IP Library › Granted Patent US 9,802,988
Granted Patent B2
US 9,802,988 · App. 14/259,413 · Granted Oct 31, 2017

Engineered type IV pilin of

Inventor: Michael Donnenberg (Baltimore, MD)
Assignee: University of Maryland, Baltimore
C07K14/33A61K39/08C07K16/1282C07K2317/20C07K2317/33G01N33/56911G01N2333/33
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Quick Facts
Patent No.
US 9,802,988
App. No.
14/259,413
Granted
Oct 31, 2017
Kind
B2
Abstract

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.

Claims (152)

1. A pharmaceutical composition comprising an isolated polypeptide comprising

i) a Clostridium difficile type IV pilin or an antigenic fragment or variant thereof, wherein the variant is recognized by an antibody that binds the C. difficile type IV pilin, wherein the isolated C. difficile type IV pilin or antigenic fragment or variant thereof is recombinantly produced, wherein the variant has at least 90% sequence identity to the type IV pilin, wherein the type IV pilin is selected from the group consisting of:

a. SEQ ID NO:46;

b. SEQ ID NO: 47;

c. SEQ ID NO:48;

d. SEQ ID NO:50;

e. SEQ ID NO:51;

f. SEQ ID NO:52;

g. SEQ ID NO:63;

h. SEQ ID NO:65;

i. SEQ ID NO: 66;

j. SEQ ID NO: 67;

k. SEQ ID NO:69;

l. SEQ ID NO: 71;

m. SEQ ID NO:73;

n. SEQ ID NO:74;

o. SEQ ID NO: 75;

p. SEQ ID NO:78;

q. SEQ ID NO:79;

r. SEQ ID NO: 81;

s. SEQ ID NO:83;

t. SEQ ID NO:85;

u. SEQ ID NO: 87;

v. SEQ ID NO:88; and

w. SEQ ID NO:89;

wherein the antigenic fragment is selected from the group consisting of:

i. an amino-terminal truncation mutant of the type IV pilin, wherein the number of amino terminal amino acids missing from the fragment ranges from 1-100 amino acids;

ii. a carboxyl-terminal truncation mutant of the type IV pilin, wherein the number of carboxyl terminal amino acids missing from the fragment ranges from 1-100 amino acids; and

iii. an internal fragment of the type IV pilin lacking both the amino and carboxyl terminal amino acids and being 10-100 amino acid residues in length; and

ii) an affinity tag sequence to facilitate purification.

2. The pharmaceutical composition of claim 1 , comprising a combination of isolated polypeptides of claim 1 .

3. The pharmaceutical composition of claim 2 , wherein said isolated polypeptides are conjugated.

4. The pharmaceutical composition of claim 2 , wherein said isolated polypeptides are encoded by nucleic acids that are conjugated genetically.

5. The pharmaceutical composition of claim 1 , wherein said isolated polypeptide comprises the C. difficile type IV pilin antigenic fragment.

6. The pharmaceutical composition of claim 1 , wherein the antigenic fragment is produced in E. coli.

7. The pharmaceutical composition claim 6 , wherein the antigenic fragment is encoded by a nucleic acid sequence optimized to increase expression in E. coli.

8. The pharmaceutical composition claim 7 , wherein the nucleic acid sequence is selected from the group consisting of:

a. SEQ ID NO:110;

b. SEQ ID NO:90;

c. SEQ ID NO:92;

d. SEQ ID NO:95;

e. SEQ ID NO:105; and

f. SEQ ID NO:98.

9. The pharmaceutical composition of claim 1 , wherein the type IV pilin is from a strain selected from the group consisting of C. difficile CD196, C. difficile CIP 107932, C. difficile QCD-32g58, C. difficile QCD-37x79, C. difficile QCD-66c26, C. difficile QCD-76w55, C. difficile QCD-97b34, C. difficile 820291, C. difficile QCD-63q42, C. difficile QCD-23m63, C. difficile 630, C. difficile ATCC 43255, C. difficile 70-100-2010, C. difficile 050-P50-2011, C. difficile 002-P50-2011, C. difficile NAP08, C. difficile NAP07, C. difficile BI1 and combinations thereof.

10. The composition of claim 6 , wherein the isolated polypeptide further comprises an enzymatic cleavage sequence.

11. A pharmaceutical composition for inducing an immune response against Clostridium difficile comprising at least one isolated polypeptide comprising

i) a C. difficile type IV pilin or an antigenic fragment or variant thereof, wherein the variant is recognized by an antibody that binds the C. difficile type IV pilin, wherein the isolated C. difficile type IV pilin or antigenic fragment or variant thereof is recombinantly produced, wherein the variant has at least 90% sequence identity to the type IV pilin, wherein the type IV pilin is selected from the group consisting of:

a. SEQ ID NO:46;

b. SEQ ID NO: 47;

c. SEQ ID NO:48;

d. SEQ ID NO:50;

e. SEQ ID NO:51;

f. SEQ ID NO:52;

g. SEQ ID NO:53;

h. SEQ ID NO: 54;

i. SEQ ID NO:55;

j. SEQ ID NO:56;

k. SEQ ID NO: 57;

l. SEQ ID NO:58;

m. SEQ ID NO:59;

n. SEQ ID NO: 60;

o. SEQ ID NO:63;

p. SEQ ID NO:65;

q. SEQ ID NO: 66;

r. SEQ ID NO:67;

s. SEQ ID NO:69;

t. SEQ ID NO: 71;

u. SEQ ID NO:73;

v. SEQ ID NO:74;

w. SEQ ID NO: 75;

x. SEQ ID NO:78;

y. SEQ ID NO:79;

z. SEQ ID NO: 81;

aa. SEQ ID NO:83;

bb. SEQ ID NO:85;

cc. SEQ ID NO: 87;

dd. SEQ ID NO:88; and

ee. SEQ ID NO:89;

wherein the antigenic fragment is selected from the group consisting of:

i. an amino-terminal truncation mutant of the type IV pilin, wherein the number of amino terminal amino acids missing from the fragment ranges from 1-100 amino acids;

ii. a carboxyl-terminal truncation mutant of the type IV pilin, wherein the number of carboxyl terminal amino acids missing from the fragment ranges from 1-100 amino acids; and

iii. an internal fragment of the type IV pilin lacking both the amino and carboxyl terminal amino acids and being 10-100 amino acid residues in length;

ii) an affinity tag sequence to facilitate purification; and

iii) an enzymatic cleavage sequence.

12. The pharmaceutical composition of claim 11 , comprising a combination of isolated polypeptides of claim 11 .

13. The pharmaceutical composition of claim 11 , wherein said isolated polypeptide comprises a C. difficile type IV pilin antigenic fragment that comprises a peptide selected from the group consisting of:

a. amino acids 35-173 of SEQ ID NO:46;

b. amino acids 35-173 of SEQ ID NO:47;

c. amino acids 35-173 of SEQ ID NO:48;

d. amino acids 31-116 of SEQ ID NO:50;

e. amino acids 31-116 of SEQ ID NO:51;

f. amino acids 32-162 of SEQ ID NO:52;

g. amino acids 31-267 of SEQ ID NO:53;

h. amino acids 36-272 of SEQ ID NO:54;

i. amino acids 36-272 of SEQ ID NO:55;

j. amino acids 36-272 of SEQ ID NO:56;

k. amino acids 31-267 of SEQ ID NO:57;

l. amino acids 31-267 of SEQ ID NO:58;

m. amino acids 31-267 of SEQ ID NO:59;

n. amino acids 31-267 of SEQ ID NO:60;

o. amino acids 34-175 of SEQ ID NO:63;

p. amino acids 34-175 of SEQ ID NO:65;

q. amino acids 34-175 of SEQ ID NO:66;

r. amino acids 34-175 of SEQ ID NO:67;

s. amino acids 34-175 of SEQ ID NO:69;

t. amino acids 42-188 of SEQ ID NO:71;

u. amino acids 43-189 of SEQ ID NO:73;

v. amino acids 42-188 of SEQ ID NO:74;

w. amino acids 42-188 of SEQ ID NO:75;

x. amino acids 40-186 of SEQ ID NO:78;

y. amino acids 40-186 of SEQ ID NO:79;

z. amino acids 40-186 of SEQ ID NO:81;

aa. amino acids 32-124 of SEQ ID NO:83;

bb. amino acids 28-120 of SEQ ID NO:85;

cc. amino acids 28-120 of SEQ ID NO:87;

dd. amino acids 33-512 of SEQ ID NO:88; and

ee. amino acids 32-174 of SEQ ID NO:89.

14. The pharmaceutical composition of claim 1 , wherein said isolated polypeptide comprises a C. difficile type IV pilin antigenic fragment that is an internal fragment of the type IV pilin lacking both the amino and carboxyl terminal amino acids and being 30-100 amino acid residues in length.

15. A pharmaceutical composition for inducing an immune response against Clostridium difficile comprising an isolated polypeptide comprising

i) an antigenic fragment of a C. difficile type IV pilin, wherein the antigenic fragment is recombinantly produced, wherein the antigenic fragment is selected from the group consisting of:

a. amino acids 35-173 of SEQ ID NO:46;

b. amino acids 35-173 of SEQ ID NO:47;

c. amino acids 35-173 of SEQ ID NO:48;

d. amino acids 31-116 of SEQ ID NO:50;

e. amino acids 31-116 of SEQ ID NO:51;

f. amino acids 32-162 of SEQ ID NO:52;

g. amino acids 31-267 of SEQ ID NO:53;

h. amino acids 36-272 of SEQ ID NO:54;

i. amino acids 36-272 of SEQ ID NO:55;

j. amino acids 36-272 of SEQ ID NO:56;

k. amino acids 31-267 of SEQ ID NO:57;

l. amino acids 31-267 of SEQ ID NO:58;

m. amino acids 31-267 of SEQ ID NO:59;

n. amino acids 31-267 of SEQ ID NO:60;

o. amino acids 34-175 of SEQ ID NO:63;

p. amino acids 34-175 of SEQ ID NO:65;

q. amino acids 34-175 of SEQ ID NO:66;

r. amino acids 34-175 of SEQ ID NO:67;

s. amino acids 34-175 of SEQ ID NO:69;

t. amino acids 42-188 of SEQ ID NO:71;

u. amino acids 43-189 of SEQ ID NO:73;

v. amino acids 42-188 of SEQ ID NO:74;

w. amino acids 42-188 of SEQ ID NO:75;

x. amino acids 40-186 of SEQ ID NO:78;

y. amino acids 40-186 of SEQ ID NO:79;

z. amino acids 40-186 of SEQ ID NO:81;

aa. amino acids 32-124 of SEQ ID NO:83;

bb. amino acids 28-120 of SEQ ID NO:85;

cc. amino acids 28-120 of SEQ ID NO:87;

dd. amino acids 33-512 of SEQ ID NO:88; and

ee. amino acids 32-174 of SEQ ID NO:89; and

ii) an affinity tag sequence to facilitate purification.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 27, 2014
From: DONNENBERG, MICHAEL
To: UNIVERSITY OF MARYLAND, BALTIMORE
Reel/Frame 032964/0645 →
CONFIRMATORY LICENSE Recorded May 6, 2014
From: THE UNIVERSITY OF MARYLAND, BALTIMORE
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 032829/0557 →
Continuity (4)
Continuation In Part 13974825 · Aug 23, 2013
Continuation 13321399
Provisional Application 61179747 · May 20, 2009
Related Publication 20140227314A1 · Aug 14, 2014