MULTIPLE VARIANTS OF MENINGOCOCCAL PROTEIN NMB1870
Meningococcal protein NMB 1870 has been described in the prior art. The inventors have found that NMB 1870 is an effective antigen for eliciting anti-meningococcal antibody responses, and that it is expressed across all meningococcal serogroups. Forty-two different NMB 1870 sequences have been identified, and these group into three variants. Serum raised against a given variant is bactericidal within the same variant group, but is not active against strains which express one of the other two variants i.e. there is intra-variant cross-protection, but not inter-variant cross-protection. For maximum cross-strain efficacy, therefore, the invention uses mixture comprising different variants of NMB 1870.
1 . A method for the heterologous expression of a protein of the invention comprising:
a) providing an E. coli host cell comprising a nucleic acid encoding a protein comprising i) a sequence having greater than 80% sequence identity to any one of SEQ ID NOs: 24 to 45; or ii) a fragment of 7 or more contiguous amino acids from any one of SEQ ID NOs: 24 to 45, wherein the E. coli host cell is capable of lipidating the protein;
b) inducing expression of the protein under conditions where the protein is lipidated.
2 . The method of claim 1 , wherein the nucleic acid is operably linked to a second nucleic acid encoding an N-terminal signal peptide.
3 . The method of claim 2 , wherein the N-terminal signal peptide is native to the protein.
4 . The method of claim 2 , wherein the N-terminal signal peptide is heterologous to the protein.
5 . The method of claim 4 , wherein the N-terminal signal peptide comprises SEQ ID 46 or the H. influenzae P4 lipoprotein leader sequence
6 . The method of claim 1 , wherein the lipid is covalently attached to an N-terminal cysteine of the protein in step b).
7 . The method of claim 1 , further comprising step c) isolating the protein from the E. coli host cell.
8 . An isolated, lipidated protein comprising:
i) a sequence having greater than 80% sequence identity to any one of SEQ ID NOs: 24 to 45; or
ii) a fragment of 7 or more contiguous amino acids from any one of SEQ ID NOs: 24 to 45, wherein the lipidated protein was expressed in E. coli.
9 . The isolated, lipidated protein of claim 8 , wherein the lipid is covalently attached to an N-terminal cysteine of the protein.
10 . An E. coli host cell containing a nucleic acid encoding the protein of claim 8 .
11 . An immunogenic composition comprising the protein of claim 8 or claim 9 .
12 . The immunogenic composition of claim 11 , further comprising a detergent to solubilize the lipidated protein.
13 . The immunogenic composition of claim 11 , further comprising an adjuvant.
14 . The immunogenic composition of claim 13 , further comprising a detergent to solubilize the lipidated protein.
15 . The immunogenic composition of claim 13 , wherein the adjuvant comprises a detergent to solubilize the lipidated protein.
16 . The immunogenic composition of claim 11 , wherein the protein is present at a concentration of at least 1 μg/ml.
17 . The immunogenic composition of claim 11 , which is sterile and/or pyrogen-free.
18 . The immunogenic composition of claim 13 , wherein the adjuvant comprises an aluminium hydroxyphosphate.
19 . The immunogenic composition of claim 11 , further comprising: (i) a saccharide antigen from N. meningitidis serogroup A, C, W135 and/or Y; and/or (ii) a saccharide antigen from Haemophilus influenzae type B; wherein the saccharide antigen(s) of (i) and/or (ii) is/are conjugated to one or more carrier proteins.