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-10420834-B2
Publication Date
2019-09-24
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 that causes repeated infections including severe lower respiratory tract disease, especially among vulnerable populations like the elderly or those with compromised cardiac, pulmonary, or immune systems. The MPV F protein is essential for viral entry and exists in a metastable prefusion conformation that upon triggering undergoes a transition to a postfusion conformation mediating membrane fusion. Although MPV F protein is the target of neutralizing antibodies and vaccine development, prior MPV F-based vaccines were unsuccessful due to structural inaccuracies in the known prefusion conformation.
A detailed re-refinement of the MPV F protein structure corrected errors in the previously published structural model, particularly at the membrane-distal apex which is a key immunodominant site. The corrected and refined structure enables the design and generation of recombinant MPV F proteins that are stabilized in the prefusion conformation and retain immunogenicity. These recombinant MPV F proteins include both F2 polypeptides and F1 ectodomains capable of trimerization and specifically binding to prefusion-specific antibodies (e.g., MPE8 monoclonal antibody).
Stabilization is achieved by one or more amino acid substitutions, including non-natural disulfide bonds (e.g., A113C and A339C), cavity-filling substitutions (e.g., T160F and I177L), mutations to reduce glycosylation at sites N57 and N172, proline substitutions to prevent formation of postfusion helix structures, and combinations thereof. Such modified MPV F proteins can be produced as soluble or membrane-anchored trimers with enhanced stability and immunogenicity, further enabling their use as immunogens to generate protective immune responses against MPV infection.
Claims Coverage
The claims disclose 40 main inventive features related to a recombinant MPV F protein immunogen stabilized in a prefusion conformation by specific amino acid substitutions. The features include structural stabilizations, binding specificities, sequence modifications, and linkage to trimerization domains or nanoparticles.
Recombinant MPV F protein stabilized by non-native disulfide bonds
The immunogen comprises a recombinant MPV F protein including one or more amino acid substitutions introducing non-native intra- or inter-protomer disulfide bonds that stabilize the MPV F protein in the prefusion conformation, involving cysteine substitutions at defined residue ranges corresponding to a reference MPV F protein sequence (SEQ ID NO: 7).
Specific binding to prefusion-specific antibodies
The stabilized recombinant MPV F protein or its immunogenic fragment specifically binds to MPV F prefusion conformation-specific antibodies, particularly to the MPE8 monoclonal antibody.
Key cysteine substitutions to stabilize disulfide bonds
Specific cysteine substitutions forming non-natural disulfide bonds include pairs such as A113C and A339C, or inter-protomer disulfides at positions like A120C with Q426C or Q428C, which stabilize the prefusion conformation.
Cavity-filling amino acid substitutions
Substitutions that fill structural cavities in the MPV F protein, notably at positions such as T160 and I177 (e.g., T160F and I177L), reduce cavity volume and stabilize the prefusion conformation.
Proline substitutions to prevent α7 helix formation
Inclusion of proline substitutions at residues 183-189 (e.g., D183P, A185P, D186P) to inhibit formation of the α7 helix associated with the postfusion conformation, thereby stabilizing the prefusion conformation.
Glycosylation modification to improve immunogenicity
Removal of native N-linked glycan sequons at N57 and/or N172 through substitutions such as N57Q and/or N172Q, or T59A and/or T174A, to reduce glycosylation and enhance immunogenicity.
Single chain MPV F proteins with linker mutations
Recombinant MPV F proteins constructed as single polypeptide chains with mutations removing protease cleavage sites between F2 and F1, linked directly or via a peptide linker (e.g., positions 95-110 with specific linker sequences), that retain the prefusion conformation.
Linkage to trimerization domains and cysteine zipper motif
The recombinant MPV F protein can be linked at the C-terminus of the F1 ectodomain to trimerization domains such as foldon, and/or to cysteine zipper motifs or oligomerization peptides to promote stable trimer formation and nanoparticle assembly.
Inclusion in protein nanoparticles or virus-like particles
The recombinant MPV F protein or fragment can be presented on protein nanoparticles (e.g., ferritin or lumazine synthase subunits) or virus-like particles to enhance immunogenicity.
Expression and composition features
The recombinant MPV F protein can have enhanced furin cleavage sites, be produced in specific cells (like GnTI−/− with deglycosylation), be soluble and stable under various conditions, and be included in immunogenic compositions with pharmaceutically acceptable carriers and optionally adjuvants for vaccination applications.
The claims define a broad array of engineered recombinant MPV F protein immunogens stabilized in their prefusion conformation by specific amino acid substitutions such as non-native disulfide bonds, cavity-filling substitutions, proline substitutions, glycosylation site removals, and single-chain designs. These immunogens are capable of specific antibody binding, improved stability, and can be included in various multimeric or nanoparticle forms suitable for vaccine compositions.
Stated Advantages
Corrected structural modeling enables production of MPV F proteins stabilized in the prefusion conformation, improving immunogen design.
The disclosed stabilized MPV F proteins elicit neutralizing immune responses against multiple MPV subgroups, including strains that cause significant disease.
Stabilizing amino acid substitutions (disulfide bonds, cavity fillers, proline mutations) enhance prefusion conformation stability and antigenicity.
Removal of specific glycosylation sites improves immunogenicity by exposing key epitopes.
Recombinant MPV F proteins with the disclosed modifications exhibit increased thermal stability and maintain antibody binding after stress conditions.
Protein nanoparticle formation with stabilized MPV F proteins can enhance presentation and immunogenicity.
Documented Applications
Use of recombinant MPV F proteins stabilized in the prefusion conformation to generate an immune response to MPV in a subject.
Therapeutic and prophylactic administration of recombinant MPV F proteins, nucleic acid molecules encoding them, or viral vectors including them to treat, inhibit, or prevent MPV infection in subjects at risk or infected.
Use of recombinant MPV F proteins or fragments for immunodiagnostic methods to detect MPV-specific antibodies in biological samples from infected subjects.
Inclusion of recombinant MPV F proteins in immunogenic compositions and vaccine formulations for managing MPV infections.
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