stability. In general trimer loss was less compared to the processed designs in Example 7. With sucrose the designs showed increasing resilience to freeze stress by MPV191391 < MPV191392 < MPV191395 < MPV191394. The result for MPV191394 could however have been influenced by some postF material in the samples as indicated with low preF binding and with a second very high meting temperature in the paragraphs above.

37°C storage stability

The purified proteins were assessed for preF trimer stability by analytical Size Exclusion Chromatography (SEC) after storage at 37 C for 2 (Figure 12G) or 14 weeks (Figure 12H) as described in Example 7.

Results and conclusion

After heat stress all proteins remained mainly trimeric (Figure 12G). Increased timer content for MPV191394 (SEQ ID NO 40) and MPV101395 (SEQ ID NO 41) could be due a higher concentration in the sample due to evaporation. Some very minor monomer content after 2 weeks was observed for MPV191807 (SEQ ID NO: 64). After 14 weeks design MPV191391 (SEQ ID NO: 62) showed the largest lost in trimer followed by design MPV191392 (SEQ ID NO: 39). Overall trimers were very stable at 37°C. EXAMPLE 13: Additional single chain protein variant

An additional single chain construct was made that had a longer deletion of the F2 C- terminus and the fusion peptide N-terminus compared to MPV191395 (Table 3). In this construct (MPV221643) amino acids 91 - 110 were replaced by a linker of 3 amino acids (GSG). Expression and antigenic analysis of HMPV F single chain constructs MPV221643 and the parent MPV191395 were performed as described in Example 2.

Results and Discussion

Expression levels of the single chain constructs MPV221643 (SEQ ID NO: 66) and MPV191395 (SEQ ID NO: 41) are shown in Figure 13A. Although the expression level of MPV221643 with the large deletion is much lower, the antigenicity is more favorable since it shows relatively lower binding to MAb MPV458, which is directed against the trimer interface in the apex (Huang et al. PLoS Pathog (2020)16(10):el008942) compared to the strongly neutralizing antibody ADI-61026 directed against site 0 in the apex (Rappazzo et al. Immunity (2022) 55: 1710-1724. e8) (Figure 13B). The deletion of F2 C-terminus and fusion peptide N-terminus apparently allows the trimer to close and have a less open structure since the interface epitope of MPV458 is less exposed. Since the apex-specific antibody directed against site 0 has a much stronger neutralizing activity compared to the apex-specific antibody directed against the interface, MPV221643 having a deletion of amino acids 91 - 110 and a linker with the sequence GSG may be a preferred vaccine antigen. Table 4. Standard amino acids, abbreviations and properties

SEQUENCES

SEQ ID NO: 1 hMPV A2 fusion protein (TN/00/3-14) full length (Signal peptide bold; wild type cleavage site region italic)

MSWKWIIFSLUTPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRE

LKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVL ATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNM PTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWY CQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVF ENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHS

SEQ ID NO: 2 foldon

GYIPEAPRDGQAYVRKDGEWVLLSTF(L)

SEQ ID NO: 3 M PV190470 (postF); optimized furin cleavage site bold; linkers italic; TEV CS bold italic; foldon bold underlined; Strep tag underlined italic; C-tag underlined

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRE LRTVSADQLAREEQIENPRQSKKRKRRVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATA VRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS AGQIKLM LENRAMVRRKGFGFLIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKENYACLLREDQGWYCQN AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSC SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIE

SEQ ID NO: 4 MPV201042 (non-stabilized sequence with 482 amino acid length; optimized furin cleavage site bold; GGGS linker italic and C-tag underlined)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA TAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP TSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFE N I E NSQALVDQSN Rl LSGGGSEPEA

SEQ ID NO: 5 M PV201043 (non-stabilized sequence with 489 amino acid length)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA TAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFE NIENSQALVDQSNRILSSAEKGNTGGGSEPEA SEQ ID NO: 6 MPV190845 (non-stabilized with 481 amino acid length; optimized furin cleavage site underlined; SAIG linker italic; foldon highlighted)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFE NIENSQALVDQSNRILSA/GGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 7 MPV190842

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFE NIENSQALVDQSNRILSA/GGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 8 MPV190843

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE NIENSQALVDQSNRILSA/GGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 9 MPV190997

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE NIENSQALVDQSNRILSA/GGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 10 MPV190856

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE NIENSQALVDQSNRILSAIGGYI PEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 11 MPV190991 MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE NIENSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 12 MPV190993

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 13 MPV191012

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE NIENSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 14 MPV191386

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 15 MPV191388

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNACKTTNEAVSTLGNGVRV

LATCVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 16 MPV191013

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVFE NIENSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 17 M PV190894 (SGGG linker italic)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 18 MPV191757

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 19 MPV200641

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYCCKVSTGRNPISMVALSPLGALVACYK

GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVF ENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 20 MPV200718

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYCCKVSTGRNPISMVALSPLGALVACYK

GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVF ENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 21 MPV201028

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 22 MPV201031

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 23 MPV201032

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRV LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN MPTSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYK

GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVF ENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 24 MPV201033

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNM

PTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWY

CQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVF ENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 25 MPV201285

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 26 MPV201027

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFE

NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 27 MPV201029

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 28 MPV201030

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 29 MPV201038

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 30 MPV201216

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFE NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 31 MPV201286

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFE NIENSQALVDQSNRILSAIGGYI PEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA SEQ ID NO: 32 MPV190444 (strep-tag underlined)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFE NIENSQALVDQSNRILSA/GGYIPEAPRDGQAYVRKDGEWVLLSTFAAWSHPQFEKGAAEPEA

SEQ ID NO: 33 MPV191769

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKKTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPQDQFNVALDQV FENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 34 MPV191806

MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRRRRFVLGAIALGVATAHAVTAGIAIAKTIRLESEVNAIKGCLKTTNECVSTLGNGVRVL

ATAVRELKEFVSKNLTSAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMNDAELARAVSYMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVINTPCWIIKAAPSCSEKDGNYACLLREDQGWYCK NAGSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSRECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVS CSTGSNQVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSNSFDPIRFPQDQFNVALDQVFES IENSQALVDQSNKILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 35 recent A2 full length wild type sequence (Yokohama.JPN/P8356/2016)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRE LKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVL ATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNM PTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWY

CQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQV FENIENSQALVDQSNRILSSAEKGNTGFIMILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHS

SEQ ID NO: 36 MPV190862

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVR

ELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAG

QIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAG STVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIG SNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQ ALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA SEQ ID NO: 37 MPV190860

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALREL

KTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVR

ELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAG

QIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAG

STVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIG

SNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENS QALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 38 MPV191031

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 39 MPV191392

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 40 MPV191394 (with A185; a wild type variant to D185)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 41 MPV191395 (with A185; a wild type variant to D185)

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 42 MPV191746 MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 43 MPV191703

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 44 MPV191708

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNA

GSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSI

GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIEN

SQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 45 MPV200620

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 46 MPV200622

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVFENIE NSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 47 MPV200631

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA VRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNA

GSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYCCKVSTGRNPISMVALSPLGALVACYKGVSCSI

GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVFENIEN

SQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 48 MPV200632

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNA

GSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYCCKVSTGRNPISMVALSPLGALVACYKGVSCSI

GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVCLDQVFENIEN

SQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 49 MPV200633

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNA

GSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKGVSCSI

GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFENIENS

QALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 50 MPV201022

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNA

GSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYCCKVSTGRHPISMVALSPLGALVACYKGVSCSI

GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFENIENS

QALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 51 MPV201222

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAISFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNA

GSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSKECNINISTTNYCCKVSTGRNPISMVALSPLGALVACYKGVSCSI

GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVCLDQVFENIENS

QALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 52 MPV191747

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGSAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 53 MPV191748

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRSVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 54 MPV191749

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPGSGSGRSVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGV

RVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVS

NMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQG

WYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVAC YKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQ VFENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 55 MPV191750

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPGSGSGSSVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGV

RVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVS

NMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQG

WYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVAC YKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQ VFENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 56 MPV191751

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE

NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA SEQ ID NO: 57 MPV191752

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRQSQAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE NSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 58 MPV191753

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRQSQGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLAT

AVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPT

SAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQ

NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVS

CSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENI ENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 59 MPV191754

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRQSQFVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 60 MPV191755

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIENPRQSQSVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRV

LATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN

MPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGW

YCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYK GVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQV FENIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 61 MPV191756

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIERQSQSVLGAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLA

TAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMP

TSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYC

QNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKG

VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFE

NIENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA SEQ ID NO: 62 MPV191391

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIE

NSQALVDQSNRILSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 63 MPV191768

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKKTNECVSTLGNGVRVLATA

VRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS

AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQN

AGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSC

SIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPQDQFNVALDQVFENI

ENSQALVDQSNRILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 64 MPV191807

MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDWTKSALR

ELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGIAIAKTIRLESEVNAIKGCLKTTNECVSTLGNGVRVLATA

VRELKEFVSKNLTSAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMNDAELARAVSYM PTSA

GQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVINTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNA

GSTVYYPNEKDCETRGDHVFCDTAAGIPVAEQSRECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCS

TGSNQVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSNSFDPIRFPQDQFNVALDQVFESIE

NSQALVDQSNKILSGGGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO: 65 recent A2 full length wild type sequence Yokohama.JPN/P8674/2016

MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRE

LKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGIAIAKTIRLESEVNAIKGALKTTNEAVSTLGNGVRVLA

TAVRELKEFVSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMNDAELARAVSYMPT

SAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVINTPCWIIKAAPSCSEKDGNYACLLREDQGWYCK

NAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVS

CSTGSNQVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSNSFDPIRFPEDQFNVALDQVFESI

ENSQALVDQSNKILNSAEKGNTGFIIVIILIAVLGLTMISVSIIIIIKKTRKPAGAPPELNGVTNGGFIPHS

SEQ ID NO: 66 MPV221643

MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALR

ELKTVSADQLARGSGGVATAHAVTAGVAIAKTIRLESEVTAIKNCLKTTNECVSTLGNGVRVLATAVRELKDFVSKNL

TRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNM PTSAGQIKLMLENRA

MVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKD

CETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQL

NKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRI

LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

Claims

75

1. Stabilized pre-fusion human pneumovirus (HMPV) F protein, comprising an Fl and an F2 domain, and comprising an amino acid sequence wherein the amino acid residue at position 69 is Y, and/or the amino acid residue at position 73 is W, and/or the amino acid residue at position 191 is I, and/or the amino acid residue at position 116 is H, and/or the amino acid residue at position 342 is P, and/or the amino acid residue at position 453 is Q, wherein the numbering of the amino acid positions is according to the numbering is amino acid residues in SEQ ID NO: 1.

2. Protein according to claim 1, further comprising one or more non-native intra- or inter-protomer disulfide bonds.

3. Protein according to claim 3, wherein the one or more disulfide bonds are selected from an intraprotomeric disulfide bond between the amino acid residues 140 and 147 and/or an intraprotomeric disulfide bond between the amino acid residues 141 or 161, and/or an intraprotomeric disulfide bond between the amino acid residues 360 and 459.

4. Protein according to claim 1, 2 or 3, wherein the amino acid residue at position 185 is P.

5. Protein according to any one of the claims 1-4, wherein the amino acid residue at position 294 is E, and/or the amino acid residue at position 368 is N. 76 Protein according to any one of the claims 1-5, comprising a non-native cleavage site between the Fl and F2 domain. Protein according to claim 6, wherein the non-native cleavage site is a furin cleavage site. Protein according to any one of claims 1-5, wherein the amino acids 97-106 have been deleted and replaced by a linking sequence of 1-10 amino acids. Protein according to any one of the claims 1-5, wherein the amino acids 91-110 have been deleted and replaced by a linking sequence of 1-10 amino acids. Protein according to any one of the preceding claims, comprising a truncated Fl domain. Protein according to claim 10, wherein the truncated Fl domain does not comprise the transmembrane and cytoplasmic regions. Protein according to claim 11, wherein the truncated Fl domain comprises the amino acids 103-481, 107-481, 111-481, 103-482, 107-482, 111-482, 103-489, 107-489, or 111-489 of the HMPV F protein. Protein according to claim 10, 11 or 12, wherein a heterologous trimerization domain is linked to the truncated Fl domain, optionally through a linking sequence. Protein according to any of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-16, 18-20, 22-24, 28-31, 33-34, SEQ ID NO: 37-64, and SEQ ID NO: 66, or fragments thereof. 77

15. Nucleic acid molecule encoding a protein according to any one of the preceding claims 1-14.

16. Nucleic acid according to claim 15, wherein the nucleic acid molecule is DNA or RNA.

17. Nucleic acid according to claim 15 or 16, encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-16, 18-20, 22-24, 28-31, 33-34, 37-64, and 66, or fragments thereof.

18. Vector comprising a nucleic acid according to claim 15, 16 or 17.

19. Pharmaceutical composition comprising a protein according to any one of the claims 1-14, a nucleic acid according to claim 15, 16 or 17 and/or vector according to claim 18.

20. A method for vaccinating a subject against HMPV, the method comprising administering to the subject a pharmaceutical composition according to claim 19.

21. A method for preventing infection and/or replication of HMPV in a subject, comprising administering to the subject a pharmaceutical composition according to claim 19.

22. An isolated host cell comprising a nucleic acid according to claim 15, 16 or 17.