Modified RSV F proteins and methods of their use
The present invention is generally related to modified or mutated respiratory syncytial virus fusion (F) proteins and methods for making and using them, including immunogenic compositions such as vaccines for the treatment and/or prevention of RSV infection.
1. A method of producing a trimeric RSV F glycoprotein, the method comprising:
(a) transforming a eukaryotic host cell to express a nucleic acid encoding an RSV F glycoprotein, wherein the glycoprotein comprises a transmembrane domain;
(b) culturing said host cell under conditions conducive to the production of said RSV F glycoprotein;
(c) extracting the RSV F glycoprotein using nonylphenol ethoxylate (NP-9); and
(d) purifying the RSV F glycoprotein with a lentil lectin affinity column wherein the purified RSV F glycoprotein is a trimer.
2. The method of claim 1 , wherein said RSV F glycoprotein comprises a deletion of one or more amino acids aligning to amino acids 137-146 of the wild-type RSV F glycoprotein, which has an amino acid sequence of SEQ ID NO: 2.
3. The method of claim 1 , wherein the RSV F glycoprotein comprises an inactivated primary cleavage site.
4. The method of claim 3 , wherein the primary cleavage site is inactivated by at least one amino acid substitution introduced at positions aligning to Arg-133, Arg-135, and Arg-136 of SEQ ID NO: 2.
5. The method of claim 3 , wherein the primary cleavage site is inactivated by at least two amino acid substitutions introduced at positions aligning to Arg-133, Arg-135, and Arg-136 of SEQ ID NO: 2.
6. The method of claim 3 , wherein the primary cleavage site is inactivated by amino acid substitutions introduced at positions aligning to Arg-133, Arg-135, and Arg-136 of SEQ ID NO: 2.
7. The method of claim 1 , wherein the RSV F glycoprotein comprises amino acid substitutions of P102A, I379V, and M447V.
8. The method of claim 1 , wherein the nucleic acid sequence is SEQ ID NO: 7.
9. The method of claim 1 , wherein the purified RSV F glycoprotein comprises or consists of an amino acid sequence of SEQ ID NO: 8, wherein said sequence is cleaved into F1 and F2 subunits which are disulfide linked.
10. The method of claim 1 , wherein the RSV F protein lacks an N-terminal signal peptide.
11. The method of claim 9 , wherein the RSV F protein lacks an N-terminal signal peptide.
12. The method of claim 3 , wherein positions aligning to Arg-133, Arg-135, and Arg-136 of SEQ ID NO: 2 are substituted with glutamine.
13. The method of claim 1 , wherein the eukaryotic cell is an insect cell.
14. The method of claim 13 , wherein the insect cell is an Sf9 cell.
15. The method of claim 1 , wherein said host cell is transfected with a baculovirus vector comprising a nucleic acid having a sequence of SEQ ID NO: 7.
16. The method of claim 13 , wherein the insect cell is an Sf21 cell.
17. The method of claim 13 , wherein the insect cell is a Trichoplusia ni cell.
18. The method of claim 17 , wherein the Trichoplusia ni cell is an High Five insect cell.
19. The method of claim 13 , wherein the insect cell is a Drosophila cell.
20. The method of claim 19 , wherein the Drosophila cell is an S2 cell.