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-11786591-B2
Publication Date
2023-10-17
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 which causes bronchiolitis and pneumonia, especially severe lower respiratory tract disease in children and the elderly. Current treatments are limited and efforts to develop MPV F protein-based vaccines have been unsuccessful due to structural misunderstandings of the MPV F protein, which plays a crucial role in viral entry and is a target of neutralizing antibodies.
This invention provides recombinant MPV F proteins stabilized in a prefusion conformation, achieved by correcting the previously published structural model of the MPV F protein through re-refinement of its structure. The corrected model reveals structural features allowing the design of MPV F proteins that remain in the prefusion state, enhancing their use as immunogens to generate an immune response against MPV. The recombinant proteins include F2 polypeptide and F1 ectodomain components that trimerize and can specifically bind to prefusion-specific antibodies such as MPE8 monoclonal antibody.
Stabilizing substitutions include non-natural disulfide bonds (e.g., A113C and A339C), cavity-filling amino acid substitutions (e.g., T160F and I177L), and modifications to remove native N-linked glycosylation sites at N57 and N172 to enhance immunogenicity. The proteins can be linked to trimerization domains or assembled into protein nanoparticles. Methods of generating immune responses, preventing or treating MPV infection, and detecting MPV binding antibodies using these stabilized proteins are also disclosed.
Claims Coverage
The patent claims cover methods of generating immune responses to MPV by administering recombinant MPV F proteins stabilized in the prefusion conformation, highlighting multiple inventive features related to structural stabilization and modifications.
Stabilization by non-native disulfide bonds
The recombinant MPV F protein includes one or more non-native intra- or inter-protomer disulfide bonds introduced by amino acid substitutions, such as A113C/A339C or A120C/Q426C (or Q428C), that stabilize the protein in the prefusion conformation.
Cavity filling amino acid substitutions to stabilize prefusion conformation
The protein includes cavity-filling substitutions at one or more of threonine 160 and isoleucine 177 cavities, such as T160F and I177L, involving substitutions of amino acids with large side chains (F, L, W, Y, H, or M).
Combination of non-native disulfide bonds and cavity-filling mutations
A combination of A113C/A339C non-native disulfide bonds and T160F/I177L cavity-filling substitutions further stabilize the prefusion conformation, possibly along with additional amino acid substitutions.
Proline substitutions to prevent α7 helix formation
Introduction of proline substitutions at positions 183-189 (e.g., A185P) reduces or prevents formation of the α7 helix, thereby stabilizing the MPV F protein prefusion conformation.
Removal of N-linked glycosylation to enhance immunogenicity
Amino acid substitutions (e.g., N57Q, N172Q, T59A, T174A) remove native N-linked glycosylation sites at positions N57 and/or N172 to improve immunogenicity.
Specific cysteine substitutions for intra- and inter-protomer disulfide bonds
Particular pairs of cysteine substitutions targeted to residues in α-helices and β-strands within the protein stabilize intra-protomer or inter-protomer interactions in the prefusion conformation.
Deletions and sequence boundaries defining F2 and F1 ectodomain
The recombinant protein includes specified residue boundaries for F2 polypeptide (positions 8-30 to 90-102) and F1 ectodomain (positions 103-130 to 470-550), with optional fusion peptide deletion (residues 103-123) to enhance stability.
Single chain MPV F proteins and linker sequences
Single polypeptide chain MPV F proteins created by removing the protease cleavage site between F2 and F1, joined directly or via peptide linkers (at specific MPV F positions), preserving the prefusion conformation.
Trimerization and nanoparticle assembly
Linkage of F1 ectodomain C-terminus to trimerization domains such as foldon sequences, transmembrane domains, or self-assembling protein nanoparticle subunits (e.g., ferritin, lumazine synthase) to enhance trimer formation and immunogenicity.
Prime-boost immunization regimens
Methods include prime-boost administration of the recombinant MPV F proteins or immunogenic fragments thereof to induce effective immune responses targeting MPV.
The claims comprehensively cover recombinant MPV F proteins stabilized in the prefusion conformation by structural modifications including disulfide bonds, cavity-filling mutations, proline substitutions, removal of glycosylation sites, deletion of fusion peptide, single-chain construction, trimerization, nanoparticle incorporation, and immunization methods using these proteins to generate protective immune responses against MPV.
Stated Advantages
Stabilized MPV F proteins in the prefusion conformation maintain epitopes that are targets for neutralizing antibodies, improving immunogenicity and vaccine efficacy.
Non-natural disulfide bonds and cavity-filling mutations increase thermal stability and maintain binding to prefusion-specific antibodies after heat stress.
Recombinant proteins can induce neutralization activity against multiple MPV subgroups (A and B), including boosting immunity after natural infection.
Deletion of glycosylation sites at N57 and N172 can increase immunogenicity by making key epitopes more accessible.
Assembly into trimers and nanoparticles enhances structural stability and antigen presentation, potentially improving immune responses.
Documented Applications
Use of stabilized recombinant MPV F proteins, nucleic acid molecules or viral vectors encoding these proteins, to generate an immune response to MPV in subjects.
Methods of treating, inhibiting, or preventing MPV infection in a subject by administering therapeutically effective amounts of recombinant MPV F ectodomain trimers, nucleic acid molecules, or protein nanoparticles.
Use of stabilized MPV F proteins in diagnostic methods to detect or isolate MPV binding antibodies from subjects infected with MPV.
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