Prefusion RSV F proteins and their use
Inventors
Kwong, Peter • Graham, Barney • McLellan, Jason • Chen, Man • Joyce, Michael Gordon • Zhang, Baoshan • Zhou, Tongqing
Assignees
US Department of Health and Human Services
Publication Number
US-9738689-B2
Publication Date
2017-08-22
Expiration Date
2034-03-12
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Abstract
Disclosed are immunogens including a recombinant RSV F protein stabilized in a prefusion conformation. Also disclosed are nucleic acids encoding the immunogens and methods of producing the immunogens. Methods for generating an immune response in a subject are also disclosed. In some embodiments, the method is a method for treating or preventing a RSV infection in a subject by administering a therapeutically effective amount of the immunogen to the subject.
Core Innovation
The invention discloses immunogens including recombinant respiratory syncytial virus (RSV) F proteins that are stabilized in a prefusion conformation. These immunogens, nucleic acids encoding them, and methods of their production are described. The use of these immunogens to generate an immune response in a subject, including methods for treating or preventing RSV infection via administration of a therapeutically effective amount of the immunogen, is also disclosed.
The problem addressed arises from the RSV F protein's metastability in its prefusion conformation, which has precluded the preparation of a homogeneous soluble prefusion RSV F protein. This instability prevented determination of the prefusion F structure and hindered identification of novel prefusion-specific antigenic sites. Existing vaccines and antibodies target postfusion antigenic sites, but prior efforts to develop RSV F protein-based vaccines have been unsuccessful due to lack of stable prefusion F protein immunogens.
Claims Coverage
The claims include one independent claim directed to an isolated immunogen with a recombinant RSV F protein stabilized by a specific intra-protomer disulfide bond, and dependent claims detailing additional protein features, modifications, and related compositions. The main inventive features focus on the stabilization of RSV F protein in the prefusion conformation and variations thereof.
RSV F protein stabilization by non-natural intra-protomer disulfide bond
An isolated immunogen comprising a recombinant RSV F protein stabilized in a prefusion conformation by a non-natural intra-protomer disulfide bond formed between cysteine residues introduced by S155C and S290C amino acid substitutions.
Use of RSV F proteins from various RSV strains with stabilization
The RSV F protein may be from human RSV subtype A or B, or bovine RSV, comprising the non-natural intra-protomer disulfide bond.
Inclusion of F2 and F1 polypeptides covering specific RSV F positions
The recombinant RSV F protein comprises a F2 polypeptide and a F1 polypeptide comprising or consisting of RSV F positions 26-109 and 137-513, respectively.
Linkage of F2 and F1 polypeptides via heterologous peptide linker
The recombinant RSV F protein comprises a F2 polypeptide and a F1 polypeptide linked through a heterologous peptide linker connecting a C-terminal residue of F2 to an N-terminal residue of F1.
Additional non-natural disulfide bond with amino acid insertions
The recombinant RSV F protein further comprises a non-natural disulfide bond between cysteine residues introduced at RSV F positions 183 and 428, including a glycine insertion between positions 182 and 183 or a cysteine insertion between positions 427 and 428.
Combination of two disulfide bonds for enhanced stabilization
The recombinant RSV F protein includes the S155C/S290C intra-protomer disulfide bond and one set of N183C/K428C or N183C/K427GC substitutions facilitating disulfide bonding.
Use of a trimerization domain linked at the C-terminus
The RSV F protein can include a C-terminal trimerization domain, such as a Foldon domain, to further stabilize and promote trimer formation in the prefusion conformation.
Linkage to nanoparticle-forming protein domains
C-terminus linkage of recombinant RSV F protein to domains such as ferritin, encapsulin, sulfur oxygenase reductase, lumazine synthase, or pyruvate dehydrogenase to form protein nanoparticles.
Incorporation in virus-like particles and protein nanoparticles
The RSV F immunogen can be presented in virus-like particles or protein nanoparticles, including ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase nanoparticles, to elicit immune responses.
Nucleic acid encoding and vectors for expression
Nucleic acid molecules encoding the immunogen, including vectors for their expression and host cells containing such vectors, supporting production of the prefusion stabilized RSV F protein.
Immunogenic compositions including adjuvants
Immunogenic compositions comprising an effective amount of the prefusion stabilized RSV F immunogen with pharmaceutically acceptable carriers and optionally adjuvants, including those promoting a Th1 biased immune response.
The independent claims cover an isolated RSV F protein immunogen stabilized in the prefusion conformation by a defined intra-protomer disulfide bond, together with various dependent claims on protein composition, linked domains, nanoparticle and virus-like particle formulations, nucleic acid encoding, host vectors, and immunogenic compositions, all directed to improved RSV vaccines and immunogenic agents.
Stated Advantages
The disclosed prefusion RSV F antigens generate RSV neutralizing immune responses many fold greater than prior RSV F protein-based immunogens.
Stabilization of RSV F in the prefusion conformation allows preparation of homogeneous soluble proteins suitable for crystallization and structural analysis.
Identification of a unique antigenic site (antigenic site Ø) at the membrane distal apex of prefusion RSV F enables the generation of potent prefusion-specific neutralizing antibodies.
The stabilized prefusion RSV F protein formats retain binding sites for potent prefusion-specific antibodies (e.g., D25, AM22) while maintaining critical antigenic sites present in both the pre- and postfusion conformations.
Formulation with trimerization domains and display on nanoparticles enhances structural stability and immunogenicity of the vaccine candidates.
Documented Applications
Use as vaccines for prophylaxis or therapy to treat or prevent respiratory syncytial virus infection in humans and animals.
Methods for generating an immune response in a subject, including induction of RSV neutralizing antibodies by administration of prefusion stabilized RSV F proteins or related immunogenic compositions.
Use in immunoassays and diagnostics for detection and quantification of RSV-specific antibodies, including prefusion-specific antibodies in sera samples.
Use as diagnostic molecules to detect and measure target antibodies in polyclonal serum responses.
Use of nucleic acids and viral vectors encoding prefusion RSV F proteins for in vivo expression to induce protective immune responses.
Display on protein nanoparticles or virus-like particles for enhanced immunogenicity and stability for vaccine formulation.
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