Recombinant metapneumovirus f proteins and their use
Inventors
Kwong, Peter • Joyce, Michael Gordon • Zhang, Baoshan • Yang, YongPing • Collins, Peter • Buchholz, Ursula • Corti, Davide • Lanzavecchia, Antonio • Stewart-Jones, Guillaume
Assignees
Institute for Research in Biomedicine IRB • US Department of Health and Human Services
Publication Number
US-12233124-B2
Publication Date
2025-02-25
Expiration Date
2035-12-24
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Abstract
Metapneumovirus (MPV) F proteins stabilized in a prefusion conformation, nucleic acid molecules and vectors encoding these proteins, and methods of their use and production are disclosed. In several embodiments, the MPV F proteins and/or nucleic acid molecules can be used to generate an immune response to MPV in a subject. In additional embodiments, the therapeutically effective amount of the MPV F ectodomain trimers and/or nucleic acid molecules can be administered to a subject in a method of treating or preventing MPV infection.
Core Innovation
Metapneumovirus (MPV) is an enveloped non-segmented negative-strand RNA virus causing bronchiolitis and pneumonia, especially in children and the elderly, with repeated infections including severe lower respiratory tract disease. The MPV F protein, essential for viral entry by mediating fusion of viral and cellular membranes, is initially expressed as a polypeptide precursor F0 that trimerizes and is proteolytically cleaved into F1 and F2 polypeptides, assembling into a trimeric F protein that adopts a metastable prefusion conformation. Prior vaccine development efforts targeting MPV F protein have been unsuccessful due to incorrect structural models and difficulties in stabilizing the immunodominant prefusion form.
The invention provides a corrected structural model of the prefusion form of the MPV F ectodomain trimer through re-refinement, enabling the design and generation of recombinant MPV F proteins stabilized in the prefusion conformation. These proteins include an F2 polypeptide and an F1 ectodomain capable of trimerizing and specifically binding to monoclonal antibodies such as MPE8. Stabilizing modifications include non-natural disulfide bonds between cysteine residues introduced by amino acid substitutions (e.g., A113C and A339C), cavity-filling substitutions (e.g., T160F and I177L), removal of glycosylation sites through substitutions (e.g., N57Q, N172Q), linking to trimerization domains (e.g., foldon), forming single-chain constructs linking F2 and F1 by peptide linkers, and incorporation into protein nanoparticles such as ferritin or lumazine synthase.
The recombinant MPV F proteins, encoding nucleic acids, expression vectors, and compositions containing these, can be used to generate an immune response against MPV, and to prevent or treat MPV infections. Methods include administration of therapeutically effective amounts of the recombinant proteins, protein nanoparticles, nucleic acids, or viral vectors to subjects at risk or infected with MPV. The disclosed proteins are soluble, stable, maintain prefusion-specific epitopes, and induce neutralizing antibodies effective against multiple MPV strains.
Claims Coverage
The claims include a method for inducing an immune response to MPV F protein by administering nucleic acid molecules encoding recombinant MPV F proteins stabilized in the prefusion conformation. Five main inventive features are addressed regarding protein stabilization and administration.
Stabilization of MPV F protein by non-native disulfide bonds
The recombinant MPV F protein is stabilized in the prefusion conformation by one or more non-native intra- or inter-protomer disulfide bonds introduced by amino acid substitutions such as A113C and A339C, or A120C combined with Q426C or Q428C cysteine substitutions, at positions corresponding to a reference MPV F sequence (SEQ ID NO: 7).
Cavity filling amino acid substitutions to stabilize prefusion conformation
The recombinant MPV F protein includes one or more cavity filling amino acid substitutions that reduce the volume of the threonine 160 and isoleucine 177 cavities, such as T160F, I177L, and substitutions at positions 162, 157, 58, 169, 54, and 55, which stabilize the protein in the prefusion conformation (positions relative to SEQ ID NO: 7).
Incorporation of proline substitutions to prevent α7 helix formation
The recombinant MPV F protein comprises proline substitutions at positions 183-189 (e.g., A185P) to reduce or prevent formation of the α7 helix, thereby stabilizing the prefusion conformation.
Removal of native glycosylation sites to improve immunogenicity
The recombinant MPV F protein lacks glycosylation at positions N57, N172, or both, accomplished by substitutions such as N57Q, N172Q, T59A, T174A, or combinations thereof, removing N-linked glycan sequons present in native MPV F.
Structural and sequence modifications and fusion constructs to enhance stability and immunogenicity
The recombinant MPV F protein includes specific combinations of cysteine substitutions, cavity-filling mutations, proline substitutions, and glycosylation site removals in defined combinations (detailed in claim 15), constructs with defined N- and C-terminal boundaries of F2 and F1 domains, deletion of fusion peptide (residues 103-123), optionally linked to trimerization domains (e.g., foldon), self-assembling protein nanoparticle subunits (e.g., ferritin), or transmembrane domains to stabilize the trimer and enhance immune response.
The claims focus on nucleic acid-mediated administration of recombinant MPV F proteins stabilized in the prefusion conformation via targeted amino acid substitutions including non-native disulfide bonds, cavity-filling substitutions, proline substitutions, and removal of select N-linked glycosylations, optionally in fusion constructs enhancing trimer formation and immunogenicity, to induce an immune response against MPV F in subjects.
Stated Advantages
Stabilization of MPV F proteins in the prefusion conformation allows for generation of immunogens that elicit neutralizing immune responses effective against multiple MPV strains.
The recombinant MPV F proteins are soluble and thermally stable, maintaining binding to prefusion-specific antibodies after incubation at elevated temperatures and multiple freeze-thaw cycles.
The corrected structural model enables design of mutations that improve expression, stability, and antigenicity, overcoming prior difficulties in MPV F-based vaccine development.
Immunization with stabilized MPV F proteins boosts neutralizing antibody titers, including in subjects previously infected with MPV.
Documented Applications
Treatment and prevention of MPV infection in subjects by administering therapeutically effective amounts of recombinant MPV F proteins stabilized in prefusion conformation, nucleic acids encoding same, protein nanoparticles, viral vectors, or virus-like particles.
Use as immunogens to induce neutralizing immune responses against MPV in subjects, including prime-boost vaccination protocols combining nucleic acid and protein immunogens.
Diagnostic assays for detecting or isolating antibodies against MPV F in biological samples by using recombinant MPV F proteins or fragments as antigens to detect binding antibodies.
Presentation of recombinant MPV F proteins on protein nanoparticles (such as ferritin or lumazine synthase nanoparticles) or incorporation into virus-like particles to enhance immune responses.
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