Prefusion rsv f proteins and their use

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.

We claim: 
     
         1 . An isolated nucleic acid molecule encoding a recombinant RSV F protein or extracellular domain thereof comprising one or more amino acid substitutions compared to a native RSV F protein that stabilize the recombinant RSV F protein in a prefusion conformation;
 wherein the one or more amino acid substitutions comprise cysteine substitutions to introduce a non-native disulfide bond that stabilizes the recombinant RSV F protein in the prefusion conformation; and wherein the cysteine substitutions comprise a first cysteine substitution at one of RSV F positions 137-216 and a second cysteine substitution at one of RSV F positions 26-61; and   wherein the RSV F positions of the first and second cysteines have a Cβ-Cα distance of from 2.0 to 5.5 angstroms in the three-dimensional structure set forth by the structural coordinates provided in Table 1.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the recombinant RSV F protein further comprises one or more additional amino acid substitutions that stabilize the recombinant RSV F protein in the prefusion conformation. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein the one or more additional amino acid substitutions comprise cavity filling amino acid substitutions at RSV F position 190, position 207, or positions 190 and 207. 
     
     
         4 . The nucleic acid molecule of  claim 3 , wherein the cavity filling amino acid substitutions are S190F and V207L amino acid substitutions. 
     
     
         5 . The nucleic acid molecule of  claim 2 , wherein the one or more additional amino acid substitutions further comprise S155C and S290C substitutions to introduce a non-native disulfide bond that stabilizes the recombinant RSV F protein in the prefusion conformation. 
     
     
         6 . The nucleic acid molecule of  claim 1 , wherein the recombinant RSV F protein is a single-chain recombinant RSV F protein. 
     
     
         7 . The nucleic acid molecule of  claim 6 , wherein:
 position 103 of an F2 polypeptide of the recombinant RSV F protein is linked to position 145 of an F1 polypeptide of the recombinant RSV F protein by a heterologous glycine-serine peptide linker; or   position 105 of an F2 polypeptide of the recombinant RSV F protein is linked to position 145 of an F1 polypeptide of the recombinant RSV F protein by a heterologous glycine-serine peptide linker.   
     
     
         8 . The nucleic acid molecule of  claim 1 , wherein the recombinant RSV F protein comprises a F2 polypeptide and a F1 polypeptide comprising or consisting of RSV F positions 26-109 and 137-513, respectively, positions 26-103 and 145-513, respectively or 26-105 and 145-513, respectively. 
     
     
         9 . The nucleic acid molecule of  claim 1 , encoding the extracellular domain of the recombinant RSV F protein, wherein the extracellular domain is soluble. 
     
     
         10 . The nucleic acid molecule of  claim 9 , wherein the extracellular domain of the recombinant RSV F protein is linked to a C-terminal trimerization domain. 
     
     
         11 . The nucleic acid molecule of  claim 10 , wherein the trimerization domain is a foldon domain. 
     
     
         12 . The nucleic acid molecule of  claim 11 , wherein:
 the recombinant RSV F protein is a single-chain protein with RSV F position 103 of an F2 polypeptide linked to RSV F position 145 of an F1 polypeptide by a heterologous glycine-serine peptide linker; and   the recombinant RSV F protein further comprises one or more additional amino acid substitutions that stabilize the recombinant RSV F protein in the prefusion conformation, wherein the one or more additional amino acid substitutions comprise:   (A) S155C and S290C substitutions to introduce a non-native disulfide bond, A149C and Y458C substitutions to introduce a non-native disulfide bond, and S190F and V207L cavity filling amino acid substitutions; or   (B) S155C and S290C substitutions to introduce a non-native disulfide bond, 183GC and 428C substitutions to introduce a non-native disulfide bond, and S190F and V207L cavity filling amino acid substitutions.   
     
     
         13 . The nucleic acid molecule of  claim 1 , wherein the recombinant RSV F protein comprises a F1 ectodomain comprising a C-terminal residue fused to a transmembrane domain. 
     
     
         14 . The nucleic acid molecule of  claim 1 , wherein the recombinant RSV F protein is a human subtype A or subtype B, or bovine RSV F protein comprising the amino acid substitutions. 
     
     
         15 . The nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule is mRNA. 
     
     
         16 . A vector comprising the nucleic acid molecule of  claim 1 . 
     
     
         17 . An isolated host cell comprising the vector of  claim 16 . 
     
     
         18 . An immunogenic composition comprising an effective amount of the nucleic acid molecule of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         19 . A method for generating an immune response to RSV F protein in a subject, comprising administering an effective amount of the nucleic acid molecule of  claim 1  to the subject to generate the immune response.