Immunogenic detoxified mutant
View Patent ↗An immunogenic detoxified protein is provided which comprises the amino acid sequence of subunit A of an E. coli heat labile toxin (LT-A) or a fragment thereof in which at least amino acid Ala-72, numbered relative to SEQ ID NO:1, of the A subunits mutated, preferably by substitution with Arg. The toxoid is useful as vaccine against an enterotoxigenic strain of E. coli and is produced by recombinant DNA means by site-directed mutagenesis. It is also an effective adjuvant.
1. A polynucleotide encoding an immunologically effective detoxified E. coli heat labile toxin (LT-A) polypeptide, wherein the polypeptide comprises SEQ ID NO:1, and further wherein the amino acid residue corresponding to Ala-72 of SEQ ID NO:1 is an arginine residue.
2. The polynucleotide of claim 1 further comprising a sequence encoding a second immunogenic antigen.
3. The polynucleotide of claim 2 wherein the second immunogenic antigen comprises a subunit B of an E. coli heat labile toxin (LT-B).
4. The polynucleotide of claim 3 , wherein the LT-A and LT-B are encoded in a polycistronic unit.
5. An expression vector comprising the polynucleotide of claim 1 .
6. An expression vector comprising the polynucleotide of claim 2 .
7. An expression vector comprising the polynucleotide of claim 3 .
8. An expression vector comprising the polynucleotide of claim 4 .
9. A host cell comprising the expression vector of claim 5 .
10. A host cell comprising the expression vector of claim 6 .
11. A host cell comprising the expression vector of claim 7 .
12. A host cell comprising the expression vector of claim 8 .
13. The host cell of claim 9 , wherein the host cell is selected from the group consisting of a bacterium, a mammalian cell, a baculovirus, an insect cell and a yeast cell.
14. The host cell of claim 13 , wherein the host cell is E. coli.
15. The host cell of claim 13 , wherein the host cell is a mammalian cell.
16. The host cell of claim 13 , wherein the host cell is an insect cell.
17. The host cell of claim 13 , wherein the host cell is a yeast cell.
18. The host cell of claim 13 , wherein the host cell produces the amino acid sequence intracellularly.
19. The host cell of claim 13 , wherein the host cell secretes the amino acid sequence.
20. The E. coli host cell of claim 14 , wherein the host cell is mutated to produce a phenotype lacking wild type LT-A.
21. A method of producing a recombinant protein comprising:
(a) providing a population of host cells according to claim 9 ; and
(b) culturing said population of cells under conditions whereby the LT-A or fragment thereof encoded by the polynucleotide in said expression vector is expressed.
22. A method of producing a recombinant protein comprising:
(a) providing a population of host cells according to claim 11 ; and
(b) culturing said population of cells under conditions whereby the LT-A or fragment thereof and the LT-B encoded by the polynucleotide in said expression vector is expressed.
23. A method of producing a recombinant protein comprising:
(a) providing a population of host cells according to claim 20 ; and
(b) culturing said population of cells under conditions whereby the LT-A or fragment thereof encoded by the polynucleotide in said expression vector is expressed.
24. A polynucleotide encoding an immunologically effective detoxified E. coli heat labile toxin (LT-A) polypeptide, wherein the polypeptide comprises SEQ ID NO:2, and further wherein the amino acid residue corresponding to Ala-72 of SEQ ID NO:1 is an arginine residue.
25. A polynucleotide encoding an immunologically effective detoxified E. coli heat labile toxin (LT-A) polypeptide, wherein the polypeptide comprises SEQ ID NO:3, and further wherein the amino acid residue corresponding to Ala-72 of SEQ ID NO:1 is an arginine residue.
26. A polynucleotide encoding an immunologically effective detoxified E. coli heat labile toxin (LT-A) polypeptide, wherein the polypeptide comprises SEQ ID NO:4, and further wherein the amino acid residue corresponding to Ala-72 of SEQ ID NO:1 is an arginine residue.