EP0119031A2
Expired / Lapsed
EP
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Avirulent Vibrio Cholerae and methods for its production
Assignee: University of Maryland, Baltimore
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Filing: 1984-02-28
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Granted: —
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Expires: 2004-02-28
Abstract
Abstract Culture of Vibrio cholerae comprising a V. cholerae strain having a restriction endonuclease DNA fragment deleted. The strain is then avirulent, while it retains capacity to colonise the intestine of a host animal. -
Inventors
- 👤 James B. Kaper
- 👤 Myron M. Levine
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-31 08:34
CPC Classifications
A; A61; A61K; A61K39/00; A61K39/02; A61K39/107; C; C07; C07K; C07K14/00; C07K14/195; C07K14/28; C12; C12N; C12N1/00; C12N1/20; C12N1/205; C12N15/00; C12N15/01; A61K2039/51; A61K2039/52; A61K2039/522; C12R; C12R2001/00; C12R2001/01; C12R2001/63; Y; Y02; Y02A; Y02A50/00; Y02A50/30; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/909
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
33 words
A culture of Vibrio cholerae comprising a Vibro cholerae strain having a restriction endonuclease fragment of DNA deleted to confer avirulence and to retain capacity to colonize the intestine of a host animal.
Claim 2
Dependent Claim
33 words
A culture of Vibrio cholerae comprising a Vibro cholerae strain having a restriction endonuclease fragment of DNA deleted to confer avirulence and to retain capacity to colonize the intestine of a host animal.
Claim 3
Dependent Claim
129 words
The culture of Vibrio cholerae according to claim 1 wherein said strain is constructed by selection of an in vivo recombinant between a virulent Vibrio cholerae and a first plasmid, said first plasmid comprising Vibrio cholerae flanking sequences of the deleted DNA and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted DNA, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination, said selection being performed by selecting for said first selectable marker and for a second selectable marker to isolate an in vivo recombinant in the presence of a second plasmid carrying the second selectable marker, said second plasmid being incompatible with the first plasmid.
Claim 4
Dependent Claim
22 words
The culture of Vibrio cholerae according to claim 2 wherein said deleted DNA codes for the Vibrio cholerae toxins, or portion thereof.
Claim 5
Dependent Claim
25 words
The culture of Vibrio cholerae according to claim 2 wherein said sequences that flank the deleted DNA are about 7,000 base pairs on each side.
Claim 6
Dependent Claim
22 words
The culture of Vibrio cholerae according to claim 2 wherein said first selectable marker and said second selectable marker are resistance genes.
Claim 7
Dependent Claim
22 words
The culture of Vibrio cholerae according to any of claims 2-5 wherein said virulent Vibrio cholerae is N16961 of the Inaba serotype.
Claim 8
Dependent Claim
17 words
The culture of Vibrio cholerae according to any of claims 2-5 wherein said first plasmid is pJBK55.
Claim 9
Dependent Claim
17 words
The culture of Vibrio cholerae according to any of claims 2-5 wherein said second plasmid is pR702.
Claim 10
Dependent Claim
18 words
The culture of Vibrio cholerae according to any of claims 2-5 wherein said in vivo recombinant is JBK56.
Claim 11
Dependent Claim
38 words
The culture of Vibrio cholerae according to any of claims 2-5 wherein the first selectable marker of the in vivo recombinant is inactivated by insertion of a third selectable marker to yield a final strain of Vibrio cholerae.
Claim 12
Dependent Claim
15 words
The culture of Vibrio cholerae according to claim 10 wherein said final strain is JBK70.
Claim 13
Dependent Claim
23 words
The culture of Vibrio cholerae according to claim 10 wherein said final strain carries a plasmid expressing the B subunit of cholera toxin.
Claim 14
Dependent Claim
15 words
The culture of Vibrio cholerae according to claim 12 wherein said final strain is JBK70(pJBK51).
Claim 15
Dependent Claim
27 words
The culture of Vibrio cholerae according to any of claims 1 and 3 wherein said restriction endonuclease fragment comprises one or more fragments of the tox gene.
Claim 16
Dependent Claim
20 words
The culture of Vibrio cholerae according to any of claims 1-5 wherein said culture is useful for vaccination against cholera.
Claim 17
Dependent Claim
17 words
The culture of Vibrio cholerae according to claim 11 wherein JBK70 is useful for vaccination against cholera.
Claim 18
Independent Claim
154 words
A method of isolating deletion mutants of Vibrio cholerae comprising the steps of (a) contructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination; and (b) mating a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid; and (c) selecting for Vibrio cholerae expressing the first selectable marker; and (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incommpatible with the first plasmid; and (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant.
Claim 19
Independent Claim
154 words
A method of isolating deletion mutants of Vibrio cholerae comprising the steps of (a) contructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination; and (b) mating a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid; and (c) selecting for Vibrio cholerae expressing the first selectable marker; and (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incommpatible with the first plasmid; and (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant.
Claim 20
Dependent Claim
61 words
A method according to claim 17 further comprising the steps of (f) mating the in vivo recombinant of step (e) of claim 17 with a third microorganism carrying a third plasmid, said third plasmid carrying a third selectable marker; and (g) selecting for Vibrio cholerae expressing the third selectable marker; and (h) curing the selected product of step (g) of plasmids.
Claim 21
Dependent Claim
31 words
A method according to claim 18 wherein the first selectable marker and the second selectable marker are antibiotic resistance genes, and the third selectable marker is resistance to a heavy metal.
Claim 22
Dependent Claim
21 words
A method according to claim 18 wherein the first microorganism, the second microorganism, and the third microorganism are E. coli strains.
Claim 23
Dependent Claim
18 words
A method according to claim 18 wherein the selected product of step (g) of claim 18 is JBK70.
Claim 24
Dependent Claim
23 words
A method according any of claims 17 and 18 wherein the selected product of step (e) of claim 17 is V. cholerae JBK56.
Claim 25
Dependent Claim
19 words
The culture of Vibrio cholerae according to claim 12 wherein said final strain is useful for vaccination against cholera.
Claim 26
Dependent Claim
19 words
The culture of Vibrio cholerae according to claim 13 wherein said final strain is useful for vaccination against cholera.
Claim 27
Dependent Claim
23 words
The culture of Vibrio cholerae according to claim 1 or 2 or 3 or 5, wherein said virulent Vibrio cholerae is Ogawa 395.
Claim 28
Dependent Claim
19 words
The culture of Vibrio cholerae according to claim 2 or 3 or 5, wherein said first plasmid is pJBK107.
Claim 29
Dependent Claim
19 words
The culture of Vibrio cholerae according to claim 2 or 3 or 5 wherein said second plasmid is pR751.
Claim 30
Dependent Claim
20 words
The culture of Vibrio cholerae according to claim 2 or 3 or 5, wherein said in vivo recombinant is JBK113.
Claim 31
Dependent Claim
15 words
The culture of Vibrio cholerae according to claim 10, wherein said final strain is CVD101.
Claim 32
Dependent Claim
17 words
The culture of Vibrio cholerae according to claim 29, wherein CVD101 is useful for vaccination against cholera.
Claim 33
Dependent Claim
38 words
A culture of Vibrio cholerae comprising a first Vibrio cholerae strain having a restriction endonuclease fragment deleted to confer avirulence and to confer substantially 100% efficacy in protecting humans from subsequent infection by a second Vibrio cholerae strain.
Claim 34
Dependent Claim
38 words
A culture of Vibrio cholerae comprising a first Vibrio cholerae strain having a restriction endonuclease fragment deleted to confer avirulence and to confer substantially 100% efficacy in protecting humans from subsequent infection by a second Vibrio cholerae strain.
Claim 35
Dependent Claim
51 words
A method according to claim 17 further comprising the steps of (f) mating the in vivo recombinant of step (e) of claim 17 with a third microorganism carrying a third plasmid, said third plasmid not carrying said first selectable marker; and (g) selecting for Vibrio cholerae lacking said first selectable marker.
Claim 36
Dependent Claim
32 words
A method according to claim 32, wherein said third plasmid lacks said first marker, said first marker having been deleted from the corresponding resistance gene to said antibiotic after in vivo recombination.
Claim 37
Dependent Claim
18 words
A method according to claim 33, wherein the selected product of step (g) of claim 32 is CVD101.
References Cited (Backward Citations)
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