Circumsporozoite proteins with increased expression in mammalian cells
Mutated and/or truncated malarial circumsporozoite proteins (CSP) and associated nucleic acids that are more stable and highly expressed in mammalian cells are described. The mutated and/or truncated CSP and associated nucleic acids can be expressed to produce malaria vaccine antigens.
1. A mutant N-terminus domain (NTD) of a circumsporozoite protein (CSP) comprising
i) a KKNSR (SEQ ID NO: 1) to SSNSS (SEQ ID NO: 2) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site of a wild-type Plasmodium falciparum CSP; or
ii) a KKNSR (SEQ ID NO: 1) to SSNSA (SEQ ID NO: 4) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site of a wild-type Plasmodium falciparum CSP;
wherein the wild-type Plasmodium falciparum CSP comprises a sequence as set forth in SEQ ID NOs: 7, 10, or 19.
2. The mutant NTD of claim 1 , further comprising a cysteine deletion.
3. The mutant NTD of claim 2 , wherein the cysteine is residue 5 of the wild-type Plasmodium falciparum CSP protein.
4. The mutant NTD of claim 1 , further comprising a cysteine to serine substitution.
5. The mutant NTD of claim 1 , expressed as a fusion protein with a secondary malaria vaccine epitope.
6. The mutant NTD of claim 5 , wherein the secondary malaria vaccine epitope is selected from RTS,S; NR2C, R2C, the junctional epitope, the minor epitope, or a junctional epitope linked to the minor epitope.
7. The mutant NTD of claim 5 , wherein the fusion protein comprises the sequence as set forth in SEQ ID NO: 12 (FL C5S-SAmut), SEQ ID NO: 13 (C5S-SAmut-23/4), SEQ ID NO: 14 (C5S-SAmut-19/3), or SEQ ID NO: 15 (C5S-SAmut-5/3).
8. A circumsporozoite protein (CSP) comprising
i) a KKNSR (SEQ ID NO: 1) to SSNSS (SEQ ID NO: 2) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site within the N-terminal domain of a wild-type Plasmodium falciparum CSP; or
ii) a KKNSR (SEQ ID NO: 1) to SSNSA (SEQ ID NO: 4) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site within the N-terminal domain of a wild-type Plasmodium falciparum CSP;
wherein the wild-type Plasmodium falciparum CSP comprises a sequence as set forth in SEQ ID NOs: 7, 10, or 19.
9. The CSP of claim 8 , further comprising a cysteine deletion within the N-terminus of the CSP.
10. The CSP of claim 9 , wherein the cysteine is residue 5 of the wild-type Plasmodium falciparum CSP protein.
11. The CSP of claim 8 , further comprising a cysteine to serine substitution within the N-terminus of the CSP.
12. The CSP of claim 8 , having the sequence SLSSNSSSLGENDD (SEQ ID NO: 8) or SLSSNSASLGENDD (SEQ ID NO: 76) in place of the wild-type SLKKNSRSLGENDD (SEQ ID NO: 7) sequence.
13. The CSP of claim 8 , having the sequence QEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLSSNSSSLGENDD (SEQ ID NO: 9) or QEYQSYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLSSNSASLGENDD (SEQ ID NO: 11) in place of the wild-type QEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGEN DD (SEQ ID NO: 10) sequence.
14. A method of stimulating an anti-malarial immune response in a subject comprising administering a therapeutically effective amount of a circumsporozoite protein (CSP) to the subject, wherein the CSP protein comprises
ii) a KKNSR (SEQ ID NO: 1) to SSNSS (SEQ ID NO: 2) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site in the N-terminal domain (NTD) of a wild-type Plasmodium falciparum CSP; or
iii) a KKNSR (SEQ ID NO: 1) to SSNSA (SEQ ID NO: 4) mutation directly upstream of a SLGENDD (SEQ ID NO: 3) cleavage site in the NTD of a wild-type Plasmodium falciparum CSP;
wherein the wild-type Plasmodium falciparum CSP comprises a sequence as set forth in SEQ ID NOs: 7, 10, or 19, thereby stimulating the anti-malarial immune response in the subject.
15. The method of claim 14 , wherein the CSP protein comprises a cysteine deletion within the N-terminus of the wild-type Plasmodium falciparum CSP.
16. The method of claim 15 , wherein the deleted cysteine was at residue 5 of the wild-type Plasmodium falciparum CSP protein.
17. The method of claim 14 , wherein the CSP protein comprises a cysteine to serine substitution.
18. The method of claim 14 , wherein the CSP comprises a malaria vaccine epitope.
19. The method of claim 18 , wherein the malaria vaccine epitope is selected from RTS,S; NR2C, R2C, the junctional epitope, the minor epitope, or a junctional epitope linked to the minor epitope.
20. The method of claim 14 , wherein the CSP comprises the sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.