Prefusion RSV F proteins and their use
Inventors
Kwong, Peter • Graham, Barney • McLellan, Jason • Chen, Man • Zhang, Baoshan • Zhou, Tongqing • Joyce, Michael Gordon • Yang, YongPing
Assignees
US Department of Health and Human Services
Publication Number
US-10858400-B2
Publication Date
2020-12-08
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 stabilized in a prefusion conformation, as well as nucleic acids encoding these immunogens and methods for their production. Methods for generating an immune response in subjects and for treating or preventing RSV infection by administering therapeutically effective amounts of these immunogens are also described. These recombinant prefusion F (PreF) antigens specifically bind prefusion-specific antibodies such as D25 or AM22, harbor a unique antigenic site Ø at the membrane-distal apex, and induce RSV neutralizing immune responses substantially greater than prior RSV F protein-based immunogens.
The problem solved addresses the challenge that prior to this invention, a homogeneous, soluble preparation of the RSV F protein in its prefusion conformation was unavailable due to its metastability and tendency to adopt the postfusion state after expression. This lack of a stable prefusion form precluded determination of its three-dimensional structure, identification of novel antigenic sites, and hampered vaccine and antibody therapy development. Prior RSV treatments relied on antibodies like palivizumab targeting antigenic sites present on postfusion F protein, which did not elicit the strongest immune responses.
The invention resulted in elucidation of the three-dimensional crystal structure of the RSV F protein in the prefusion conformation bound to antibodies such as D25, revealing the newly identified antigenic site Ø responsible for potent neutralizing responses. Structure-guided modifications, including amino acid substitutions to introduce disulfide bonds, cavity filling mutations, and linking trimerization domains, were engineered to stabilize the RSV F protein in its prefusion conformation. The stabilized proteins retain key neutralizing epitopes, bind to prefusion-specific antibodies, and are used as immunogens for eliciting highly effective RSV neutralizing antibody responses. Additionally, epitope-scaffold proteins and protein nanoparticles presenting these antigens are also disclosed.
Claims Coverage
The patent claims cover a total of 19 inventive features related to isolated immunogens comprising recombinant RSV F proteins with specific stabilizing substitutions and configurations, including nucleic acids, vectors, host cells, virus-like particles, protein nanoparticles, and immunogenic compositions.
Recombinant RSV F protein stabilized in a prefusion conformation by specific amino acid substitutions
An isolated immunogen comprising a recombinant RSV F protein with S190F and V207L amino acid substitutions compared to native RSV F protein, which stabilizes it in a prefusion conformation including antigenic site Ø (residues 62-69 and 196-209) that specifically binds to D25 or AM22 antibodies after incubation in physiological conditions.
Additional amino acid substitutions on RSV F protein
The RSV F protein can further comprise one or more additional amino acid substitutions beyond S190F and V207L to enhance properties while retaining prefusion stabilization and antibody binding.
RSV F protein variants from different strains
The recombinant RSV F protein includes human subtype A or B, or bovine RSV F protein containing the S190F and V207L substitutions.
Inclusion of F2 and F1 polypeptides with defined positions
The recombinant RSV F protein comprises F2 and F1 polypeptides consisting of RSV F positions 26-109 and 137-513, respectively.
Linkage of F2 and F1 polypeptides by a heterologous peptide linker
The recombinant RSV F protein can have a C-terminal residue of F2 linked to the N-terminal residue of F1 via a heterologous peptide linker.
Defined F1 polypeptide sequence inclusion
The recombinant RSV F protein includes an F1 polypeptide comprising residues 137-513 of SEQ ID NO: 191.
Soluble recombinant RSV F protein with trimerization domain linked to F1 ectodomain
The RSV F protein is soluble and comprises an F1 ectodomain with a C-terminal residue linked to a trimerization domain.
Use of Foldon domain as trimerization domain
The trimerization domain linked to the F1 ectodomain is a Foldon domain that stabilizes trimer formation.
Specific amino acid sequence including Foldon domain
The recombinant RSV F protein linked to the Foldon domain comprises residues 26-109 and 137-544 of SEQ ID NO: 191.
C-terminal linkage to protein nanoparticle domains
The C-terminus of recombinant RSV F protein is linked to domains from ferritin, encapsulin, Sulfur Oxygenase Reductase (SOR), lumazine synthase, or pyruvate dehydrogenase, forming protein nanoparticles.
Transmembrane domain linkage to F1 ectodomain
The recombinant RSV F protein includes an F1 ectodomain comprising a C-terminal residue linked to a transmembrane domain, enabling membrane anchoring.
Virus-like particle comprising the stabilized immunogen
A virus-like particle comprises the immunogen with stabilized prefusion RSV F protein as disclosed.
Protein nanoparticle comprising the stabilized immunogen
A protein nanoparticle comprises the immunogen including stabilized prefusion RSV F protein.
Protein nanoparticle types
The protein nanoparticle is selected from ferritin, encapsulin, Sulfur Oxygenase Reductase (SOR), lumazine synthase, or pyruvate dehydrogenase nanoparticles.
Nucleic acid molecule encoding the immunogen
A nucleic acid molecule encodes the recombinant RSV F protein immunogen with stabilizing S190F and V207L substitutions and prefusion conformation.
Vector comprising the nucleic acid encoding the immunogen
A vector contains the nucleic acid molecule encoding the stabilized recombinant RSV F protein immunogen.
Isolated host cell containing the vector
An isolated host cell is transformed with the vector expressing the recombinant RSV F protein immunogen.
Pharmaceutical immunogenic composition
An immunogenic composition comprises an effective amount of the immunogen and a pharmaceutically acceptable carrier.
Method for generating immune response to RSV F protein
A method comprises administering an effective amount of the disclosed immunogen to a subject to generate an immune response against RSV F protein.
The claims broadly cover immunogens containing a recombinant RSV F protein stabilized in a prefusion conformation via S190F and V207L substitutions and related modifications. The claims extend to nucleic acids, vectors, host cells, compositions, various nanoparticle and virus-like particle formats, and methods for generating immune responses against RSV.
Stated Advantages
The disclosed recombinant RSV F proteins generate RSV neutralizing immune responses many fold greater than prior RSV F protein-based immunogens.
Stabilization of the prefusion conformation enables identification of novel antigenic sites specific to this conformation, such as antigenic site Ø.
The recombinant RSV F proteins specifically bind prefusion-specific antibodies (like D25 and AM22), enabling their use as vaccines and diagnostic molecules.
The stabilized prefusion RSV F proteins can be formulated in soluble, trimeric forms suitable for pharmaceutical use, maintaining known neutralizing epitopes.
Presentation of the prefusion RSV F protein on protein nanoparticles or virus-like particles allows enhanced immune stimulation.
Documented Applications
Use of recombinant RSV F proteins stabilized in prefusion conformation as vaccines for prevention or treatment of RSV infection in humans, cattle, and other subjects.
Use of the recombinant RSV F proteins as diagnostic molecules to detect RSV binding antibodies in sera.
Use of viral vectors encoding the stabilized RSV F proteins for immunization and immune response generation in subjects.
Formulation of stabilized RSV F proteins on protein nanoparticles such as ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase nanoparticles for enhanced immunogenicity.
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