Prefusion RSV F proteins and their use
Inventors
Kwong, Peter D. • Graham, Barney S. • McLellan, Jason S. • Boyington, Jeffrey • Chen, Lei • Chen, Man • Chuang, Gwo-Yu • Georgiev, Ivelin Stefanov • Gorman, Jason • Joyce, Michael Gordon • Kanekiyo, Masaru • Ofek, Gilad • Pancera, Marie • Sastry, Mallika • Soto, Cinque • Srivatsan, Sanjay • Stewart-Jones, Guillaume • Yang, YongPing • Zhang, Baoshan • Zhou, Tongqing
Assignees
US Department of Health and Human Services
Publication Number
US-11981707-B2
Publication Date
2024-05-14
Expiration Date
2034-03-13
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Abstract
Disclosed are Respiratory Syncytial Virus (RSV) antigens including a recombinant RSV F protein stabilized in a prefusion conformation. Also disclosed are nucleic acids encoding the antigens and methods of producing the antigens. 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 antigen to the subject.
Core Innovation
The invention provides recombinant Respiratory Syncytial Virus (RSV) F proteins stabilized in a prefusion conformation, termed PreF antigens, which exhibit enhanced immunogenicity and induce RSV neutralizing immune responses many fold greater than those achieved with prior RSV F protein-based immunogens. The three-dimensional structure of the RSV F protein in its prefusion conformation was elucidated, revealing a unique antigenic site Ø at its membrane distal apex that is specifically recognized by potent neutralizing antibodies such as D25, AM22, and 5C4. Using this structural information, stabilized forms of prefusion RSV F protein antigens were engineered, including modifications such as amino acid substitutions introducing non-natural disulfide bonds and cavity-filling substitutions, to lock the protein in the prefusion state, maintaining antigenic site Ø and the capacity for specific binding to prefusion-specific antibodies.
The problem being solved addresses the instability and metastability of the native RSV F protein's prefusion conformation, which has hindered the development of effective RSV vaccines. Prior RSV F protein-based vaccine efforts were unsuccessful because the unstable prefusion conformation readily adopts the postfusion state upon expression and purification, which lacks highly protective neutralizing epitopes. By stabilizing RSV F in the prefusion conformation that presents the antigenic site Ø, the invention overcomes these limitations, enabling the production of highly immunogenic antigens that elicit potent neutralizing antibodies, providing improved prophylactic and therapeutic options against RSV infection, including in human and veterinary subjects.
Claims Coverage
The claims disclose one independent claim defining an isolated immunogen comprising a recombinant RSV F protein or extracellular domain thereof with a specific substitution that stabilizes it in a prefusion conformation. Several dependent claims elaborate on features such as binding to prefusion-specific antibodies, inclusion of F1 and F2 polypeptides, linker types in single-chain proteins, multimerization, and inclusion of trimerization and transmembrane domains.
Stabilization by S190I substitution
The immunogen comprises a recombinant RSV F protein or extracellular domain with a S190I amino acid substitution that stabilizes the protein in a prefusion conformation, relative to a reference RSV F protein sequence (SEQ ID NO: 124).
Prefusion-specific antibody binding
The immunogen specifically binds to the prefusion-specific antibodies D25 or AM22, which do not bind the postfusion conformation of RSV F protein.
F1 and F2 polypeptides with substantial identity
The recombinant RSV F protein or extracellular domain comprises an F2 polypeptide and an F1 polypeptide with at least 90% identity to residues 26-109 and 137-513 or 26-103 and 145-513 of SEQ ID NO: 910.
Single chain protein configuration
The recombinant RSV F protein can be a single chain protein formed by linking F2 and F1 polypeptides with either a heterologous peptide linker or direct linkage, for example, position 105 of F2 linked to position 145 of F1 by a Gly-Ser linker.
Multimerization and trimerization
The immunogen can be administered as a multimer and is linked to a trimerization domain, such as a Foldon domain, optionally linked at the C-terminus of the F1 polypeptide.
Inclusion of transmembrane domain and viral particles
The recombinant RSV F protein or extracellular domain includes a C-terminal residue of the F1 polypeptide linked to a transmembrane domain, and can be included in virus-like particles or protein nanoparticles including ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase nanoparticles.
Nucleic acid and vector encoding the immunogen
Claims also cover nucleic acid molecules encoding the immunogen or precursor protein thereof, including RNA molecules and vectors, such as viral vectors, and host cells transformed therewith.
Use in immunogenic compositions and methods
The immunogen is included in immunogenic compositions with pharmaceutically acceptable carriers and can be administered to subjects to generate an immune response to RSV F protein, including methods of treatment and prevention of RSV infection.
The claims encompass recombinant RSV F proteins or extracellular domains stabilized in prefusion conformation by S190I substitution, featuring F1 and F2 polypeptides, optionally linked as single chains or multimers, including trimerization and transmembrane domains, with specific binding to prefusion-specific antibodies. Further claims encompass nucleic acids, vectors, viral particles, immunogenic compositions, and methods for generating immune responses or treating RSV infections.
Stated Advantages
PreF antigens elicit RSV neutralizing immune responses many fold greater than prior RSV F protein-based immunogens.
Stabilization of RSV F in prefusion conformation preserves antigenic site Ø, a supersite targeted by highly potent neutralizing antibodies.
PreF antigens are specifically bound by prefusion-specific antibodies and further target novel epitopes not present in postfusion F.
PreF antigens exhibit enhanced physical stability under various conditions including temperature, pH, osmolarity, and freeze-thaw cycles compared to prior RSV F constructs.
Vaccination with PreF antigens induces protective neutralizing antibody titers in animal models effective against RSV infection.
PreF immunogens elicit higher and more durable neutralizing responses in both mice and non-human primates compared to postfusion forms of RSV F.
The use of protein nanoparticles displays PreF antigens for improved antigen presentation and immunogenicity.
Documented Applications
Use as immunogens to induce RSV neutralizing immune responses in mammalian subjects (e.g., humans and cattle) for prevention or treatment of RSV infection.
Use in vaccination compositions including adjuvants for immunization protocols including prime-boost regimens targeting RSV subtypes A and B.
Use in methods of treating or preventing RSV infection in subjects at risk of or infected with RSV.
Use for detection or isolation of RSV F prefusion-specific antibodies from biological samples for diagnostic or research purposes.
Use of recombinant RSV F proteins stabilized in prefusion conformation in virus-like particles and protein nanoparticles for enhanced immunogenicity.
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