Prefusion PIV F immunogens and their use
Inventors
Zhang, Baoshan • Stewart-Jones, Guillaume • Mascola, John • Xu, Kai • Chuang, Gwo-Yu • Ou, Li • Kwong, Peter • Tsybovsky, Yaroslav • Kong, Wing-pui • Druz, Aliaksandr • Corti, Davide • Lanzavecchia, Antonio
Assignees
Institute for Research in Biomedicine IRB • US Department of Health and Human Services
Publication Number
US-11845778-B2
Publication Date
2023-12-19
Expiration Date
2037-10-25
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Abstract
Embodiments of a recombinant human Parainfluenza Virus (hPIV) F ectodomain trimer stabilized in a prefusion conformation are provided. Also disclosed are nucleic acids encoding the hPIV F ectodomain trimer and methods of producing the hPIV F ectodomain trimer. Methods for inducing an immune response in a subject are also disclosed. In some embodiments, the method can be a method for treating or inhibiting a hPIV infection in a subject by administering a effective amount of the recombinant hPIV F ectodomain trimer to the subject.
Core Innovation
Human parainfluenza viruses (hPIVs) are significant causes of childhood illness and hospitalization worldwide, with four common types: hPIV1, hPIV2, hPIV3, and hPIV4. The hPIV envelope protein, F, forms a mature trimer adopting a metastable "prefusion" conformation that undergoes conformational changes to fuse viral and target-cell membranes. Despite the disease burden caused by hPIVs, vaccines are not available.
The invention relates to recombinant hPIV1, hPIV2, hPIV3, and hPIV4 F ectodomain trimers with protomers containing amino acid substitutions or deletions that stabilize the ectodomain trimer in its prefusion conformation. These prefusion-stabilized F ectodomain trimers elicit a superior immune response in animal models compared to non-stabilized counterparts. Various specific amino acid modifications are described for each hPIV type to achieve stabilization, including disulfide bonds and cavity-filling substitutions.
Protomers of the recombinant F ectodomain trimers can be linked to trimerization domains (such as GCN4) to promote trimerization or to transmembrane domains for membrane anchoring. The compositions can include such recombinant F ectodomain trimers individually or in combination, with or without adjuvants, and may be presented on protein nanoparticles or virus-like particles. Additionally, nucleic acids encoding these trimers and vectors for their expression are disclosed. Methods for inducing immune responses, as well as treating or inhibiting hPIV infection by administering an effective amount of these compositions, are also provided.
Claims Coverage
The patent includes one independent claim centered on a recombinant human parainfluenza virus 1 F ectodomain trimer with specific prefusion stabilizing mutations.
Prefusion stabilization of hPIV1 F ectodomain trimer
A recombinant hPIV1 F ectodomain trimer comprising protomers with A466I and S473I cavity-filling amino acid substitutions that stabilize the trimer in a prefusion conformation, with amino acid numbering according to SEQ ID NO: 2.
Mutation inhibiting F2/F1 protease cleavage
Introduction of mutations to inhibit protease cleavage at the F2/F1 site, including deletion of residues 113 and 114 with residues 112 and 115 linked by a GS peptide linker.
Non-native disulfide bonds for prefusion stabilization
Additional amino acid substitutions forming non-native disulfide bonds between specific cysteine pairs (175C-241C, 173C-245C, 206C-233C, 88C-225C, 219C-224C, or 165C-171C) for further stabilizing the prefusion conformation.
C-terminal residue positioning
The C-terminal residue of protomers is positioned within hPIV1 F residues 473 to 497.
Protomers sequence composition
Protomers consist essentially of residues 22-112 and 115-479 linked by a heterologous peptide linker, or an amino acid sequence at least 90% identical to residues 1-458 of SEQ ID NO: 4, including the one or more amino acid substitutions.
Linkage to trimerization domain
C-terminal residue linkage of the protomers to a trimerization domain, optionally a GCN4 trimerization domain, directly or via a peptide linker.
Solubility and alternate linking
The recombinant hPIV1 F ectodomain trimer is soluble, and the C-terminal residue can alternatively be linked to a transmembrane domain or protein nanoparticle subunit.
Compositions and methods of use
Immunogenic compositions, protein nanoparticles, virus-like particles, nucleic acid molecules encoding the immunogen; and methods of inducing an immune response by administering the immunogen or nucleic acid molecule.
The claims cover recombinant hPIV1 F ectodomain trimers stabilized in the prefusion conformation by defined amino acid substitutions, including cavity filling and non-native disulfide bonds, methods of reducing protease cleavage, linkages to trimerization domains or other anchoring moieties, compositions comprising these trimers, and methods for eliciting immune responses and treating hPIV1 infection.
Stated Advantages
Prefusion-stabilized hPIV F ectodomain trimers produce a superior immune response in animal models compared to corresponding non-stabilized trimers.
Recombinant F ectodomain trimers exhibit enhanced stability of the prefusion conformation, including retention of conformation upon storage and under physical stress.
Protein nanoparticles displaying recombinant F trimers self-assemble and facilitate targeted immune responses.
Compositions comprising prefusion stabilized trimers induce potent neutralizing antibody titers against multiple hPIV types in animal models, including mice and non-human primates.
Documented Applications
Inducing an immune response in subjects, including humans, to hPIV1, hPIV2, hPIV3, and/or hPIV4 F proteins.
Vaccination for the prophylaxis and treatment of hPIV infections by administering recombinant prefusion-stabilized hPIV F ectodomain trimers, nucleic acids, vectors, protein nanoparticles, or virus-like particles.
Use in prime-boost vaccination regimens, including DNA prime and protein boost protocols.
Formulation as subunit vaccines, membrane-anchored vaccines, or nanoparticle-based vaccines suitable for administration to newborns, infants, pregnant women, or at-risk populations.
Production of recombinant hPIV F trimers in cell culture for use as immunogens.
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