Prefusion RSV F proteins and their use
Inventors
Kwong, Peter • Graham, Barney • Mascola, John • Ou, Li • Druz, Aliaksandr • Chen, Man • Kong, Wing-pui • Georgiev, Ivelin Stefanov • Rundlet, Emily • Joyce, Michael Gordon • Tsybovsky, Yaroslav • Thomas, Paul • Pancera, Marie • Sastry, Mallika • Soto, Cinque • Van Galen, Joseph • Stewart-Jones, Guillaume • Yang, YongPing • Zhang, Baoshan • Baxa, Ulrich
Assignees
US Department of Health and Human Services
Publication Number
US-11878998-B2
Publication Date
2024-01-23
Expiration Date
2037-03-29
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Abstract
Embodiments of a recombinant Respiratory Syncytial Virus (RSV) F ectodomain trimer stabilized in a prefusion conformation are provided. Also disclosed are nucleic acids encoding the RSV F ectodomain trimer and methods of producing the RSV F ectodomain trimer. Methods for inducing an immune response in a subject are also disclosed. In some embodiments, the method can be a method for treating or preventing a RSV infection in a subject by administering a therapeutically effective amount of the recombinant RSV F ectodomain trimer to the subject.
Core Innovation
The invention provides recombinant Respiratory Syncytial Virus (RSV) F ectodomain trimers stabilized in a prefusion conformation through structure-based design. These recombinant proteins comprise three modified F2-F1 ectodomain protomers genetically linked via a glycine-serine peptide linker replacing RSV F positions 104-144, including specific amino acid substitutions and disulfide bonds to enhance stability and antigenic presentation. The stabilization maintains antigenic site Ø, allowing specific binding to prefusion-specific antibodies such as D25, AM22, and 5C4.
The problem addressed is that RSV is a leading cause of severe respiratory disease, especially in infants and the elderly, and that prior RSV F protein immunogens such as DS-Cav1, while effective, may not elicit immune responses sufficient to prevent the most severe disease or support maternal immunization protocols for passive immunity in infants. The invention solves this by iterative structure-based design yielding RSV F immunogens with up to four-fold increased neutralizing antibody titers compared to DS-Cav1, improved thermal and antigenic stability, and elimination of the requirement for furin cleavage.
Claims Coverage
The patent contains multiple independent claims focused on recombinant RSV F ectodomain trimers, nucleic acid molecules encoding the protomers, vectors including such nucleic acids, immunogenic compositions, host cells, production methods, and methods of inducing immune responses. The main inventive features relate to specific structural modifications stabilizing RSV F in the prefusion conformation with enhanced immunogenicity and stability.
Recombinant RSV F ectodomain trimer stabilized in prefusion conformation
The trimer consists of three recombinant F2-F1 ectodomain protomers each comprising a deletion of RSV F positions 104-144, a glycine-serine peptide linker between positions 103 and 145, and amino acid substitutions including a non-native disulfide bond between engineered cysteines at 155C and 290C, 190F and 207L substitutions, and at least one additional non-native inter-protomer disulfide bond, for example between cysteines at 98C and 361C or 183GC and 428C, based on a reference sequence SEQ ID NO: 57.
Specific amino acid substitutions for stabilization and expression
Protomers comprise specific substitutions such as S155C and S290C for the non-native disulfide bond, S190F and V207L cavity-filling substitutions, Q98C and Q361C substitutions for inter-protomer disulfide bond, with option to include L373R substitution that enhances protein yield in single-chain constructs.
Retention and binding to prefusion-specific antigenic site Ø
The recombinant RSV F ectodomain trimer is stabilized in prefusion conformation preserving antigenic site Ø and specifically binds prefusion specific antibodies including D25, AM22, 5C4, and MPE8.
Use of trimerization domains for stabilization
The protomers can be linked at their C-terminus to trimerization domains like T4 Fibritin foldon (sequence GYIPEAPRDGQAYVRKDGEWVLLSTF, SEQ ID NO: 66), directly or via a peptide linker, linked to RSV F positions near 511, 513 or 523 to promote trimerization and stability.
Nucleic acids, vectors, and production methods
Isolated nucleic acid molecules encoding these recombinant F2-F1 ectodomain protomers, including precursors with signal peptides and expressing these proteins in host cells, as well as viral vectors containing these nucleic acids, are covered. Methods of producing and purifying the stable prefusion trimer are included.
Immunogenic compositions and methods of inducing immune responses
Immunogenic compositions comprising the recombinant RSV F ectodomain trimers and methods of administering effective amounts to subjects, including humans and bovine subjects, to induce neutralizing immune responses to RSV F are claimed.
The claims cover recombinant RSV F ectodomain trimers with specific deletions, linkers, and carefully designed amino acid substitutions and disulfide bonds to stabilize the protein in a prefusion conformation with improved immunogenicity and stability, nucleic acids and vectors encoding these protomers, production methods, immunogenic compositions including these proteins, and methods for inducing protective immune responses in subjects.
Stated Advantages
The novel RSV F immunogens produce a 4-fold higher neutralizing antibody titer than previous DS-Cav1 immunogens.
They do not require furin cleavage, simplifying production.
They have increased antigenic stability against heat inactivation, with stability at 60° C. being over 10-fold higher than DS-Cav1 in several embodiments.
The recombinant RSV F ectodomain trimers have enhanced solubility and can remain stable in solution for extended times.
Documented Applications
Inducing an immune response in a subject to prevent or reduce RSV infection, including maternal immunization to provide passive immunity to infants.
Administering immunogenic compositions comprising the recombinant RSV F ectodomain trimer to humans or bovine subjects for vaccination.
Using nucleic acid molecules or viral vectors encoding the recombinant RSV F protomers for in vivo expression to induce immunity.
Including recombinant RSV F ectodomain trimers in virus-like particles or protein nanoparticles for use as vaccines.
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