Prefusion PIV F immunogens and their use
Inventors
Zhang, Baoshan • Stewart-Jones, Guillaume • Zhou, Tongqing • Mascola, John • Xu, Kai • Yang, YongPing • Thomas, Paul • 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-11078239-B2
Publication Date
2021-08-03
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
Embodiments of recombinant human Parainfluenza Virus (hPIV) F ectodomain trimers stabilized in their prefusion conformation are provided, comprising protomers with one or more amino acid substitutions or deletions. These recombinant F ectodomain trimers include those from hPIV1, hPIV2, hPIV3, and hPIV4. Corresponding nucleic acids encoding these trimers and methods for their production are also provided. Methods for inducing an immune response or treating hPIV infection by administering these recombinant trimers to a subject are disclosed.
The problem addressed arises from the significant human disease burden caused by hPIV infections, particularly among children worldwide, and the lack of available vaccines for hPIV1, hPIV2, hPIV3, and hPIV4. The native hPIV F protein is a metastable membrane-anchored trimer that undergoes a conformational change from the prefusion to postfusion state during viral membrane fusion, complicating stable presentation of the prefusion antigen for immune recognition. Due to the sequence diversity, prior structural stabilization methods based on related viruses like RSV and PIV5 were insufficient for hPIV F proteins. Thus, there is a need for recombinant hPIV F ectodomain trimers stabilized specifically in the prefusion conformation to elicit a superior immune response.
The invention provides strategies to stabilize the prefusion conformation of hPIV F ectodomain trimers through specific amino acid substitutions or deletions, including non-native disulfide bonds and cavity filling substitutions. These modifications enhance solubility, stability, expression, and immunogenicity of the recombinant ectodomains. Furthermore, the recombinant trimers may be linked to trimerization domains such as GCN4, fused to transmembrane domains for membrane anchoring, or presented on protein nanoparticles like ferritin. Such stabilized recombinant hPIV F trimers induce superior neutralizing immune responses in animal models compared to unstabilized or postfusion conformations.
Claims Coverage
The independent claims focus on immunogens based on recombinant hPIV F ectodomain trimers stabilized in a prefusion conformation, nucleic acid molecules encoding such trimers, vectors including these nucleic acids, and methods of inducing an immune response or treating hPIV infection utilizing them. The main inventive features include specific amino acid substitutions for stabilization, molecular constructs linking trimers to trimerization or membrane domains, and applications in vaccines and immunization protocols.
Stabilization of hPIV3 F ectodomain trimer in prefusion conformation
Immunogens comprising recombinant hPIV3 F ectodomain trimers with protomers containing amino acid substitutions including 463V and/or 474Y cavity filling substitutions and/or one or more sets of cysteine substitutions (162C-168C, 170C-242C, 213C-230C, 216C-221C, 85C-222C, 172C-238C) that form disulfide bonds to stabilize the prefusion conformation, with numbering according to SEQ ID NO: 9.
Combination of cavity filling and multiple disulfide bonds for enhanced stabilization
The combination of 463V and/or 474Y cavity filling substitutions together with two or more disulfide bond-forming cysteine substitution pairs among the 162C-168C, 170C-242C, 213C-230C, 216C-221C, and 85C-222C sets to further stabilize the hPIV3 F ectodomain trimer in the prefusion conformation.
Mutation to inhibit protease cleavage site
Inclusion of mutations such as K108E that inhibit cleavage of the F2/F1 protease cleavage site in the hPIV3 F ectodomain protomers to aid stability and retain prefusion conformation.
Molecular constructs linking protomers to trimerization domains or transmembrane domains
Linkage of a C-terminal residue of the protomers in the recombinant hPIV F ectodomain trimer to a trimerization domain (e.g., a GCN4 trimerization domain) by a direct or peptide linker connection, or alternatively linking the protomers to a transmembrane domain for membrane anchoring.
Formation of immunogenic compositions comprising one or more or all four of hPIV1, hPIV2, hPIV3, and hPIV4 F ectodomain trimers stabilized in prefusion conformation
Immunogenic compositions containing recombinant trimers from multiple hPIV subtypes stabilized in the prefusion conformation for broad protective vaccination.
Nucleic acids and vectors encoding the stabilized protomers
Isolated nucleic acid molecules encoding the protomers with the stabilizing mutations, optionally linked to expression control sequences or included in viral vectors for expression and immunization.
Methods of inducing hPIV immune response and treatment by administration of these immunogens
Methods of treating, inhibiting, or preventing hPIV infection in a subject by administering an effective amount of the stabilized recombinant hPIV F ectodomain trimers, their encoding nucleic acids or vectors, or immunogenic compositions comprising them.
The claims cover stabilized recombinant hPIV F ectodomain trimers for all hPIV subtypes with specific amino acid substitutions that maintain the prefusion conformation, molecular fusions to trimerization or transmembrane domains, nucleic acids and vectors encoding these structures, pharmaceutical compositions including one or more subtype trimers, and methods of inducing immune responses or treating hPIV infections using such compositions.
Stated Advantages
The recombinant prefusion-stabilized hPIV1, hPIV2, hPIV3, and hPIV4 F ectodomain trimers produce a superior immune response in animal models compared to unstabilized or postfusion conformations.
The stabilizing mutations improve solubility, stability, expression, and physical stability of the recombinant F ectodomain trimers.
The prefusion stabilizing modifications enable retention of the prefusion conformation for extended periods and under stress conditions, which increases the effectiveness of the immunogen.
The recombinant ectodomain trimers can be produced as soluble proteins or membrane anchored proteins, increasing versatility of vaccine design.
The recombinant hPIV F ectodomain trimers can be effectively presented on protein nanoparticles or virus-like particles to enhance immune presentation.
The immunogenic compositions including combinations of hPIV1-4 prefusion-stabilized F proteins induce broad and potent neutralizing immune responses against multiple hPIV types.
Documented Applications
Use as immunogens for inducing an immune response to parainfluenza virus in subjects, including humans.
Vaccination compositions for preventing or treating hPIV1, hPIV2, hPIV3, and/or hPIV4 respiratory infections.
Protein subunit vaccines comprising prefusion-stabilized recombinant hPIV F ectodomain trimers.
Prime-boost vaccination regimens using DNA, viral vector, protein, or nanoparticle-based vaccines encoding or comprising stabilized hPIV F ectodomain trimers.
Protein nanoparticle presentations, such as ferritin nanoparticles linked to recombinant hPIV F trimers for enhanced immunogenicity.
Virus-like particles displaying membrane-anchored prefusion stabilized hPIV F proteins for immunization.
Immunization of pregnant subjects to provide passive immunity to newborns to inhibit early postnatal hPIV infection.
Treatment or inhibition of hPIV infection in subjects at risk or infected with hPIV using compositions comprising the recombinant prefusion-stabilized ectodomains or encoding nucleic acids or viral vectors.
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