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-11130785-B2
Publication Date
2021-09-28
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 relates to recombinant Respiratory Syncytial Virus (RSV) F proteins stabilized in a prefusion conformation, including isolated recombinant RSV F proteins and nucleic acids encoding the antigens. The prefusion stabilization preserves a unique antigenic site designated antigenic site Ø, located at the membrane distal apex of the RSV F protein trimer. Stabilization includes amino acid substitutions such as the introduction of non-natural disulfide bonds and cavity-filling mutations, and can be combined with methods such as appending a trimerization domain or fusion to protein nanoparticles.
The problem solved by the invention is the metastable nature of the RSV F protein prefusion conformation, which in soluble form readily adopts the postfusion conformation, thereby abrogating the presentation of key neutralizing epitopes, particularly antigenic site Ø. Prior vaccines using RSV F protein have been ineffective due to the lack of stabilization of the prefusion state and the resulting failure to elicit highly neutralizing immune responses. The invention addresses this by engineering and producing stabilized prefusion RSV F proteins that induce significantly greater neutralizing antibody responses and protection against RSV infection in animal models compared to postfusion F or other immunogens.
Claims Coverage
The patent claims cover a set of inventive features focused on isolated immunogens comprising recombinant RSV F proteins stabilized in a prefusion conformation, including specific amino acid substitutions that introduce non-natural disulfide bonds, and additional stabilizing mutations.
Prefusion stabilization of recombinant RSV F protein by non-native disulfide bonds
The recombinant RSV F protein has one or more amino acid substitutions including cysteine substitutions that form a non-native disulfide bond between a first cysteine at RSV F positions 137-216 and a second cysteine at RSV F positions 26-61 with a Cβ-Cβ distance of 2.0 to 5.5 angstroms in the prefusion structure. This confers binding of the antigenic site Ø epitope and specific binding by D25 or AM22 antibodies after incubation without the antibody.
Additional disulfide bond substitutions for further stabilization
Additional stabilizing cysteine substitutions include pairs at positions 149C and 458C, 183C and 428C, 369C and 455C, 99C and 361C, or 99C and 362C to form non-native disulfide bonds.
Additional stabilizing mutations in the prefusion conformation
The recombinant RSV F protein may include further amino acid substitutions that stabilize the protein, such as cavity filling mutations at positions 190 and 207. Notably, S190F and V207L cavity filling substitutions are exemplified.
Single-chain RSV F proteins
The recombinant RSV F protein may be single-chain in format, wherein position 103 or 105 of the F2 polypeptide is linked to position 145 of the F1 polypeptide by a heterologous glycine-serine peptide linker.
Composition and multimeric forms of recombinant RSV F protein
The recombinant RSV F protein includes F2 and F1 polypeptides comprising RSV F positions 26-109 and 137-513 or close variants. The extracellular domain can be soluble and linked to a C-terminal trimerization domain, such as a foldon domain. The protein can form trimers stabilized by intraprotomer disulfide bonds.
Virus like particles, protein nanoparticles, nucleic acids, vectors, and immunogenic compositions
The claims cover immunogens comprising RSV F protein variants in virus-like particles and protein nanoparticles, nucleic acid molecules encoding any such proteins, and vectors comprising these nucleic acids, as well as immunogenic compositions containing these materials.
The claims cover isolated immunogens comprising recombinant RSV F proteins stabilized in a prefusion conformation by specific non-natural disulfide bonds and cavity-filling substitutions, including single-chain and trimeric forms, with associated nucleic acid and vector constructs, multimers including nanoparticles or virus-like particles, and immunogenic compositions useful for generating RSV neutralizing immune responses.
Stated Advantages
The prefusion stabilized RSV F proteins elicit RSV neutralizing immune responses many fold greater than those elicited by prior RSV F protein-based immunogens.
The stabilized prefusion F proteins maintain the key neutralizing antigenic site Ø and are stable under various physical conditions such as temperature, pH, osmolality, and freeze-thaw cycles.
The stabilized prefusion F proteins provide protection against RSV challenge in animal models including mice and non-human primates.
The stabilized prefusion F proteins can be formulated with various adjuvants including alum and poly I:C, retaining immunogenicity.
The stabilized prefusion RSV F proteins can be used to design vaccines that elicit broad neutralizing antibodies to RSV subtypes A and B.
Documented Applications
Use of recombinant RSV F proteins stabilized in a prefusion conformation as vaccines to elicit an immune response conferring protection against RSV infection in subjects such as humans and bovines.
Use in methods for treating, inhibiting or preventing RSV infection in subjects by administering the recombinant RSV F antigen.
Use of these proteins, nucleic acids, or antibodies specific to prefusion RSV F in diagnostic assays to detect RSV binding antibodies in biological samples.
Use as immunogenic compositions for prime-boost immunization regimens including administration of subtype A and subtype B RSV F proteins stabilized in a prefusion conformation.
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