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-11027007-B2
Publication Date
2021-06-08
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) F proteins stabilized in a prefusion conformation, nucleic acid molecules and vectors encoding these proteins, and methods of their use and production are disclosed. The MPV F proteins and/or nucleic acid molecules can be used to generate an immune response to MPV in a subject. They can also be administered to a subject in a therapeutically effective amount to treat or prevent MPV infection.
MPV is an enveloped negative-strand RNA virus causing bronchiolitis and pneumonia, especially severe in children and the elderly, with existing treatment limited to Ribaviran. The MPV F protein is crucial for viral entry, existing initially as a monomer (F0) processed into F1 and F2 subunits that assemble into a trimeric protein adopting a metastable prefusion conformation capable of inducing membrane fusion. Prior efforts to develop MPV F-based vaccines were unsuccessful due to structural inaccuracies.
A detailed re-refinement of a previously published MPV F structure (PDB No. 4DAG) revealed the original model of the membrane-distal aspect was incorrect. This correction yielded a substantially refined structural model of the prefusion MPV F ectodomain trimer, enabling, for the first time, the design and generation of recombinant MPV F proteins stabilized in the prefusion conformation. These stabilized proteins can be used as immunogens to generate an immune response to MPV F in a subject.
Claims Coverage
The claims include one independent claim directed to a nucleic acid molecule encoding a stabilized recombinant MPV F protein and comprise multiple inventive features related to amino acid substitutions for stabilization.
Stabilization by non-native intra- or inter-protomer disulfide bonds
A nucleic acid molecule encoding a recombinant MPV F protein or immunogenic fragment thereof stabilized in a prefusion conformation by one or more amino acid substitutions that introduce non-native intra- or inter-protomer disulfide bonds, alone or with other modifications, to stabilize the MPV F protein.
Non-natural disulfide bond between A113C and A339C
The recombinant MPV F protein comprises a non-natural disulfide bond formed by cysteine substitutions at positions A113C and A339C that stabilize the prefusion conformation of the protein.
Non-natural disulfide bonds between A120C and Q426C or Q428C
The recombinant MPV F protein includes a non-natural disulfide bond between cysteine substitutions A120C and Q426C, or A120C and Q428C, stabilizing the prefusion conformation.
Cavity filling amino acid substitutions reducing volume of T160 and/or I177 cavities
The recombinant MPV F protein includes one or more cavity filling amino acid substitutions at positions that reduce the volume of a threonine 160 cavity and/or an isoleucine 177 cavity in the MPV F protein, stabilizing it in the prefusion conformation.
Specific cavity filling substitutions at multiple positions
Substitutions at positions 160, 162, and/or 157 reduce the threonine 160 cavity and substitutions at positions 177, 58, 169, 54, and/or 55 reduce the isoleucine 177 cavity, stabilizing the protein.
Types of amino acid substitutions for cavity filling
The cavity filling substitutions can be phenylalanine, leucine, tryptophan, tyrosine, histidine, or methionine.
Combination of disulfide bonds and cavity filling substitutions
The recombinant MPV F protein includes the A113C/A339C disulfide bond and T160F/I177L cavity filling substitutions that stabilize the protein in the prefusion conformation.
Proline substitutions to prevent formation of α7 helix
The protein can include a proline substitution at one of positions 183-189 to prevent or reduce α7 helix formation, thereby stabilizing the prefusion conformation.
Removal of N-linked glycosylation at N57 and/or N172
The recombinant MPV F protein is not glycosylated at N57, N172, or both, via substitutions such as N57Q, N172Q, T59A, and/or T174A to remove respective N-glycan sequons, enhancing immunogenicity.
Specific cysteine substitutions introducing intra- and inter-protomer disulfide bonds
The recombinant MPV F protein includes cysteine substitutions introducing intra-protomer disulfide bonds between defined amino acid ranges, and inter-protomer disulfide bonds between other specified amino acid ranges, stabilizing the protein in the prefusion conformation.
Combinations of multiple amino acid substitutions
Specific combinations of mutations including A113C, A339C, A120C, Q426C, Q428C, T160F, I177L, proline substitutions D183P, A185P, D186P, and glycosylation site removals N57Q, N172Q, T59A, T174A that stabilize the prefusion conformation.
Polypeptide segment boundaries of F2 and F1 ectodomain
The recombinant MPV F protein comprises an F2 polypeptide with N- and C-terminal residues between MPV F positions 8-30 and 90-102, respectively; and an F1 ectodomain with N- and C-terminal residues between positions 103-130 and 470-550.
Deletion of fusion peptide
The recombinant MPV F protein includes deletion of residues 103-123 to remove the fusion peptide from F1 ectodomain, contributing to stabilization.
Sequence identity to human MPV subgroups
The F2 polypeptide and/or F1 ectodomain are at least 80% identical to corresponding sequences from human A1, A2, B1, or B2 MPV strains.
Forming trimers stabilized in prefusion conformation
The recombinant MPV F protein forms trimers stabilized in the prefusion conformation.
Linkage to trimerization domains
A C-terminal residue of the F1 ectodomain is linked, directly or by peptide linker, to a trimerization domain, such as a foldon domain.
Linkage to transmembrane or protein nanoparticle subunits
The F1 ectodomain C-terminus can be linked to a transmembrane domain or a self-assembling protein nanoparticle subunit, such as ferritin or lumazine synthase.
Encoding by nucleic acid and expression vector
Nucleic acid molecules encoding the stabilized recombinant MPV F proteins are included, as are expression vectors (including viral vectors) operably linked to promoters for expression.
The claims cover nucleic acid sequences encoding recombinant MPV F proteins stabilized in a prefusion conformation by amino acid substitutions that form non-native disulfide bonds, cavity filling mutations, proline substitutions preventing α7 helix formation, and removal of specific glycosylation sites. Additional features include specific sequence ranges for F2 and F1 ectodomains, deletion of fusion peptide, trimer formation, linkage to trimerization or nanoparticle domains, and inclusion in expression vectors.
Stated Advantages
The disclosed recombinant MPV F proteins are stabilized in the prefusion conformation, preserving epitopes targeted by neutralizing antibodies.
The stabilized MPV F proteins exhibit increased thermal stability, maintaining specific antibody binding after heat incubation.
Such stabilized proteins can elicit potent neutralizing immune responses in animal models, including cross-neutralization of multiple MPV subgroups.
They can boost immunity following natural MPV infection, enhancing neutralizing antibody titers.
Documented Applications
Use of recombinant MPV F proteins stabilized in prefusion conformation as immunogens to generate an immune response to MPV in a subject.
Administration of therapeutically effective amounts of the recombinant MPV F proteins or nucleic acid molecules to treat or prevent MPV infection in subjects at risk or infected.
Use in vaccine compositions for immunization regimens including prime-boost protocols with protein or nucleic acid vaccines.
Design and use of protein nanoparticles and virus-like particles incorporating recombinant MPV F proteins for enhanced immunogenicity.
Use of recombinant MPV F proteins as diagnostic reagents for detecting MPV binding antibodies in biological samples.
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