Prefusion coronavirus spike proteins and their use
Inventors
Graham, Barney • McLellan, Jason • Ward, Andrew • Kirchdoerfer, Robert • Cottrell, Christopher • Joyce, Michael Gordon • Kanekiyo, Masaru • Wang, Nianshuang • Pallesen, Jesper • Yassine, Hadi • Turner, Hannah • Corbett, Kizzmekia
Assignees
Dartmouth College • Scripps Research Institute • US Department of Health and Human Services
Publication Number
US-10960070-B2
Publication Date
2021-03-30
Expiration Date
2037-10-25
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Abstract
Coronavirus S ectodomain trimers 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 coronavirus S ectodomain trimers and/or nucleic acid molecules can be used to generate an immune response to coronavirus in a subject. In additional embodiments, the therapeutically effective amount of the coronavirus S ectodomain trimers and/or nucleic acid molecules can be administered to a subject in a method of treating or preventing coronavirus infection.
Core Innovation
The invention relates to recombinant coronavirus spike (S) proteins, specifically alphacoronavirus and betacoronavirus S ectodomain trimers that are stabilized in a prefusion conformation by introducing one or more proline substitutions at or near the junction between the heptad repeat 1 (HR1) and the central helix domains. These modifications prevent the conformational change of the S protein from its prefusion to postfusion conformation, thereby enhancing stability and immunogenic properties of the S protein trimers.
The problem addressed is the need for an effective vaccine and therapeutic agents against highly pathogenic coronaviruses like MERS-CoV and SARS-CoV, which have caused outbreaks with high case-fatality rates. Prior coronavirus vaccine approaches had limitations, and coronavirus S proteins tend to undergo conformational changes reducing stability and immunogenicity. The absence of prophylactic or therapeutic measures and the high pathogenicity and transmissibility of such coronaviruses underline the urgent need for immunogens that induce effective immune responses.
The disclosed invention provides immunogens comprising recombinant coronavirus S ectodomain trimers with stabilizing mutations, such as double proline substitutions, that retain the prefusion conformation and produce superior immune responses in animal models compared to non-stabilized forms. The invention also covers nucleic acid molecules and vectors encoding these proteins, methods of producing them, and methods of using these immunogens to induce immune responses, treat, inhibit, or prevent coronavirus infections in subjects.
Claims Coverage
The patent includes 23 claims with multiple inventive features focusing on recombinant coronavirus S ectodomain trimers stabilized in a prefusion conformation, nucleic acid molecules encoding them, methods of production, and methods of inducing immune responses using these immunogens.
Prefusion stabilization of coronavirus S ectodomain trimers by proline substitutions
An immunogen comprising a recombinant coronavirus S ectodomain trimer with protomers containing one or two proline substitutions at the junction between heptad repeat 1 (HR1) and the central helix which stabilize the trimer in a prefusion conformation.
Use of consecutive proline substitutions for stabilization
The recombinant coronavirus S ectodomain trimer comprises two consecutive proline substitutions at the junction between the HR1 and the central helix.
Applicability to multiple coronaviruses
The immunogen is applicable to coronaviruses including MERS-CoV, SARS-CoV, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV, or SDCV, and specifically to betacoronaviruses.
Additional amino acid modifications for stabilization and cleavage site mutations
Protomers may contain additional amino acid substitutions that stabilize the prefusion conformation, and mutations at S1/S2 and/or S2' protease cleavage sites to inhibit cleavage.
Structural and solubility features
The recombinant coronavirus S ectodomain trimer is soluble and protomers may be linked at their C-terminus to a transmembrane domain or a protein nanoparticle subunit, such as ferritin, by peptide linkers or direct linkage.
Incorporation into nanoparticles and virus-like particles
The immunogen can be incorporated into protein nanoparticles (e.g., ferritin nanoparticles) or virus-like particles.
Nucleic acid molecules and vectors
Isolated nucleic acid molecules encoding a protomer of the stabilized recombinant coronavirus S ectodomain trimer, operably linked to promoters, including RNA molecules, and vectors including viral vectors for expression.
Methods for production and immune response induction
Methods of producing the stabilized recombinant coronavirus S ectodomain trimer by expressing nucleic acid molecules or vectors in host cells and purifying the trimer; and methods of generating immune responses or treating coronavirus infections by administering an effective amount of the immunogen.
The claims cover recombinant coronavirus S ectodomain trimers stabilized in the prefusion conformation by proline mutations, their encoding nucleic acids and vectors, methods for their production, and their use in eliciting protective immune responses and treating or preventing coronavirus infections, including configurations linked to transmembrane domains, nanoparticles, and incorporation into virus-like particles.
Stated Advantages
Prefusion-stabilized coronavirus S ectodomain trimers exhibit higher expression levels compared to wild-type S proteins.
The double proline substitutions provide increased thermal stability to the S ectodomain trimers.
Prefusion stabilization results in homogeneous trimeric structures maintaining the prefusion conformation.
Immunogens based on prefusion-stabilized trimers produce superior immune responses, including higher neutralizing antibody titers, even at lower doses compared to non-stabilized S proteins.
Vaccination with prefusion stabilized S proteins protects against lethal coronavirus challenge in animal models.
Documented Applications
Use of recombinant coronavirus S ectodomain trimers stabilized in the prefusion conformation as immunogens to generate an immune response in a subject.
Use of immunogenic compositions comprising the stabilized S ectodomain trimers for administration to subjects to treat, inhibit, or prevent coronavirus infections.
Production of recombinant coronavirus S ectodomain trimers by expressing nucleic acids or vectors in host cells and purifying the proteins.
Incorporation of recombinant coronavirus S ectodomain trimers into protein nanoparticles and virus-like particles for vaccine development.
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