Engineered type IV pilin of
The present invention relates to engineered Clostridium difficile type IV pilin (tfp) genes, type IV pilin proteins which can serve as a diagnostic marker for identification of patients infected with C. difficile , and vaccines comprising type IV pilin proteins, antigenic fragments and variants thereof for therapeutic interventions.
1. An immunogenic composition comprising an isolated polypeptide comprising i) a C. difficile type IV pilin or an antigenic fragment or variant thereof, and ii) an affinity tag sequence to facilitate purification, wherein the antigenic fragment or variant thereof comprises a polypeptide sequence that has at least 90% sequence identity to a polypeptide sequence selected from the group consisting of:
a. amino acids 35-173 of SEQ ID NO:2;
b. amino acids 35-173 of SEQ ID NO:6;
c. amino acids 35-173 of SEQ ID NO:10;
d. amino acids 35-171 of SEQ ID NO:14;
e. amino acids 35-170 of SEQ ID NO:18;
f. amino acids 34-119 of SEQ ID NO:22;
g. amino acids 31-116 of SEQ ID NO:26;
h. amino acids 32-164 of SEQ ID NO:30;
i. amino acids 32-164 of SEQ ID NO:34;
j. amino acids 32-162 of SEQ ID NO:38;
k. amino acids 36-272 of SEQ ID NO:42;
l. SEQ ID NO:2;
m. SEQ ID NO:6;
n. SEQ ID NO:10;
o. SEQ ID NO:14;
p. SEQ ID NO:18;
q. SEQ ID NO:22;
r. SEQ ID NO:26;
s. SEQ ID NO:30;
t. SEQ ID NO:34; and
u. SEQ ID NO:38.
2. The composition of claim 1 , comprising a combination of the isolated polypeptides.
3. The composition of claim 2 , wherein said isolated polypeptides are conjugated.
4. The composition of claim 2 , wherein said isolated polypeptides are conjugated genetically to form a fusion protein, wherein the fusion protein is produced from a nucleic acid sequence encoding the combination.
5. The composition of claim 2 , wherein the combination of isolated polypeptides comprises a C. difficile type IV pilin selected from the group consisting of:
a. SEQ ID NO:2;
b. SEQ ID NO:6;
c. SEQ ID NO:10;
d. SEQ ID NO:14;
e. SEQ ID NO:18;
f. SEQ ID NO:22;
g. SEQ ID NO:26;
h. SEQ ID NO:30;
i. SEQ ID NO:34;
j. SEQ ID NO:38;
k. SEQ ID NO:42; and
l. combinations thereof.
6. The composition of claim 2 , wherein the combination of isolated polypeptides comprises a C. difficile type IV pilin antigenic fragment, wherein the antigenic fragment comprises a polypeptide sequence selected from the group consisting of:
a. amino acids 35-173 of SEQ ID NO:2;
b. amino acids 35-173 of SEQ ID NO:6;
c. amino acids 35-173 of SEQ ID NO:10;
d. amino acids 35-171 of SEQ ID NO:14;
e. amino acids 35-170 of SEQ ID NO:18;
f. amino acids 34-119 of SEQ ID NO:22;
g. amino acids 31-116 of SEQ ID NO:26;
h. amino acids 32-164 of SEQ ID NO:30;
i. amino acids 32-164 of SEQ ID NO:34;
j. amino acids 32-162 of SEQ ID NO:38; and
k. amino acids 36-272 of SEQ ID NO:42.
7. The composition of claim 6 , wherein the antigenic fragment is recombinantly produced.
8. The composition of claim 7 , wherein the antigenic fragment is produced in E. coli.
9. The composition of claim 8 , wherein the polypeptide is encoded by a nucleic acid sequence optimized to increase expression in E. coli using codons that are preferred in E. coli , wherein the nucleic acid sequence is selected from the group consisting of:
a. SEQ ID NO:3;
b. SEQ ID NO:7;
c. SEQ ID NO:11;
d. SEQ ID NO:15;
e. SEQ ID NO:19;
f. SEQ ID NO:23;
g. SEQ ID NO:27;
h. SEQ ID NO:31;
i. SEQ ID NO:35;
j. SEQ ID NO:39; and
k. SEQ ID NO:43.
10. The composition of claim 2 , wherein the combination comprises SEQ ID NO:2 or an antigenic fragment thereof, wherein the antigenic fragment comprises amino acids 35-173 of SEQ ID NO:2.
11. The composition of claim 10 , wherein the combination further comprises a type IV pilin selected from the group consisting of:
a. SEQ ID NO:6;
b. SEQ ID NO:10;
c. SEQ ID NO:14;
d. SEQ ID NO:18; and
e. combinations thereof.
12. The composition of claim 11 , wherein the polypeptide further comprises an enzymatic cleavage sequence.