Stabilized pre-fusion HMPV fusion protein

Abstract

The present invention relates to a stabilized prefusion human metapneumovirus (HMPV) F protein, a nucleic acid molecule encoding the HMPV F protein, and their use.

Description

Technical Field

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

Background Art

[0002] Background of the Invention Human metapneumovirus (HMPV) belongs to the family Pneumoviridae, which also includes the respiratory syncytial virus (RSV). Genetic analysis of HMPV isolates has shown two major groups, A and B, and four subgroups (A1, A2, B1, and B2), mainly based on the diversity of the attachment protein (G) and the fusion protein (F) (van Hoogen et al., Emerg. Infect. Dis. 10(4):658-666, 2004). Recently, Noa et al. (Microorganisms. 2020 Aug 21;8(9):1280) have described the subdivision of A2 into A2a and A2b, and currently the latter is spreading.

[0003] To infect host cells, like other enveloped viruses such as influenza virus, RSV, and HIV, HMPV requires fusion of the viral membrane with the host cell membrane. In the case of HMPV, the conserved fusion protein (HMPV F protein) mediates the fusion of the viral membrane with the host cell membrane. The HMPV F protein initially folds into a "pre-fusion" conformation. This metastable structure has recently been elucidated (Battles et al., Nat Commun. Nov 16;8(1):1528, 2017). During cell entry, the pre-fusion conformation undergoes refolding and a conformational change 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 structure that leads to membrane fusion, which occurs by connecting irreversible protein refolding to membrane juxtaposition, which occurs by first folding into a metastable form (pre-fusion conformation) and then undergoing a discrete / stepwise conformational change to a lower energy conformation (post-fusion conformation).

[0004] Human metapneumovirus (HMPV) was first identified in 2001 in clinical samples from pediatric patients with diseases similar to those caused by human respiratory syncytial virus (RSV), in samples from individuals in whom RSV could not be identified (van den Hoogen et al., Nat. Med. 7(6):719-724, 2001). Subsequent studies have shown that HMPV is a major cause of both upper and lower respiratory tract infections in infants, young children, the elderly, and immunocompromised individuals or those with underlying chronic medical conditions. The clinical symptoms of HMPV infection are similar to those caused by RSV and range from mild respiratory illness to bronchiolitis and pneumonia. HPMV infection appears to be ubiquitous, as virtually all children become seropositive by the age of 5 years. Epidemiological studies to date have suggested that HMPV infection causes lower respiratory tract infections in 5-15% of otherwise healthy infants (Falsey et al., J. Infect. Dis. 187:785-790, 2003). HMPV was detected in 4.9% of children under 5 years of age admitted to the hospital with acute respiratory infection or fever, a number similar to that for influenza and higher than that for parainfluenza virus type 3 (PIV-3) (Williams et al., J Infect Dis. 201(12):1890-1898, 2010). The incidence of disease is highest in children less than 0-6 months of age. Reinfection with HMPV has also been reported.

[0005] Currently, most of the infection data has been obtained from studies in children, but there is accumulating evidence that HMPV can also cause severe diseases in adults. For influenza and RSV, infections in adults are most severe in the elderly and patients with chronic underlying medical conditions. The incidence of symptomatic infections in the adult population is typically less than 5% in most studies (Falsey, Pediatr. Infect. Dis. J. 27:S80 - 83, 2008). Influenza A, influenza B and RSV are the main viral causes of respiratory diseases in the elderly (over 65 years old), followed by HMPV, which has been quantified by Gaunt et al. to contribute 2.1 disability - adjusted life - years (DALYs) per 1,000 hospital admissions (Gaunt et al., J. Clin. Virol. 52(3):215 - 221, 2011).

[0006] Currently, there are no approved therapeutic or preventive measures to address HMPV infection. Since HPMV is the major cause of acute viral respiratory infections after RSV, an effective therapy or vaccine against HMPV is needed.

Summary of the Invention

[0007] The present invention provides a recombinant stabilized trimeric pre-human metapneumovirus (HMPV) fusion (F) protein, i.e., a recombinant HMPV F protein stabilized in the pre-fusion conformation. The HMPV F protein of the present invention comprises at least one epitope specific for the pre-fusion conformation of the F protein. The present invention provides both full-length HMPV F protein and soluble HMPV F protein. In certain embodiments, the pre-fusion hMPV F protein is a soluble protein (i.e., not membrane-bound and lacking transmembrane and cytoplasmic regions). The present invention also provides a nucleic acid molecule encoding a pre-fusion HMPV F protein according to the present invention, and a vector comprising such a nucleic acid molecule. The present invention also relates to a pharmaceutical composition, preferably a vaccine composition, comprising one or more HMPV F proteins, nucleic acid molecules and / or vectors according to the present invention, and its use for inducing an immune response against the HMPV F protein, in particular its use as a vaccine. The present invention also relates to a method for inducing an anti-human metapneumovirus (HMPV) immune response in a subject, comprising administering to the subject an effective amount of a pre-fusion HMPV F protein, a nucleic acid molecule encoding the HMPV F protein and / or a vector comprising the nucleic acid molecule. Preferably, the induced immune response is characterized by neutralizing antibodies against HMPV F, T cells, and / or protective immunity against HMPV. In certain aspects, the present invention relates to a method for inducing anti-human metapneumovirus (HMPV) F protein neutralizing 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 the HMPV F protein and / or a vector comprising the nucleic acid molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing general description and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the exact embodiments shown in the drawings.

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[0009] Detailed Description of the Invention The fusion protein (F) of human metapneumovirus (HMPV or hMPV) is a trimeric class I fusion protein involved in the fusion of the viral and host cell membranes, which is required for infection. HPMV F mRNA is translated into a 539 amino acid precursor protein designated F0. The precursor protein contains a signal peptide sequence at the N-terminus (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 signal peptidase in the endoplasmic reticulum. The precursor F0 has to be proteolytically cleaved (processed) by a cellular protease to generate two domains (F1 and F2) in a metastable disulfide-linked heterodimer (F1+F2) for activation. The newly formed N-terminus of F1 is thought to be the fusion peptide. Three F2-F1 dimers associate to form the mature F protein, which adopts a metastable fusogenic ("pre-fusion") conformation that is induced to undergo a conformational change upon contact with the target cell membrane. This conformational change exposes the fusion peptide, which associates with the host cell membrane and promotes fusion of the viral or infected cell membrane with the target cell membrane. Immediately adjacent to the fusion peptide and transmembrane domain (see Figure 1) are two heptad repeat (HR) regions, HR1 and HR2, respectively. When cleaved, HMPV F can be induced to undergo an essentially irreversible and energetically favorable conformational change from the pre-fusion form to the post-fusion state by the release potential energy that drives membrane fusion.

[0010] The F1 domain (corresponding to amino acid residues 103 to 539 of SEQ ID NO: 1) contains a hydrophobic fusion peptide of 23 amino acids at the N-terminus (corresponding to amino acids 103 to 126 of SEQ ID NO: 1), a refolding region 2 (RR2) (corresponding to amino acids 426 to 491 of SEQ ID NO: 1) (accompanied by HR2 containing amino acids 453 to 484), and the C-terminus contains a transmembrane region (TM) (corresponding to amino acid residues 492 to 513 of SEQ ID NO: 1) and a cytoplasmic region (corresponding to amino acid residues 514 to 539) (Ulbrandt et al., Journal of General Virology (2008), 89, 3113 - 3118).

[0011] After cleavage, the F2 domain (corresponding to amino acid residues 19 to 102 of SEQ ID NO: 1) is covalently bound to F1 by two disulfide bonds (Ulbrandt et al., Journal of General Virology (2008), 89, 3113 - 3118). The F1 - F2 heterodimer is assembled as a homotrimer in the virion.

[0012] Currently, there is no vaccine available against HMPV infection, and such a vaccine is desired. One potential approach for manufacturing a vaccine is a subunit vaccine based on a purified HMPV F protein. However, in this approach, it is desirable that the purified HMPV F protein has a conformation similar to the pre-fusion conformation of the HMPV F protein, is stable over time, and can be produced in sufficient quantities. Also, in the case of a subunit-based vaccine, the HMPV F protein needs to be truncated by deletion of the transmembrane (TM) region and cytoplasmic region such that a soluble secreted F protein (F or sF) is produced. Since the TM region provides membrane anchoring and trimerization, soluble F proteins without an anchor are considerably more unstable than the full-length protein and readily refold to the post-fusion end state. Thus, to obtain a soluble F protein in a stable pre-fusion conformation that exhibits high expression levels and high stability, the pre-fusion conformation needs to be stabilized. Since the full-length (membrane-bound) HMPV F protein is also metastable, stabilization of the pre-fusion conformation is also desirable for any live attenuated, vector-based or RNA vaccine approach using the full-length HMPV F protein, i.e., including the TM and cytoplasmic regions.

[0013] The present invention provides a trimeric recombinant prefusion HMPV F protein, i.e., an HMPV F protein stabilized in the prefusion conformation. In the research leading to the present invention, in order to obtain the stable prefusion HMPV F protein, several modifications were introduced compared to the amino acid sequence of the wild-type HMPV F protein, particularly the amino acid sequence of SEQ ID NO: 1, such as amino acid mutations, deletions, insertions and / or fusions. The stable prefusion HMPV F protein of the present invention is in the prefusion conformation, i.e., it contains (presents) at least one epitope specific to the prefusion conformation F protein. An epitope specific to the prefusion conformation F protein is an epitope that is not present in the postfusion conformation. Without being bound by any particular theory, the prefusion conformation of the HMPV F protein may contain the same epitopes as those present on the HMPV F protein expressed on native HMPV virions, and thus may provide an advantage in inducing protective neutralizing antibodies. In certain embodiments, the protein of the present invention contains at least one epitope recognized by a prefusion-specific anti-HMPV monoclonal antibody. Examples of such prefusion HMPV antibodies include MPE8 (Corti et al., Nature 50(7467):439-443, 2013) and ADI-14448 (Gilman et al., Sci Immunol. 2016 Dec 16;1(6):eaaj1879.doi:10.1126 / SciImmunol.aaj1879.Epub 2016 Dec 9). In certain embodiments, the recombinant prefusion HMPV F protein contains at least one epitope recognized by at least one prefusion-specific monoclonal antibody as described above and is trimeric. In certain embodiments, the stable prefusion HMPV F protein according to the present invention is soluble and thus contains a truncated F1 domain [i.e., the transmembrane and cytoplasmic regions are (partially) deleted].

[0014] The present invention particularly provides a prefusion human metapneumovirus (HMPV) F precursor F0 protein comprising an F1 domain and an F2 domain, and comprising at least one modification in the amino acid sequence of the F1 domain and / or the F2 domain.

[0015] In certain embodiments, said at least one modification stabilizes the prefusion conformation and / or enhances trimer formation and / or (thermal) stability of the HMPV F protein.

[0016] For the primary amino acid sequence of the HMPV F0 protein, the following terms are used to describe the structural features of the F protein. The term F0 means the full-length translated HMPV F protein precursor. During maturation, the F0 polypeptide undergoes proteolytic cleavage at a cleavage site located between F2 and F1, i.e., between amino acid 102 and amino acid 103.

[0017] As used herein, the F2 domain comprises at least a portion of amino acids 19 - 102, and the F1 domain comprises at least a portion of amino acids 103 - 539. The soluble F protein comprises the F2 domain and the F1 domain of the HMPV F protein and does not comprise the transmembrane domain of the HMPV F protein. The soluble portion of the F1 domain comprises at least a portion to all of amino acids 103 - 491 of the F0 protein. As noted above, these amino acid positions (and all subsequent amino acid positions shown herein) are shown based on the exemplary HMPV F protein precursor polypeptide (F0) of SEQ ID NO: 1.

[0018] According to the present invention, the HMPV F protein comprises at least one modification that stabilizes the prefusion conformation of the F protein such that the HMPV F protein retains at least one immunodominant epitope of the prefusion conformation of the F protein and / or enhances trimer formation and / or (thermal) stability of the HMPV F protein.

[0019] In certain embodiments, said at least one modification is the introduction of at least one non-natural cleavage site. Thus, in certain embodiments, the invention provides a stabilized pre-human metapneumovirus (HMPV) F precursor (F0) protein comprising an F1 domain and an F2 domain, and having, for example, a first non-natural cleavage site at the C-terminus of the F2 domain, between the F1 domain and the F2 domain.

[0020] Additionally or alternatively, the C-terminus of the F2 domain may be truncated. Thus, in certain embodiments, the first non-natural cleavage site is located at the C-terminus of the truncated F2 domain. For example, the F2 domain may be truncated at the C-terminus after position 89, and the first non-natural cleavage site is located after the C-terminal amino acid residue at position 89. In studies leading to the present invention, it has been shown that the cleavage site is present at residues 92-93. Thus, for example, when a furin site containing four residues is added after residue 89, the terminal position is approximately position 92, but the last residue at the C-terminus is not natural.

[0021] According to the present invention, it has been shown that by introducing a non-natural cleavage site between the F1 domain and the F2 domain, the processing (cleavage) of the HMPV F0 protein is improved compared to the HMPV F protein having a natural cleavage site. According to the present invention, the improved processing has been shown to enhance the stability and antigenicity of the HMPV F protein.

[0022] According to the present invention, the term non-natural cleavage site means a cleavage site that does not exist in the naturally occurring HMPV F protein.

[0023] The cleavage site is a proteolytic site utilized by cellular proteases that activate a broad precursor protein including a class I viral fusion protein such as HMPV F. Thus, it is known that various cellular proteases that catalyze the proteolytic activation process, such as furin, TMPRSS2, cathepsin, and other transmembrane serine proteases, cleave various viral cell surface proteins required for viral entry into host cells. The cleavage site typically comprises or consists of a specific amino acid sequence recognized by a cellular protease. Thus, the protease recognizes the site at which it cleaves via a specific amino acid sequence along the polypeptide chain. For example, furin-like proteases preferably cleave the protein immediately following a basic amino acid target sequence (typically Arg-X-(Arg / Lys)-Arg). According to the present invention, the non-native cleavage site can be introduced at the N-terminus of the native cleavage site, i.e., in addition to the native cleavage site. In a preferred embodiment, the native cleavage site is replaced by a first non-native cleavage site.

[0024] In certain embodiments, the protein of the present invention further comprises a second non-native cleavage site within the F2 domain. According to the present invention, the second non-native cleavage site is introduced into the F2 domain located on the N-terminal side of the first non-native cleavage site, and a spacer sequence is present between the first non-native cleavage site and the second non-native cleavage site. Thus, the second non-native cleavage site is introduced into the F2 domain, located on the N-terminal side of the first native or non-native cleavage site, and a spacer sequence is present between the first non-native cleavage site and the second non-native cleavage site, i.e., the first cleavage site and the second cleavage site are separated by the spacer sequence and are not directly adjacent. The spacer sequence can be part of the native sequence of the F2 domain or can be a heterologous sequence separating the first non-native cleavage site and the second non-native cleavage site.

[0025] In certain embodiments, the second non-native cleavage site is located after amino acid residue 88, or after amino acid residue 89, or after amino acid residue 90 of the F2 domain, and the first non-native cleavage site is located at the C-terminal position of the F2 domain. In preferred embodiments, the second non-native cleavage site is located after amino acid 88 or 89 of the F2 domain. In preferred embodiments, the second non-native cleavage site is located after amino acid 88 or 89 of the F2 domain and is cleaved after residue 92 or 93.

[0026] In certain embodiments, the first non-native cleavage site comprises the amino acid sequence RX1X2R, where X1 and X2 can be any amino acid.

[0027] In preferred embodiments, the first non-native cleavage is a furin cleavage site comprising the sequence RX1[K / R]R.

[0028] In certain preferred embodiments, the F0 protein comprises the p27 peptide of the RSV F protein between the F1 domain and the (truncated) F2 domain, where the p27 peptide comprises the first cleavage site at its C-terminus. Thus, in this case, the spacer sequence is (part of) the p27 peptide sequence. The p27 peptide can be any p27 peptide of any RSV F protein, or a variant thereof. In certain embodiments, the p27 peptide is from RSV A and has the amino acid sequence ELPRFMNYTLNNAKKTNVTLSKK RKRR(SEQ ID NO: 2; the cleavage site is indicated by an underline), or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2. In certain embodiments, the p27 peptide has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99% sequence identity to SEQ ID NO: 2. In these embodiments, the p27 peptide contains a first non-natural cleavage site at its C-terminus, and the second non-natural cleavage site is located on the N-terminal side of the p27 peptide. Also in this case, the F2 domain may be truncated at its C-terminal side. The F domain may be truncated after the amino acid residue at position 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 101. In a preferred embodiment, the F2 domain is truncated after the amino acid residue at position 89, followed by a cleavage site, such that Arg is included at position 91 of the F2 C-terminus.

[0029] In other embodiments, the p27 peptide is derived from RSV B and has the amino acid sequence EAPQYMNYTINTTKNLNVSISKK RKRR (SEQ ID NO: 150), or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 150. In certain embodiments, the p27 peptide has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99% sequence identity to SEQ ID NO: 150.

[0030] In certain embodiments, the F0 protein contains an optimized variant of the p27 peptide of the RSV F protein located between the F1 domain and the (truncated) F2 domain, wherein the p27 peptide contains a first cleavage site at its C-terminus.

[0031] In certain embodiments, the p27 peptide is modified by deleting one or more of the glycosylation sites in the p27 peptide sequence. The p27 peptide typically has two or three N-linked glycosylation sites. The glycosylation site is typically the "NXS / T" motif that enables N-linked glycosylation in mammalian cells. The motif can be adjacent or not adjacent to a short linker (Gly / Ser). Preferably, all glycosylation sites in the p27 sequence are deleted. Deletion of the glycosylation site can be accomplished by mutating specific amino acids in the NXS / T motif such that the glycosylation site is no longer present, or by deleting one or more amino acids such that the glycosylation site is no longer present.

[0032] According to the present invention, it has been shown that using a deglycosylated mutant of the p27 peptide improves cleavage without requiring the addition of furin.

[0033] In other embodiments, the p27 peptide is modified by deleting 1 to 11 amino acids, preferably 9 or 11 amino acids, from the p27 sequence.

[0034] In certain preferred embodiments, the p27 peptide comprises an amino acid sequence selected from SEQ ID NOs: 185 and 186.

[0035] In certain embodiments, the second non-native cleavage site comprises the sequence RX1X2R, where X1 and X2 can be any amino acid (https: / / doi.org / 10.1371 / journal.pone.0054290).

[0036] In a preferred embodiment, the second non-native cleavage is a furin cleavage site comprising the sequence RX1[K / R]R, preferably RRRR.

[0037] Additionally or alternatively, the protein may include an F1 domain with a truncated C-terminus. Thus, the TM and cytoplasmic regions may be removed to obtain the soluble F protein and to obtain the HMPV F ectodomain. Thus, in certain embodiments, the protein includes a truncated F1 domain. In certain embodiments, the truncated F1 domain does not include a transmembrane region and a cytoplasmic region. The F1 domain may be truncated after the amino acid at position 481, 482, 483, 484, 485, 486, 487, 488, or 489. In certain embodiments, the F1 domain is truncated after the amino acid residue at position 481 or 489. Preferably, the F1 domain is truncated after the amino acid residue at position 489. Thus, in a preferred embodiment, the truncated F1 domain comprises, or consists of, amino acids 103-481 or 103-489, preferably amino acids 103-489 of the HMPV F protein.

[0038] Throughout this application, the position of an amino acid residue (or amino acid position) is shown with reference to the sequence of the HMPV F protein of SEQ ID NO:1. Thus, the expression "amino acid residue at position 88, for example, of the HMPV F protein" as used in the present invention means the amino acid corresponding to the amino acid at position 88 in the HMPV F protein of SEQ ID NO:1. It should be noted that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature F0 protein (SEQ ID NO:1), i.e., including the signal peptide. When using an HMPV strain other than the strain TN / 00 / 3-14 of SEQ ID NO:1, the amino acid positions of the F protein are numbered based on the numbering of the F protein of the strain of SEQ ID NO:1 by inserting gaps as necessary to align the sequences of the other HMPV strains with the F protein of SEQ ID NO:1. The sequence alignment can be performed using methods well known in the art, for example, by CLUSTALW, Bioedit, or CLC Workbench.

[0039] The amino acids according to the present invention can be any of the 20 naturally occurring amino acids (or "standard" amino acids) or variants thereof, such as D-amino acids (the D-enantiomers of amino acids having a chiral center), or any variant not naturally present in proteins (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. For example, cysteine can form disulfide covalent bonds (or disulfide bridges) with other cysteine residues, proline induces rotation of the protein backbone, and glycine is more flexible than other amino acids. Table 2 shows the abbreviations and properties of the standard amino acids. Those skilled in the art will understand that mutations can be introduced into proteins by conventional molecular biology methods.

[0040] As described above, in certain embodiments (e.g., in the case of soluble F proteins), the proteins of the present invention contain a truncated F1 domain. As used herein, a "truncated" F1 domain means an F1 domain that is not the full-length F1 domain, i.e., an F1 domain in which one or more amino acid residues at the C-terminus are deleted. In certain embodiments, at least the transmembrane domain and the cytoplasmic domain are deleted so as to enable expression as a soluble ectodomain. Since the TM region is involved in membrane anchoring and trimerization, an "anchorless" (i.e., without the TM and cytoplasmic domains) soluble F protein is monomeric and exhibits low expression. Therefore, in order to obtain a soluble trimeric F protein with a stable pre-fusion conformation, the pre-fusion conformation needs to be stabilized. To promote trimerization, it is known to replace the TM / CT region with a heterologous trimerization domain (e.g., the foldon domain).

[0041] According to the present invention, there is provided a soluble trimeric HMPV prefusion F protein having no heterologous trimerization domain. Thus, in certain embodiments, the protein according to the present invention comprises one or more stabilizing amino acid residues in the HR2 domain, and the HR2 domain comprises amino acids 453-484 of the HMPV F precursor (F0) protein. According to the present invention, surprisingly, due to the presence of one or more stabilizing mutations in the HR2 domain, even in the absence of a heterologous trimerization domain, compared to the HMPV F protein having no one or more stabilizing mutations in the HR2 domain, it has been found that the trimer content increases.

[0042] As described above, the HMPV F protein is typically a homotrimer, i.e., a macromolecular complex usually formed by three non-covalently bound protein monomers (or protomers). In certain embodiments, the one or more stabilizing amino acids in the HR2 domain optimize the inter-protomer interactions between one or more amino acid residues in the HR2 domains of different HMPV F protomers in the trimer.

[0043] In a preferred embodiment, the amino acid at position 477 is I, V, L, F or M.

[0044] In certain embodiments, further, the amino acid residue at position 473 is I, F or W, and / or the amino acid residue at position 474 is I, and / or the amino acid residue at position 475 is R, and / or the amino acid residue at position 476 is K, and / or the amino acid residue at position 478 is D, and / or the amino acid residue at position 479 is E, and / or the amino acid residue at position 480 is L, and / or the amino acid residue at position 484 is I, and / or the amino acid residue at position 488 is I.

[0045] In a preferred embodiment, the amino acid residue at position 473 is W, the amino acid residue at position 477 is I, and the amino acid residue at position 484 is I.

[0046] In another preferred embodiment, the amino acid residue at position 473 is W, the amino acid residue at position 476 is K, the amino acid residue at position 477 is F, and the amino acid residue at position 484 is I.

[0047] In yet another specific embodiment, the amino acid at position 477 is I, V, L, F or M, the amino acid residue at position 473 is I, F or W, the amino acid residue at position 475 is R, the amino acid residue at position 476 is K, the amino acid residue at position 478 is D, the amino acid residue at position 479 is E, and the amino acid residue at position 484 is I.

[0048] In certain embodiments, to further stabilize the trimeric pre-fusion HMPV F protein, the amino acid residue at position 112 is R and / or the amino acid residue at position 209 is E and / or the amino acid residue at position 453 is P or Q.

[0049] In further embodiments, the amino acid residue at position 149 is Y and / or the amino acid residue at position 313 is W and / or the amino acid residue at position 445 is Y.

[0050] In a preferred embodiment, the amino acid residue at position 112 is R, the amino acid residue at position 209 is E, and the amino acid residue at position 453 is P or Q.

[0051] According to the present invention, the HMPV F protein is stabilized (in the trimeric pre-fusion conformation) by introducing one or more modifications, such as one or more amino acid additions, deletions or substitutions. One such stabilizing modification is the addition of at least one non-native cleavage site in the amino acid sequence of the HMPV F0 protein.

[0052] Another stabilizing modification is the introduction of one or more stabilizing amino acids in the HR2 domain.

[0053] Accordingly, the present invention provides a stabilized trimeric HMPV protein in the pre-fusion conformation. The modifications according to the present invention preferably result in an enhanced stabilization of the pre-fusion conformation of the HMPV F trimer as compared to the HMPV F protein without these modifications. The modifications according to the present invention preferably result in a reduced binding to antibodies (such as DS7) against the post-fusion conformation and / or a reduced binding to the tip-border binding antibodies MPV458 and MPV465, and generate a stable "closed" pre-fusion F trimer as compared to the HMPV F protein without the modifications. Additionally or alternatively, the modifications according to the present invention result in an increase in trimer content, melting temperature and / or trimer stability after storage at 4 °C and / or 37 °C for 2 weeks or after freeze-thaw cycles as compared to the HMPV F protein without these modifications. In particular, the modifications according to the present invention result in an increase in trimer content and trimer stability after storage at 4 °C for at least 6 months as compared to the HMPV F protein without these modifications.

[0054] Additionally or alternatively, the modifications of the present invention result in an increase in the expression level of the pre-fusion HMPV F trimer as compared to the HMPV F protein without these modifications.

[0055] Accordingly, according to the present invention, the presence of specific amino acids at the indicated positions has been demonstrated to enhance the stability of the trimeric protein in the pre-fusion conformation. According to the present invention, the specific amino acids can be already present at the indicated positions (e.g., naturally occurring variants of the HMPV F protein), or can be introduced by substitution (mutation) of the amino acid at that position with a specific amino acid residue according to the present invention. According to the present invention, the protein contains one or more mutations in its amino acid sequence, i.e., one or more naturally occurring amino acids at the indicated positions are replaced by another amino acid.

[0056] Certain stabilizing amino acid residues present in the naturally occurring HMPV F protein include 231I, 404P, and 368N. Thus, in certain embodiments, the amino acid residue at position 231 is I, the amino acid residue at position 404 is P, and / or the amino acid residue at position 368 is N.

[0057] The protein according to the invention may further comprise one or more additional stabilizing mutations, for example, one or more of the stabilizing mutations described in co-pending patent application EP21215259. Thus, in certain embodiments, the amino acid residue at position 69 is Y or W, and / or the amino acid residue at position 73 is W, and / or the amino acid residue at position 185 is P, 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.

[0058] Additionally or alternatively, the protein may further comprise one or more non-natural intramolecular or intermolecular disulfide bonds as described in EP21215259. In certain embodiments, the one or more disulfide bonds are selected from an intramolecular disulfide bond between amino acid residues 140 and 147, and / or an intramolecular disulfide bond between amino acid residues 141 or 161, and / or an intramolecular disulfide bond between amino acid residues 360 and 459.

[0059] Any and / or all of the stabilizing modifications can be used individually and / or in combination with any of the other stabilizing modifications disclosed herein to obtain the HMPV F protein according to the present invention. In an exemplary embodiment, the HMPV F protein comprises at least one non-native cleavage site. Additionally or alternatively, the HMPV F protein comprises one or more stabilizing mutations in the HR2 domain. Additionally or alternatively, the HMPV F protein has an amino acid residue at position 112 that is R, and / or an amino acid residue at position 209 that is E, and / or an amino acid residue at position 453 that is P or Q, and / or an amino acid residue at position 149 that is Y, and / or an amino acid residue at position 313 that is W, and / or an amino acid residue at position 445 that is Y.

[0060] It should be noted again that with respect to the positions of the amino acid residues, SEQ ID NO: 1 is used as a reference. Those skilled in the art will be able to determine the corresponding amino acid residues in the F proteins of other HMPV strains.

[0061] As described above, in certain embodiments (e.g., in the case of soluble F proteins), the protein of the present invention comprises a truncated F1 domain. As used herein, a "truncated" F1 domain means an F1 domain that is not the full-length F1 domain, i.e., an F1 domain in which one or more amino acid residues at the C-terminus are deleted. In certain embodiments, at least the transmembrane domain and the cytoplasmic domain are deleted so as to enable expression as a soluble ectodomain. In certain embodiments, the F1 domain is truncated after amino acid residues 481, 482, 483, 484, 485, 486, 487, 488 or 489.

[0062] In a preferred embodiment, the truncated F1 domain comprises or consists of amino acids 103-489 of the HMPV F protein.

[0063] In a preferred embodiment, the soluble protein of the present invention does not contain a heterologous trimerization domain. However, according to the present invention, the heterologous trimerization domain may be linked to the truncated F1 domain via a linker sequence if desired. In other specific embodiments, the heterologous trimerization domain is a foldon domain comprising the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 136).

[0064] In a preferred embodiment, the HMPV F0 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 135, SEQ ID NOs: 151 to 159, SEQ ID NOs: 161 to 182, and SEQ ID NO: 184, or a fragment thereof. In a particularly preferred embodiment, the HMPV F0 protein comprises the amino acid sequences of SEQ ID NOs: 30, 70, 81, 82, 83, 96, 111, 119, 131, 132, 133, 134, 135, 155, 156, 159, 170, 180, and 184, or a fragment thereof. In a particularly preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 111, 159, 180, or 184, or a fragment thereof. In a preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 111, or a fragment thereof. In a preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 159, or a fragment thereof. In another preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 180, or a fragment thereof. In another preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 184, or a fragment thereof. In a preferred embodiment, the amino acid sequence does not contain any heterologous C-terminal tag sequence.

[0065] The present invention further provides an HMPV F protein comprising at least one modification in the amino acid sequence of the F1 and / or F2 domain, wherein the protein is cleaved at said one or more cleavage sites such that the F2 domain and the F1 domain covalently linked by one or more native disulfide bonds are produced, and the protein is a trimer. As described above, the present invention provides an immature (or inactive) HMPV F0 protein. The protein typically needs to be cleaved (or processed) by a protease in order to be activated after expression. The present invention also includes a processed HMPV F protein, i.e., an HMPV F protein after cleavage (or processing). Thus, the present invention also provides a processed HMPV F protein based on the above-described HMPV F0 protein, wherein the processed protein is cleaved at one or more cleavage sites such that an HMPV F protein comprising a (truncated) F2 domain and a (truncated) F1 domain covalently linked by one or more disulfide bonds to form an F1-F2 dimer is produced. Then three F1-F2 dimers (each F1-F2 dimer is a protomer) form a homotrimer. In embodiments where the inactive F0 protein comprises a second non-native cleavage site, it will be understood that the spacer sequence between the first non-native site and the second non-native site is removed by cleavage.

[0066] It will also be understood that the mature HMPV F protein does not contain a signal sequence.

[0067] In a preferred embodiment, the processed HMPV F protein of the present invention comprises an F1 domain comprising amino acids 103-481, preferably amino acids 103-489 of the HMPV F0 protein, and an F2 domain comprising amino acids 19-88 of the HMPV F0 protein. In a particularly preferred embodiment, the processed HMPV F protein of the present invention comprises an F1 domain comprising amino acids 103-489 of the HMPV F0 protein and an F2 domain comprising amino acids 19-89 of the HMPV F0 protein.

[0068] In certain embodiments, the F1 domain consists of amino acids 103-489 of the HMPV F0 protein, and the F2 domain consists of amino acids 19-89 of the HMPV F0 protein. According to the present invention, the amino acid sequence of the F1 and / or F2 domain contains one or more modifications compared to the amino acid sequence of the F1 and / or F2 domain of the wild-type HMPV F protein.

[0069] According to the present invention, when the F2 domain contains amino acids 19-89, it has been found that the amino acid at position 91 (after cleavage) is preferably R (the same as in the native sequence). Thus, in certain embodiments, the amino acid at position 89+2 is R, and the F2 domain is truncated after the amino acid at position 89, but the addition of a non-native cleavage site (such as RRRR) preferably results in the retention of the R at position 91.

[0070] In certain embodiments, the processed HMPV F protein is derived from an HMPV F0 sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182, and SEQ ID NOs: 184, i.e., it contains the F1 and F2 domains from an F0 sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182, and SEQ ID NOs: 184.

[0071] In particularly preferred embodiments, the processed HMPV F protein contains an F2 domain comprising amino acids 1-89, preferably amino acids 19-89, of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 70, 81, 82, 83, 96, 111, 119, 131, 132, 133, 134, 135, 155, 156, 159, 170, 180, and 184, and an F1 domain comprising amino acids 121-507. In particularly preferred embodiments, the HMPV F protein contains an F2 domain comprising amino acids 1-89, preferably amino acids 19-89, of an amino acid sequence of SEQ ID NOs: 111, 159, 180, or 184, and an F1 domain comprising amino acids 121-507.

[0072] Throughout the present application, nucleotide sequences are presented in the 5' to 3' direction, as is customary in the art, and amino acid sequences are presented from the N-terminus to the C-terminus. Those skilled in the art will understand that the protein can be mutated by conventional molecular biology methods.

[0073] As described above, according to the present invention, the HMPV F protein is stabilized in the pre-fusion conformation, as measured, for example, by a reduction in binding to a post-fusion specific HMPV F antibody (e.g., DS7) and / or a reduction in binding to an open tip border conjugate (e.g., MPV458 and MPV465), compared to the HMPV F protein without the modification of the present invention.

[0074] Additionally or alternatively, the trimer content of the HMPV F protein according to the present invention is increased compared to the HMPV F protein without the modification of the present invention, as measured, for example, by an increase in the trimer content in the supernatant detected by analytical SEC or an increase in the trimer yield of the purified protein.

[0075] Additionally or alternatively, the thermal stability of the HMPV F protein is higher compared to the HMPV F protein without the modification of the present invention, as measured, for example, by the trimer content after heat stress or the melting temperature in the supernatant or the melting temperature of the purified protein.

[0076] The present invention further provides a nucleic acid molecule encoding the HMPV F protein according to the present invention. The nucleic acid molecule can be a DNA or RNA polynucleotide. In a preferred embodiment, the nucleic acid molecule encoding the protein according to the present invention is codon-optimized for expression in mammalian cells, preferably human cells. Methods for codon optimization are known and have been previously described (e.g., WO 96 / 09378). A sequence is considered codon-optimized if at least one non-preferred codon has been replaced by a more preferred codon as compared to the wild-type sequence. Here, a non-preferred codon is a codon that is used in an organism with a lower frequency than another codon encoding the same amino acid, and a more preferred codon is a codon that is used in an organism with a higher frequency than the non-preferred codon. The frequency of codon usage in a particular organism can be found, for example, in the codon frequency table at http: / / www.kazusa.or.jp / codon. Preferably, a plurality of non-preferred codons, preferably most or all non-preferred codons, are replaced by more preferred codons. Preferably, the codons most frequently used in the organism are used in the codon-optimized sequence. Substitution with preferred codons generally results in higher expression.

[0077] As a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that a number of different polynucleotides and nucleic acid molecules can encode the same protein. Also, those skilled in the art will understand that nucleotide substitutions can be made using conventional techniques without affecting the protein sequence encoded by the nucleic acid molecule so as to reflect the codon usage of any particular host organism in which the protein will be expressed. Thus, unless otherwise specifically indicated, "nucleotide sequences or nucleic acid molecules encoding an amino acid sequence" includes all nucleotide sequences or nucleic acid molecules that are degenerate forms of one another and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may or may not contain introns.

[0078] In certain embodiments, the nucleic acid molecule of the present invention encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 135, SEQ ID NOs: 151 to 159, SEQ ID NOs: 161 to 182, and SEQ ID NO: 184, or a processed HMPV F protein derived therefrom, or a fragment thereof. In particularly preferred embodiments, the nucleic acid molecule encodes a protein comprising an amino acid sequence comprising the amino acid sequences of SEQ ID NOs: 30, 70, 81, 82, 83, 96, 111, 119, 131, 132, 133, 134, 135, 155, 156, 159, 170, 180, and 184, or a processed HMPV F protein derived therefrom, or a fragment thereof. In particularly preferred embodiments, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 111, 159, 180 or 184, or a processed HMPV F protein derived therefrom. In preferred embodiments, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 111, or a processed HMPV F protein derived therefrom. In preferred embodiments, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 159, or a processed HMPV F protein derived therefrom. In another preferred embodiment, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 180, or a processed HMPV F protein derived therefrom. In another preferred embodiment, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 184, or a processed HMPV F protein derived therefrom.

[0079] The nucleic acid sequence can be cloned using conventional molecular biology techniques or can be generated de novo by DNA synthesis, which can be performed by service companies (e.g., GeneArt, GenScripts, Invitrogen, Eurofins) operating in the field of DNA synthesis and / or molecular cloning using conventional methods.

[0080] As described above, the nucleic acid molecules described herein can be RNA polynucleotides (or RNAs). The RNA can be mRNA, modified mRNA, self-replicating RNA or circular mRNA.

[0081] Preferred RNAs are self-replicating. A self-replicating RNA molecule (replicon), when delivered into vertebrate cells, can result in the production of multiple daughter RNAs by transcription from itself (through the antisense copies it generates from itself) even in the absence of proteins. Self-replicating RNA molecules are typically + strand molecules that can be directly translated after delivery into cells, and this translation provides an RNA-dependent RNA polymerase, which then generates both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs and collinear subgenomic transcripts are themselves translated to result in in situ expression of the encoded immunogen, or are transcribed to generate additional transcripts having the same sense as the delivered RNA, which can be translated to result in in situ expression of the immunogen. The overall result of this series of transcriptions is a huge amplification in the number of introduced replicon RNAs, and thus, the encoded immunogen becomes the major polypeptide product of the cell.

[0082] One suitable system for achieving self - replication is the use of RNA replicons based on alphaviruses. These + - strand replicons are translated after delivery into cells to produce replicase (or replicase - transcriptase). The replicase is translated as a polyprotein, which autocleaves to produce a replication complex that generates genomic - strand copies of the + - strand delivered RNA. These - strand transcripts are themselves transcribed to produce further copies of the + - strand parental RNA and also produce sub - genomic transcripts that encode immunogens. Thus, translation of the sub - genomic transcripts results in the in - situ expression of immunogens by infected cells. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild - type virus sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used in replicons.

[0083] Accordingly, preferred self - replicating RNA molecules encode (i) an RNA - dependent RNA polymerase capable of transcribing RNA from the self - replicating RNA molecule, and (ii) the HPMV F protein according to the present invention. The polymerase is an alphavirus replicase and can include, for example, one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0084] The native alphavirus genome encodes structural virion proteins in addition to non-structural replicase polyproteins, but the self-replicating RNA molecules of the present invention preferably do not encode alphavirus structural proteins. Thus, preferred self-replicating RNAs can result in the production of genomic RNA copies of themselves intracellularly, but cannot result in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecules cannot persist in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are not present in the self-replicating RNAs of the present invention, and their positions are occupied by genes encoding immunogens of interest, such that the subgenomic transcripts encode immunogens rather than structural alphavirus virion proteins.

[0085] Thus, self-replicating RNA molecules useful in the present invention can have two open reading frames. The first (5') open reading frame encodes a replicase and the second (3') open reading frame encodes an immunogen. In some embodiments, the RNA can have additional (e.g., downstream) open reading frames that encode, for example, additional immunogens (see below) or auxiliary polypeptides.

[0086] Self-replicating RNA molecules can have a 5' sequence that is compatible with the encoded replicase. Self-replicating RNA molecules can have various lengths, but are typically 5000-25000 nucleotides in length, for example, 8000-15000 nucleotides in length, or 9000-12000 nucleotides in length.

[0087] RNA molecules useful in the present invention can have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful in the present invention can have a 5' triphosphate group. In capped RNA, this can be linked to 7-methylguanosine via a 5' to 5' bridge.

[0088] RNA molecules can have a 3' polyA tail. It can also contain a polyA polymerase recognition sequence (e.g., AAUAAA) near its 3' end. RNA molecules useful in the present invention are typically single-stranded. Single-stranded RNA can generally exhibit an adjuvant effect by binding to TLR7, TLR8, RNA helicase, and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be activated by dsRNA formed during the replication of single-stranded RNA or within the secondary structure of single-stranded RNA.

[0089] RNA molecules useful in the present invention can be conveniently prepared by in vitro transcription (IVT). IVT can use a (cDNA) template that is prepared and propagated in plasmid form in bacteria or synthesized (e.g., by gene synthesis and / or polymerase chain manipulation methods). For example, a DNA-dependent RNA polymerase (e.g., bacteriophage T7, T3, or SP6 RNA polymerase) can be used to transcribe RNA from a DNA template. If necessary, appropriate capping and polyA addition reactions can be used (however, the polyA of the replicon is usually encoded within the DNA template). These RNA polymerases may have strict requirements regarding the transcribed 5' nucleotide, and in some embodiments, these requirements need to be consistent with those of the encoded replicase so that it is ensured that the IVT-transcribed RNA can function efficiently as a substrate for its self-encoded replicase.

[0090] Self-replicating RNAs can contain one or more nucleotides with modified nucleobases (in addition to any 5’ cap structure). Thus, the RNAs can contain: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2’-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2’-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6.-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p) (2’-O-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m’lm (1,2’-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4 acetyl 2TO methylcytidine); k2C (lysidine); mlG (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2’-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2’-O-dimethylguanosine); m22Gm (N2,N2,2’-O-trimethylguanosine); Gr(p) (2’-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW *(undermodified hydroxywibutosin); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosylqueuosine); manQ (mannosylqueuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G *(Archaeosine); D (Dihydrouridine); m5Um (5,2’-O-Dimethyluridine); s4U (4-Thiouridine); m5s2U (5-Methyl-2-thiouridine); s2Um (2-Thio-2’-O-methyluridine); acp3U (3-(3-Amino-3-carboxypropyl)uridine); ho5U (5-Hydroxyuridine); mo5U (5-Methoxyuridine); cmo5U (Uridine 5-oxyacetic acid); mcmo5U (Uridine 5-oxyacetic acid methyl ester); chm5U (5-(Carboxyhydroxymethyl)uridine)); mchm5U (5-(Carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-Methoxycarbonylmethyluridine); mcm5Um (S-Methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-Methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-Aminomethyl-2-thiouridine); mnm5U (5-Methylaminomethyluridine); mnm5s2U (5-Methylaminomethyl-2-thiouridine); mnm5se2U (5-Methylaminomethyl-2-selenouridine); ncm5U (5-Carbamoylmethyluridine); ncm5Um (5-Carbamoylmethyl-2’-O-methyluridine); cmnm5U (5-Carboxymethylaminomethyluridine); cnmm5Um (5-Carboxymethylaminomethyl-2-L-O methyluridine); cmnm5s2U (5-Carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-Dimethyladenosine); Tm (2’-O-Methylinosine); m4C (N4-Methylcytidine); m4Cm (N4,2-O-Dimethylcytidine); hm5C (5-Hydroxymethylcytidine); m3U (3-Methyluridine); cm5U (5-Carboxymethyluridine); m6Am (N6,T-O-Dimethyladenosine); rn62Am (N6,N6,0-2-Trimethyladenosine); m2’7G (N2,7-Dimethylguanosine); m2’2’7G (N2,N2,7-Trimethylguanosine); m3Um (3,2T-O-Dimethyluridine); m5D (5-Methyldihydrouridine); f5Cm (5-Formyl-2’-O-methylcytidine); mlGm (1,2’-O-Dimethylguanosine); m’Am (1,2-O-dimethyladenosine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14 (4-demethylguanosine); imG2 (isoguanosine); or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6) alkyluracil, 5-methyluracil, 5-(C2-C6) alkenyluracil, 5-(C2-C6) alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxylcytosine, 5-(C1-C6) alkylcytosine, 5-methylcytosine, 5-(C2-C6) alkenylcytosine, 5-(C2-C6) alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6) alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or abasic nucleotide. For example, self-replicating RNA can contain one or more modified pyrimidine nucleobases such as pseudouridine and / or 5-methylcytosine residues. However, in some embodiments, the RNA can be free of modified nucleobases and also free of modified nucleotides, i.e., all of the nucleotides in the RNA are standard A, C, G, and U ribonucleotides (except for a 5' cap structure that can include 7'-methylguanosine). In other embodiments, the RNA can include a 5' cap that includes 7'-methylguanosine, and the first, second, or third 5' ribonucleotide can be methylated at the 2' position of the ribose. The RNA used in the present invention preferably contains only phosphodiester bonds between nucleosides, but in some embodiments, it can contain phosphoramidate, phosphorothioate, and / or methylphosphonate bonds. Preferably, the liposome contains fewer than 10 (e.g., 5, 4, 3, or 2) different RNA species, and most preferably, the liposome contains a single RNA species, i.e., all of the RNA molecules in the liposome have the same sequence and the same length.,

[0091] In certain embodiments, the nucleic acid molecule is an RNA polynucleotide having an open reading frame encoding the HPMV F protein according to the present invention or a fragment thereof, and can be formulated in a cationic lipid nanoparticle, a cationic nanoemulsion, or a polymer-based formulation, or in any combination or alternative formulation suitable for introducing saRNA into human or animal cells in vitro or in vivo in various species including humans.

[0092] In some embodiments, the RNA replicons of the present disclosure can be formulated using one or more liposomes, lipoplexes, and / or lipid nanoparticles. In some embodiments, the liposome or lipid nanoparticle formulations described herein can include a polycationic composition. In some embodiments, formulations containing a polycationic composition can be used to deliver the RNA replicons described herein in vivo and / or in vitro.

[0093] Accordingly, the present invention further provides an RNA replicon encoding a recombinant pre-fusion HMPV F protein or a fragment thereof, wherein the HMPV F protein comprises an amino acid sequence selected from SEQ ID NO: 180 or 184, or a fragment or variant thereof.

[0094] In certain embodiments, the RNA replicon, in the 5' to 3' direction, (1) a 5' untranslated region (5'-UTR) required for RNA virus non-structural protein-mediated amplification, (2) a polynucleotide sequence encoding at least one, preferably all, of the RNA virus non-structural proteins, (3) an RNA virus subgenomic promoter, (4) a polynucleotide sequence encoding a recombinant pre-fusion HMPV F protein or a fragment or variant thereof, and (5) a 3' untranslated region (3'-UTR) required for RNA virus non-structural protein-mediated amplification is included.

[0095] In certain embodiments, the RNA replicon, in the 5' to 3' direction, (1) an alphavirus 5' untranslated region (5'-UTR), (2) the 5' replication sequence of the alphavirus non-structural gene nsp1, (3) the downstream loop (DLP) motif of the virus species, (4) a polynucleotide sequence encoding an autocatalytic protease peptide, (5) A polynucleotide sequence encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3, and nsp4, (6) An alphavirus subgenomic promoter, (7) A polynucleotide sequence encoding a recombinant prefusion HMPV F protein or a fragment thereof, (8) An alphavirus 3' untranslated region (3'UTR), and (9) A polyadenosine sequence that may optionally be included comprises.

[0096] In certain embodiments, this specification, in the direction from the 5' end to the 3' end, (1) A 5'-UTR having the polynucleotide sequence of SEQ ID NO: 136, (2) A 5' replication sequence having the polynucleotide sequence of SEQ ID NO: 137, (3) A DLP motif comprising the polynucleotide sequence of SEQ ID NO: 138, (4) A polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 139, (5) Polynucleotide sequences encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3, and nsp4 having the nucleic acid sequences of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, and SEQ ID NO: 143, respectively, (6) A subgenomic promoter having the polynucleotide sequence of SEQ ID NO: 144, (7) A polynucleotide sequence encoding a prefusion HMPV F protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 180 and SEQ ID NO: 184, or a fragment or variant thereof, and (8) A 3'UTR having the polynucleotide sequence of SEQ ID NO: 145 comprises.

[0097] In certain embodiments, (a) the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 146, and the RNA replicon further comprises a polyadenosine sequence, preferably, the polyadenosine sequence has SEQ ID NO: 147 at the 3' end of the replicon.

[0098] In certain embodiments, the RNA replicon comprises the polynucleotide sequence of SEQ ID NO: 187 or 188. In preferred embodiments, nucleotides 7972-9649 of the polynucleotide sequence of SEQ ID NO: 187 are the coding sequence for the HMPV F protein (including the stop codon), particularly the HMPV protein of SEQ ID NO: 184. In another preferred embodiment, nucleotides 7972-9500 of the polynucleotide sequence of SEQ ID NO: 188 are the coding sequence for the HMPV F protein (including the stop codon), particularly the HMPV protein of SEQ ID NO: 184.

[0099] Also provided is a nucleic acid comprising a DNA sequence encoding the RNA replicon described herein, preferably, the nucleic acid further comprises a T7 promoter operably linked to the 5' end of the DNA sequence, more preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 148.

[0100] As used herein, the term "fragment" refers to a protein or (poly)peptide having amino-terminal and / or carboxy-terminal and / or internal deletions, but the remaining amino acid sequence is identical to the corresponding position in the sequence of the HMPV F protein, e.g., the full-length sequence of the HMPV F protein. It will be understood that for inducing an immune response and generally for the purpose of vaccination, the protein need not be full-length and need not have all of its wild-type functions, and fragments of the protein are equally useful.

[0101] Fragments according to the invention are immunologically active fragments and typically comprise at least 15 or at least 30 amino acids of the HMPV F protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 550 amino acids of the HMPV F protein.

[0102] Furthermore, for example, those skilled in the art will understand that proteins can be modified, for example, by amino acid substitution, deletion, addition, etc. using ordinary molecular biology methods. Generally, conservative amino acid substitutions can be applied without accompanying a decrease in the function or immunogenicity of the polypeptide. This can be easily confirmed according to ordinary methods well-known to those skilled in the art.

[0103] As a result of the degeneracy of the genetic code, those skilled in the art will understand that a number of different nucleic acids can encode the same polypeptide or protein. Also, those skilled in the art understand that nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acid molecule can be made using ordinary techniques so as to reflect the codon usage of any particular host organism in which the polypeptide will be expressed. Thus, unless otherwise indicated, the "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may or may not contain introns.

[0104] Nucleic acid sequences can be cloned using ordinary molecular biology techniques or can be generated de novo by DNA synthesis, which can be carried out using ordinary methods by service companies (e.g., GeneArt, GenScripts, Invitrogen, Eurofins) operating in the field of DNA synthesis and / or molecular cloning.

[0105] The present invention also provides a vector comprising the nucleic acid molecule described above. Thus, in certain embodiments, the nucleic acid molecule according to the invention is part of a vector. Such vectors can be readily manipulated by methods well known to those skilled in the art and can be designed, for example, to be capable of replication in prokaryotic and / or eukaryotic cells. In addition, many vectors can be used for the transformation of eukaryotic cells and are integrated either wholly or partially into the genome of such cells to generate stable host cells containing the desired nucleic acid within their genomes. The vector used can be any vector suitable for DNA cloning that can be used for the transcription of the nucleic acid of interest.

[0106] Preferably, the vector is a self-replicating RNA replicon.

[0107] As used herein, the term "self-replicating RNA molecule" is used interchangeably with "self-amplifying RNA molecule" or "RNA replicon" or "replicon RNA" or "saRNA", and refers to an RNA molecule engineered from the genome of a positive-strand RNA virus that contains all of the genetic information necessary to direct its own amplification or self-replication in a permissive cell. The self-replicating RNA molecule is similar to mRNA. It is single-stranded, 5'-capped, 3'-polyadenylated, and of positive orientation. The RNA molecule encodes a polymerase, replicase, or other protein that can interact with a protein, nucleic acid, or ribonucleoprotein from a virus or host cell to catalyze an RNA amplification process in order to direct its own replication, and contains cis-acting RNA sequences necessary for the replication and transcription of subgenomic replicon-encoded RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs and collinear subgenomic transcripts are themselves translated to result in in situ expression of a gene of interest, or are transcribed to produce additional transcripts having the same sense as the delivered RNA, which can be translated to result in in situ expression of a gene of interest. The overall result of this series of transcriptions is a huge amplification in the number of introduced replicon RNAs, and thus the encoded gene of interest becomes the major polypeptide product of the cell.

[0108] In certain embodiments, the RNA replicon of the present application comprises, in the direction from the 5'-end to the 3'-end, (1) a 5'-untranslated region (5'-UTR) necessary for RNA virus non-structural protein-mediated amplification, (2) a polynucleotide sequence encoding at least one, preferably all, of the non-structural proteins of an RNA virus, (3) a subgenomic promoter of an RNA virus, (4) a polynucleotide sequence encoding a recombinant fusion pre-HMPV F protein or a fragment or variant thereof, and (5) a 3'-untranslated region (3'-UTR) necessary for RNA virus non-structural protein-mediated amplification.

[0109] In certain embodiments, the self-replicating RNA molecule encodes an enzyme complex (replicase polyprotein) for self-amplification that includes RNA-dependent RNA polymerase function, helicase, capping, and polyadenylation activities. Viral structural genes downstream of the replicase under the control of a subgenomic promoter can be replaced by the prefusion HMPV F protein or a fragment or variant thereof described herein. The replicase, when transfected, is immediately translated and interacts with the 5' and 3' ends of the genomic RNA to synthesize complementary genomic RNA copies. These serve as templates for the synthesis of capped and polyadenylated genomic copies of the new plus strand and subgenomic transcripts. Amplification ultimately results in a very high RNA copy number of up to 2×10 5 copies per cell. Thus, much less saRNA is sufficient to achieve efficient gene delivery and protective vaccination compared to normal mRNA (Beissert et al., Hum Gene Ther. 2017, 28(12):1138-1146).

[0110] Subgenomic RNA is an RNA molecule of shorter length or smaller size than the genomic RNA from which it is derived. Viral subgenomic RNA can be transcribed from an internal promoter having a sequence present in the genomic RNA or its complement. Transcription of subgenomic RNA can be mediated by viral-encoded polymerases associated with proteins, ribonucleoproteins, or combinations thereof encoded by the host cell. Many RNA viruses produce subgenomic mRNAs (sgRNAs) for the expression of their 3'-proximal genes.

[0111] In some embodiments of the present disclosure, the pre-fusion HMPV F protein or fragment thereof described herein is expressed under the control of a subgenomic promoter. In certain embodiments, instead of the native subgenomic promoter, the subgenomic RNA can be placed under the control of an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV), bovine viral diarrhea virus (BVDV), poliovirus, foot-and-mouth disease virus (FMD), enterovirus 71, or hepatitis C virus. Subgenomic promoters range from 24 nucleotides (Semliki Forest virus) to over 100 nucleotides (turnip yellow mosaic virus) and are typically found upstream of the transcription start point.

[0112] In some embodiments, the RNA replicon comprises the coding sequences of at least one, at least two, at least three, or at least four non-structural viral proteins (e.g., nsP1, nsP2, nsP3, nsP4). The genome of an alphavirus encodes the non-structural proteins nsP1, nsP2, nsP3, and nsP4, which are produced as a single polyprotein precursor sometimes referred to as P1234 (or nsP1-4 or nsP1234), which is cleaved into mature proteins by proteolytic processing. nsP1 can have a size of about 60 kDa, can have methyltransferase activity, and can be involved in the capping reaction of the virus. nsP2 has a size of about 90 kDa and can have helicase and protease activity, while nsP3 is about 60 kDa and contains three domains: a macrodomain, a central (or alphavirus-specific) domain, and a hypervariable domain (HVD). nsP4 has a size of about 70 kDa and contains the core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection, the alphavirus genomic RNA is translated to produce the P1234 polyprotein, which is cleaved into individual proteins. Disclosure of a nucleic acid or polypeptide sequence herein, e.g., the sequences of nsP1, nsP2, nsP3, nsP4, also discloses sequences that are considered to be based on or derived from the original sequence.

[0113] In some embodiments, the RNA replicon comprises a coding sequence of a portion of at least one of the non-structural viral proteins described above. For example, the RNA replicon can comprise about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the coding sequence of at least one non-structural viral protein, or a range between any two of these values. In some embodiments, the RNA replicon can comprise a coding sequence of a substantial portion of at least one non-structural viral protein. As used herein, a "substantial portion" of a nucleic acid sequence encoding a non-structural viral protein includes a nucleic acid sequence encoding a non-structural viral protein that is sufficient to achieve an approximate identification of that protein, by manual evaluation of the sequence by one of ordinary skill in the art or by computer automated sequence comparison and identification using an algorithm such as BLAST (see, e.g., "Basic Local Alignment Search Tool"; Altschul S F et al., J. Mol. Biol. 215:403-410, 1993). In some embodiments, the RNA replicon can comprise the entire coding sequence of at least one of the non-structural proteins described above. In some embodiments, the RNA replicon comprises substantially all of the coding sequence of a native viral non-structural protein. In certain embodiments, the one or more non-structural viral proteins described above are from the same virus. In other embodiments, the one or more non-structural proteins described above are from different viruses.

[0114] An RNA replicon can be derived from any suitable plus-strand RNA virus, such as an alphavirus or a flavivirus. Preferably, the RNA replicon is derived from an alphavirus. The term "alphavirus" means an enveloped, single-stranded plus-sense RNA virus of the family Togaviridae. The genus Alphavirus contains approximately 30 members, which can infect humans and other animals. Alphavirus particles typically have a diameter of 70 nm, tend to be spherical or slightly pleomorphic, and have an isometric nucleocapsid of 40 nm. The total genome length of alphaviruses ranges from 11,000 to 12,000 nucleotides and has a 5' cap and a 3' polyA tail. Two open reading frames (ORFs) [non-structural (ns) and structural] are present within the genome. The nsORF encodes the proteins (nsP1-nsP4) required for viral RNA transcription and replication. The structural ORF encodes three structural proteins, namely the core nucleocapsid protein C, and the envelope proteins P62 and E1 that associate as a heterodimer. The surface glycoprotein anchored to the viral membrane results in receptor recognition and entry into target cells by membrane fusion. The four ns protein genes are encoded by genes in the 5'-proximal two-thirds of the genome, and the three structural proteins are translated from a subgenomic mRNA that is colinear with the 3'-proximal one-third of the genome.

[0115] In some embodiments, the self-replicating RNA useful in the present invention is an RNA replicon derived from an alphavirus viral species. In some embodiments, the alphavirus RNA replicon is from an alphavirus belonging to the VEEV / EEEV group or the SF group or the SIN group. Non-limiting examples of SF group alphaviruses include Semliki Forest virus, O'nyong'nyong virus, Ross River virus, Middelburg virus, Chikungunya virus, Barmah Forest virus, Getah virus, Mayaro virus, Sagiyama virus, Bebaru virus, and Una virus. Non-limiting examples of SIN group alphaviruses include Sindbis virus, Gwaltney SA virus, South African arbovirus 86, Ockelbo virus, Aura virus, Babanki virus, Wataroa virus, and Kyzylagach virus. Non-limiting examples of VEEV / EEEV group alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mukambo virus (MUCV), Pixuna virus (PIXV), Middelburg virus (MIDV), Chikungunya virus (CHIKV), O'nyong'nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (UNAV).

[0116] Non-limiting examples of alphavirus species include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mukambo virus (MUCV), Semliki Forest virus (SFV), Pixuna virus (PIXV), Middelburg virus (MIDV), Chikungunya virus (CHIKV), O'nyong'nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Wataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highlands J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) and Baggie Creek virus. Both toxic and non-toxic alphavirus strains are suitable. In some embodiments, the alphavirus RNA replicon is that of Sindbis virus (SIN), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV) or Eastern equine encephalitis virus (EEEV). In some embodiments, the alphavirus RNA replicon is that of Venezuelan equine encephalitis virus (VEEV).

[0117] In certain embodiments, the self-replicating RNA molecule comprises a polynucleotide encoding one or more non-structural proteins nsp1-4, a subgenomic promoter, such as the 26S subgenomic promoter, and a gene of interest encoding the pre-fusion HMPV F protein or a fragment thereof as described herein.

[0118] The self-replicating RNA molecule can have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA.

[0119] The 5'-nucleotides of the self-replicating RNA molecules useful in the present invention may have a 5'-triphosphate group. In capped RNA, this can be linked to 7-methylguanosine via a 5'-to-5' bridge. The 5'-triphosphate can enhance RIG-I binding.

[0120] The self-replicating RNA molecule may have a 3'-polyA tail. It may also contain a polyA polymerase recognition sequence (e.g., AAUAAA) near its 3'-end.

[0121] In any of the embodiments of the present disclosure, the RNA replicon can be devoid (or contain no) of the coding sequence of at least one (or all) of the structural viral proteins (e.g., nucleocapsid protein C and envelope proteins P62, 6K, and E1). In these embodiments, the sequence encoding one or more structural genes can be replaced with one or more heterologous sequences such as, for example, the coding sequence of the prefusion HMPV F protein or a fragment thereof described herein. In a preferred embodiment, the RNA replicon is devoid (or contains no) of all of the coding sequences of the structural viral proteins.

[0122] In certain embodiments, the self-replicating RNA vector of the present application includes one or more features for conferring resistance to translational repression by the innate immune system or for enhancing the expression of the GOI (e.g., the prefusion HMPV F protein or a fragment or variant thereof described herein).

[0123] In certain embodiments, the RNA sequence can be codon-optimized to improve translation efficiency. The RNA molecule can be modified by any method known in the art for enhancing stability and / or translation in view of the present disclosure, for example, by adding a polyA tail of at least 30 adenosine residues and / or by capping the 5'-end with a modified ribonucleotide that can be incorporated during RNA synthesis or enzymatically manipulated post-transcriptionally (e.g., a 7-methylguanosine cap).

[0124] In certain embodiments, the RNA replicon of the present application, in the direction from the 5' end to the 3' end, comprises (1) an alphavirus 5' untranslated region (5'-UTR), (2) the 5' replication sequence of the alphavirus non-structural gene nsp1, (3) the downstream loop (DLP) motif of the viral species, (4) a polynucleotide sequence encoding an autoprotease peptide, (5) a polynucleotide sequence encoding the alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4, (6) an alphavirus subgenomic promoter, (7) a polynucleotide sequence encoding a recombinant prefusion HMPV F protein or a fragment or variant thereof, (8) an alphavirus 3' untranslated region (3'UTR), and (9) an optional polyadenosine sequence that may be included if desired.

[0125] In certain embodiments, the self-replicating RNA vector of the present application includes a downstream loop (DLP) motif of a viral species. As used herein, "downstream loop" or "DLP motif" means a polynucleotide sequence comprising at least one RNA stem-loop, which, when placed downstream of the start codon of an open reading frame (ORF), results in enhanced ORF translation compared to other constructs that are otherwise identical but do not have the DLP motif. As an example, members of the genus Alphavirus are able to resist activation of the antiviral RNA-activated protein kinase (PKR) by a prominent RNA structure present within the viral 26S transcript, which allows for eIF2-independent translation initiation of these mRNAs. This structure, termed the downstream loop (DLP), is located downstream of the AUG of the SINV 26S mRNA. The DLP is also detected in Semliki Forest virus (SFV). Similar DLP structures have been reported to be present in at least 14 other members of the genus Alphavirus, including members of the New World [e.g., MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV] and Old World (SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV). The putative structures of these alphavirus 26S mRNAs were constructed based on SHAPE (selective 2'-hydroxyl acylation and primer extension) data [Toribio et al., Nucleic Acids Res. May 19;44(9):4368-80, 2016, the contents of which are incorporated herein by reference]. In all cases except CHIKV and ONNV, a stable stem-loop structure was detected, although MAYV and EEEV showed DLP with lower stability (Toribio et al., 2016 supra). In the case of Sindbis virus, the DLP motif is found in the first 150 nt of the Sindbis subgenomic RNA. The hairpin is located downstream of the Sindbis capsid AUG start codon (AUG corresponds to nt50 of the Sindbis subgenomic RNA).Previous studies of array comparison and structural RNA analysis have shown the evolutionary conservation of DLP in SINV and inferred the existence of equivalent DLP structures in many members of the genus Alphavirus (see, for example, Ventoso, J. Virol. 9484-9494, Vol. 86, September 2012). Examples of self-replicating RNA vectors containing the DLP motif are described in US Patent Application Publication US2018 / 0171340 and International Patent Application Publication WO2018106615 (the entire contents of which are incorporated herein by reference). In some embodiments, the replicon RNA of the present application contains a DLP motif that exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 138.

[0126] In one embodiment, the self-replicating RNA molecule also contains a coding sequence for a self-protease peptide that is functionally linked upstream of the coding sequence of a non-structural protein (e.g., one or more of nsp1-4) or a gene of interest (e.g., the prefusion HMPV F protein or a fragment thereof described herein) downstream of the DLP motif. Examples of self-protease peptides include, but are not limited to, peptide sequences selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Theiler's murine encephalomyelitis virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), and combinations thereof. In some embodiments, the replicon RNA of the present application contains the coding sequence of P2A having the amino acid sequence of SEQ ID NO: 139. Preferably, the coding sequence exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 146.

[0127] All replicons of the present invention may also include 5' and 3' untranslated regions (UTRs). The UTRs can be wild-type New World or Old World alphavirus UTR sequences or sequences derived from any of them. In various embodiments, the 5' UTR can be of any suitable length, such as about 60 nt, 50 - 70 nt or 40 - 80 nt. In some embodiments, the 5' UTR can also have a conserved primary or secondary structure (e.g., one or more stem-loops) and can be involved in the replication of alphavirus or replicon RNA. In some embodiments, the 3' UTR is up to several hundred nucleotides, for example, it can be 50 - 900, or 100 - 900, or 50 - 800, or 100 - 700, or 200 nt - 700 nt. The 3' UTR can also have a secondary structure, such as a stem-loop, followed by a polyadenylic acid tract or poly(A) tail. In any of the embodiments of the present invention, the 5' and 3' untranslated regions can be operably linked to any of the other sequences encoded by the replicon. The UTRs can be operably linked to a sequence encoding a heterologous protein or peptide and / or a promoter by providing the sequences and spacings necessary for the recognition and transcription of the other coding sequences. In view of the present disclosure, any polyadenylation signal known to those skilled in the art can be used. For example, the polyadenylation signal can be the SV40 polyadenylation signal, the LTR polyadenylation signal, the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal or the human β-globin polyadenylation signal.

[0128] In another embodiment, the self-replicating RNA replicon of the present application includes a modified 5' untranslated region (5'-UTR), and preferably, the RNA replicon lacks at least a part of the nucleic acid sequence encoding the viral structural protein. For example, the modified 5'-UTR may include one or more nucleotide substitutions at positions 1, 2, 4, or combinations thereof. Preferably, the modified 5'-UTR includes a nucleotide substitution at position 2, and more preferably, the modified 5'-UTR has a substitution from U to G or from U to A at position 2. Examples of such self-replicating RNA molecules are described in US Patent Application Publication US2018 / 0104359 and International Patent Application Publication WO2018075235 (the entire contents of which are incorporated herein by reference). In some embodiments, the replicon RNA of the present application includes a 5'-UTR that exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 136.

[0129] In some embodiments, the RNA replicons of the present application include a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream or at the 5' end of the polynucleotide sequence encoding the pre-fusion HMPV F protein or a fragment thereof. Signal peptides typically direct protein localization, facilitate secretion of the protein from the cell in which it is produced, and / or improve antigen expression and cross-presentation to antigen-presenting cells. The signal peptide may be present at the N-terminus of the pre-fusion HMPV F protein or a fragment thereof when expressed from the replicon, but is cleaved off by signal peptidase, for example, upon secretion from the cell. The expressed protein from which the signal peptide has been cleaved is often referred to as the "mature protein". In view of the present disclosure, any signal peptide known in the art may be used. For example, the signal peptide may be the cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG, the Ig heavy chain epsilon signal peptide SPIgE, or the short leader peptide sequence of HMPV. An exemplary nucleic acid sequence encoding the signal peptide is shown in SEQ ID NO: 149.

[0130] In various embodiments, the RNA replicons disclosed herein can be engineered, synthetic, or recombinant RNA replicons. By way of non-limiting example, the RNA replicon can be one or more of the following: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques, such as by utilization of chemical nucleic acid synthesis or use of enzymes for replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (including methylation) or recombination (including homologous recombination and site-specific recombination); 2) linked nucleotide sequences that are not naturally linked; 3) engineered using molecular cloning techniques and lacking one or more nucleotides compared to a naturally occurring nucleotide sequence; and 4) engineered using molecular cloning techniques and having one or more sequence changes or rearrangements compared to a naturally occurring nucleotide sequence.

[0131] Any component or sequence of an RNA replicon can be operably linked to any other component or sequence. Components or sequences of an RNA replicon can be operably linked for the expression of a gene of interest in a host cell or treated organism and / or for the self-replicating ability of the replicon. As used herein, the term “operably linked” should be construed in its broadest reasonable context and means the linkage of polynucleotide elements in a functional relationship. A polynucleotide is said to be “operably linked” when it is placed in a functional relationship to another polynucleotide. For example, a promoter or UTR operably linked to a coding sequence can effect the transcription and expression of the coding sequence in the presence of appropriate enzymes. The promoter need not be adjacent to the coding sequence so long as it functions to direct the expression of the coding sequence. Thus, a functional linkage between an RNA sequence encoding a heterologous protein or peptide and a regulatory sequence (e.g., a promoter or UTR) is a functional linkage that enables the expression of the polynucleotide of interest. Functional linkages also mean that sequences such as RdRp (e.g., nsP4), nsP1-4, UTRs, promoters, and other coding sequences in an RNA replicon are linked such that they enable the transcription or translation of the pre-fusion HMPV F protein and / or replication of the replicon. UTRs can be operably linked by providing the sequences and spacing necessary for the recognition and translation of other coding sequences by ribosomes.

[0132] The immunogenicity of the pre-fusion HMPV F protein or fragments or variants thereof expressed by an RNA replicon can be determined by several assays known to those of skill in the art in view of the present disclosure.

[0133] Another general aspect of the present application relates to a nucleic acid comprising a DNA sequence encoding the RNA replicon of the present application. The nucleic acid can be, for example, a DNA plasmid or a fragment of a linearized DNA plasmid. Preferably, the nucleic acid further comprises a promoter, such as a T7 promoter, operably linked to the 5' end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 148. The nucleic acid can be used in the production of the RNA replicon of the present application using methods known in the art in view of the present disclosure. For example, the RNA replicon can be obtained by in vivo or in vitro transcription of the nucleic acid.

[0134] Host cells containing the RNA replicon of the present application or a nucleic acid encoding the RNA replicon also form part of the present invention. The HMPV F protein or a fragment or variant thereof can be produced by recombinant DNA technology, including the expression of the molecule in host cells such as 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, etc., or transgenic animals or plants. In certain embodiments, the cells are derived from multicellular organisms, and in certain embodiments, the cells are derived from vertebrates or invertebrates. In certain embodiments, the cells are mammalian cells, such as human cells, or insect cells. Generally, the production of a recombinant protein, such as the HMPV F protein or a fragment or variant thereof of the present invention, in a host cell involves introducing a heterologous nucleic acid molecule encoding the protein in an expressible form into the host cell, culturing the cell under conditions suitable for the expression of the nucleic acid molecule, and expressing the protein or a fragment or variant thereof in the cell. The nucleic acid molecule encoding the protein in an expressible form can be in the form of an expression cassette and usually requires sequences capable of effecting the expression of the nucleic acid, such as enhancers, promoters, polyadenylation signals, etc. Those skilled in the art recognize that various promoters can be used to express genes in host cells. The promoter can be constitutive or regulatable and can be obtained from various sources, including viruses, prokaryotes, or eukaryotes, or can be artificially designed.

[0135] In certain embodiments of the invention, the vector is an adenovirus vector. The adenovirus according to the invention belongs to the family Adenoviridae and preferably belongs to the genus Mastadenovirus. It can be a human adenovirus, but it is also possible that it is an adenovirus that infects other species, including bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), canine adenovirus (e.g., CAdV2), porcine adenovirus (e.g., PAdV3 or 5) or simian adenovirus (including simian adenovirus and ape adenovirus, e.g., chimpanzee adenovirus or gorilla adenovirus), but is not limited thereto. Preferably, the adenovirus is a human adenovirus (HAdV or AdHu) or a simian adenovirus, such as a chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV) or a rhesus adenovirus (RhAd). In the present invention, when referred to as Ad without indication of the species, it means a human adenovirus. For example, the shorthand notation "Ad26" means the same as HAdV26, which is human adenovirus serotype 26. Also, the notation "rAd" used herein means a recombinant adenovirus. For example, "rAd26" means recombinant human adenovirus 26.

[0136] The most advanced research has been conducted using human adenoviruses, and in certain embodiments of the invention, human adenoviruses are preferred. In certain preferred embodiments, the recombinant adenoviruses according to the invention are based on human adenoviruses. In a preferred embodiment, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the invention, the adenovirus is a human adenovirus of serotype 26. The advantages of these serotypes include low seroprevalence and / or low existing neutralizing antibody titers in the human population, as well as experience of use in human subjects in clinical trials. In some embodiments, the adenovirus is replication-deficient, for example because it contains a deletion in the E1 region of the genome. In the case of adenoviruses derived from non-group C adenoviruses such as Ad26 or Ad35, it is common to replace the E4-orf6 coding sequence of the adenovirus with that of a human subgroup C adenovirus such as Ad5. This enables the growth of such adenoviruses in well-known complementing (or packaging) cell lines that express the E1 gene of Ad5, such as 293 cells, PER.C6 cells, etc. (see, for example, Havenga et al., 2006, J Gen Virol 87:2135-43; WO 03 / 104467). However, such adenoviruses are unable to replicate in non-complementing cells that do not express the E1 gene of Ad5.

[0137] Host cells containing a nucleic acid molecule encoding a pre-fusion HMPV F protein also form part of the present invention. The pre-fusion HMPV F protein can be produced by recombinant DNA techniques including expression of the molecule in a host cell, such as 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, etc., or transgenic animals or plants. In certain embodiments, the cell is derived from a multicellular organism, and in certain embodiments, it is derived from a vertebrate or invertebrate. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. Generally, the production of a recombinant protein, such as the pre-fusion HMPV F protein of the present invention, in a host cell involves introducing a heterologous nucleic acid molecule encoding the protein in an expressible form into the host cell, culturing the cell under conditions suitable for expression of the nucleic acid molecule, and expressing the protein in the cell. The nucleic acid molecule encoding the protein in an expressible form can be in the form of an expression cassette and typically requires sequences capable of effecting expression of the nucleic acid, such as enhancers, promoters, polyadenylation signals, etc. Those skilled in the art will recognize that various promoters can be used to express genes within host cells. The promoter can be constitutive or regulatable and can be obtained from various sources including viruses, prokaryotes or eukaryotes, or can be artificially designed.

[0138] Cell culture media are available from various vendors, and a suitable medium for host cells expressing the protein of interest (here, the pre-fusion HMPV F protein) can routinely be selected. The suitable medium may or may not contain serum.

[0139] A "heterologous nucleic acid molecule" (also referred to herein as a "transgene") is a nucleic acid molecule that does not naturally occur in a host cell. It is introduced, for example, into a vector by standard molecular biology techniques. A transgene is generally operably linked to expression control sequences. This can be done, for example, by placing the nucleic acid encoding the transgene under the control of a promoter. Further regulatory sequences may be added. A number of promoters are available for the expression of transgenes and are known to those skilled in the art and may include, for example, viral, mammalian, synthetic promoters, etc. Non-limiting examples of suitable promoters for achieving expression in eukaryotic cells include the CMV promoter (US 5,385,839), such as the CMV immediate early promoter, which includes, for example, nt. -735 to +95 from the CMV immediate early gene enhancer / promoter. A polyadenylation signal, such as the bovine growth hormone polyA signal (US 5,122,458), may be present after the transgene. Alternatively, several widely used expression vectors are available in the art and from commercial suppliers and include, for example, the pcDNA and pEF vector series from Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc., which can be used to recombinantly express a protein of interest or to obtain appropriate promoter and / or transcription terminator sequences, polyA sequences, etc.

[0140] 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 microcarrier) and suspension culture. Most large-scale suspension cultures are carried out as batch or fed-batch processes because they are the easiest to implement and scale up. Today, continuous processes based on perfusion principles are becoming more common and are equally suitable. Appropriate culture media are also well known to those skilled in the art and are generally available in large quantities from commercial suppliers or can be specially prepared according to standard protocols. Culturing can be carried out, for example, in dishes, roller bottles or bioreactors, using batch, fed-batch, continuous systems, etc. Appropriate conditions for culturing cells are known (see, for example, Tissue Culture, Academic Press, edited by Kruse and Paterson (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)).

[0141] The present invention further provides a pharmaceutical composition comprising a pre-fusion HMPV F protein and / or a fragment thereof and / or a nucleic acid molecule and / or a vector as described herein. Thus, the present invention provides a composition comprising a pre-fusion HMPV F protein or a fragment thereof that presents epitopes that are present in the pre-fusion conformation of the HMPV F protein but not in the post-fusion conformation. The present invention also provides a composition comprising a nucleic acid molecule and / or a vector encoding such a pre-fusion HMPV F protein or fragment. In particular, the present invention provides a pharmaceutical composition, such as a vaccine composition, comprising the pre-fusion HMPV F protein, the HMPV F protein fragment and / or the nucleic acid molecule and / or the vector and one or more pharmaceutically acceptable excipients.

[0142] The present invention also provides the use of the stabilized pre-fusion HMPV F protein (fragment), nucleic acid molecule and / or vector according to the invention for vaccinating a subject against HMPV.

[0143] The present invention also provides the use of the stabilized pre-fusion HMPV F protein (fragment), nucleic acid molecule, and / or vector according to the invention for inducing an immune response against the HMPV F protein in a subject. Further, provided is a method for inducing an immune response against the HMPV F protein in a subject, comprising administering to the subject the pre-fusion HMPV F protein (fragment) and / or nucleic acid molecule and / or vector according to the invention. Further, provided is the use of the pre-fusion HMPV F protein (fragment) and / or nucleic acid molecule and / or vector according to the invention for the manufacture of a medicament used for inducing an immune response against the HMPV F protein in a subject. In particular, the present invention provides the pre-fusion HMPV F protein (fragment) and / or nucleic acid molecule and / or vector according to the invention for use as a vaccine.

[0144] The pre-fusion HMPV F protein (fragment), nucleic acid molecule or vector of the present invention can be used for the prevention (defense) and / or treatment of HMPV infection. In certain embodiments, the prevention and / or treatment can be directed to patient groups susceptible to HMPV infection. Such patient groups can include, for example, the elderly (e.g., 50 years or older, 60 years or older, preferably 65 years or older), the young (e.g., 5 years or younger, 1 year or younger), pregnant women (for maternal immunity) and hospitalized patients, as well as patients who have been treated with antiviral compounds but exhibit an inadequate antiviral response, but are not limited thereto.

[0145] The pre-fusion HMPV F protein, fragment, nucleic acid molecule and / or vector according to the invention can be used in the sole treatment and / or prevention of diseases or conditions caused by HMPV, or in combination with other prophylactic and / or therapeutic measures, such as (existing or future) vaccines, antiviral agents and / or monoclonal antibodies.

[0146] The present invention further provides a method for preventing and / or treating HMPV infection in a subject using the pre-fusion HMPV F protein or a fragment thereof, a nucleic acid molecule and / or a vector according to the present invention. In certain embodiments, the method for preventing and / or treating HMPV infection in a subject comprises administering to a subject in need thereof an effective amount of the pre-fusion HMPV F protein (fragment), nucleic acid molecule and / or vector. A therapeutically effective amount means an amount of a protein, nucleic acid molecule or vector that is effective to prevent, ameliorate and / or treat a disease or condition resulting from infection with HMPV. Prevention includes suppression or reduction of the spread of HMPV, or suppression or reduction of the onset, occurrence or progression of one or more symptoms associated with HMPV infection. As used herein, amelioration may mean reduction of a visible or perceptible disease symptom, viremia or any other measurable sign of HMPV infection.

[0147] For administration to a subject such as a human, the present invention can use a pharmaceutical composition comprising a pre-fusion HMPV F protein (fragment), a nucleic acid molecule, and / or a vector described herein and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that, at the dosages and concentrations used, the carrier or excipient causes no undesirable or harmful effects in the subject to which it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington’s Pharmaceutical Sciences, 18th edition, edited by A.R. Gennaro, Mack Publishing Company

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

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

[2000] ). The HMPV F protein or nucleic acid molecule is preferably formulated as a sterile solution for administration, although it is also possible to utilize a lyophilized formulation. The sterile solution is prepared by sterile filtration or other methods known per se in the art. The solution is then lyophilized or filled into a pharmaceutical administration container. The pH of the solution is generally in the range of pH 3.0 to 9.5, for example pH 5.0 to 7.5. The HMPV F protein is typically present in a solution containing a suitable pharmaceutically acceptable buffer, and the composition may also contain salts. Optionally, a stabilizer such as albumin may be present. In certain embodiments, a surfactant is added. In certain embodiments, the HMPV F protein can be formulated into an injectable preparation.

[0148] In certain embodiments, the compositions according to the invention further comprise one or more adjuvants. Adjuvants are known in the art to further enhance the immune response to the antigenic determinants to which they are applied. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the HMPV F protein of the invention. Examples of suitable adjuvants include the following: aluminum salts such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions) such as squalene-water emulsions such as MF59 (see, for example, WO90 / 14837); saponin formulations such as QS21 and immunostimulating complexes (ISCOMs) (see, for example, US5,057,540; WO90 / 03184, WO96 / 11711, WO2004 / 004762, WO2005 / 002620); bacterial or microbial derivatives such as monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG motif-containing oligonucleotides, ADP-ribosylated bacterial toxins or mutants thereof such as Escherichia coli (E. coli) heat-labile enterotoxin LT, cholera toxin CT, etc.; eukaryotic proteins [such as antibodies or fragments thereof (such as those against the antigen itself or CD1a, CD3, CD7, CD80) and ligands for receptors (such as CD40L, GMCSF, GCSF, etc.)] (these stimulate the immune response upon interaction with recipient cells). In certain embodiments, the compositions of the invention comprise aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate or combinations thereof, at a concentration of 0.05 to 5 mg of aluminum content per dose, for example 0.075 to 1.0 mg.

[0149] In other embodiments, the composition does not contain an adjuvant.

[0150] In certain embodiments, the present invention provides a method for manufacturing a vaccine against human metapneumovirus (HMPV), comprising providing an HMPV F protein (fragment), nucleic acid or vector according to the present invention and formulating it in a pharmaceutically acceptable composition. The term "vaccine" means a substance or composition containing an active ingredient effective to induce a certain degree of immunity in a subject against a particular pathogen or disease, which results in at least a reduction (up to complete disappearance) in the severity, duration or other symptoms associated with infection by the pathogen or disease. In the present invention, the vaccine comprises an effective amount of a pre-fusion HMPV F protein (fragment) and / or a nucleic acid molecule encoding a pre-fusion HMPV F protein and / or a vector containing the nucleic acid molecule, which results in an effective immune response against HMPV. This provides a method for preventing severe lower respiratory tract diseases leading to hospitalization and results in a decrease in the frequency of complications such as pneumonia and bronchiolitis due to HMPV infection and replication in the subject. The term "vaccine" according to the present invention means that it is a pharmaceutical composition and thus typically contains a pharmaceutically acceptable diluent, carrier or excipient. It may or may not contain further active ingredients. In certain embodiments, it may be a combined vaccine (combination vaccine) further containing, for example, other components that induce an immune response against other proteins of HMPV and / or against other infectious agents such as RSV, HMPV and / or influenza. Administration of the further active ingredient may be carried out, for example, by separate administration or by administration of a combined product of the vaccine of the present invention and the further active ingredient.

[0151] Administration of the composition according to the present invention can be carried out using standard routes of administration. Non-limiting embodiments include intramuscular injection.

[0152] The subject used herein is preferably a mammal, such as a rodent, such as a mouse, cotton rat, or non-human primate, or a human. Preferably, the subject is a human subject.

[0153] Furthermore, the protein of the present invention can be used as a diagnostic means for testing the immune state of an individual, for example, by determining whether an antibody capable of binding to the protein of the present invention is present in the serum of the individual. Accordingly, the present invention also relates to an in vitro diagnostic method for detecting the presence of HMPV infection in a patient, comprising a) a step of contacting a biological sample obtained from the patient with the protein according to the present invention, and b) a step of detecting the presence of an antibody-protein complex.

[0154] The present invention is further illustrated in the following examples. These examples do not limit the present invention in any way. They are merely for clarifying the present invention.

[0155] Examples Example 1 : Introduction of a non-native F2-F1 cleavage site The schematic structures of full-length and soluble HMPV F (HMPV F ectodomain) are shown in FIGS. 1a and 1b, respectively (for numbering, see SEQ ID NO: 1). The F1 domain in the soluble variant has a truncated C-terminus and may (optionally) have a foldon trimerization domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 2) fused to the F ectodomain, similar to many other soluble trimeric viral fusion proteins.

[0156] Based on the F protein of the wild-type HMPV strain TN / 00 / 3-14 (SEQ ID NO: 1), a DNA fragment encoding the hMPV protein was synthesized (Genscript, Piscataway, NJ) and cloned into the pcDNA2004 expression vector (a modified pcDNA3 plasmid with an enhanced CMV promoter).

[0157] A natural variant H368N with an F2 C-terminus defined in FIG. 2A (found in TN / 85 / 6-3; Genbank ID ACJ53577.1), and a plasmid corresponding to the HMPV F ectodomain of the TN / 00 / 3-14 strain having a C-terminal foldon domain and four stabilizing mutations and non-native disulfide bridges (T69Y, A116H, A140C, A147C, D185P and E453Q) described in co-pending application EP21215259 were used to transfect HEK293 cells. Modification from the native TMPRSS2 cleavage site (RQSR) to a non-native polybasic furin site (RRRR), and truncation of the F2 C-terminus (after position 90) were applied to increase the cleavage efficiency of the HMPV F0 protein. In some of these proteins, a second non-native polybasic furin site (RRRR) cleavage site was introduced within the F2 domain, which was located N-terminal to the first cleavage site and separated by a spacer sequence of one or more amino acid residues from the F2 domain.

[0158] The expression platform used was the 96-well format Expi293F expression system (Thermo Fisher Scientific, Waltham, USA). The plasmid encoding HMPV F was co-transfected with the plasmid encoding furin at a 5:1 HMPV F-furin DNA ratio. The constructs were transfected in duplicates or quadruplicates.

[0159] After 3 days, the supernatant was collected and analyzed on a 4-12% (w / v) Bis-Tris NuPAGE gel in 1× MOPS (Life Technologies) under reducing conditions, and then Western blotting was performed to detect the effectiveness of cleavage (Figure 2B). Western blot analysis was performed as follows. Semi-dry blotting was performed using iBlot2: program P0 according to the manufacturer's recommendations. Blocking was performed for 1 hour with Odyssey blocking buffer, incubated for 1 hour with a 1:10,000 primary antibody (anti-hMPV-F A2 / B2 polyclonal serum; generated after immunization of rabbits with peptides at Genscript, Piscataway, NJ) in Odyssey blocking buffer, and incubated for 1 hour with a secondary antibody [1:5000 α-Rabbit IRDYE CW800 (Rockland Immunochemicals, Inc., Limerick, PA, US) in Odyssey blocking buffer]. All incubations were performed at room temperature on a roller platform. After the primary and secondary antibodies, the blot was washed 3 times for 5 minutes each with 50 ml of TBS / 0.05% Tween20, and then a final wash was performed with 50 ml of 1× PBS. The blot was visualized by scanning with an Odyssey scanner using both the 700CW and 800CW channels.

[0160] Furthermore, the recovered crude cell supernatant was analyzed for trimer content and trimer stability after heat stress by 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 4.6×150 mm 1.8 μm column [Sepax (231300-4615), injection volume 20 μL, flow rate 0.35 mL / min]. Thermal stability was shown as the ratio of trimers after heat stress compared to a 4°C control. Constructs transfected in duplicate or quadruplicate were pooled for SEC analysis. Elution was monitored by a UV detector. The SEC profile was analyzed by Chromeleon software version (version 7.2.7, Thermo Fisher Scientific). Plots were plotted in GraphPad Prism (version 9.0.0, GraphPad Software) (Figure 2C).

[0161] Also, the antigenicity of the HMPV F protein in the crude cell supernatant was evaluated using the Biolayer Interference method in Octet (ForteBio, Portsmouth, UK), and using the monoclonal antibodies ADI-14448 (Gilman et al., Sci Immunol. 2016 Dec 16;1(6):eaaj1879.doi:10.1126 / sciimmunol.aaj1879. 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 2D). ADI-14448 has been described as a cross-neutralizing antibody against RSV and HMPV, which is pre-fusion specific and binds to antigenic site III of RSV preF. Thus, ADI-14448 binding serves as an indicator of the pre-fusion conformation. The X-ray structure of MAb DS7 has been described as a complex with the HMPV fusion protein (Wen et al., Nat Struct Mol Biol. 2012 Mar 4;19(4):461-3.doi:10.1038 / nsmb.2250). The HMPV F + DS7 complex shows that the heptad repeat 2 (HR2), which is part of the refolding region 2 (RR2), is in a non-preF conformation. This is because beta sheet 22 is detached, which suggests a post-fusion conformation rather than a pre-fusion conformation. Thus, DS7 binding indicates a non-pre-fusion or post-fusion conformation.

[0162] Thus, for the preferred pre-fusion HPMV F protein according to the present invention, DS7 binding is undesirable. In the Octet assay, the antibody was immobilized on an anti-human Fc biosensor (ForteBio, Portsmouth, UK). After equilibrating the sensor in kinetic buffer (ForteBio, Portsmouth, UK) for 600 seconds, the sensor was transferred to kinetic buffer containing 5 μg / ml of the desired antibody. Then, another equilibration step in simulated cell culture medium was added. Finally, the sensor was transferred to a solution of cell culture supernatant containing pre-fusion HMPV F protein. The initial gradient (also referred to as the association phase; curve fitting was performed for the first 300 seconds) and the binding (nm) at 300 seconds are shown. Data analysis was performed using ForteBio Data Analysis 8.2 software (ForteBio, Portsmouth, UK). Bar plots were plotted in GraphPad Prism (version 9.0.0, GraphPad Software).

[0163] All proteins showed expression (Figure 2B) and were trimers according to analytical SEC (Figure 2C). Detection by Western blot showed that processing of all proteins was incomplete (Figure 2B).

[0164] By introducing stabilizing mutations and modifying the native TMPRSS2-like cleavage site to a non-native furin cleavage site by substituting QS with RR, the expression level (Figure 2C, left histogram panel) and thermal stability (Figure 2C, right histogram panel) of MPV210531 were enhanced compared to MPV201285 without stabilizing mutations. The expression levels of proteins with shorter (truncated) F2-C termini (MPV210502, MPV210500, MPV210507, and MPV210509) were decreased compared to MPV210531. The thermal stability at 60 °C of MPV210500, MPV210507, and MPV210509 with short F2 C termini was enhanced compared to MPV210531 with a long F2 C terminus.

[0165] In the octet, the protein showed both anti-pre-F binding (binding to ADI-14448) and anti-non-pre-F binding (binding to DS7), indicating that the protein was not in a fully pre-fusion trimer conformation (Figure 2D). In designs with a truncated F2 C-terminus and two non-native cleavage sites (MPV210500, MPV210507, and MPV210509), anti-non-pre-F binding was reduced.

[0166] Stabilized HMPV designs with a truncated F2 C-terminus and an additional furin cleavage site showed improved cleavage of F0 (Figure 2B), improved thermal stability (Figure 2C), and reduced binding to the postF-specific MAb DS7 (Figure 2D).

[0167] Example 2 : Optimization of F0 processing Improved processing and truncation of the F2 C-terminus improved the stability and quality of the pre-fusion F protein of HMPV (e.g., improved cleavage of F0, improved thermal stability, reduced binding to the post-F-specific antibody DS7), so the F2 C-terminus was systematically truncated from position 102 to position 88. Also, the p27 peptide (including the first non-native cleavage site) and the second non-native cleavage site were introduced between the (truncated) F2 domain and the F1 domain.

[0168] Use of the p27 region of RSV F, i.e., the amino acid sequence ELPRFMNYTLNNAKKTNVTLSKK RKRR (SEQ ID NO: 2; the cleavage site is underlined) has been shown to improve F0 processing. The p27 peptide of RSV F is known to be cleaved very efficiently by furin-like proteases (Krarup et al., Nat Commun. 2015 Sep 3;6:8143.doi:10.1038 / ncomms9143.; Gonzales-Reyes et al. 2001, Proc Natl Acad Sci USA 98:9859-9864).

[0169] In the present invention, according to the design listed in FIG. 3A, both the furin site RRRR and the RSV F p27 domain were introduced between the (truncated) F2 domain and the F1 domain. HEK293 cells were transfected with the plasmid corresponding to the HMPV F ectodomain described in Example 1 and the F2 truncation according to FIG. 3A. The addition of the RSV p27 domain at the C-terminus to the introduced furin site showed a very efficient increase in the cleavage of HMPV F as determined by Western blot after reducing SDS-PAGE (FIG. 3B). According to analytical SEC, in terms of the remaining trimer content after heat stress, the thermal stability of MPV210498 and MPV210751 at 60°C was the highest (right in FIG. 3C). Also, the antigenicity (i.e., pre-fusion conformation) of these two mutants was the highest. This is because they showed the lowest binding to the postF-specific Mab DS7 in Octet (FIG. 3D).

[0170] Conclusion: The introduction of a second furin cleavage site and the RSV-p27 domain (including the first non-native cleavage site) between F1 and F2 resulted in successful complete processing. Also, truncation of the F2 C-terminus after residue 89 or 90 (in addition to the second furin cleavage site of four arginines (R) and the RSV-p27 domain) resulted in the most stable HMPV preF protein with the highest pre-fusion quality, as indicated by low DS7 binding.

[0171] Example 3 : Stabilization of the HR2 region It is known to trimerize the soluble prefusion F ectodomain of HMPV with a heterotrimerization domain, such as Foldon. However, the addition of Foldon introduces an additional heterologous protein domain that does not confer additional advantages regarding vaccine immunogens other than F trimerization. Since immunogens in other vaccines or other vaccine components can also utilize Foldon for trimerization, the preferred vaccine components are based solely on viral proteins and do not contain any additional heterologous non-viral protein domains. The present invention provides a trimer conformation of soluble HMPV protein in the absence of Foldon by optimizing the interaction in the HR2 region of the prefusion stem of the F protein. In mutants with optimized F2 truncation (after amino acid residue 89), a furin cleavage site introduced at the truncated F2 terminus, and the RSV F p27 domain for optimal cleavage, the stabilization of the preF trimer by the stabilization of the HR2 stem region was evaluated. Furthermore, the F1 C-terminus was truncated after position 481 or 489. The protein also had a C-tag and a linker for purification purposes. When the Foldon domain was deleted (MPV211241), almost no trimers were detected by analytical SEC (left histogram panel of Figure 4A, left histogram panel of Figure 5A). However, several different stabilization strategies in HR2 (by the mutations in Figures 4 and 5A) restored and even improved trimer expression in the absence of Foldon. Trimers in the absence of Foldon remained relatively stable after storage at 4 °C for 6 - 8 weeks (left histogram panel of Figure 4) and after heating at 58 °C for 30 minutes (right histogram panel of Figure 4 and right histogram panel of Figure 5A).

[0172] To measure antigenicity, supernatants were tested using the Octet with anti-preF (ADI-14448), anti-postF (DS7), and MAb against the boundary of the tip of prefusion F that can only be recognized when the trimer is open or "breathing" (temporarily open or partially open) (MPV458; Huang et al., PLoS Pathog. 2020 Oct 9;16(10):e1008942.doi:10.1371 / journal.ppat.1008942.eCollection 2020 Oct.). HMPV F mutants with stabilized HR2 showed anti-preF, anti-tip boundary (MPV458), and little anti-non-pre-F binding (binding to DS7) (Figure 5B).

[0173] Conclusion: Mutations that stabilize HR2 enhance trimerization in the absence of foldon and further enhance trimer expression.

[0174] Example 4 : Substitutions that stabilize the prefusion conformation of HMPV In this example, alternative stabilizing mutations were evaluated in the backbone corresponding to the designs of MPV211287 and MPV211918 in the absence of the stabilizing substitutions T69Y, A116H, disulfide A140C+A147C, and D185P (mutations described in co-pending application EP21215259). The backbone contains an F2 truncation after amino acid position 89, an introduced furin cleavage site, and p27 of RSV (including the furin cleavage site). The protein further contains H368N, the stabilizing mutation E453Q, substitutions that stabilize HR2 (L473W, D475R, Q476K, S477F, N478D, R479E, A484I) or substitutions that stabilize HR2 (L473W, S477I, A484I), a linker, and a C-tag. One stabilizing substitution (V231I) was obtained from strain B2 / 3817 / 04 (Genbank ID AGL74059.1). One new stabilizing substitution (E453P) replaced the stabilizing substitution E453Q (already described in co-pending application EP21215259). Two other new stabilizing substitutions were V112R and D209E. Reverting the previous stabilizing substitutions at positions 69, 116, 140, 147, and 185 (described in co-pending application EP21215259) to wild type (MPV211940 in Figure 6A and MPV211942 in Figure 7A) by mutation resulted in loss of preF trimer expression. All single stabilizing substitutions except Q453P, particularly V112R, enhanced trimer expression. Combinations of substitutions, particularly the addition of the D209E substitution, further enhanced expression and improved thermal stability (Figures 6A, 7A). Octet analysis showed that mutants with the new stabilizing substitutions had improved antigenicity with relatively low binding to DS7 compared to the reference MPV211940 and MPV211942 (Figures 6B, 7B). In the present invention, improved antigenicity means pre-fusion properties measured by enhanced binding of pre-F-specific antibodies (e.g., ADI-14448) and reduced binding of post-F-binding antibodies (e.g., DS7) and anti-border antibodies (e.g., MPV458). These last two antibodies have very low neutralizing activity.

[0175] Since some of the binding to DS7 or MPV458 may also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced and purified on a larger scale for further thorough analysis (see Example 8).

[0176] Example 5 : Additional substitutions to stabilize the pre-fusion conformation of HMPV Additional stabilizing mutations T69W, S149Y, N313W and S445Y and the natural mutation N404P were introduced into the MPV212033 backbone containing only the stabilizing mutation V112R, F2 truncation after amino acid 89, the p27 + furin site of RSV, the wild-type mutation H368N, the stabilizing mutation E453Q, HR2 stabilization (L473W, D475R, Q476K, S477F, N478D, R479E, A484I) and the linker and C-tag and evaluated. Combinations of substitutions further enhanced expression and improved thermal stability (Figures 8A and C) (see Example 1 for methods). Differential scanning fluorimetry (DSF) of cell culture supernatants showed that all additional substitutions except N404P increased the melting temperature (Tm50) of HMPV F measured by DSF (see Example 4 for method details). Octet analysis showed that mutants with the additional substitutions S149Y, T69W, S445Y and N313W had improved antigenicity with relatively low binding to DS7 compared to the reference MPV212033 (Figure 8B).

[0177] Since some of the binding to DS7 may also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced and purified on a larger scale for further analysis (see Example 8).

[0178] Example 6 : HR2 mutations in the backbone containing V112R, D209E, V231I and E453P Furthermore, HR2 variants were evaluated in a backbone with F2 truncation after amino acid 89, an introduced furin cleavage site, RSV p27, F1 truncation after amino acid 489, and the absence of foldon. The protein further contained the H368N, V112R, D209E, V231I, and E453P mutations.

[0179] All mutants tested showed enhanced trimer expression compared to MPV210571 (Figure 9A) (see Example 1 for methods). Compared to MPV212047 with the HR2-stabilizing mutations L473W, D475R, Q476K, S477F, N478D, R479E, A484I (see Example 3), the trimer contents of MPV212032 (L473W, S477I, A484I), MPV220115 (S477I), MPV220120 (L473Y, S477I, A484I), and MPV220121 (L473I, S477I, A484I) were in a similar range. The binding profiles in the octet were equivalent to those of MPV210751 and MPV212047 (Figure 9B). Differential scanning fluorimetry (DSF) of cell culture supernatants showed that all HR2 designs (except MPV220123, for which the Tm50 could not be determined due to low signal) had improved thermal stability, shown as the melting temperature in the DSF of the supernatant (see Example 4 for method details) compared to MPV210571.

[0180] Conclusion: The HR2-stabilizing mutations in MPV212032 (L473W, S477I, A484I), MPV220115 (S477I), MPV220120 (L473Y, S477I, A484I), and MPV220121 (L473I, S477I, A484I) improved trimer content and thermal stability compared to MPV210751. Since some of the binding to DS7 may also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced and purified on a larger scale for further analysis (see Example 8).

[0181] Example 7 : Further stabilization of the HR2 variant containing S477I and A484I The HR2 stabilizing mutations S477I, A484I, and F2 truncation after amino acid position 89, introduced furin cleavage sites, RSV p27, absence of foldon, and MPV220116 with mutations H368N, V112R, D209E, V231I and E453P (+ linker and C-tag) were selected to test for additional stabilizing mutations T69W, S149Y, N313W and S445Y as well as the natural mutation N404P.

[0182] The combination of substitutions further enhanced expression (Figure 10A) (see Example 1 for methods). Octet analysis showed that variants containing the additional substitutions S149Y, T69W, S445Y and N313W had improved antigenicity with binding to a relatively low DS7 as compared to the reference MPV220116 (Figure 10B). Differential scanning fluorimetry (DSF) of cell culture supernatants showed that all additional substitutions improved the melting temperature (Tm50) of HMPV F thermal stability as shown by the melting temperature in the DSF of the supernatant (see Example 4 for method details) (Figure 10C).

[0183] Conclusion: The additional substitutions S149Y, T69W, S445Y and N313W improved trimer content, antigenicity and thermal stability. Since part of the binding to DS7 can also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced and purified on a larger scale for further analysis (see Example 8).

[0184] Example 8 : Production and purification of the selected proteins Manufacture and purification Several HMPV proteins in Table 1 were produced and purified. Most of the selected HMPV F proteins are described in the above examples: MPV210530 (Figure 3), MPV210751 (Figure 4), MPV211918 (Figure 4), MPV212017, MPV212047 (Figure 6), MPV212043 (Figure 6), MPV212044 (Figure 6), MPV212045 (Figure 6), MPV212046 (Figure 6), MPV220087 and MPV220092.

[0185] At a scale of 300 ml, ExpiFectamine 293 (Thermo Fisher Scientific, Waltham, USA) was used according to the manufacturer's instructions to transiently transfect cells, which were then cultured in a shaking incubator at 37 °C and 10% CO2 for 5 days. The culture supernatant was collected, centrifuged at 600 rpm for 10 minutes, and filtered through a 0.22 μm PVDF filter to remove cells and cell debris.

[0186] The protein was purified using a 2- or 3-step protocol. First, the harvested and clarified culture supernatant was loaded onto a prepacked C-tagXL 5 ml column (Thermo Fisher Scientific, cat# 494307205, Waltham, USA). This column was prepacked with an affinity resin (Capture Select) consisting of a C-tag specific single domain antibody immobilized on agarose-based beads. This resin is highly specific for binding proteins with a C-tag. Elution of the C-tagged protein was performed using a TRIS buffer containing 2M MgCl2. Based on the UV signal (A280), the elution fractions were pooled and concentrated using an Amicon Ultracel 50 kDa MWCO centrifugal filter device (Merck Millipore, cat# UFC805024, Darmstadt, Germany). The protein was purified using a 2- or 3-step protocol. First, the harvested and clarified culture supernatant was loaded onto a prepacked CaptureSelect C-tagXL 5 ml column (Thermo Fisher Scientific, cat# 494307205, Waltham, USA). This column was prepacked with an affinity resin (Capture Select) consisting of a C-tag specific single domain antibody immobilized on agarose-based beads. This resin is highly specific for binding proteins with a C-tag. Elution of the C-tagged protein was performed using a TRIS buffer containing 2M MgCl2. Based on the UV signal (A280), the elution fractions were pooled and concentrated using an Amicon Ultracel 50 kDa MWCO centrifugal filter device (Merck Millipore, cat# UFC805024, Darmstadt, Germany).Next, the concentrated and collected elution peaks were applied to a Superdex 200 Increase 10 / 300 column (Cytiva, cat# 28-9909-44, Marlborough, Massachusetts, United States) equilibrated in running buffer (20 mM Tris, 150 mM NaCl, pH 7.4) for purification purposes (i.e., to remove the minimum amount of multimeric and monomeric proteins). The three-step protocol included additional operations on a Superose 6 Increase 10 / 300 column (Cytiva, cat# 29-0915-96, Marlborough, Massachusetts, United States) for the C-tag pool (applied only to MPV210751, MPV211918, MPV212017, and MPV212047) prior to Superdex 200. The quality after purification of this design showed that this step was unnecessary as no larger aggregates were present. Therefore, it was not applied to other purifications.

[0187] Also, the postF hMPV protein (SEQ ID NO: 3) described in Mas et al., PLoS Pathog. 2016 Sep 9;12(9):e1005859. doi:10.1371 / journal.ppat.1005859. eCollection 2016 Sep. was produced and purified. An expression plasmid encoding the recombinant postfusion hMPV F protein was produced as described in Example 2. Cells were transiently transfected on a 300 ml scale and then purified by a two-step protocol (see above for details). TEV cleavage was then performed to remove the foldon and c-tag. 1 μL of TEV (10,000 units / mL) was used for 15 μg of the protein. The protein-TEV mixture was incubated overnight at 4°C. The TEV-His protease was removed from the protein sample by Ni Sepharose Excel beads (GE Healthcare, 17-3712-03) pulldown. Ni Sepharose Excel beads were added to the protein-TEV mixture and incubated at room temperature for 2 hours. The flow-through was collected via a Micro Bio-Spin column (BioRad, 7326204). The cleaved protein sample was subjected to a heat shock at 45°C for 30 minutes (Hsieh et al., Nat Commun 2022 Mar 14;13(1):1299. doi:10.1038 / s41467-022-28931-3). The protein sample was then applied to a Superose 6 Increase 10 / 300 column (GE Healthcare, Chicago, USA) equilibrated with running buffer (20 mM Tris, 150 mM NaCl, pH 7.4) for purification purposes (i.e., to remove the minimum amount of multimeric and monomeric proteins).

[0188] The protein was then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Figure 11A). The protein was stained with Instant Blue and visualized on the gel.

[0189] Results and Conclusions The yields of various mutants are shown in Table 1. Truncation of the F2 C-terminus decreased the yield, while HR2 stabilization and deletion of the foldon increased the yield. Introduction of V112R, D209E, V231I, and E453P (MPV212047) resulted in improved expression. Furthermore, introduction of S149Y, N404P (MPV220087), followed by introduction of S445P, N313W (MPV220092), resulted in enhanced expression for MPV220092. The major band on reducing SDS-PAGE corresponded to the F1 domain in the absence of residual F0 (Figure 11A), indicating a fully processed protein. [Table 1]

[0190] Trimer content The purified protein was evaluated by size-exclusion chromatography (SEC) for analytical use after purification and after storage at 4 °C to examine the trimer content after purification (Figure 11B). SEC was performed using ultra-high performance liquid chromatography (UHPLC) with a Sepax Unix-C SEC-300 4.6×150 mm 1.8 μm column [Sepax (231300-4615), injection volume 20 μL, flow rate 0.3 mL / min] and a Vanquish system (ThermoFisher Scientific). Elution was monitored with a UV detector (Thermo Fisher Scientific), a μDawn light scattering (LS) detector (Wyatt Technologies), a μT-rEx refractive index (RI) detector (Wyatt Technologies), and a Nanostar dynamic light scattering (DLS) detector (Wyatt Technologies). The SEC profile was analyzed using the Astra 7.3.2.19 software package (Wyatt Technology). Chromatograms were plotted in Graph Pad Prism (version 9.0.0, GraphPad Software).

[0191] Results and conclusions In analytical SEC, all purified proteins were highly trimerized after purification (Figure 11B) and had the predicted molecular weight of the trimeric protein (Table 1). After 7 weeks of storage, a high trimer content was maintained in all proteins. MPV212044 showed a peak adjacent to the trimer after storage at 37 °C for 7 weeks, which is a peak of a species with a molecular weight smaller than the trimer.

[0192] In conclusion, all purified proteins are trimers (Figure 11B and Table 1), and MPV212047 shows a high trimer content after 7 weeks at 4 °C.

[0193] Stability at 37°C The purified proteins were aliquoted, and the aliquots were incubated at 37 °C (control samples for all proteins and time points were maintained at 4 °C) and then analyzed by SEC-MALS (see the above section for methods). Table 1 shows the relative trimer content of the samples stored at 37 °C compared to the control samples. Chromatograms were plotted in GraphPad Prism (version 9.0.0, GraphPad Software).

[0194] Results and conclusions Chromatograms of the control samples and samples stored at 37 °C for 2 weeks are overlaid for each purified protein (Figure 11C). MPV210530 shows a reduction in trimer stability after 2 weeks at 37 °C, as indicated by a reduction in the trimer peak and additional peaks reflecting aggregates, compared to the design with truncated F2 after position 89. All proteins with a truncated F2 C-terminus after position 89 showed the same trimer content after storage at 37 °C compared to the control. All proteins were stable at 37 °C for at least 2 weeks.

[0195] Freeze stability The protein was instantaneously frozen once, five times, or ten times. After thawing, the trimer content was compared with that of the non-frozen sample by SEC-MALS (see the above section for the method). Table 1 shows the relative trimer content of the stressed sample with respect to the control sample. Chromatograms were plotted in GraphPad Prism (version 9.0.0, GraphPad Software).

[0196] Results and conclusions The protein was tested in a buffer without cryoprotectant (buffer composition: 20 mM Tris, 150 mM NaCl, pH 7.4), and after a single instantaneous freeze, all proteins remained highly trimeric. After 10 freeze-thaw cycles (Figure 11D), differences in freeze stability were observable. Mutations enhancing freeze / thaw stability could be ranked in the following order: D209E > V112R > V321I > Q453P. In MPV212044, some aggregates were detected. In conclusion, the HMPV F protein of the present invention exhibits high freeze stability.

[0197] Thermal stability of the protein The thermal stability of the purified pre-fusion HMPV F protein was measured by differential scanning fluorimetry (DSF) by monitoring the fluorescence emission of Sypro Orange Dye (ThermoFisher Scientific) in a 96-well optical qPCR plate. A protein solution of 66.67 μg / ml at 15 μl per well was used (buffer described in Example 2) (Figure 11E). To each well, 5 μl of 20× Sypro orange solution was added. Upon a gentle temperature increase from 25°C to 95°C (0.015°C / second), the protein unfolds and the fluorescent dye binds to the exposed hydrophobic residues, resulting in a characteristic change in luminescence. A ViiA7 real-time PCR instrument (Applied BioSystems) was used to measure the melting curve. The first derivative of the fluorescence signal (a.u.) with respect to temperature (°C) and the average melting curve of three individual samples (technical triplicates) were plotted using Graphpad Prism software (DanDiego, CA, US). The Tm50 (the lowest point on the curve) was subtracted from the average melting curve. The Tm50 value represents the temperature at which 50% of the protein unfolds and thus serves as a measure of the protein's thermal stability.

[0198] Results and conclusions A design (MPV210751) with a truncated F2 C-terminus at position 89, further comprising a furin site, and RSV p27, and T69Y, A116H, A140C, A147C, D185P, H368N, E453Q, showed an increase in melting temperature of approximately 5 °C each compared to the design MPV210530 which has a long F2 C-terminus (Figure 11E). Removal of the foldon and introduction of HR2 stabilizing mutations in MPV211918 decreased the melting temperature by approximately 3 °C compared to MPV210751. Introduction of the stabilizing substitutions V112R, D209E, V231I and 453P (MPV212017) increased the melting temperature by approximately 10 °C compared to MPV211918. The disulfide bridge 140-147 and the stabilizing substitutions T69Y, A116H and D185P did not result in additional stabilizing effects and removal did not decrease the melting temperature (when MPV212047 was compared to MPV212017).

[0199] Conclusion: HMPV F with a truncated F2 C-terminus, together with stabilization of HR2 and addition of new stabilizing mutations, resulted in a high yield of very stable trimers in the absence of the foldon domain.

[0200] In vitro antigenicity The in vitro antigenicity of the selected purified protein was tested using the bio-layer interferometry technology by octet (Figure 11F). In addition to the antibodies described in Example 2, anti-tip border conjugate MPV465 (Huang et al., PLoS Pathog. 2020 Oct 9;16(10):e1008942.doi:10.1371 / journal.ppat.1008942.eCollection 2020 Oct.), and antibody ADI-18992 that is described to bind to site IV and recognize the preF and postF conformations were also used (Gilman et al., Sci Immunol. 2016 Dec 16;1(6):eaaj1879.doi:10.1126 / sciimmunol.aaj1879.Epub 2016 Dec 9). The antibodies were immobilized as described in Example 2. After equilibrating the sensor in kinetic buffer (ForteBio) for 600 seconds, the sensor was transferred to kinetic buffer containing 5 μg / ml of the desired antibody. Then, another equilibration step was included in the kinetic buffer. Finally, the sensor was transferred to a protein solution (20 μg / mL in 1×KB). The analysis was performed as described in Example 2.

[0201] Results and Conclusions In the case of the HMPV F mutant (MPV210530) where the F2 C-terminus was not truncated, the binding to non-pre-F antibodies and anti-border antibodies was higher compared to the purified protein with F2 truncation. The stabilized pre-F proteins MPV212017 and MPV212047 with new stabilizing mutations showed the most favorable binding and bound only to pre-F specific antibodies. This proves that the decrease in antigenicity in the supernatant, for example regarding MPV212047, observed in Figure 5B was caused by impurities.

[0202] Structural characterization by nsTEM The purified protein was analyzed by negative stain transmission electron microscopy (nsTEM) (Figure 11G).

[0203] A continuous carbon grid (copper, EMS) was glow-discharged in an Easiglow plasma cleaner for 30 seconds. 4 μL of a sample diluted to a concentration in the range of 5 - 25 μg / ml (20 mM Tris, 150 mM NaCl, pH 7.4) was applied to the glow-discharged grid and incubated for 1 minute. The sample solution was partially absorbed by gentle side blotting, and the grid was immediately stained by placing it on a 40 μl drop of 2% (w / v) uranyl acetate solution for a total of 1 minute. After staining, the grid was blot-dried and stored at room temperature prior to imaging. The prepared grid was imaged in a Talos L120C TEM (Thermo Fisher Scientific) equipped with a Ceta camera. The resulting pixels were in the range of 2.4 - 2.8 Å / pixel depending on the imaging conditions. The parameters of the contrast transfer function (CTF) were estimated for each micrograph using CTFFIND4, and the remaining processing (selection and 2D classification) was performed in RELION (version 3 or 4).

[0204] Results and Conclusions Two-dimensional (2D) class averages obtained by electron microscopy (EM) analysis of negatively stained samples using acidic staining of purified MPV212047 showed regular homogeneous closed trimers (Figure 11G).

[0205] Structural characterization of MPV212047 by cryo-electron microscopy The cryo-electron microscopy (Cryo-EM) structure of HMPV F design MPV212047 was elucidated to confirm the correct trimeric pre-fusion conformation of this protein (Figure 12). For this purpose, 3.5 μL of purified preF protein (MPV212047) complex at 0.8 - 1.0 mg / ml was applied to a plasma-cleaned (Gatan Solarus) Quantifoil 1.2 / 1.3 holey gold grid and then vitrified using a Vitrobot Mark IV (FEI Company). The cryo-grid was loaded into a Glacios transmission electron microscope (ThermoFisher Scientific) equipped with a Falcon IV direct electron detector and operating at 200 keV in nanoprobe mode. Images were recorded in counting mode of EPU with a pixel size of 0.948 Å and a nominal defocus range of -1.8 to -1.2 μm. Images were recorded with an exposure of 5.7 s in EER format corresponding to a total dose of approximately 40 electrons per square Å. During data acquisition, the movies were subjected to beam-induced motion correction, contrast transfer function (CTF) parameter estimation, automatic reference particle picking, particle extraction with a box size of 280 pixels, and two-dimensional (2D) classification in CryoSPARC Live. Particle images with distinct HA features were merged and subjected to ab initio 3D reconstruction and then 3D heterogeneous refinement with C3 symmetry in CryoSPARC. Then, the particles were refined using heterogeneous (UN) refinement in CryoSPARC with C3 symmetry. Before visualization, all density maps were sharpened by applying various negative temperature factors using an automated method and used for model building together with the half maps. The local resolution was determined using ResMap. The initial template of the HMPV F trimer was derived from SWISS-MODEL. The model was docked into the EM density map using Chimera and then manually adjusted using COOT. The shape of the model was further improved using Rosetta. The shape parameters of the final model were verified in COOT as well as using MolProbity and EMRinger. These refinements were repeated until no further improvement was seen.The overfitting of the model was evaluated through its refinement against one cryo-EM half-map. FSC curves were calculated for cross-validation between the obtained model and the working half-map, and between the obtained model and the free half-map and full map. Drawings were created using PyMOL (Figure 12). One protomer is shown as a cartoon representation and two protomers are shown as surface representations.

[0206] In confirmation with previous SEC-MALS, BLI and nsTEM data (Figure 11), Cryo-EM analysis of the MPV212047 design demonstrated the presence of pre-fusion trimers with the tip (distal of HR2) in a closed conformation.

[0207] Example 9 : Immunogenicity of the closed pre-fusion HMPV F protein MPV212047 The adjuvant-added pre-fusion HMPV F protein is more immunogenic and effective than the post-fusion HMPV F in naive cotton rats. The immunogenicity of pre-fusion HMPV F (preF) MPV212047 was compared to that of post-fusion HMPV F (postF) MPV190470. This was done by intramuscularly immunizing naive cotton rats with 16, 3 or 0.6 μg of preF or 10 μg of postF protein adjuvanted with 50 μL of AS01B per animal (n = 8 per dose). Proteins were purified as described in Example 8. As a control, cotton rats were injected intramuscularly with phosphate-buffered saline (PBS) (negative control, n = 8) or 10 4 plaque-forming units (PFU) of HMPV A2 (positive control, n = 6). Animals were immunized twice at the indicated doses on days 0 and 21. The positive control group was immunized once on day 0. Animals were sacrificed on day 42 with 10 5Challenged with HMPV A2 of PFU and sacrificed on day 46. The ELISA binding antibody titer was measured against HMPV preF (site-specific biotinylated MPV212047:MPV220554) in the serum isolated on day 42 (Figure 13A). The titer is shown as the log10 of the relative potency. The assay is exploratory and the LLoD is based on the 99% quartile limit using all expected negative samples. Each dot represents the value of an individual animal, and the horizontal line represents the median response of the group. The neutralizing antibody titer was measured against HMPV A2 (TN / 94-49) in the serum isolated on day 42 by plaque reduction neutralization test (PRNT) (Figure 13B). The titer is shown as the log2 of the 50% inhibitory concentration (IC50). Each dot represents the value of an individual animal, and the horizontal line represents the median response of the group. The amount of HMPV A2 virus on day 4 (day 46) post-infection was measured in nasal homogenates by plaque assay and is expressed as log10 pfu per gram of tissue (Figure 13C). Each dot represents an individual animal, and the median response per group is indicated by the horizontal line. Open symbols indicate animals that did not have a detectable level of preF-binding antibody in the serum on day 42.

[0208] The level of HMPV preF-binding antibody was significantly higher in animals immunized with 3 μg of preF protein compared to the 10 μg of postF protein group, and correspondingly, the level of neutralizing antibody was also significantly higher (Figure 13A, B). Also, the protection against intranasal HMPV A2 virus was significantly lower when animals were immunized with postF protein compared to preF protein (Figure 13C).

[0209] In summary, the adjuvant-added HMPV preF protein is more immunogenic and effective than the HMPV postF protein in naive cotton rats.

[0210] The adjuvant-added closed pre-fusion HMPV F protein is more immunogenic than the open pre-fusion HMPV F protein. The in vivo immunogenicity of MPV212047 (closed F), a closed prefusion HMPV F, was compared to that of an HMPV F protein with an open conformation (MPV220215; open F). For this purpose, the MPV220215 HMPV F trimer was purified by C-tag affinity purification and SEC, and the HMPV F trimer was characterized as described in Example 8. MPV220215 was designed as a single-chain construct with a foldon trimerization domain and stabilizing substitutions V112R, S149Y, V231I, and E453P. The trimer conformation was confirmed by SEC-MALS analysis, eluting with a retention time of approximately 4 minutes and a size of 161 kDa (Figure 14A). The open prefusion conformation of MPV220215 was confirmed by BLI by detection of ADI-14448 prefusion-specific antibody binding and by enhanced MPV458 and MPV465 apical border binding compared to the closed MPV212047 HMPV F (Figure 14B).

[0211] The immunogenicity of the purified open MPV220215 and closed MPV212047 HMPV prefusion F trimers was evaluated by intramuscular immunization of female Balb / C mice (n = 5) with 1.5, 5, or 15 μg of protein or PBS immunization negative control group (n = 3) per animal with 10 μL of AS01B adjuvant added on days 0 and 28. In sera isolated 2 weeks after the second immunization (day 42), ELISA binding antibody titers were measured against HMPV preF as described above (Figure 14C). Upon immunization with both proteins, a dose-dependent increase in HMPV preF binding antibody levels was observed, reaching significantly higher levels with the closed MPV212047 protein than with the open MPV220215 variant. Furthermore, upon immunization with the closed prefusion HMPV F, a significantly higher HMPV A2 neutralizing antibody titer was detected compared to MPV220215 (Figure 14D).

[0212] These results indicate that the closed (closed-form) pre-fusion HMPV F conformation induces more potent neutralizing antibodies in mice than the open (open-form) HMPV F protein conformation.

[0213] Immunogenicity of HMPV pre-fusion F in pre-exposed mice The immunogenicity of HMPV pre-fusion F (MPV212047) was evaluated in the context of pre-exposure to HMPV A2 by intramuscularly immunizing female Balb / C mice (n = 7) with 15 μg of adjuvant-free MPV212047 protein purified as described in Example 8. The mice were pre-exposed on day 0 to at least 1 × 10 3 ~3 × 10 5 PFU of HMPV HMPV A2 and immunized with MPV212047 at week 12. In sera isolated 2 and 4 weeks after immunization (weeks 14 and 16), the ELISA binding antibody titers against HMPV pre-F were determined as described in Example 9 (Figure 15). The levels of HMPV preF-binding antibodies increased dramatically upon immunization with MPV212047, with the geometric mean of the titers increasing 186-fold. This demonstrates the in vivo immunogenicity of adjuvant-free HMPV pre-fusion F in the context of pre-exposure.

[0214] Example 10 : Introduction of stabilizing substitutions into HMPV F A2, B1, and B2 strain variants Pre-fusion HMPV subtype A2(2000)F protein with minimal amino acid substitutions To generate an HMPV prefusion F protein with minimal HR2 stabilization substitutions, several HR2 amino acids were reverted to wild type in the stabilizing backbone MPV212047. The resulting construct, MPV220558, contained the HR2 substitutions L473W, Q476K, S477F, and A484I, but had the wild-type amino acids D475, N478, and R479. Also, from variant MPV220558, the surface-exposed H368N substitution was reverted to wild type to obtain MPV220647. In summary, MPV220647 is based on the HMPV A2 strain TN / 00 / 3-14 (A22000), contains the p27 peptide, has the F2 domain truncated at amino acid 89, and contains the following combination of stabilizing mutations, namely, V112R, D209E, V231I, and E453P, along with the four stabilizing HR2 mutations L473W, Q476K, S477F, and A484I.

[0215] As described in Example 1, HMPV F trimer expression was evaluated upon transient expression of MPV212047, MPV220558, and MPV220647. The melting temperature (Tm50) of the HMPV F trimer in the supernatant was measured by differential scanning fluorimetry (DSF). For this, the fluorescence emission of Sypro Orange Dye (ThermoFisher Scientific) added to the HMPV F protein in solution was monitored. Measurements were taken at an initial temperature of 25°C and a final temperature of 95°C (increase of 54°C / hour). A ViiA7 real-time PCR instrument (Applied Biosystems) was used to measure the melting curve, and the Tm50 value was derived from the negative first derivative as described previously (Rutten et al. (2020) Cell Rep 30:4540-4550).

[0216] The effects on trimer peak height were observed by subsequent reversal of the three HR2 stabilizing substitutions and by reversal from H368N to wild type (Figure 16A, black bars). However, the melting temperatures of the three HMPV F proteins were not negatively affected, as indicated by the average Tm50 of MPV212047, MPV220558, and MPV220647 being 72.3 °C, 73.2 °C, and 73.5 °C, respectively (Figure 16A, gray bars).

[0217] Introduction of stabilizing substitutions into HMPV F strain variants Next, the introduction of stabilizing substitutions in MPV220647 to more recent A2 strains as well as to recent subtype B1 and B2 strains was evaluated as described above (Figure 16B). The stabilizing effect of these mutations was demonstrated by comparing wild-type B2 (MPV23362) to the stabilized B2 mutant MPV220641. Wild-type HMPV F B2 eluted at the expected monomer retention time in analytical SEC (Figure 16C, gray line), while the stabilized HMPV F B2 MPV220641 was expressed at a significantly higher level and eluted as a trimer with a retention time of approximately 4.5 minutes (Figure 16C, black line). Consistent with this, the average melting temperature of wild-type HMPV F B2 increased from 57.9 °C to 72.7 °C in the stabilized MPV220641 as measured by DSF (Figure 16D). Finally, prefusion HMPV F stabilization was confirmed in biolayer interferometry (BLI) measurements using a quantitative Octet with prefusion (ADI-61026), tip-border (MPV458), and pre-nonfusion (DS7) antibodies as described in Example 1.

[0218] Compared to wild-type HMPV F B2, the stabilized HMPV F B2 MPV220641 showed improved binding to the prefusion-specific ADI-61026 as well as reduced binding to the tip-border MPV458 and pre-nonfusion DS7 antibodies (Figure 16E).

[0219] Trimer expression patterns were also observed in stabilized HMPV F A2 2019 (MPV220639) and HMPV F B1 2020 (MPV220640), although the expression levels were slightly lower compared to A2 2000 (MPV220647). The melting temperatures of recent HMPV F A2 and B1 strains were equal to or higher than that of MPV220639 (73.6 °C and 75.8 °C for MPV220640 and MPV22647, respectively) (Figure 16D). All stabilized HMPV F proteins were expressed in the prefusion conformation, as confirmed by the low binding of MPV458 and DS7 in the Octet and prefusion antibody binding of ADI-61026 (Figure 16E).

[0220] In conclusion, the stabilized HMPV F substitutions found in the HMPV A2 (2000) backbone were successfully introduced into other more recent HMPV subtypes, generating stable trimeric prefusion HMPV F proteins.

[0221] Example 11 .Stabilization, purification, and immunogenicity of the HMPV subtype A2 (2019) prefusion F protein Improvement of trimer expression of the more stabilized tag-free HMPV F variant MPV221190 The stabilized HMPV subtype A2 (2019) prefusion F variant MPV220639 from Example 10 was generated without the C-tag purification tag to obtain variant MPV220759. In this backbone, additional substitutions S149Y and N404P were introduced to obtain variant MPV221190, and their effects on trimer expression were evaluated after transient transfection in Expi293F cells as described in Example 1. SEC analysis of the cell culture supernatant showed enhanced trimer expression in MPV221190 containing both S149Y and N404P compared to the backbone MPV220759 (Figure 17A).

[0222] Purification and characterization of tag-free MPV220759 and MPV221190 HMPV F trimers Next, both the MPV220759 and MPV221190 HMPV F variants were transiently transfected into Expi293F cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured at 37 °C and 10% CO2 for 5 days. The culture supernatant was harvested, centrifuged at 600 g for 10 minutes to remove cells and cell debris, and then sterile filtered using a 0.22 μm vacuum filter. The HMPV F protein was purified using a two-step purification protocol including ion exchange (cation) purification at pH 5.0 and purification by size exclusion chromatography using a Superdex 200 16 / 600 pg column. The trimer fractions were pooled and further characterized by analytical SEC-MALS using a μDAWNTREOS instrument (Wyatt) connected to an Optilab μ T-rEX refractive index detector (Wyatt) combined with an in-line Nanostar DLS reader (Wyatt) and an ultra-high performance liquid chromatography system (Vanquish, Thermo Scientific). The protein was loaded onto a Unix-C SEC-300 15 cm column (Sepax Technologies) equipped with a corresponding guard column (Sepax Technologies) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL / min. Analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package, and the molecular weight of the HMPV F trimer was calculated by Astra software and compared with the calculated weight to confirm the trimer conformation (Figures 17B, C).

[0223] The melting temperature (Tm50) of the purified HMPV F trimer was determined by DSF as described in Example 10. MPV221190 containing the substitutions S149Y and N404P had a higher melting temperature of 75.5 °C compared to the Tm50 of 73.5 °C of MPV220759 that did not have these substitutions (Figure 17D).

[0224] The pre-fusion conformation of the purified HMPV F protein was confirmed in biolayer interferometry (BLI) measurements using a quantitative Octet with HMPV F pre-fusion (ADI-14448 and ADI-61026), apex border (MPV458), and non-pre-fusion (DS7) antibodies. Antibodies were immobilized on anti-human IgG sensors at a concentration of 5 μg / ml, and the initial binding rate of HMPV F at 20 μg / ml for 300 seconds of association was plotted as the mean + SD of 3 - 6 individual measurements (Figure 17E). Binding of both HMPV F proteins to the pre-fusion specific ADI-14448 and ADI-61026 antibodies was confirmed, and binding to the apex border binding antibody MPV458 and non-pre-fusion DS7 was not confirmed.

[0225] The stress resistance of both purified HMPV F proteins MPV220759 and MPV221190 to repeated instant freezing (SF) cycles in liquid nitrogen and to supercooling stress to -20°C was evaluated by measuring the relative trimer content in analytical SEC compared to untreated protein (Figure 17F). Incremental SF stress decreased the trimer content of both proteins, but less significantly for MPV221190 (Figure 17F, filled bars). Consistent with this, the average decrease in trimer content over 5 supercooling stress measurements was 23% for MPV220759 and 11% for MPV221190 (Figure 17F, open bars).

[0226] In conclusion, two stable trimeric HMPV pre-fusion F proteins based on the A2(2019) sequence without a trimerization domain or purification tag were produced. Variant MPV221190 with additional stabilizing substitutions S149Y and N404P showed higher trimer expression and improved temperature stress resistance compared to MPV220759.

[0227] Immunogenicity of HMPV pre-fusion F MPV220759 and MPV221190 The in vivo immunogenicity of the purified MPV220759 and MPV221190 HMPV pre-fusion F trimers was evaluated by intramuscular immunization of female Balb / C mice (n = 8) with 1.5, 5 or 15 μg of protein adjuvanted with 10 μL of AS01B per animal on days 0 and 28, and compared to 15 μg of AS01B-adjuvanted MPV212047 (n = 8) or a PBS-immunized control group (n = 3). In sera isolated 2 weeks after the second immunization (day 43), ELISA binding antibody titers against HMPV preF were determined as described in Example 9 (Figure 17G). Upon immunization with MPV220759 and MPV221190, a dose-dependent increase in the levels of HMPV preF-binding antibodies was observed, reaching levels equivalent to MPV212047 at the 15 μg protein dose. This illustrates the immunogenicity of these HMPV pre-fusion F proteins in mice.

[0228] Example 12 . In vitro comparison of differently stabilized HMPV pre-fusion F proteins Pre-fusion stabilized HMPV F proteins are described by Hsieh et al. (Nat Commun. 2022 Mar 14;13(1):1299). Expression of this so-called DS-CavEs2 design (MPV220552) was compared to MPV221190 after transient transfection in Expi293F cells as described in Example 1. DS-CavEs2 (MPV220552) has a foldon trimerization domain and eluted after approximately 4 retention times, while foldon-free MPV221190 eluted later at approximately 4.5 retention times, showing significantly enhanced trimer expression (Figure 18A).

[0229] Next, as described in Example 11, after transient expression in Expi293F cells, the MPV220552 trimer was purified by StrepTag affinity chromatography (GE Healthcare, 28-9075), followed by purification by size exclusion chromatography using a Superose 6 (GE Healthcare) column. The trimer fractions were pooled and further characterized by SEC-MALS, DSF, and Octet as described in Example 11. Equivalent to previous reports (Hsieh et al., 2022), the trimer conformation of the DS-CavEs2 design was confirmed at a melting temperature of 71.1 °C, lower than that of the alternatively stabilized HMPV F MPV221190 (Figures 17D, 18B, C). Both proteins bound to the ADI-14448 and ADI-61026 prefusion antibodies but not to the unfused pre-DS7. However, MPV220552 showed high binding to the tip-border antibody MPV458, indicating a more open tip structure compared to MPV221190 (Figure 18D).

[0230] The stability of both purified proteins was evaluated by long-term incubation at 4 °C or 37 °C followed by SEC for analysis. Examination of the elution pattern of HMPV F showed a stable trimer peak for MPV221190, but a decrease in the trimer peak was observed for MPV220552, along with the appearance of a smaller species eluting at a retention time of 4.5 - 5 minutes upon storage at 37 °C for 2 weeks (Figure 18E).

[0231] In summary, the MPV221190 HMPV prefusion F stabilized trimer has higher expression levels, a more closed trimer structure in the determination by BLI, and enhanced stability in the determination by DSF and stress resistance compared to the previously published DS-CavEs2 (MPV220552) HMPV prefusion F.

[0232] Example 13 . Stabilization of the HMPV prefusion F trimer in the absence of a heterologous trimerization domain and in the absence of stabilization of the HR2 region To examine the need for HR2 stabilization in the presence of multiple alternative stabilizing substitutions in the head domain, trimeric expression of the pre-fusion F protein of HMPV was evaluated in a backbone in the absence of a heterologous trimerization domain and in the absence of stabilizing substitutions in the HR2 region. This backbone was equivalent to MPV212047 except for the introduction of seven HR2 substitutions. Briefly, backbone MPV220847 had an F2 truncation after amino acid 89, an introduced furin cleavage site, and p27 of RSV, and also included a furin cleavage site and a C-terminal truncation at position 489. The variant further included the substitutions V112R, D209E, V231I, H368N, E453P, a linker, and a C-tag.

[0233] The trimeric expression of MPV220847 was compared with that of two variants with the S477I (MPV220115) or S477L (MPV220851) substitution in the HR2 region, which have previously been shown to improve the trimer content of HMPV F (Figure 4). As described in Example 1, upon transient expression in Expi293F cells, all three HMPV F variants showed a trimer peak eluting with a retention time of approximately 4.5 minutes (Figure 19A). This indicates that HR2 stabilization is not required when sufficient alternative substitutions have been introduced. However, although a single melting event for all three HMPV F variants was measured at around 70 °C, the melting temperatures of MPV220115 and MPV220851 were increased in DSF compared to the backbone MPV220847, so the stabilizing effect of the S447I and S477L substitutions was still evident (Figure 19B). The pre-fusion conformations of all three HMPV F variants were confirmed by BLI by the binding of the pre-fusion-specific antibody ADI-14448 and the lack of binding to the non-pre-fusion antibody DS7 (Figure 19C) (DSF and BLI were performed as described in Example 11).

[0234] In conclusion, the prefusion F protein of HMPV can be stabilized without the need for a heterologous trimerization domain and without the need for stabilization of the HR2 region, although the latter confers an obvious advantage in terms of the thermal stability of the HMPV F protein.

[0235] Example 14 Stabilization of the prefusion F trimer of HMPV without a heterologous trimerization domain by stabilization of the HR2 region at position 477 Stabilization of pre-fusion HMPV F by optimization of the HR2 stem region identified position S477 as an appropriate target for stabilization (Figure 4). In the backbone MPV211241, which has previously been shown to express little trimeric HMPV F, the effects of hydrophobic amino acid substitutions were systematically evaluated (left histogram panel of Figure 4A, left histogram panel of 5A). For this purpose, HMPV F variants containing wild-type S477, or any of S477I, S477L, S477F, S477V, S477M, S477Y or S477W were expressed in Expi293F cells and analyzed as described in Example 1. To assess the stability of HMPV F trimer expression, culture supernatants were subjected to incubation at 58 °C for 30 minutes and evaluated by analytical SEC. As previously shown, the backbone MPV211241 containing wild-type S477 did not express HMPV F trimers but eluted with a retention time of 4.5 - 5 minutes corresponding to the HMPV F monomer (Figure 20, black line). In the case of heat stress, aggregates appeared with a retention time of approximately 3 minutes (Figure 20, grey line). Substitutions S477I, S477L, S477F, S477V, S477M (MPV211247, MPV211249, MPV23278, MPV211248, MPV23279 respectively) successfully restored HMPV F trimer expression, while substitutions S477Y and S477W (MPV23280 and MPV23281) partially restored HMPV F trimer expression and were expressed as a mixture of trimer and monomer (Figure 20, black line). Upon heat stress at 58 °C, HMPV F trimers expressed from variants containing either the S477I or S477L substitution remained as trimers (MPV211247 and MPV211249, Figure 20, grey line; Figure 4A), while all other S477 variants were affected, with a decrease in trimer content and the presence of aggregates being shown (Figure 20, grey line).

[0236] In summary, introduction of hydrophobic amino acid residues at position 477 in the HR2 region of HMPV F enhanced trimer expression, and it was demonstrated that the S477I and S477L substitutions gave the most stable trimers.

[0237] Example 15 . Stabilization of the full-length prefusion HMPV F trimer The effects of stabilization substitutions in the head domain of full-length HMPV F (the "head domain" is defined herein as the portion of the mature processed protein that is N-terminal to the HR2 stem region) and in the HR2 region were evaluated by flow cytometry. For this purpose, full-length wild-type HMPV A2(2019)F with F2 truncation after amino acid 89, an introduced furin cleavage site, and p27 of RSV (including the furin cleavage site) was designed (MPV221364). This wild-type backbone was compared to MPV221376, which additionally contains four HR2 substitutions, namely L473W, Q476K, S477F, and A484I. Similarly, the wild-type backbone was compared to MPV221371, which has head domain substitutions V112R, D209E, V231I, and E453P but no HR2 stabilization. Finally, the mutant MPV221377 had both head and HR2 stabilizations as described above.

[0238] Plasmids encoding HMPV F were co-transfected into Expi293F cells at a ratio of HMPV F:furin:GFP plasmid DNA of 3:2:5. Two days after transfection, the cells were harvested, washed, stained, fixed, and then subjected to flow cytometry (FACS Canto II, Becton Dickinson). The staining process included live / dead Violet staining (ThermoFisher) as well as staining with HMPV F antibodies AD-61026 and DS7, followed by staining with an Alexa Fluor647-labeled anti-human IgG detection antibody. The median fluorescence intensity (MFI) of the HMPV F antibody signal was determined by applying a single-cell, live, GFP-positive cell gate. The pre-fusion F-specific antibody ADI-61026 against HMPV was detected at comparable levels in all four HMPV F variants. This indicates that full-length membrane-expressed processed HMPV F is present in a pre-fusion conformation on the surface of transfected Expi293F cells (Figure 21). However, the presence of non-pre-fusion HMPV F has also been confirmed in these cells, and the highest DS7 binding was detected in the wild-type backbone MPV221364. The introduction of the HR2 substitution reduced DS7 binding, as did the introduction of the head domain substitution. The combination of substitutions in both the head domain and the HR2 region resulted in the most significant reduction in DS7 binding, confirming the stabilizing effect of substitutions in both HMPV F protein regions in full-length HMPV F (Figure 21).

[0239] Example 16 .Alternative p27 sequences improve HMPV F processing without the need for exogenous furin co-expression The introduction of a second furin cleavage site and the RSV p27 domain between F1 and F2 of HMPV resulted in complete processing of F0 in the case of co-transfection with furin (Example 2). The RSV p27 sequence was optimized to achieve complete processing while eliminating the need for co-transfection with exogenous furin.

[0240] The RSV p27 variants contain sequences based on either the representative RSV A (MPV23259) or B (MPV23260) sequences, which were introduced in the stabilized backbone MPV221190 (Figure 22A). The plasmid encoding HMPV F was co-transfected with the plasmid encoding furin, at a 5:1 HMPV F:furin DNA ratio (20% furin), as described in Example 1, or with the plasmid encoding the carrier plasmid, at a 5:1 HMPV F:carrier DNA ratio (0% furin), and trimer expression was evaluated by analytical SEC as described in Example 1. The backbone MPV221190 and the p27 RSV A and RSV B variants (MPV23259 and MPV232660, respectively) eluted as trimers with a retention time of approximately 4.5 minutes when co-transfected with 20% furin (Figure 22B, gray line). However, in the absence of co-transfection with furin, the elution pattern shifted to a shorter retention time (Figure 22B, black line), indicating that HMPV F was not fully processed. The latter was confirmed by detection of HMPV F1 and F2 by Western blot analysis of cell lysates under reducing conditions, as described in Example 1. In the absence of co-transfection with furin, an additional band was detected between the F1 and F2 fragments, which likely corresponded to F2 with an attached p27 fragment. This additional band was not present in the HMPV F cell lysates co-transfected with 20% furin (Figure 22C).

[0241] Additional mutations on the RSV p27 fragment are based on the backbone MPV23259 with the p27 RSV A sequence and included a systematic deletion of glycosylation sites in the p27 sequence (MPV23261 - MPV23267) and deletions of the p27 sequence (MPV23268, MPV23269), as shown in Figure 22A. When these mutants were evaluated as described above, equivalent trimer elution patterns were shown when co-transfected with 20% furin (Figure 22B, gray line), but different elution profiles were shown when transfected in the absence of additional furin (Figure 22C, black line). Expression of the p27 mutants in the absence of co-transfection with furin resulted in incomplete processing of HMPV F in mutants with one or two glycan deletions (MPV23261 - MPV23266) (Figure 22B, C). However, removal of all three glycosylation sites in MPV23267 resulted in the same trimer retention time regardless of whether this mutant was co-transfected with furin (Figure 22B) and showed complete HMPV F processing in Western blot without requiring exogenous furin expression (Figure 22C). Removal of "NNTKNTNVTLS" from the p27 sequence in mutant MPV23269 resulted in an equivalent effect, but removal of "LPRFMNYTL" in MPV23268 was not successful.

[0242] In conclusion, optimization of the RSV p27 sequence inserted between F1 and F2 of HMPV F can achieve complete HMPV F processing without requiring exogenous furin expression.

Table 2

[0243] Sequence

Table 3

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Claims

1. A pre-fusion human pneumovirus (HMPV) F precursor F0 protein comprising an F1 domain and an F2 domain, and comprising at least one modification in the amino acid sequence of the F1 domain and / or the F2 domain, wherein the at least one modification stabilizes the pre-fusion conformation and / or enhances trimer formation, and the at least one modification is the introduction of at least one non-native cleavage site, the HR2 domain of F0 comprises amino acids 453-484 of the HMPV F precursor (F0) protein, the amino acid residue at position 473 is W, the amino acid residue at position 476 is K, the amino acid residue at position 477 is F, and the amino acid residue at position 484 is I, wherein the positions of the above amino acids are shown with reference to the sequence of Sequence ID No.

1.

2. The protein according to claim 1, further comprising a second unnatural cleavage site in the F2 domain located N-terminal to a first cleavage site, wherein a spacer sequence exists between the first unnatural cleavage site and the second unnatural cleavage site, the first and / or second unnatural cleavage sites comprising the amino acid sequence RXXR, and the spacer being a p27 peptide of an RSV A or RSV F protein comprising an amino acid sequence selected from SEQ ID NOs. 185 and SEQ ID NOs.

186.

3. The protein according to claim 2, wherein the p27 peptide sequence comprises a deletion of 1 to 11 amino acids from the p27 peptide sequence.

4. A protein according to any one of claims 1 to 3, comprising an F2 domain whose C-terminus is truncated.

5. The protein according to claim 4, wherein the F2 domain is truncated after the amino acid at position 89.

6. A protein according to any one of claims 1 to 3, comprising an F1 domain whose C-terminus is truncated.

7. The protein according to claim 6, wherein the F1 domain is truncated after the amino acid residue at position 489 of the HMPV F precursor (F0) protein.

8. The protein according to claim 1, wherein the amino acid at position 474 is I, and / or the amino acid at position 475 is R, and / or the amino acid residue at position 478 is D, and / or the amino acid residue at position 479 is E, and / or the amino acid residue at position 480 is L, and / or the amino acid residue at position 488 is I.

9. The protein according to any one of claims 1 to 3, wherein the amino acid residue at position 112 is R, and / or the amino acid residue at position 209 is E, and / or the amino acid residue at position 453 is P or Q.

10. The protein according to any one of claims 1 to 3, wherein the amino acid residue at position 149 is Y, and / or the amino acid residue at position 313 is W, and / or the amino acid residue at position 445 is Y.

11. The protein according to any one of claims 1 to 3, wherein the amino acid residue at position 231 is I.

12. The protein according to any one of claims 1 to 3, wherein the amino acid residue at position 404 is P.

13. The protein according to any one of claims 1 to 3, wherein the amino acid residue at position 368 is N.

14. The protein according to any one of claims 1 to 3, wherein the amino acid residue at position 69 is Y or W, and / or the amino acid residue at position 73 is W, and / or the amino acid residue at position 185 is P, 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.

15. The protein according to any one of claims 1 to 3, further comprising one or more non-natural disulfide bonds within or between protomers.

16. The protein according to claim 15, wherein the one or more disulfide bonds are selected from an intraprotomer disulfide bond between amino acid residues 140 and 147, and / or an intraprotomer disulfide bond between amino acid residues 141 or 161, and / or an intraprotomer disulfide bond between amino acid residues 360 and 459.

17. A protein according to any one of claims 1 to 3, comprising an amino acid sequence or fragment thereof selected from the group consisting of SEQ ID NOs: 4 to 135, SEQ ID NOs: 151 to 159, SEQ ID NOs: 161 to 182, and SEQ ID NO:

184.

18. The protein according to claim 17, comprising the amino acid sequence or fragment thereof of SEQ ID NO: 111, SEQ ID NO: 159, SEQ ID NO: 180, or SEQ ID NO:

184.

19. The protein according to any one of claims 1 to 3, wherein the protein is cleaved at one or more cleavage sites such that an F2 domain and an F1 domain are covalently bonded by one or more natural disulfide crosslinks, and the protein is a trimer.

20. The protein according to claim 19, wherein the F1 domain contains amino acids 103 to 489 of the HMPV F0 protein, and the F2 domain contains amino acids 19 to 88 of the HMPV F0 protein.

21. A nucleic acid molecule encoding a protein according to any one of claims 1 to 3.

22. The nucleic acid according to claim 21, wherein the nucleic acid molecule is DNA or RNA.

23. The nucleic acid according to claim 21, comprising an amino acid sequence or fragment thereof selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182, and SEQ ID NO: 184, which encodes a protein.

24. The nucleic acid according to claim 23, which encodes a protein comprising the amino acid sequence of SEQ ID NO: 111, the amino acid sequence of SEQ ID NO: 159, the amino acid sequence of SEQ ID NO: 180, or the amino acid sequence of SEQ ID NO:

184.

25. A vector comprising the nucleic acid described in claim 21.

26. A pharmaceutical composition comprising the protein according to any one of claims 1 to 3.

27. A pharmaceutical composition comprising the nucleic acid described in claim 21.

28. A pharmaceutical composition comprising the vector according to claim 25.

29. A use of the pharmaceutical composition according to claim 26 in the manufacture of a pharmaceutical for vaccinating a target against HMPV, wherein the pharmaceutical is administered to the target.

30. Use of the pharmaceutical composition according to claim 27 in the manufacture of a pharmaceutical for vaccinating a target against HMPV, wherein the pharmaceutical is administered to the target.

31. A use of the pharmaceutical composition according to claim 28 in the manufacture of a pharmaceutical for vaccinating a target against HMPV, wherein the pharmaceutical is administered to the target.

32. Use of the pharmaceutical composition according to claim 26 in the manufacture of a pharmaceutical for preventing infection and / or replication of HMPV in a subject, wherein the pharmaceutical is administered to the subject.

33. Use of the pharmaceutical composition according to claim 27 in the manufacture of a pharmaceutical for preventing infection and / or replication of HMPV in a subject, wherein the pharmaceutical is administered to the subject.

34. Use of the pharmaceutical composition according to claim 28 in the manufacture of a pharmaceutical for preventing infection and / or replication of HMPV in a subject, wherein the pharmaceutical is administered to the subject.

35. An isolated host cell containing the nucleic acid described in claim 21.

Images