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-10017543-B2
Publication Date
2018-07-10
Expiration Date
2034-03-13
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 isolated immunogens comprising recombinant Respiratory Syncytial Virus (RSV) F proteins stabilized in a prefusion conformation by specific amino acid substitutions, including non-natural disulfide bonds and cavity-filling substitutions. These prefusion-stabilized RSV F proteins exhibit antigenic site Ø, a unique membrane-distal apex epitope present only in the prefusion state, which binds prefusion-specific antibodies such as D25, AM22, 5C4, and MPE8, and elicits significantly greater neutralizing immune responses than previous RSV F protein-based immunogens.
The problem addressed involves the metastability of the RSV F protein prefusion conformation, which has impeded the development of effective vaccines. RSV causes significant morbidity and mortality, particularly among infants and the elderly, and prior vaccine efforts based on RSV F protein have proven unsuccessful because the native RSV F protein readily converts to a postfusion conformation lacking key neutralizing epitopes. There is a need to produce a recombinant RSV F antigen that is stably locked in the prefusion conformation exposing antigenic site Ø to elicit potent and protective neutralizing immune responses.
The disclosure solves this problem by engineering recombinant RSV F proteins stabilized in the prefusion conformation through one or more amino acid substitutions, such as disulfide bonds between cysteines introduced at positions 155 and 290 and cavity-filling substitutions at positions 190 and 207. These modifications stabilize the prefusion conformation, preserving antigenic site Ø and enabling high-yield, homogenous recombinant protein production. The resulting antigens induce immune responses generating RSV-neutralizing antibodies that are significantly more potent and protective than those elicited by postfusion F proteins or previous vaccine candidates.
Claims Coverage
The independent claims encompass isolated RSV F immunogens stabilized in a prefusion conformation describing key amino acid substitutions, sequences, and structural features. The main inventive features include specific stabilizing disulfide bonds and cavity-filling mutations, preservation of antigenic site Ø, and recombinant RSV F protein constructs with defined polypeptide compositions and linkages.
Recombinant RSV F protein stabilization with non-natural disulfide bond and cavity-filling substitutions
Isolated immunogen comprising a recombinant RSV F protein stabilized in prefusion conformation by a non-natural disulfide bond via cysteine substitutions at positions 155 and 290, and a cavity-filling substitution at position 190, 207, or both, with amino acids selected from F, L, W, Y, H, or M.
Preservation of antigenic site Ø comprising residues 62-69 and 196-209
The recombinant RSV F protein in prefusion conformation comprises antigenic site Ø defined by residues 62-69 and 196-209 corresponding to native RSV F sequences (e.g., SEQ ID NOs: 1-184), critical for binding prefusion-specific antibodies.
Specific binding to prefusion-specific antibodies
The immunogen specifically binds prefusion antibodies D25, AM22, 5C4, or MPE8 that do not bind postfusion conformations, enabling identification and stability of prefusion conformation.
Recombinant RSV F protein with defined F1 and F2 polypeptides but optionally lacking pep27
Recombinant protein comprises F1 and F2 polypeptides derived from defined residue ranges correlating to RSV F reference sequences, with optional deletion of pep27 peptide segment to facilitate maturation and stability in prefusion condition.
Single-chain RSV F protein embodiments with F1 and F2 polypeptides linked by peptide linkers
Single-chain constructs link F1 and F2 polypeptides (e.g., via Gly-Ser peptide linkers at defined residue positions such as between 103/105 and 145) to form stable prefusion conformation monomers or trimers.
Recombinant RSV F proteins linked to trimerization domains or nanoparticles
Stabilized prefusion RSV F proteins can be linked C-terminally to trimerization domains such as Foldon or linked to protein nanoparticles (e.g., ferritin, encapsulin), supporting assembly into trimers or higher order multimeric particles maintaining prefusion stability and antigenic properties.
The claims define RSV F immunogens that are stabilized in the prefusion conformation via specified disulfide bonds and cavity-filling substitutions, preserving neutralization-sensitive antigenic site Ø, encompassing monomeric and trimeric recombinant proteins, single-chain constructs, and various multimeric antigen formulations linked to trimerization or nanoparticle domains, all specifically bound by prefusion-specific neutralizing antibodies.
Stated Advantages
Prefusion-stabilized RSV F proteins elicit RSV-neutralizing immune responses vastly stronger than postfusion RSV F or previous vaccine candidates, providing significant improvements in vaccine efficacy.
Stabilization modifications (disulfide bonds, cavity-filling substitutions) improve recombinant protein expression yield and stability under diverse physicochemical conditions, allowing homogeneous preparations suitable for vaccine development.
The disclosed immunogens maintain critical prefusion-specific antigenic sites, enabling induction and detection of highly potent neutralizing antibodies that target a viral vulnerability supersite.
Combination of structure-guided design and biophysical characterization accelerates vaccine development and enables rational engineering of stabilized prefusion RSV F antigens.
Documented Applications
Use as immunogens and vaccines for eliciting robust neutralizing immune responses to RSV in human and veterinary subjects, including prevention and treatment of RSV infection.
Use in prime-boost immunization protocols, including co-administration or sequential administration of RSV F subtype A and B antigens stabilized in prefusion conformations.
Use in recombinant viral vectors or nucleic acid formulations encoding prefusion-stabilized RSV F proteins for gene-based vaccination.
Use as antigen components for protein nanoparticles or virus-like particles presenting prefusion RSV F trimers to enhance immunogenicity.
Use for detection, isolation, and quantification of RSV F prefusion-specific neutralizing antibodies in biological samples for diagnostic or research purposes.
Interested in licensing this patent?