Algal produced malarial transmission blocking vaccines
The present invention relates to the production of malaria transmission blocking vaccines in single-celled green algae, particularly algae of the genus Chlamydomonas , e.g., Chlamydomonas reinhardtii ; the immunogenic Plasmodium polypeptides produced and compositions comprising them; and methods for preventing, ameliorating, reducing, delaying, treating and blocking the transmission of malaria by administration of immunogenic Plasmodium polypeptides produced in an algal host cell.
1. An algal cell comprising a polynucleotide comprising a nucleic acid sequence having at least about 90% sequence identity to the full length of SEQ ID NO:13 or SEQ ID NO:2, wherein the nucleic acid sequence encodes a Plasmodium surface protein P25.
2. An algal cell comprising the expression cassette comprising a promoter that drives expression of a coding sequence in an algal host cell, wherein the promoter is operably linked to a polynucleotide comprising a nucleic acid sequence having at least about 90% sequence identity to the full length of SEQ ID NO:13 or SEQ ID NO:2, wherein the nucleic acid sequence encodes a Plasmodium surface protein P25.
3. A method of producing an algal cell of claim 1 , comprising expressing in an algal cell a polynucleotide encoding a nucleic acid sequence having at least about 90% sequence identity to the full length of SEQ ID NO:13 or SEQ ID NO:2, wherein the nucleic acid sequence encodes a Plasmodium surface protein P25.
4. The algal cell of claim 1 , wherein the algal cell is a Chlamydomonas reinhardtii cell.
5. The algal cell of claim 1 , wherein the algal cell comprises a polynucleotide comprising a nucleic acid sequence having at least about 95% sequence identity to the full length of SEQ ID NO:13 or SEQ ID NO:2.
6. The algal cell of claim 1 , wherein the algal cell comprises a polynucleotide of SEQ ID NO:13 or SEQ ID NO:2.
7. The algal cell of claim 1 , wherein the Plasmodium surface protein P25 comprises at least one EGF domain and does not comprise a Plasmodium signal peptide or transmembrane domain.
8. The algal cell of claim 1 , wherein the Plasmodium surface protein P25 is a Plasmodium falciparum surface protein.
9. The algal cell of claim 1 , wherein the nucleic acid sequence is operably linked to a tag selected from the group consisting of an affinity tag, a purification tag, an immunogenicity tag, a delivery tag and a stability tag.
10. The algal cell of claim 1 , wherein the nucleic acid sequence is operably linked to a nucleic acid sequence encoding one or more tags selected from the group consisting of poly-His, Mistic, serum amyloid A (SAA), Tic40, small ubiquitin modifier peptide (SUMO), Streptavidin-Binding Peptide (SBP), green fluorescent protein (GFP), FLAG, cholera toxin beta subunit (CTB), and heat-labile enterotoxin beta subunit (LTB).
11. The algal cell of claim 1 , wherein the nucleic acid sequence is operably linked to a nucleic acid sequence encoding a cholera toxin beta subunit (CTB).
12. The algal cell of claim 11 , wherein the nucleic acid sequence encoding a cholera toxin beta subunit (CTB) has at least 60% sequence identity to SEQ ID NO:32.
13. The algal cell of claim 1 , wherein the algal cell further comprises one or more polynucleotides encoding one or more Plasmodium polypeptides selected from the group consisting of CDPK4, HAP2, MAPK-2, MDV 1/Peg3, P47, P48/45, P230, PKG, AP2-0, DOZI, HMGP2, Nek-4, CelTOS, CDPK3, Chitinase, CTRP, IMC1b, MAOP, P28, SOAP, Cap380, CSP, ECP1, IMC1a, LAP1/CCp3/SR, LAP2/CCp1, LAP3/CCp5, LAP4/CCp2, LAPS/FNPA, LAP6/CCp4, transglutaminase, CSP, CRMP1, CRMP2, MAEBL, TRAP, and UOS3/TREP/S6.
14. The algal cell of claim 2 , wherein the algal cell is a Chlamydomonas reinhardtii cell.
15. The algal cell of claim 2 , wherein the promoter is selected from the group consisting of psbA and psbD.
16. The algal cell of claim 2 , wherein the algal cell comprises a polynucleotide comprising a nucleic acid sequence having at least about 95% sequence identity to the full length of SEQ ID NO:13 or SEQ ID NO:2.
17. The algal cell of claim 2 , wherein the algal cell comprises a polynucleotide of SEQ ID NO:13 or SEQ ID NO:2.
18. The algal cell of claim 2 , wherein the Plasmodium surface protein P25 comprises at least one EGF domain and does not comprise a Plasmodium signal peptide or transmembrane domain.
19. The algal cell of claim 2 , wherein the Plasmodium surface protein P25 is a Plasmodium falciparum surface protein.
20. The algal cell of claim 2 , wherein the nucleic acid sequence is operably linked to a tag selected from the group consisting of an affinity tag, a purification tag, an immunogenicity tag, a delivery tag and a stability tag.
21. The algal cell of claim 2 , wherein the nucleic acid sequence is operably linked to a nucleic acid sequence encoding one or more tags selected from the group consisting of poly-His, Mistic, serum amyloid A (SAA), Tic40, small ubiquitin modifier peptide (SUMO), Streptavidin-Binding Peptide (SBP), green fluorescent protein (GFP), FLAG, cholera toxin beta subunit (CTB), and heat-labile enterotoxin beta subunit (LTB).
22. The algal cell of claim 2 , wherein the nucleic acid sequence is operably linked to a nucleic acid sequence encoding a cholera toxin beta subunit (CTB).
23. The algal cell of claim 22 , wherein the nucleic acid sequence encoding a cholera toxin beta subunit (CTB) has at least 60% sequence identity to SEQ ID NO:32.
24. The algal cell of claim 2 , wherein the algal cell further comprises one or more polynucleotides encoding one or more Plasmodium polypeptides selected from the group consisting of CDPK4, HAP2, MAPK-2, MDV 1/Peg3, P47, P48/45, P230, PKG, AP2-0, DOZI, HMGP2, Nek-4, CelTOS, CDPK3, Chitinase, CTRP, IMC1b, MAOP, P28, SOAP, Cap380, CSP, ECP1, IMC1a, LAP1/CCp3/SR, LAP2/CCp1, LAP3/CCp5, LAP4/CCp2, LAPS/FNPA, LAP6/CCp4, transglutaminase, CSP, CRMP1, CRMP2, MAEBL, TRAP, and UOS3/TREP/S6.
25. A method of producing an algal cell of claim 2 , comprising expressing in an algal cell a polynucleotide comprising a nucleic acid sequence having at least about 90% sequence identity to the full length of SEQ ID NO:13 or SEQ ID NO:2, wherein the nucleic acid sequence encodes a Plasmodium surface protein P25.