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EP4448802A1 - Protéines de fusion hmpv pré-hybrides stabilisées - Google Patents

Protéines de fusion hmpv pré-hybrides stabilisées

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Publication number
EP4448802A1
EP4448802A1 EP22834561.7A EP22834561A EP4448802A1 EP 4448802 A1 EP4448802 A1 EP 4448802A1 EP 22834561 A EP22834561 A EP 22834561A EP 4448802 A1 EP4448802 A1 EP 4448802A1
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EP
European Patent Office
Prior art keywords
seq
hmpv
amino acid
protein
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP22834561.7A
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German (de)
English (en)
Inventor
Johannes Petrus Maria Langedijk
Mark Johannes Gerardus BAKKERS
Tina RITSCHEL
Jaroslaw JURASZEK
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Janssen Vaccines and Prevention BV
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Janssen Vaccines and Prevention BV
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Application filed by Janssen Vaccines and Prevention BV filed Critical Janssen Vaccines and Prevention BV
Publication of EP4448802A1 publication Critical patent/EP4448802A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • the present invention relates to the field of medicine.
  • the invention in particular relates to recombinant pre-fusion HMPV F proteins and to fragments thereof and to nucleic acid molecules encoding the HMPV F proteins and fragments thereof, and to uses thereof, e.g. in vaccines.
  • HMPV Human metapneumovirus
  • G attachment protein
  • F fusion protein
  • HMPV To infect a host cell, HMPV, like other enveloped viruses such as influenza virus, RSV and HIV, requires fusion of the viral membrane with a host cell membrane.
  • HMPV F protein conserved fusion protein
  • the HMPV F protein initially folds into a "pre-fusion” conformation. This metastable structure has recently been solved (Battles et al., Nat Commun. Nov 16;8(1): 1528, 2017.)
  • the prefusion conformation undergoes refolding and conformational changes to its "post-fusion" conformation (McLellan, J. Virol.
  • the HMPV F protein is a metastable protein that drives membrane fusion by coupling irreversible protein refolding to membrane juxtaposition by initially folding into a metastable form (pre-fusion conformation) that subsequently undergoes discrete/stepwise conformational changes to a lower energy conformation (post-fusion conformation).
  • HMPV was first identified in 2001 in clinical samples from pediatric patients who had disease resembling that of human Respiratory Syncytial Virus (RSV) but in samples from whom RSV could not be identified (van den Hoogen et al., Nat. Med. 7(6): 719-724, 2001).
  • HMPV is a major cause of both upper and lower respiratory tract infections in infants, young children, the elderly and among immunocompromised persons or those with underlying chronic medical conditions.
  • the clinical manifestation of HMPV infections is similar to that caused by RSV, ranging from mild respiratory illness to bronchiolitis and pneumonia.
  • HPMV infections appear to be ubiquitous since virtually all children are seropositive by the age of 5 years.
  • Previous epidemiological studies have suggested that HMPV infections cause lower respiratory tract infection in 5-15% of otherwise healthy infants (Falsey et al., J. Infect. Dis. 187: 785-790, 2003).
  • HMPV parainfluenza virus type 3
  • the present invention provides recombinant stabilized pre-fusion human pneumovirus (HMPV) fusion (F) proteins, i.e. recombinant HMPV F proteins that are stabilized in the prefusion conformation.
  • HMPV F proteins of the invention comprise at least one epitope that is specific to the pre-fusion conformation of the F protein.
  • the prefusion hMPV F proteins are soluble proteins (i.e. are not membrane-bound, and lack the transmembrane and cytoplasmic regions).
  • the invention also provides nucleic acid molecules encoding the pre-fusion HMPV F proteins according to the invention and vectors comprising such nucleic acid molecules.
  • the invention also relates to pharmaceutical compositions, preferably vaccine compositions, comprising one or more HMPV F proteins, nucleic acid molecules and/or vectors according to the invention, and to the use thereof in inducing an immune response against HMPV F protein, in particular to the use thereof as a vaccine.
  • the invention also relates to methods for inducing an anti-human pneumovirus (HMPV) immune response in a subject, comprising administering to the subject an effective amount of a prefusion HMPV F protein, a nucleic acid molecule encoding said HMPV F proteins, and/or a vector comprising said nucleic acid molecule.
  • HMPV anti-human pneumovirus
  • the induced immune response is characterized by neutralizing antibodies to HMPV, T cells and/or protective immunity against HMPV.
  • the invention relates to a method for inducing neutralizing antihuman pneumovirus (HMPV) F protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a pre-fusion HMPV F protein, a nucleic acid molecule encoding said HMPV F protein, and/or a vector comprising said nucleic acid molecule.
  • HMPV neutralizing antihuman pneumovirus
  • FIG. 1 Simplified schematic drawing of soluble versions of protein monomers of the invention. The residue position is numbered as in the full-length wild type protein including signal peptide. Processed protein designs (a and b) and single chain protein design (c) are depicted. Fl and F2 domains are indicated, as well as F2 C-terminal amino acid after processing, fusion peptide (FP), fibritin trimerization domain (foldon), and the linker (GSGSGR) in single chain proteins (c) between F2 and Fl. The depicted proteins are examples.
  • FIG. 2 Expi-HEK expressed proteins corresponding to the ectodomain of HMPV F of strain TN/00/3-14 with furin cleavage site between F2 and Fl.
  • FIG. 3 Expi-HEK expressed proteins corresponding to the ectodomain of HMPV F with furin cleavage site between F2 and Fl and C-terminal foldon trimerization domain.
  • P360C/A459C The location of P360C/A459C is indicated with spheres on the cartoon representation of a hMPV preF monomer.
  • C459, located in P-sheet 23, is in the refolding region 2 (RR2; dark gray) and once the disulfide bridge is formed to C360, located in the loop between a8 and P 14, the RR2 is locked in preF conformation,
  • RR2 refolding region 2
  • One preF monomer of the trimeric x-ray structure with RR2 in dark gray and the monomer+DS7 with missing RR2 are shown as cartoons. The location of the disulfide bridge is not in the footprint of DS7.
  • FIG. 6 Expi-HEK expressed proteins with furin cleavage site between F2 and Fl, foldon trimerization domain and SAIG or SGGG linker.
  • FIG. 7 Purification and characterization of selected preF hMPV proteins with furin cleavage site between F2 and Fl, foldon trimerization domain and SAIG or SGGG linker.
  • PostF was produced as control protein, (a) After harvest the proteins are purified first via affinity chromatography C-tagXL 5ml column using the C-terminal C-tag (data not shown) followed by size exclusion chromatography on a Superose 6 (10/300) column. A representative example of MPV190444 is depicted. Fractions between the dashed lines corresponding to HMPV F trimer were pooled, (b) SDS-PAGE analysis under reduced conditions for selected preF proteins (Coomassie stained).
  • Binding to pre-fusion specific antibodies ADI-14448 and ADI-15614, pre and post- binding antibody ADI-18992 and non-preF specific antibody DS7 are measured using biolayer interferometry on the Octet, (e) Melting temperature of the HMPV-F proteins measured by DSF. For each sample the derivative is plotted, and the assigned melting points are indicated in degree Celsius. HMPV-F proteins can have multiple melting temperatures, (f) Trimer content and stability after freezing evaluated by SEC-MALS in a buffer without (left) and with (right) sucrose.
  • Reference sample storage at 4°C (light gray line) compared to one-time (dashed line), 5 times (dotted line) and 10 times (dash-dotted line) snap freezing, (g) Stability and trimer content after 2 (g) and 14 (h) weeks at 37°C evaluated by SEC-MALS.
  • Reference sample stored at 4°C (dark gray) is compared to samples incubated at 37°C (light gray). Aggregates (A) and Trimer (T) are indicated.
  • FIG. 8 Expi-HEK expression of stabilized single chain HMPV F proteins with a linker between F2 and Fl, foldon trimerization domain and an SAIG linker between Fl and foldon.
  • (b) The trimer content and retention time of the trimer are analyzed in analytical SEC. Trimer elutes between 4.2 and 4.35 min.
  • FIG. 9 Expi-HEK expression of stabilized single chain HMPV F proteins with a linker between F2 and Fl, foldon trimerization domain and a SAIG or SGGG liker between Fl and foldon.
  • FIG. 10 Expi-HEK expression of stabilized single chain HMPV F proteins with a linker between F2 and Fl, foldon trimerization domain and a SGGG liker between Fl and foldon.
  • FIG. 12 Purification and characterization of selected preF hMPV single chain proteins with foldon trimerization domain and SAIG or SGGG linker between Fl and foldon.
  • PostF was added as control protein, (a) After harvest the proteins are purified first via affinity chromatography C-tagXL 5ml column using the C-terminal C-tag (data not shown) followed by size exclusion chromatography on a Superose 6 (10/300) column. A representative example is depicted, (b) SDS-PAGE analysis under reduced conditions (Coomassie stained), (c) Trimer content of purified hMPV proteins analyzed by SEC-MALS. Samples were analyzed after purification and after storage at 4°C for 6 months or longer.
  • HMPV-F proteins In vitro antigenicity of purified HMPV-F proteins (as Figure 7).
  • FIG. 13 A. Expi-HEK expression of stabilized HMPV F proteins without cleavage site between F2 and Fl. Amino acids of F2 are joined to Fl to create single chain proteins comprising different linkers, (a) The trimer content and retention time of the trimer are analyzed in analytical SEC in crude supernatant at the day of harvest.
  • the fusion protein (F) of the human pneumovirus (HMPV or hMPV) is involved in fusion of the viral membrane with a host cell membrane, which is required for infection.
  • the HPMV F mRNA is translated into a 539 amino acid precursor protein designated F0, which contains a signal peptide sequence at the N-terminus (i.e. amino acid residues 1-18 of SEQ ID NO: 1) (Ulbrandt et al, Journal of General Virology (2008), 89, 3113-3118) which is removed by a signal peptidase in the endoplasmic reticulum.
  • F0 is cleaved by cellular proteases generating two domains or subunits designated Fl and F2.
  • the Fl domain (corresponding to amino acid residues 103-539 of SEQ ID NO: 1) contains a 23 hydrophobic fusion peptide at its N-terminus (corresponding to amino acids 103-126 of SEQ ID NO: 1), the refolding region 2 (RR2) (corresponding to amino acids 426-491 of SEQ ID NO: 1) and the C-terminus contains the transmembrane region (TM) (corresponding to amino acid residues 492-513 of SEQ ID NO: 1) and the cytoplasmic region (corresponding to amino acid residues 514 - 539) (Ulbrandt et al, Journal of General Virology (2008), 89, 3113-3118).
  • the F2 domain (corresponding to amino acid residues 19-102 of SEQ ID NO: 1) is covalently linked to Fl by two disulfide bridges (Ulbrandt et al, Journal of General Virology (2008), 89, 3113-3118).
  • the F1-F2 heterodimers are assembled as homotrimers in the virion.
  • a vaccine against HMPV infection is not currently available but is desired.
  • One potential approach to producing a vaccine is a subunit vaccine based on purified HMPV F protein.
  • the purified HMPV F protein is in a conformation which resembles the conformation of the pre-fusion state of HMPV F protein, and which is stable over time, and can be produced in sufficient quantities.
  • the HMPV F protein needs to be truncated by deletion of the transmembrane (TM) and the cytoplasmic region to create a soluble secreted F protein (F or sF).
  • the anchorless soluble F protein is considerably more labile than the full-length protein and will readily refold into the post-fusion end-state. In order to obtain soluble F protein in the stable pre-fusion conformation that shows high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized.
  • the present invention now provides recombinant stable pre-fusion HMPV F proteins, i.e. HMPV F proteins that are stabilized in the pre-fusion conformation.
  • HMPV F proteins that are stabilized in the pre-fusion conformation.
  • modifications such as mutations, deletions, insertions, and fusions of amino acids as compared to the amino acid sequence of a wild-type HMPV F protein, were introduced in order to obtain said stable pre-fusion HMPV F proteins.
  • the stable pre-fusion HMPV F proteins of the invention are in the pre-fusion conformation, i.e. they comprise (display) at least one epitope that is specific to the pre-fusion conformation F protein.
  • an epitope that is specific to the pre-fusion conformation F protein is an epitope that is not present in the post-fusion conformation.
  • the pre-fusion conformation of HMPV F protein may contain epitopes that are the same as those present on the HMPV F protein expressed on natural HMPV virions, and therefore may provide advantages for eliciting protective neutralizing antibodies.
  • the proteins of the invention comprise at least one epitope that is recognized by a pre-fusion specific anti-HMPV monoclonal antibody. Examples of such pre-fusion HMPV antibodies are MPE8 (Corti et.
  • the recombinant pre-fusion HMPV F proteins comprise at least one epitope that is recognized by at least one pre-fusion specific monoclonal antibody as described above and are trimeric.
  • the stable pre-fusion HMPV F proteins according to the invention are soluble and thus comprise a truncated Fl domain (i.e. the transmembrane and cytoplasmic region have been (partially) deleted).
  • the present invention in particular provides stabilized pre-fusion human pneumovirus (HMPV) F proteins, comprising an Fl and an F2 domain, comprising an amino acid sequence wherein the amino acid residue at position 69 is Y, and/or the amino acid residue at position 73 is W, and/or the amino acid residue at position 191 is I, and/or the amino acid residue at position 116 is H, and/or the amino acid residue at position 342 is P, and/or the amino acid residue at position 453 is Q, wherein the numbering of the amino acid positions is according to the numbering is amino acid residues in SEQ ID NO: 1.
  • HMPV human pneumovirus
  • the position of the amino acid residues are given in reference to the sequence of the HMPV F protein of SEQ ID NO: 1.
  • the wording “the amino acid residue at position “x” of the HMPV F protein thus means the amino acid corresponding to the amino acid at position “x” in the HMPV F protein of SEQ ID NO: 1. It is noted that, in the numbering system used throughout this application 1 refers to the N-terminal amino acid of an immature F0 protein (SEQ ID NO: 1), i.e. including the signal peptide.
  • the amino acid positions of the F protein are to be numbered with reference to the numbering of the F protein of the strain of SEQ ID NO: 1, by aligning the sequences of the other HMPV strain with the F protein of SEQ ID NO: 1 with the insertion of gaps as needed. Sequence alignments can be done using methods well known in the art, e.g. by CLUSTALW, Bioedit or CLC Workbench.
  • An amino acid according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as e.g. D-amino acids (the D- enantiomers of amino acids with a chiral center), or any variants that are not naturally found in proteins, such as e.g. norleucine.
  • the standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions.
  • amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the protein backbone, and glycine that is more flexible than other amino acids.
  • Table 4 shows the abbreviations and properties of the standard amino acids. It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology procedures.
  • novel trimeric HMPV proteins in the pre-fusion conformation have been generated.
  • the modifications according to the invention preferably result in increased expression levels and/or increased stabilization of the pre-fusion HMPV F trimers as compared to HMPV F proteins that do not comprise these modification(s).
  • the modifications according to the invention result in increased trimer content and trimer stability after storage at 4°C as compared to HMPV F proteins that do not comprise these modification(s).
  • the modifications according to the invention result in increased trimer content and trimer stability after storage at 4°C for at least 6 months as compared to HMPV F proteins that do not comprise these modification(s).
  • the proteins further comprise one or more non-native intra- or inter-protomer disulfide bonds that further stabilize the proteins in the pre-fusion conformation.
  • the one or more disulfide bonds are selected from an intraprotomeric disulfide bond between the amino acid residues 140 and 147 and/or an intraprotomeric disulfide bond between the amino acid residues 141 or 161, and/or an intraprotomeric disulfide bond between the amino acid residues 360 and 459.
  • the presence of specific amino acids and/or one or more disulfide bridges at the indicated positions increase the stability of the proteins in the pre-fusion conformation.
  • the specific amino acids or disulfide bridges are introduced by substitution (mutation) of the amino acid at that position into a specific amino acid according to the invention.
  • the proteins thus comprise one or more mutations in their amino acid sequence, i.e. the naturally occurring amino acids at the indicated positions have been substituted with another amino acid.
  • the amino acid residue at position 185 is P.
  • amino acid residue at position 294 is E, and/or the amino acid residue at position 368 is N.
  • the proteins comprise a non-native cleavage site between the Fl and F2 domain.
  • the non-native cleavage site is a furin cleavage site, e.g. RRRR or RRAR, in order to improve the processing of the protein in the cells that are used to produce the proteins.
  • the F2 and Fl domains are directly linked through a linking sequence, in order create a single chain polypeptide.
  • part of the Fl (at the N-terminus) and/or F2 domain (at the C-terminus) have been deleted and replaced by a linking sequence.
  • at least the amino acids 97-106 have been deleted and replaced by a linking sequence of 1-10 amino acids.
  • the amino acids 91-110 have been deleted and replaced by a linking sequence of 1-10 amino acids
  • the pre-fusion HMPV F proteins are soluble proteins, i.e. not membrane bound.
  • the proteins comprise a truncated Fl domain.
  • the truncated Fl domain does not comprise the transmembrane and cytoplasmic regions.
  • the Fl domain may be truncated after the amino acid at position 481, 482, 483, 484, 485, 486, 487, 488 or 489.
  • the truncated Fl domain comprises the amino acids 103-481, 107-481, 111- 481, 103-482, 107-482, 111-482, 103-489, 107-489, or 111-489 of the HMPV F protein.
  • a heterologous trimerization domain is linked to the truncated Fl domain, optionally through a linking sequence.
  • HMPV F proteins are provided that show high expression and that bind to pre-fusion-specific antibodies, indicating that the proteins are in the pre-fusion conformation.
  • the HMPV F proteins remain stabilized in the pre-fusion conformation, i.e. even after processing of the proteins (i.e. after cleaving into Fl and F2) the proteins still bind to the pre-fusion specific antibodies, indicating that the pre-fusion specific epitope is retained after processing of the proteins.
  • a fibritin - based trimerization domain is fused to the C- terminus of the soluble HMPV-F.
  • This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was described earlier as an artificial natural trimerization domain (Letarov et al., Biochemistry Moscow 64: 817-823 (1993); S-Guthe et al., J. Mol. Biol. 337: 905-915. (2004)).
  • the Fl domain is truncated at position 481, 482 or 489 and fused C terminally to the heterologous trimerization domain using an amino acid linker, e.g. SAIG or SGGG.
  • the heterologous trimerization domain is the fibritin domain comprising the amino acid sequence: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 2).
  • the pre-fusion HMPV F proteins thus comprise one or more mutations (as compared to the wild-type HMPV F protein, in particular the HMPV F protein of SEQ ID NO: 1) selected from the group consisting of: a) a mutation of the amino acid residue T at position 69; b) a mutation of the amino acid residue L at position 73; c) a mutation of the amino acid residue A at position 116; d) a mutation of the amino acid residue A at position 140; e) a mutation of the amino acid residue L at position 141; f) a mutation of the amino acid residue A at position 147; g) a mutation of the amino acid residue A at position 161; h) a mutation of the amino acid residue D at position 185; i) a mutation of the amino acid residue V at position 191; j) a mutation of the amino acid residue N at position 342; k) a mutation of the amino acid residue P at position 360; l) a mutation of the amino acid residue of the amino
  • the pre-fusion HMPV F proteins comprise one or more further mutations (as compared to the wild-type HMPV F protein, in particular the HMPV F protein of SEQ ID NO: 1) selected from the group consisting of: a) a mutation of the amino acid residue T at position 69 into Y; b) a mutation of the amino acid residue L at position 73 into W; c) a mutation of the amino acid residue A at position 116 into H; d) a mutation of the amino acid residue A at position 140 into C; e) a mutation of the amino acid residue L at position 141 into C; f) a mutation of the amino acid residue A at position 147 into C; g) a mutation of the amino acid residue A at position 161 into C h) a mutation of the amino acid residue D at position 185 into P; i) a mutation of the amino acid residue V at position 191 into I; j) a mutation of the amino acid residue N at position 342 into P; k) a mutations (as
  • the pre-fusion HMPV F proteins comprise at least two mutations (as compared to a wild-type HMPV F protein). In certain embodiments, the proteins comprise at least three mutations. In certain embodiments, the proteins comprise at least four, five or six mutations.
  • the proteins of the invention comprise a truncated Fl domain.
  • a “truncated” Fl domain refers to a Fl domain that is not a full length Fl domain, i.e. wherein either N- terminally or C-terminally one or more amino acid residues have been deleted.
  • at least the transmembrane domain and cytoplasmic domain have been deleted to permit expression as a soluble ectodomain.
  • the Fl domain is truncated after amino acid residue 481, 482, 483, 484, 485, 486, 487, 488 or 489.
  • the HMPV F protein comprises an amino acid sequence wherein the amino acid at position 73 is W, the amino acid at position 116 is H, the amino acid at position 185 is P, the amino acid at position 432 is P, and the amino acid at position 368 is N, the amino acid at position 453 is Q, and comprises an intraprotomeric disulfide bond between the amino acid residues 140 and 147.
  • the HMPV F protein comprises a truncated Fl domain consisting of the amino acids 103-481 and an F2 domain, and an amino acid sequence wherein the amino acid at position 73 is W, the amino acid at position 116 is H, the amino acid at position 185 is P, the amino acid at position 432 is P, and the amino acid at position 368 is N, the amino acid at position 453 is Q, and comprises an intraprotomeric disulfide bond between the amino acid residues 140 and 147.
  • the proteins according to the invention comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8-16, 18-20, 22-24, 28-31,
  • the protein according to the invention comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 66.
  • the proteins do not comprise a signal sequence.
  • nucleotide sequences are provided from 5’ to 3’ direction, and amino acid sequences from N-terminus to C-terminus, as custom in the art. It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology procedures.
  • the modifications according to the invention preferably result in increased expression levels and/or increased stabilization of the prefusion HMPV F proteins as compared to HMPV F proteins that do not comprise these modifcation(s).
  • the present invention further provides nucleic acid molecules encoding the HMPV F proteins according to the invention.
  • the nucleic acid molecules encoding the proteins according to the invention are codon-optimized for expression in mammalian cells, preferably human cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378). A sequence is considered codon- optimized if at least one non-preferred codon as compared to a wild-type sequence is replaced by a codon that is more preferred.
  • a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a nonpreferred codon.
  • the frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon.
  • more than one nonpreferred codon, preferably most or all non-preferred codons are replaced by codons that are more preferred.
  • the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons generally leads to higher expression.
  • the nucleic acid molecule may be DNA or RNA.
  • the RNA is mRNA, modified mRNA, self-replicating (or self-amplifying) RNA, or circular mRNA.
  • the present invention thus also encompasses RNA molecules, e.g. self-amplifying RNA molecules (or replicons) encoding a protein as described herein.
  • nucleic acid sequence or nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences or nucleic acid molecules that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.
  • nucleic acid molecules according to the invention encode a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-16, 18-20, 22-24, 28-31, 33-34, 37-64, and 66, or fragments thereof.
  • Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScripts, Invitrogen, Eurofins).
  • the invention also provides vectors comprising a nucleic acid molecule as described above.
  • a nucleic acid molecule according to the invention thus is part of a vector.
  • the vector is an adenovirus vector.
  • An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), a canine adenovirus (e.g., CAdV2), a porcine adenovirus (e.g., PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus).
  • a bovine adenovirus e.g., bovine adenovirus 3, BAdV3
  • the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd).
  • a human adenovirus is meant if referred to as Ad without indication of species, e.g., the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26.
  • the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.
  • a recombinant adenovirus according to the invention is based upon a human adenovirus.
  • the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc.
  • an adenovirus is a human adenovirus of serotype 26.
  • serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials.
  • Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g., US6083716; WO 2005/071093; WO 2010/086189; WO 2010/085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine 24: 849-62; and review by Lasar
  • the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus.
  • the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P.
  • the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see, e.g., WO 2012/172277), or ChAdOx 2 (see, e.g., WO 2018/215766).
  • the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see, e.g., WO 2019/086466).
  • the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see, e.g., WO 2019/086456), or BZ1 (see, e.g., WO 2019/086466).
  • BLY6 see, e.g., WO 2019/086456
  • BZ1 see, e.g., WO 2019/086466
  • the adenoviral vectors comprise capsid proteins from rare serotypes, e.g. including Ad26.
  • the vector is an rAd26 virus.
  • An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining the serotype and/or tropism of a particular adenovirus.
  • Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins.
  • a “capsid protein” for a particular adenovirus such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein.
  • the capsid protein is an entire capsid protein of Ad26.
  • the hexon, penton, and fiber are of Ad26.
  • a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of a first serotype with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like. See for example WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also e.g.
  • WO 2019/086461 for chimeric adenoviruses Ad26HVRPtrl, Ad26HVRPtrl2, and Ad26HVRPtrl3, that include an Ad26 virus backbone having partial capsid proteins of Ptrl, Ptrl2, and Ptrl3, respectively)
  • the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26).
  • the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome.
  • non-group C adenovirus such as Ad26 or Ad35
  • rAd26 vectors Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63.
  • Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792.
  • Examples of vectors useful for the invention for instance include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.
  • a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector).
  • a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector).
  • the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention.
  • the nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically.
  • the DNA can be double-stranded or single-stranded.
  • the adenovirus vectors useful in the invention are typically replication deficient.
  • the virus is rendered replication deficient by deletion or inactivation of regions critical to replication of the virus, such as the El region.
  • the regions can be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding the stabilized pre-fusion HMPV F protein (usually linked to a promoter), or a gene encoding the pre-fusion HMPV F protein fragment (usually linked to a promoter) within the region.
  • the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions.
  • E2- and/or E4-complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication.
  • a packaging cell line is typically used to produce sufficient amounts of adenovirus vectors for use in the invention.
  • a packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell.
  • Suitable packaging cell lines for adenoviruses with a deletion in the El region include, for example, PER.C6, 911, 293, and El A549.
  • the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the El region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p. 4654-63, which is incorporated herein by reference.
  • the nucleic acid sequence encoding the pre-fusion HMPV F protein is cloned into the El and/or the E3 region of the adenoviral genome.
  • Host cells comprising the nucleic acid molecules encoding the pre-fusion HMPV F proteins form also part of the invention.
  • the pre-fusion HMPV F proteins may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants.
  • the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin.
  • the cells are mammalian cells.
  • the cells are human cells.
  • the production of a recombinant proteins, such the pre-fusion HMPV F proteins of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in said cell.
  • the nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like.
  • promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.
  • Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the pre-fusion HMPV F proteins.
  • the suitable medium may or may not contain serum.
  • a “heterologous nucleic acid molecule” (also referred to herein as ‘transgene’) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into for instance a vector by standard molecular biology techniques.
  • a transgene is generally operably linked to expression control sequences. This can for instance be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added.
  • Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like.
  • a non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter.
  • a polyadenylation signal for example the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s).
  • several widely used expression vectors are available in the art and from commercial sources, e.g.
  • the cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up.
  • continuous processes based on perfusion principles are becoming more common and are also suitable.
  • Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley -Liss Inc., 2000, ISBN 0-471-34889-9)).
  • the invention further provides pharmaceutical compositions comprising a pre-fusion HMPV F protein, and/or fragment thereof, and/or a nucleic acid molecule, and/or a vector, as described herein.
  • the invention thus provides compositions comprising a pre-fusion HMPV F protein, or fragment thereof, that displays an epitope that is present in a pre-fusion conformation of the HMPV F protein but is absent in the post-fusion conformation.
  • the invention also provides compositions comprising a nucleic acid molecule and/or a vector, encoding such pre-fusion HMPV F protein or fragment.
  • the invention in particular provides pharmaceutical compositions, e.g. vaccine compositions, comprising a pre-fusion HMPV F protein, a HMPV F protein fragment, and/or a nucleic acid molecule, and/or a vector, as described above and one or more pharmaceutically acceptable excipients.
  • the invention also provides the use of a stabilized pre-fusion HMPV F protein (fragment), a nucleic acid molecule, and/or a vector, according to the invention, for vaccinating a subject against HMPV.
  • the invention also provides the use of a stabilized pre-fusion HMPV F protein (fragment), a nucleic acid molecule, and/or a vector, according to the invention inducing an immune response against HMPV F protein in a subject.
  • methods for inducing an immune response against HMPV F protein in a subject comprising administering to the subject a pre-fusion HMPV F protein (fragment), and/or a nucleic acid molecule, and/or a vector, according to the invention.
  • prefusion HMPV F protein fragments
  • nucleic acid molecules and/or vectors according to the invention for the manufacture of a medicament for use in inducing an immune response against HMPV F protein in a subject.
  • the invention in particular provides pre-fusion HMPV F protein (fragments), and/or nucleic acid molecules, and/or vectors according to the invention for use as a vaccine.
  • the pre-fusion HMPV F protein (fragments), nucleic acid molecules, or vectors of the invention may be used for prevention (prophylaxis) and/or treatment of HMPV infections.
  • the prevention and/or treatment may be targeted at patient groups that are susceptible HMPV infection.
  • patient groups include, but are not limited to e.g., the elderly (e.g. > 50 years old, > 60 years old, and preferably > 65 years old), the young (e.g. ⁇ 5 years old, ⁇ 1 year old), pregnant women (for maternal immunization), and hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.
  • the pre-fusion HMPV F proteins, fragments, nucleic acid molecules and/or vectors according to the invention may be used in stand-alone treatment and/or prophylaxis of a disease or condition caused by HMPV, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.
  • the invention further provides methods for preventing and/or treating HMPV infection in a subject utilizing the pre-fusion HMPV F proteins or fragments thereof, nucleic acid molecules and/or vectors according to the invention.
  • a method for preventing and/or treating HMPV infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion HMPV F protein (fragment), nucleic acid molecule and/or a vector, as described above.
  • a therapeutically effective amount refers to an amount of a protein, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by HMPV.
  • Prevention encompasses inhibiting or reducing the spread of HMPV or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by HMPV.
  • Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of HMPV infection.
  • the invention may employ pharmaceutical compositions comprising a pre-fusion HMPV F protein (fragment), a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered.
  • pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L.
  • the HMPV F proteins, or nucleic acid molecules preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers.
  • the pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5.
  • the HMPV F proteins typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt.
  • stabilizing agent may be present, such as albumin.
  • detergent is added.
  • the HMPV F proteins may be formulated into an injectable preparation.
  • a composition according to the invention further comprises one or more adjuvants.
  • Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant.
  • the terms “adjuvant” and “immune stimulant” are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system.
  • an adjuvant is used to enhance an immune response to the HMPV F proteins of the invention.
  • suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see e.g.
  • WO 90/14837 saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g.
  • compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.
  • the compositions comprise a combination of adjuvants, e.g. alum and CpG.
  • compositions do not comprise adjuvants.
  • the invention provides methods for making a vaccine against respiratory syncytial virus (HMPV), comprising providing an HMPV F protein (fragment), nucleic acid or vector according to the invention and formulating it into a pharmaceutically acceptable composition.
  • HMPV respiratory syncytial virus
  • the term "vaccine” refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease.
  • the vaccine comprises an effective amount of a pre-fusion HMPV F protein (fragment) and/or a nucleic acid molecule encoding a prefusion HMPV F protein, and/or a vector comprising said nucleic acid molecule, which results in an effective immune response against HMPV.
  • a pre-fusion HMPV F protein fragment
  • a nucleic acid molecule encoding a prefusion HMPV F protein
  • a vector comprising said nucleic acid molecule
  • it may be a combination vaccine that further comprises other components that induce an immune response, e.g. against other proteins of HMPV and/or against other infectious agents, e.g. against RSV, HMPV and/or influenza.
  • the administration of further active components may for instance be done by separate administration or by administering combination products of the vaccines of the invention and the further active components.
  • compositions according to the invention can be performed using standard routes of administration.
  • Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transcutaneous, or mucosal administration, e.g. intranasal, oral, and the like.
  • a composition is administered by intramuscular injection.
  • the skilled person knows the various possibilities to administer a composition, e.g. a vaccine in order to induce an immune response to the antigen(s) in the vaccine.
  • a subject as used herein preferably is a mammal, for instance a rodent, e.g. a mouse, a cotton rat, or a non-human-primate, or a human.
  • the subject is a human subject.
  • the proteins, fragments, nucleic acid molecules, vectors, and/or compositions may also be administered, either as prime, or as boost, in a homologous or heterologous primeboost regimen.
  • a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as ‘priming vaccination’).
  • the administration comprises a prime and at least one booster administration.
  • the invention further provides methods for making a vaccine against HMPV, comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a pre-fusion HMPV F protein or fragment thereof as described herein, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition.
  • provided herein are methods of producing an adenoviral particle comprising a nucleic acid molecule encoding a HMPV F protein or fragment thereof (transgene).
  • the methods comprise (a) contacting a host cell of the invention with an adenoviral vector of the invention and (b) growing the host cell under conditions wherein the adenoviral particle comprising the transgene is produced.
  • Recombinant adenovirus can be prepared and propagated in host cells, according to well- known methods, which entail cell culture of the host cells that are infected with the adenovirus.
  • the cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.
  • the invention further provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a HMPV F protein or fragment thereof, as described herein.
  • the proteins of the invention may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the protein of the invention.
  • the invention thus also relates to an in vitro diagnostic method for detecting the presence of an HMPV infection in a patient said method comprising the steps of a) contacting a biological sample obtained from said patient with a protein according to the invention; and b) detecting the presence of antibody-protein complexes.
  • EXAMPLE 1 Design of a soluble proteins
  • the cleavage site between F2 and Fl was changed to a polybasic cleavage site, e.g. RRRR, for proteins based on Figure la and lb (e.g. SEQ ID NO: 4).
  • Alternative single chain designs, that are not processed (i.e. not cleaved between Fl and F2) were also designed.
  • An example with the linker sequence GSGSGR to replace the amino acids 97-106 is depicted in Fig. 1C.
  • the designs may further have a linker and C-tag, C-terminal to the foldon sequence to allow affinity purification (e.g. SEQ ID NO: 4).
  • point mutations and combinations of point mutations were introduced, e.g one or more of the following mutations T69Y, L73W, A116H, A140C/A147C, L141C/A161C, D185P, V191I, N342P, P360C/A459C, H368N and E453Q (wherein the numbering is according to the numbering of amino acid positions in SEQ ID NO: 1).
  • the DNA fragments encoding for proteins MPV201042 (SEQ ID NO: 4) and MPV201043 (SEQ ID NO: 5) with furin cleavage site and truncation after position 482 or 489 were synthesized (Genscript, Piscataway, NJ) and cloned in the pcDNA2004 expression vector (modified pcDNA3 plasmid with an enhanced CMV promotor).
  • the expression platform used is the Expi293F expression system (Thermo Fisher Scientific, Waltham, USA) in 96- well format.
  • both proteins showed both anti pre-F and anti non-pre-F binding indicating that the protein is not fully in the prefusion trimeric conformation.
  • the retention time (about 4.8-4.9 min) in analytical SEC showed that the proteins are monomeric (Figure 2b). Further design strategies were applied to obtain trimeric pre-F proteins, in a backbone which is C-terminally truncated at position 481.
  • a foldon trimerization domain was added via a 4 residue SAIG linker (MPV190845; SEQ ID NO: 6).
  • one or more of stabilizing mutations D185P (MPV190842; SEQ ID NO: 7), E453Q (MPV190843; SEQ ID NO: 8), Al 16H (MPV190997 SEQ ID NO: 9), H368N (MPV190856; SEQ ID NO: 10), N342P (MPV190991; SEQ ID NO: 11), L73W (MPV19993; SEQ ID NO: 12), A140C/A147C (MPV191012; SEQ ID NO: 13) and P360C/A459C (MPV191013; SEQ ID NO: 16) were introduced (see Figure 3a for the mutations) Furthermore, additional combinations of the stabilizing mutations were evaluated (MPV191386 SEQ ID NO: 14 (with L73W, Al 16
  • EXAMPLE 4 Comparing different linkers between Fl and foldon and additional combinations of stabilizing mutations.
  • EXAMPLE 5 Stabilizing the trimer apex HMPV F protein with furin cleavage site, foldon, SGGG linker between Fl and foldon and with stabilizing mutation D185P and disulfide A360P/A489C (MPV201028; SEQ ID NO: 21) was further modified by introduction of mutations in the protomer interface in the apex: MPV201031 (SEQ ID NO: 22) with T69Y, MPV201032 (SEQ ID NO: 23) with T69Y and L73W mutations and MPV201033 (SEQ ID NO: 24) with T69Y, L73W, and V191I mutations. Expression and analysis of HMPV F proteins were performed as described in example 2.
  • the introduction of the dimer interface mutations showed a reduction of the non-preF antibody binding according to the endpoint binding in Octet (DS7 Binding after 300s in Figure 5a).
  • Introduction of T69Y resulted in a more uniform trimer peak according to analytical SEC without the shoulder at 4.35 minutes ( Figure 5b). The peak at 4.35 minutes is likely a more open trimer species which has a shorter retention time (see also example 8).
  • T69Y is improving the closed trimer quality. Further improvement by L73W and L191I was not detected in the protein analysis of the supernatant.
  • the T69Y mutation at position 69 resulted in a more compact trimer with a higher ratio of preF -binding and non- preF binding and therefore improved the quality of the trimer.
  • HMPV F constructs were evaluated with a SAIG- linker between the Fl domain and foldon (MP VI 90845; SEQ ID NO: 6 and MPV201286; SEQ ID NO: 31).
  • HMPV F constructs were performed as described in Example 2.
  • the crude supernatant was additionally incubated for 30 minutes at 50, 60 and 70 °C.
  • linker SAIG non-stabilized: MPV190845; SEQ ID NO: 6 and stabilized: MPV201286; SEQ ID NO: 31
  • linker SGGG non-stabilized: MPV201285 SEQ ID NO: 25 and stabilized MPV201216 SEQ ID NO: 30
  • HMPV proteins based on a processed design with furin cleavage site and foldon were produced and purified. Designs were explored of F proteins derived from several HMPV strains, i.e. strain TN/00/3-14 (SEQ ID NO: 1), Yokohama. JPN/P8356/2017 (SEQ ID NO: 35) and Yokohama.JPN/P8674/2017 (SEQ ID NO: 65).
  • the linker between Fl and foldon was SAIG or SGGG.
  • MPV190444 SEQ ID NO: 32
  • MPV190444 has an additional strep-tag before the C-tag (see Table 1 for detailed list of mutation included per design).
  • the cells were transiently transfected using ExpiFectamine 293 (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions and cultured in a shaking incubator for 5 days at 37°C and 10% CO2.
  • ExpiFectamine 293 Thermo Fisher Scientific, Waltham, USA
  • MPV201031 SEQ ID NO: 22
  • MPV201216 SEQ ID NO: 30
  • the culture supernatants were harvested, centrifuged for 10 min at 600rpm and filtered over a 0.22pm PVDF filter to remove cells and cellular debris.
  • the proteins were purified by means of a two-step protocol. First, the harvested and clarified culture supernatant was loaded on a pre-packed C-tagXL 5- or 6-ml column
  • a postF hMPV protein (SEQ ID NO: 3) as described by Mas et. al., PLoS Pathog. 2016 Sep 9;12(9):el005859. doi: 10.1371/joumal.ppat.1005859. eCollection 2016 Sep.) was produced and purified.
  • Expression plasmid encoding the recombinant post-fusion hMPV F protein are prepared as in in Example 2. On 300ml-scale the cells are transiently transfected and subsequently purified by means of a two-step protocol (see details above). Subsequently, TEV cleavage was performed to remove the foldon and c-tag.
  • TEV TEV
  • pg of protein 1 pL of TEV (10 000 Units/mL) was used.
  • the protein-TEV mixture was incubated overnight at 4°C.
  • the TEV-His protease was removed from the protein sample by a Ni Sepharose excel beads (GE Healthcare, 17-3712-03) pull down.
  • Ni Sepharose excel beads were added to the protein-TEV mixture and incubated for 2 hours at room temperature. Flow through was collected via a micro bio-spin column (Bio Rad, 7326204). The cleaved protein sample was heat-shocked for 30 minutes at 45°C.
  • the protein sample was applied to a Superose 6 Increase 10/300 column (GE Healthcare, Chicago, USA) equilibrated in running buffer (20mM Tris, 150mM NaCl, pH7.4) for polishing purpose, i.e. remove the minimal amount of multimeric and monomeric protein. Proteins were subsequently analyzed on Sodium Dodecyl Sulfate Polyacrylamide Gel
  • MPV191757 shows minimal unprocessed material indicated with F0.
  • the purified proteins were assessed by analytical Size Exclusion Chromatography (SEC) to study trimer content after purification and trimer content and stability after storage at 4°C.
  • SEC was performed with an Ultra High-Performance Liquid Chromatography (UHPLC) using a Vanquish system (ThermoFisher Scientific) with a Sepax Unix-C SEC-300 4.6X150mm 1.8 pm column (Sepax (231300-4615), injection volume 20pL, flow 0.3mL/min.).
  • the elution was monitored by a UV detector (Thermo Fisher Scientific), a pDawn Light Scatter (LS) detector (Wyatt Technologies), a pT-rEx Refractive Index (RI) detector (Wyatt Technologies) and a Nanostar Dynamic Light Scattering (DLS) detector (Wyatt Technologies).
  • the SEC profiles were analyzed by the Astra 7.3.2.19 software package (Wyatt Technology). Due to the long-time span between the sample measurements the samples were run on column with different lot numbers. A small shift in retention time for the standard was observed and used to correct the retention times of the proteins, allowing superposition of the chromatograms. Chromatograms were plotted in GraphPad Prism (version 9.0.0, GraphPad Software)
  • proteins of the invention show a high trimer content after purification and the best storage stability was observed for MPV191757 with mutations L73W, Al 16H, A140C/A147C, D185P, N342P, H368N and E453Q.
  • the binding of the purified proteins is summarized in Figure 7d.
  • the reference postF protein was binding only minimally to the pre-fusion specific antibodies in the initial slope but the binding after 300 seconds indicated there may be minimal amounts of protein in preF conformation in the sample.
  • All pre-fusion HMPV-F proteins showed binding to the preF-specific antibodies ADI- 18444 and ADI-15614.
  • the preF proteins showed reduced non-preF binding (DS7 binding) compared to the postF reference according to the initial slope signal. However, the binding after 300 seconds showed a signal for non-preF, indicating that a subpopulation may be in non-preF trimeric conformation.
  • the variant that showed full processing after purification (MPV190444, Figure 7b) showed non-preF binding.
  • Thermo-stability of the purified pre-fusion HMPV F proteins was determined by Differential Scanning Fluorimetry (DSF) by monitoring the fluorescent emission of Sypro Orange Dye (ThermoFisher Scientific) in a 96 well optical qPCR plate. 15 pl of a 66.67pg/ml protein solution was used per well (buffer as described in Example 2; for MPV190856 5% sucrose was added to the buffer). To each well, 5 pl of 20x Sypro orange solution was added. Upon gradual increase of the temperature, from 25°C to 95°C (0.015°C/s), the proteins unfold and the fluorescent dye binds to the exposed hydrophobic residues leading to a characteristic change in emission.
  • DSF Differential Scanning Fluorimetry
  • the melting curves were measured using a ViiA7 real time PCR machine (Applied BioSystems).
  • the Tm50 values represent the temperature at which 50% of the protein is unfolded and thus are a measure for the temperature stability of the proteins.
  • SEQ ID NO: 35 (MPV191769 SEQ ID NO: 33) and recent B2 strain Yokohama.JPN/P8674/2016 (SEQ ID NO: 65) (MPV191806 SEQ ID NO: 34) showed a similar melting temperature compared to designs in reference strain (TN/00/3-14, SEQ ID NO: 1), showing that the mutations are transferable to other strains.
  • the purified proteins were snap frozen in liquid nitrogen once, 5 times and 10 times and assessed by analytical Size Exclusion Chromatography (SEC) (as described above for Figure 7A). As control a sample stored at 4°C was measure.
  • SEC Size Exclusion Chromatography
  • HMPV F protein ( Figure 7F) with D185P, H368N and E453Q (MPV190856 SEQ ID NO: 10) was least stable after 10-time snap freezing.
  • a construct with an alternative disulfide bridge (MPV191388 SEQ ID NO: 15) showed similar results. With the addition of sucrose to the buffer all designs showed increased freezing stability. For the designs with disulfide bridge quality was maintained best after 10-times snap freezing.
  • the purified proteins were assessed for preF trimer stability by analytical Size Exclusion Chromatography (SEC) after storage at 37°C for 2 or 14 weeks (as described above for Figure 7a).
  • SEC Size Exclusion Chromatography
  • the stabilizing mutations were transferred into a single chain, i.e. a non-processed polypeptide (Figure 1C) (as described in WO 2014/174018; Krarup et. al., 2015 (Nat Commun. 2015 Sep 3;6:8143. doi: 10.1038/ncomms9143.)).
  • the designs all had a C-terminal foldon domain and a SAIG linker between Fl and foldon.
  • HMPV F constructs Single chain protein non-stabilized (MPV190862; SEQ ID NO: 36) and constructs stabilized with D185P and E453Q (MPV190860 SEQ ID NO: 37) and D185P, E453Q and L73W (MPV191031 SEQ ID NO: 38) were compared.
  • Expression and analysis of HMPV F constructs were performed as described in Example 2. Purification was performed as in Example 7. The purified proteins were analyzed by negative stain Transmission Electron Microscopy (nsTEM).
  • Continuous carbon grids (copper, EMS) were glow discharged for 30 seconds in a easiglow plasma cleaner.
  • the sample solution was partially absorbed by gentle side blotting, and the grid was immediately stained with by depositing it on top of a 40 pl drop of a 2% (w/v) uranyl acetate solution for a total of 1 min. After staining, the grid was blotted dry and stored at room temperature prior to imaging.
  • the prepared grids were imaged in a Talos L120C TEM (Thermo Fisher Scientific) equipped with a Ceta camera.
  • Resulting pixel ranged from 2.4 to 2.8 ang per pixel depending on imaging conditions.
  • CTF Contrast Transfer Function
  • MPV191703 (SEQ ID NO: 43) with T69Y, L73W, D185P, H368N and E453Q and MPV191708 (SEQ ID NO: 44) with L73W, D185P, V191I, H368N and E453Q were tested to evaluate apex mutations (example 5) in amino acid position 69 and 191.
  • HMPV F constructs were performed as described in Example 2.
  • a SGGG linker (MPV191746; SEQ ID NO: 42) increased pre-F binding and trimer content compared to a SAIG linker (MPV191392; SEQ ID NO: 39), however the retention time was minimally reduced (Figure 9B).
  • Single chain designs with additional apex mutations in amino acid position 69 (MPV191703: SEQ ID NO: 43) and 191 (MPV191708; SEQ ID NO: 44) and a SGGG linker had longer retention times (MPV191708 ⁇ MPV191703). Additionally, a slight reduction in un-favorable binding to DS7 (Figure 9A) was observed.
  • a single chain HMPV F protein was stabilized by the introduction of T69Y, L73W, A140C/A147C, D185P, H368H and E453Q (MPV200620; SEQ ID NO: 45; numbering of the positions is based on SEQ ID NO: 1). Subsequently, disulfide P360C/A459C (MPV200622; SEQ ID NO: 46), mutation V191I (MPV200631; SEQ ID NO: 47) and mutation N342P (MPV200632: SEQ ID NO: 48) were added.
  • the single chain backbone MPV191746 (SEQ ID NO: 42) was compared to other single chain designs (Figure 11 A). Level of non-preF trimer binding was comparable for the different constructs. As described in Example 8 and 9, a later retention time indicates a closed trimer ( Figure 11B). MPV191748 (SEQ ID NO: 53) and MPV191756 (SEQ ID NO: 61) had similar retention times as the original design (MPV191746), indicating a correctly closed trimer. However, MPV191748 showed reduced preF -binding and MPV191748 and MPV191756 both showed reduced expression levels compared to MPV191746 ( Figure
  • EXAMPLE 12 Production, purification and characterization of selected single chain proteins
  • Proteins were produced and purified as described in Example 7. (MPV201222 was produced on an increased 400ml transfection scale in HEK-E cells for 6 days).
  • the stabilizing mutations increased expression and stabilized preF of several diverse HMPV strains (MPV191746, MPV191768, MPV191807). Designs with disulfide bridge P360C/A459C show reduced yields. Table 3.: Purified proteins of example 12 and yields
  • the purified proteins were assessed by analytical Size Exclusion Chromatography (SEC) to study trimer content after purification and trimer content and stability after storage at 4°C ( Figure 12C) as described in Example 7.
  • SEC Size Exclusion Chromatography
  • the SGGG linker variant (MPV191746; SEQ ID NO: 42) of MPV191392 (SEQ ID NO: 39) showed a smaller shift in retention time after 6 months, indicative of a more compact trimer conformation (see Example 8).
  • Introducing the set of stabilizing mutations into a recent A2 (MPV191768; SEQ ID NO: 42) of MPV191392 (SEQ ID NO: 39) showed a smaller shift in retention time after 6 months, indicative of a more compact trimer conformation (see Example 8).
  • Thermo-stability of several the purified pre-fusion HMPV F proteins were determined by Differential Scanning Fluorimetry (DSF) as described in Example 7. Melting temperatures of MPV191703, MPV200620, MPV200622 and MPV200632 were measured after 6 months storage of the purified proteins at 4°C.
  • the purified proteins were tested for freezing stability as described in example 7.
  • the purified proteins were assessed for preF trimer stability by analytical Size Exclusion Chromatography (SEC) after storage at 37 C for 2 ( Figure 12G) or 14 weeks ( Figure 12H) as described in Example 7.
  • SEC Size Exclusion Chromatography
  • SEQ ID NO: 35 recent A2 full length wild type sequence (Yokohama.JPN/P8356/2016)
  • SEQ ID NO: 65 recent A2 full length wild type sequence Yokohama.JPN/P8674/2016

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Abstract

La présente invention concerne des protéines F du pneumovirus humain (HMPV) stabilisées en pré-fusion, des molécules d'acide nucléique codant pour lesdites protéines HMPV F, ainsi que l'utilisation de celles-ci.
EP22834561.7A 2021-12-16 2022-12-08 Protéines de fusion hmpv pré-hybrides stabilisées Pending EP4448802A1 (fr)

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