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Patent 2284736 Summary

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(12) Patent Application: (11) CA 2284736
(54) English Title: MYCOBACTERIUM RECOMBINANT VACCINES
(54) French Title: VACCINS RECOMBINES A BASE DE MYCOBACTERIES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/74 (2006.01)
  • A61K 38/19 (2006.01)
  • A61K 39/04 (2006.01)
  • C12N 1/20 (2006.01)
  • C12N 15/87 (2006.01)
  • C12N 15/90 (2006.01)
(72) Inventors :
  • LABIDI, ABDEL HAKIM (United States of America)
(73) Owners :
  • CYTOCLONAL PHARMACEUTICS, INC. (United States of America)
(71) Applicants :
  • CYTOCLONAL PHARMACEUTICS, INC. (United States of America)
(74) Agent: KIRBY EADES GALE BAKER
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1998-03-27
(87) Open to Public Inspection: 1998-10-08
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1998/006056
(87) International Publication Number: WO1998/044096
(85) National Entry: 1999-09-27

(30) Application Priority Data:
Application No. Country/Territory Date
60/042,849 United States of America 1997-03-28

Abstracts

English Abstract




Mycobacterium recombinant vaccines for treatment of intracellular diseases
have been developed utilizing an antigen delivery system in the form of
Mycobacterium strains, a genetic transfer system in the form of cloning
nonpathogenic and expression vectors, and related technologies to provide
products combining nontoxic immuno-regulating Mycobacterium adjuvants,
nontoxic immuno-stimulating exogenous antigens specific for a variety of
diseases, and nontoxic amounts of cytokines that boost the TH-1 pathway.
Cloning and expression Mycobacterium vectors include both extra-nuclear and
integrative vectors.


French Abstract

Cette invention se rapporte à des vaccins recombinés à base de mycobactéries destinés au traitement de maladies intracellulaires, que l'on a développé au moyen d'un système d'administration d'antigènes se présentant sous la forme de souches de mycobactéries, d'un système de transfert génétique se présentant sous la forme de vecteurs d'expression et de clonage non pathogènes et de techniques associées. On a ainsi développé des produits combinant des adjuvants mycobactériens immuno-régulateurs, non toxiques, des antigènes exogènes immuno-stimulateurs, non toxiques, spécifiques d'une variété de maladies et des quantités non toxiques de cytokines qui renforcent la voie T¿H?-1. Les vecteurs mycobactériens d'expression et de clonage incluent à la fois des vecteurs extra-cellulaires et des vecteurs d'intégration.

Claims

Note: Claims are shown in the official language in which they were submitted.



83
WE CLAIM:
1. A pharmaceutical composition for administration to a human or animal
providing a continuous source of a protein of interest into said human or
animal
upon administration thereto and stimulating the cellular immunity of said
human or
animal upon administration thereto made according the steps of:
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture at about 4°C to obtain a pellet of
live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold glycerol and
centrifuging at about 4°C to obtain electro-competent Mycobacterium
cells;
(f) mixing said electro-competent Mycobacterium cells with integrating
vector DNA, said integrating vector DNA comprising a first coding region for a
protein of interest cloned under the control of a promoter sequence recognized
in
Mycobacterium, a second coding region for an attachment site and an integrase
gene from mycobacteriophage D29, and a third coding region for a selection
marker suitable for said Mycobacterium strain to form a transformation
mixture;
(g) performing electroporation on said transformation mixture to form an
electroporated culture comprising transformed Mycobacterium cells, said
transformed Mycobacterium cells having said integrating vector DNA
incorporated
into the genome of said Mycobacterium strain at a transformation efficiency
rate of
greater than or equal to 10 7 transformants per microgram of vector DNA and
being
capable of expressing said protein of interest after administration into said
human
or animal; and
(h) isolating said transformed Mycobacterium cells from non-transformed
Mycobacterium cells by growing said electroporated culture in the presence of
a



84

substance which said selection marker permits transformed Mycobacterium cells
to
be distinguished from non-transformed Mycobacterium cells.
2. The composition of claim 1, made by a method further comprising the
step of transferring the transformed Mycobacterium cell culture of step (h)
from
said culture medium to a liquid protein-free culture medium and cultivating
said
transformed Mycobacterium cells in said liquid protein-free culture medium
under
appropriate conditions for vaccine production.
3. The composition of claim 1, wherein said second coding region for said
attachment site and integrase gene from said mycobacteriophage D29 essentially
consisting of the sequence provided in SEQ ID No:5.
4. The composition of claim 1, wherein said second coding region for said
attachment site and integrase gene from said mycobacteriophage D29 essentially
consisting of the sequence provided in SEQ ID No:6.
5. The composition of claim 1, wherein said second coding region for said
attachment site and integrase gene from said mycobacteriophage D29 essentially
consisting of the sequence provided in SEQ ID No:7.
6. The composition of claim 1, wherein said third coding region for said
selection marker suitable for said Mycobacterium strain comprising a kanamycin
selection marker essentially consisting of the sequence provided in SEQ ID
No:2.
7. The composition of claim 1, wherein said second coding region for an
attachment site and an integrase gene from a mycobacteriophage D29 variant.
8. The composition of claim 1, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium


85

triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium
chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and
Mycobacterium vaccae.
9. The composition of claim 1, further comprising a cytokine associated
with cellular immunity.
10. The composition of claim 1, further comprising a chemotherapeutic
agent.
11. A pharmaceutical composition fir administration to a human or animal
providing a continuous source of a protein of interest into said human or
animal
upon administration thereto and stimulating the cellular immunity of said
human or
animal upon administration thereto made according the steps of:
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture at about 4°C to obtain a pellet of
live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold glycerol and
centrifuging at about 4°C to obtain electro-competent Mycobacterium
cells;
(f) mixing said electro-competent N ycobacterium cells with integrating
vector DNA, said integrating vector DNA comprising a first coding region for a
protein of interest cloned under the control of a promoter sequence recognized
in



86

Mycobacterium, a second coding region for the minimal functional component of
an attachment site and an integrase gene from mycobacteriophage D29, and a
third
coding region for a selection marker suitable for said Mycobacterium strain to
form
a transformation mixture;
(g) performing electroporation on said transformation mixture to form an
electroporated culture comprising transformed Mycobacterium cells, said
transformed Mycobacterium cells having said integrating vector DNA
incorporated
into the genome of said Mycobacterium strain at a transformation efficiency
rate of
greater than or equal to 10 7 transformants per microgram of vector DNA and
being
capable of expressing said protein of interest after administration into said
human
or animal; and
(h) isolating said transformed Mycobacterium cells from nontransformed
Mycobacterium cells by growing said electroporated culture in the presence of
a
substance which said selection marker permits transformed Mycobacterium cells
to
be distinguished from non-transformed Mycobacterium cells.
12. The composition of claim 11, made by a method further comprising the
step of transferring the transformed Mycobacterium cell culture of step (h)
from
said culture medium to a liquid protein-free culture medium and cultivating
said
transformed Mycobacterium cells in said liquid protein-free culture medium
under
appropriate conditions for vaccine production.
13. The composition of claim 11, wherein said second coding region for
said minimal functional component of said attachment site and integrase gene
from
said mycobacteriophage D29 essentially consisting of the sequence provided in
SEQ ID No:5.
14. The composition of claim 11, wherein said second coding region for
said minimal functional component of said attachment site and integrase gene
from


87

said mycobacteriophage D29 essentially consisting of the sequence provided in
SEQ ID No:6.
15. The composition of claim 11, wherein said second coding region for
said minimal functional component of said attachment site and integrase gene
from
said mycobacteriophage D29 essentially consisting of the sequence provided in
SEQ ID No:7.
16. The composition of claim 11, wherein said second coding region for an
attachment site and an integrase gene from a mycobacteriophage D29 variant.
17. The composition of claim 11, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium
triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium
chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and
Mycobacterium vaccae.
18. The composition of claim 11, further comprising a cytokine associated
with cellular immunity.
19. The composition of claim 11, funkier comprising a chemotherapeutic
agent.
20. A pharmaceutical composition for administration to a human or animal
providing a continuous source of a protein of interest into said human or
animal
upon administration thereto and stimulating the cellular immunity of said
human or
animal upon administration thereto made according the steps of:
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a




88

commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture at about 4 ° C to obtain a pellet
of live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold glycerol and
centrifuging at about 4°C to obtain electro-competent Mycobacterium
cells;
(f) mixing said electro-competent Mycobacterium cells with integrating
vector DNA, said integrating vector DNA comprising a first coding region for a
protein of interest cloned under the control of a promoter recognized in
Mycobacterium, a second coding region for an attachment site and integrase
gene
from mycobacteriophage D29, and the minimal functional component of a third
coding region for a selection marker suitable for said Mycobacterium strain to
form
a transformation mixture;
(g) performing electroporation on said transformation mixture to form an
electroporated culture comprising transformed Mycobacterium cells, said
transformed Mycobacterium cells having said integrating vector DNA
incorporated
into the genome of said Mycobacterium strain at a transformation efficiency
rate of
greater than or equal to 10 7 transformants per microgram of vector DNA and
being
capable of expressing said protein of interest after administration into said
human
or animal; and
(h) isolating said transformed Mycobacterium cells from nontransformed
Mycobacterium cells by growing said electroporated culture in the presence of
a
substance which said selection marker permits transformed Mycobacterium cells
to
be distinguished from non-transformed Mycobacterium cells.
21. The composition of claim 20, made by a method further comprising the
step of transferring the transformed Mycobacterium cell culture of step (h)
from






89

said culture medium to a liquid protein-free culture medium and cultivating
said
transformed Mycobacterium cells in said liquid protein-free culture medium
under
appropriate conditions for vaccine production.
22. The composition of claim 20, wherein said third coding region for said
minimal functional component of said selection marker suitable for said
Mycobacterium strain comprising a kanamycin selection marker essentially
consisting of the sequence provided in SEQ ID No:2.
23. The composition of claim 20, wherein said second coding region for an
attachment site and an integrase gene from mycobacteriophage D29 variant.
24. The composition of claim 20, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium
triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium
chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and
Mycobacterium vaccae.
25. The composition of claim 20, further comprising a cytokine associated
with cellular immunity.
26. The composition of claim 20, further comprising a chemotherapeutic
agent.
27. A pharmaceutical composition for administration to a human or animal
providing a continuous source of a protein of interest into said human or
animal
upon administration thereto and stimulating the cellular immunity of said
human or
animal upon administration thereto made according the steps of:



90
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture about about 4°C to obtain a pellet
of live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold glycerol and
centrifuging at about 4°C to obtain electro-competent Mycobacterium
cells;
(f) mixing said electro-competent Mycobacterium cells with an
extra-chromosomal DNA vector, said extra-chromosomal DNA vector comprising a
first
coding region for a protein of interest cloned under the control of a promoter
sequence recognized in Mycobacterium, a second region supporting replication
in
Mycobacterium, and a third coding region for a selection marker suitable for
said
Mycobacterium strain to form a transformation mixture;
(g) performing electroporation on said transformation mixture to form an
electroporated culture comprising transformed Mycobacterium cells, said
Mycobacterium cells comprising said extra-chromosomal DNA vector transformed
at a transformation efficiency rate of greater than or equal to 10 7
transformants per
microgram of vector DNA and being capable of expressing said protein of
interest
after administration into said human or animal; and
(h) isolating said transformed Mycobacterium cells from nontransformed
Mycobacterium cells by growing said electroporated culture in the presence of
a
substance which said selection marker permits transformed Mycobacterium cells
to
be distinguished from non-transformed Mycobacterium cells.
28. The composition of claim 27, made by a method further comprising the
step of transferring the transformed Mycobacterium cell culture of step (h)
from



91
said culture medium to a liquid protein-free culture medium and cultivating
said
transformed Mycobacterium cells in said liquid protein-free culture medium
under
appropriate conditions for vaccine production.
29. The composition of claim 27, wherein said second region supporting
replication in Mycobacterium essentially consisting of the sequence of an
origin of
replication of a Mycobacterium plasmid provided in SEQ ID No:3.
30. The composition of claim 27, wherein said second region supporting
replication in Mycobacterium essentially consisting of the sequence of an
origin of
replication of a Mycobacterium plasmid provided in SEQ ID No:4.
31. The composition of claim 27, wherein said coding region for a
selection marker suitable far said Mycobacterium strain comprising a kanamycin
selection marker essentially consisting of the sequence provided in SEQ ID
No:2.
32. The composition of claim 27, wherein said Mycobacterium plasmid is
pAL 5000.
33. The composition of claim 27, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium
triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium
chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and
Mycobacterium vaccae.
34. The composition of claim 27, further comprising a cytokine associated
with cellular immunity.




92



35. The composition of claim 27, further comprising a chemotherapeutic
agent.
36. A pharmaceutical composition for administration to a human or animal
providing a continuous source of a protein of interest into said human or
animal
upon administration thereto and stimulating the cellular immunity of said
human or
animal upon administration thereto made according the steps of:
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture at about 4°C to obtain a pellet of
live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold glycerol and
centrifuging at about 4°C to obtain electro-competent Mycobacterium
cells;
(f) mixing said electro-competent Mycobacterium cells with an
extra-chromosomal DNA vector, said extra-chromosomal DNA vector comprising a
first
coding region for a protein of interest cloned under the control of a promoter
sequence recognized in Mycobacterium, a second region for the minimal
functional
component supporting replication in Mycobacterium, and a third coding region
for
a selection marker suitable for said Mycobacterium strain to form a
transformation
mixture;
(g) performing electroporation on said transformation mixture to form an
electroporated culture comprising transformed Mycobacterium cells, said
Mycobacterium cells comprising said extra-chromosomal DNA vector transformed
at a transformation efficiency rate of greater than or equal to 10 7
transformants per




93



microgram of vector DNA and being capable of expressing said protein of
interest
after administration into said human or animal; and
(h) isolating said transformed Mycobacterium cells from nontransformed
Mycobacterium cells by growing said electroporated culture in the presence of
a
substance which said selection marker permits transformed Mycobacterium cells
to
be distinguished from non-transformed Mycobacterium cells.
37. The composition of claim 36, made by a method further comprising the
step of transferring the transformed Mycobacterium cell culture of step (h)
from
said culture medium to a liquid protein-free culture medium and cultivating
said
transformed Mycobacterium cells in said liquid protein-free culture medium
under
appropriate conditions for vaccine production.
38. The composition of claim 36, wherein said second region for the
minimal functional component supporting replication in Mycobacterium
essentially
consisting of the sequence of an origin of replication of a Mycobacterium
plasmid
provided in SEQ ID No:3.
39. The composition of claim 36, wherein said second region for the
minimal functional component supporting replication in Mycobacterium
essentially
consisting of the sequence of an origin of replication of a Mycobacterium
plasmid
provided in SEQ ID No:4.
40. The composition of claim 36, wherein said Mycobacterium plasmid is
pAL 5000.
41. The composition of claim 36, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium
triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium
chitae, Mycobacterium duvalti, Mycobacterium flavescens, Mycobacterium




94



nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and,
Mycobacterium vaccae.
42. The composition of claim 36, further comprising a cytokine associated
with cellular immunity.
43. The composition of claim 36, further comprising a chemotherapeutic
agent.
44. A pharmaceutical composition for administration to a human or animal
providing a continuous source of a protein of interest into said human or
animal
upon administration thereto and stimulating the cellular immunity of said
human, or
animal upon administration thereto made according the steps of:
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture at about 4°C to obtain a pellet of
live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold glycerol and
centrifuging at about 4°C to obtain electro-competent Mycobacterium
cells;
(f) mixing said electro-competent Mycobacterium cells with an
extra-chromosomal DNA vector, said extra-chromosomal DNA vector comprising a
first
coding region for a protein of interest cloned under the control of a promoter
sequence recognized in Mycobacterium, a second region supporting replication
in
Mycobacterium, and a third coding region for the minimal functional component
of




95
a selection marker suitable for said Mycobacterium strain to form a
transformation
mixture;
(g) performing electroporation on said transformation mixture to form an
electroporated culture comprising transformed Mycobacterium cells, said
Mycobacterium cells comprising said extra-chromosomal DNA vector transformed
at a transformation efficiency rate of greater than or equal to 10 7
transformants per
microgram of vector DNA and being capable of expressing said protein of
interest
after administration into said human or animal; and
(h) isolating said transformed Mycobacterium cells from nontransformed
Mycobacterium cells by growing said electroporated culture in the presence of
a
substance which said selection marker permits transformed Mycobacterium cells
to
be distinguished from non-transformed Mycobacterium cells.
45. The composition of claim 44, made by a method further comprising the
step of transferring the transformed Mycobacterium cell culture of step (h)
from
said culture medium to a liquid protein-free culture medium and cultivating
said
transformed Mycobacterium cells in said liquid protein-free culture medium
under
appropriate conditions for vaccine production.
46. The composition of claim 44, wherein said coding region for the
minimal functional component of said selection marker suitable for said
Mycobacterium strain comprising a kanamycin selection marker essentially
consisting of the sequence provided in SEQ ID No:2.
47. The composition of claim 44, wherein said Mycobacterium plasmid is
pAL 5000.
48. The composition of claim 44, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium
triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium




96
chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and
Mycobacterium vaccae.
49. The composition of claim 44, further comprising a cytokine associated
with cellular immunity.
50. The composition of claim 44, further comprising a chemotherapeutic
agent.
51. A pharmaceutical composition for administration to a human or
animal, said composition capable of stimulating the cellular immunity of said
human or animal upon administration into said human or animal comprising the
steps of:
(a) cultivating an inoculum of at least one live Mycobacterium strain,
which is nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strain at an appropriate temperature to obtain a liquid cell
culture;
(b) cooling said culture to about 4°C;
(c) centrifuging said cooled culture to obtain a pellet of live
Mycobacterium cells and a supernatant;
(d) separating said pellet from said supernatant;
(e) washing said pellet by suspending in sterile cold saline and centrifuging
at about 4°C to obtain washed live Mycobacterium cells; and
(f) mixing said live Mycobacterium cells with an antigen of interest.
52. The composition of claim 51, wherein said Mycobacterium strain is
selected from the group consisting of Mycobacterium gastri, Mycobacterium
triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium




chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and
Mycobacterium vaccae.
53. The composition of claim 51, further comprising a cytokine associated
with cellular immunity.
54. The composition of claim 51, further comprising a chemotherapeutic
agent.
55. A pharmaceutical composition for administration to a human or
animal, said composition capable of stimulating the cellular immunity of said
human or animal upon administration into said human or animal comprising the
steps of:
(a) cultivating an inoculum of two or more live Mycobacterium strains,
which are nonpathogenic to said human or animal and capable of sustaining a
commensal symbiotic relationship with macrophages in said human or animal, in
a
liquid culture medium capable of providing sufficient nutrients for growth of
said
Mycobacterium strains at an appropriate temperature to obtain a liquid cell
culture;
(b) centrifuging said culture to obtain a pellet of live Mycobacterium cells
and a supernatant;
(c) separating said pellet from said supernatant;
(d) washing said pellet and centrifuging to obtain washed Mycobacterium
cells;
(e) killing said washed Mycobacterium cells to obtain Mycobacterium
adjuvant; and
(f) mixing said Mycobacterium adjuvant with an antigen of interest.
56. The composition of claim 55, wherein said Mycobacterium strains are
selected from the group consisting of Mycobacterium gastri, Mycobacterium




98

triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium
chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium
nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium vaccae, and
Mycobacterium neoaurum.
57. The composition of claim 55, further comprising a cytokine associated
with cellular immunity.
58. The composition of claim 55, further comprising a chemotherapeutic
agent.
59. A shuttle vector comprising a first region supporting replication in
Escherichia coli, a second coding region for an attachment site and an
integrase
gene from mycobacteriophage D29, and a third coding region for a selection
marker suitable for both Escherichia coli and Mycobacterium, said first region
supporting replication in Escherichia coli comprising the minimal functional
component of said origin of replication for Escherichia coli, wherein said
shuttle
vector is capable of transforming bacteria at a transformation efficiency rate
of
greater than or equal to 10 7 transformants per microgram of vector DNA.
60. The shuttle vector according to claim 59, wherein said minimal
functional component of said origin of replication for Escherichia coli
essentially
consisting of the sequence provided in SEQ ID NO:1.
61. The shuttle vector according to claim 59, wherein said second coding
region for an attachment site and an integrase gene from mycobacteriophage D29
variant.
62. A shuttle vector comprising a first region supporting replication for
Escherichia coli, a second coding region for an attachment site and an
integrase




99

gene from mycobacteriophage D29, and a third coding region for a selection
marker suitable for both Escherichia coli and Mycobacterium, said second
coding
region for said attachment site and integrase gene from said mycobacteriophage
D29 comprising the minimal functional component of said attachment site and
integrase gene from said mycobacteriophage D29, wherein said shuttle vector is
capable of transforming bacteria at a transformation efficiency rate of
greater than
or equal to 10 7 transformants per microgram of vector DNA.
63. The shuttle vector according to claim 62, wherein minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID NO:5.
64. The shuttle vector according to claim 62, wherein minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID NO:6.
65. The shuttle vector according to claim 62, wherein minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID NO:7.
66. The shuttle vector according to claim 62, wherein said second coding
region for an attachment site and an integrase gene from a mycobacteriophage
D29
variant.
67. A shuttle vector comprising a first region supporting replication in
Escherichia coli, a second coding region, for an attachment site and an
integrase
gene from mycobacteriophage D29, and a third coding region for a selection.
marker suitable for both Escherichia coli and Mycobacterium, said third coding
region for said selection marker suitable for both Escherichia coli and
Mycobacterium comprising the minimal functional component of said selection




100



marker suitable for both Escherichia coli and Mycobacterium, wherein said
shuttle
vector is capable of transforming bacteria at a transformation efficiency rate
of
greater than or equal to 10 7 transformants per microgram of vector DNA.
68. The shuttle vector according to claim 67, wherein said minimal
functional component of said selection marker suitable for both Escherichia
coli
and Mycobacterium comprising a kanamycin selection marker essentially
consisting of the sequence provided in SEQ ID NO:2.
69. The shuttle vector according to claim 67, wherein said second coding
region for an attachment site and an integrase gene from mycobacteriophage D29
variant.
70. A shuttle vector comprising a first region supporting replication in
Escherichia coli, a second region supporting replication in Mycobacterium, and
a
third coding region for a selection marker suitable for both Escherichia coli
and
Mycobacterium, said first region supporting replication in Escherichia coli
comprising the minimal functional component of said origin of replication for
Escherichia coli, wherein said shuttle vector is capable of transforming
bacteria at
a transformation efficiency rate of greater than or equal to 10 7
transformants per
microgram of vector DNA.
71. The shuttle vector according to claim 70, wherein said minimal
functional component of said origin of replication for Escherichia coli
essentially
consisting of the sequence provided in SEQ ID No:1.
72. A shuttle vector comprising a first region supporting replication in
Escherichia coli, a second region supporting replication in Mycobacterium, and
a
third coding region for a selection marker suitable for both Escherichia coli
and
Mycobacterium, said second region supporting replication in Mycobacterium




101


essentially comprising the minimal functional. component of said origin of
replication of said Mycobacterium plasmid, wherein said shuttle vector is
capable
of transforming bacteria at a transformation efficiency rate of greater than
or equal
to 10 7 transformants per microgram of vector DNA.
73. The shuttle vector according to claim 72, wherein said minimal
functional component of said origin of replication of said Mycobacterium
plasmid
essentially consisting of the sequence provided in SEQ ID No:3.
74. The shuttle vector according to claim 72, wherein said minimal
functional component of said origin of replication of said Mycobacterium
plasmid
essentially consisting of the sequence provided in SEQ ID No:4.
75. A shuttle vector comprising a first region supporting replication in
Escherichia toll, a second region supporting replication in Mycobacterium, and
a
third coding region for a selection marker suitable for both Escherichia toll
and
Mycobacterium, said third coding region for a selection marker suitable for
both
Escherichia coli and Mycobacterium comprising the minimal functional
component of said selection marker suitable for both Escherichia coli and
Mycobacterium, wherein said shuttle vector is capable of transforming bacteria
at a
transformation efficiency rate of greater than or equal to 10 7 transformants
per
microgram of vector DNA.
76. The shuttle vector according to claim 75, wherein said minimal
functional component of said selection marker suitable for both Escherichia
toll
and Mycobacterium comprising a kanamycin selection marker essentially
consisting of the sequence provided in SEQ ID NO:2.
77. A vector comprising a first coding region for an attachment site and
integrase gene from mycobacteriophage D29 and a second coding region for a




102

selection marker suitable for Mycobacterium, said first coding region for said
attachment site and integrase gene from said mycobacteriophage D29 comprising
the minimal functional component of said attachment site and integrase gene
from
said mycobacteriophage D29, wherein said vector is capable of transforming
bacteria at a transformation efficiency rate of greater than or equal to 10 7
transformants per microgram of vector DNA.
78. The vector according to claim 77, wherein said minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID No:5.
79. The vector according to claim 77, wherein said minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID No:6.
80. The vector according to claim 77, wherein said minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID No:7.
81. The shuttle vector according to claim 77, wherein said second coding
region for an attachment site and an integrase gene from a mycobacteriophage
D29
variant.
82. A vector comprising a first coding region for an attachment site and
integrase gene from mycobacteriophage D29 and a second coding region for a
selection marker suitable for Mycobacterium; said second coding region for
said
selection marker suitable for said Mycobacterium strain comprising the minimal
functional component of said selection marker suitable for said Mycobacterium
strain, wherein said shuttle vector is capable of transforming bacteria at a




103

transformation efficiency rate of greater than or equal to 10 7 transformants
per
microgram of vector DNA.
83. The vector according to claim 82, wherein said minimal functional
component of said selection marker suitable for said Mycobacterium strain
comprising a kanamycin selection marker essentially consisting of the sequence
provided in SEQ ID No:2.
84. The shuttle vector according to claim 82, wherein said second coding
region for an attachment site and an integrase gene from mycobacteriophage D29
variant.
85. A vector for carrying and expressing selected genes of a
Mycobacterium strain comprising a first coding region for an attachment site
and
integrase gene from mycobacteriophage D29 and a second coding region for a
selection marker suitable for Mycobacterium, said first coding region for said
attachment site and integrase gene from said mycobacteriophage D29 comprising
the minimal functional component of said attachment site and integrase gene
from
said mycobacteriophage D29, wherein said vector is capable of transforming
bacteria at a transformation efficiency rate of greater than or equal to 10 7
transformants per microgram of vector DNA.
86. The vector according to claim 85, wherein said minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID No:5.
87. The vector according to claim 85, wherein said minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID No:6.



104

88. The vector according to claim 85, wherein said minimal functional
component of said attachment site and integrase gene from said
mycobacteriophage
D29 essentially consisting of the sequence provided in SEQ ID No:7.
89. The shuttle vector according to claim 85, wherein said second coding
region for an attachment site and an integrase gene from a mycobacteriophage
D29
variant.
90. A vector for carrying and expressing selected genes of a
Mycobacterium strain comprising a first coding region for an attachment site
and
integrase gene from mycobacteriophage D29 and a second coding region for a
selection marker suitable for Mycobacterium, said second coding region for
said
selection marker suitable for said Mycobacterium strain comprising the minimal
functional component of said selection marker suitable for said Mycobacterium
strain, wherein said vector is capable of transforming bacteria at a
transformation
efficiency rate of greater than or equal to 10 7 transformants per microgram
of
vector DNA.
91. The vector according to claim 90, wherein said minimal functional
component of said selection marker suitable for said Mycobacterium strain
comprising a kanamycin selection marker essentially consisting of the sequence
provided in SEQ ID No:2.
92. The shuttle vector according to claim 90, wherein said second coding
region for an attachment site and an integrase gene from mycobacteriophage D29
variant.
93. A vector comprising a first region supporting replication in
Mycobacterium and a second coding region for a selection marker suitable for
Mycobacterium, said first region supporting replication in Mycobacterium




105

comprising the minimal functional component of said origin of replication of
said
Mycobacterium plasmid, wherein said vector is capable of transforming bacteria
at
a transformation efficiency rate of greater than or equal to 10 7
transformants per
microgram of vector DNA.
94. The vector according to claim 93, wherein said minimal functional
component of origin of replication of said Mycobacterium plasmid essentially
consisting of the sequence provided in SEQ ID No:3.
95. The vector according to claim 93, wherein said minimal functional
component of origin of replication of said Mycobacterium plasmid essentially
consisting of the sequence provided in SEQ ID No:4.
96. A vector comprising a first region supporting replication in
Mycobacterium and a second coding region for a selection marker suitable for
Mycobacterium, said second coding region for said selection marker suitable
for
said Mycobacterium comprising the minimal functional component of said
selection marker suitable for said Mycobacterium, wherein said vector is
capable of
transforming bacteria at a transformation efficiency rate of greater than or
equal to
10 7 transformants per microgram of vector DNA.
97. The vector according to claim 96, wherein said minimal functional
component of said selection marker suitable for said Mycobacterium comprising
a
kanamycin selection marker essentially consisting of the sequence provided in
SEQ
ID No:2.
98. A vector comprising a first region supporting replication in Escherichia
coli and a second coding region for a selection marker suitable for said
Escherichia
coli, said first coding region for an origin replication for Escherichia coli



106


comprising the minimal functional component of said origin of replication for
said
Escherichia coli.
99. The vector according to claim 98, wherein said minimal functional
component of said origin of replication of said Escherichia coli essentially
consisting of the sequence provided in SEQ ID No:1.
100. A vector comprising a first region supporting replication in
Escherichia coli and a second coding region for a selection marker suitable
for said
Escherichia coli, said second coding region for a selection marker suitable
for said
Escherichia coli comprising the minimal functional component of said selection
marker suitable for said Escherichia coli, wherein said vector is capable of
transforming bacteria at a transformation efficiency rate of greater than or
equal to
7 transformants per microgram of vector DNA.
101. The vector according to claim 100, wherein said minimal functional
component of said selection marker suitable for said Escherichia coli
comprising a
kanamycin selection marker for said Escherichia coli essentially consisting of
the
sequence provided in SEQ ID No:2.
102. A vector for carrying and expressing selected genes in Escherichia
coli comprising a first region supporting replication in Escherichia coli and
a
second coding region for a selection marker suitable for said Escherichia
coli, said
first region supporting replication in Escherichia coli comprising the minimal
functional component of said origin of replication for said Escherichia coli,
wherein said vector is capable of transforming bacteria at a transformation
efficiency rate of greater than or equal to 10 7 transformants per microgram
of
vector DNA.




107



103. The vector according to claim 102, wherein said minimal functional
component of said origin of replication of said Escherichia coli essentially
consisting of the sequence provided in SEQ ID No: 1.
104. A vector for carrying and expressing selected genes in Escherichia
coli comprising a first region supporting replication in Escherichia coli and
a
second coding region for a selection marker suitable for said Escherichia coli
strain, said second coding region for a selection marker suitable for said
Escherichia coli comprising the minimal functional component of said selection
marker suitable for said Escherichia coli, wherein said vector is capable of
transforming bacteria at a transformation efficiency rate of greater than or
equal to
7 transformants per microgram of vector DNA.
105. The vector according to claim 104, wherein said minimal functional
component of said selection marker suitable for said Escherichia coli
comprising a
kanamycin selection marker for said Escherichia coli essentially consisting of
the
sequence provided in SEQ ID No:2.
I
106. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 1 to stimulate cellular
immunity in said human or animal.
107. The method according to claim 106, further comprising
administration of a cytokine associated with cellular immunity.
108. The method according to claim 106, further comprising
administration of a chemotherapeutic agent.




108


109. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 11 to stimulate cellular
immunity in said human or animal.
110. The method according to claim 109, further comprising
administration of a cytokine associated with cellular immunity.
111. The method according to claim 109, further comprising
administration of a chemotherapeutic agent.
112. A method of administering to a human or animal an effective amount,
of the pharmaceutical composition according to claim 20 to stimulate cellular
immunity in said human or animal.

113. The method according to claim 112, further comprising
administration of a cytokine associated with cellular immunity.
114. The method according to claim 112, further comprising
administration of a chemotherapeutic agent.
115. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 27 to stimulate cellular
immunity in said human or animal.
116. The method according to claim 115, further comprising
administration of a cytokine associated with cellular immunity.
117. The method according to claim 115, further comprising
administration of a chemotherapeutic agent.




109

118. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 36 to stimulate cellular
immunity in said human or animal.
119. The method according to claim 118, further comprising
administration of a cytokine associated with cellular immunity.
120. The method according to claim 118, further comprising
administration of a chemotherapeutic agent.
121. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 44 to stimulate cellular
immunity in said human or animal.
122. The method according to claim 121, further comprising
administration of a cytokine associated with cellular immunity.

123. The method according to claim 121, further comprising
administration of a chemotherapeutic agent.
124. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 51 to stimulate cellular
immunity in said human or animal.
125. The method according to claims 124, further comprising
administration of a cytokine associated with cellular immunity.
126. The method according to claim 124, further comprising
administration of a chemotherapeutic agent:




110
127. A method of administering to a human or animal an effective amount
of the pharmaceutical composition according to claim 55 to stimulate cellular
immunity in said human or animal.
128. The method according to claim 127, further comprising
administration of a cytokine associated with cellular immunity.
129. The method according to claim 127, further comprising
administration of a chemotherapeutic agent.
130. A culture medium comprising about 0.25% proteose peptone; about
0.2% nutrient broth; about 0.075% pyruvic acid; about 0.05% sodium glutamate;
about 0.5% albumin fraction V; about 0.7% dextrose; about 0.0004% catalase;
about 0.005% oleic acid; L(-) amino-acid complex ( about 0.126% alanine; about
0.097% leucine; about 0.089% glycine; about 0.086% valine; about 0.074%
arginine; about 0.06% threonine; about 0.059% aspartic acid; about 0.057%
serine;
about 0.056% proline; about 0.05% glutamic acid; about 0.044% isoleucine;
about
0.033% glutamine; 0.029% phenylalanine; about 0.025% asparagine; about
0.024% lysine; about 0.023% histidine; about 0.021% tyrosine; about 0.02%
methionine; about 0.014% tryptophan; and about 0.01% cysteine); about 0.306%
Na2HPO4; about 0.055% KH2PO4; about 0.05% NH4Cl; about 0.335% NaCl; about
0.0001% ZnSO4; about 0.0001% CuSO4; about 0.0001% FeCl3; about 0.012%
MgSO4; and about 0.05% Tween 80; wherein the pH of said medium is about 7.
131. The culture medium according to claim 130, further comprising about
0.8% glycerol.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
MYCOBACTERIUM RECOMBINANT VACCINES
TECHNICAL FIELD OF THE INVENTION
The present invention relates to DNA constructs for cloning and methods of
cloning mycobacterium genes.


CA 02284736 1999-09-27
WO 98!44096 PCT/US98/06056
2
BACKGROUND OF THE INVENTION
The mammalian immune system comprises both humoral and cellular
components which are interrelated but have different roles. Although both
arms of the immune system involve helper T cells, the outcome of the immune
response depends on which subclass of T cells is involved. Helper T
lymphocytes are produced by two maturation pathways (TH-1 and TH-2), are
grouped according to cluster differentiation (CD4 and CD8), and secrete
different cytokines. Both components of the immune system constantly scan
and survey what is displayed in association with the molecules of the major
histocompatibility complex (MHC), at the cell surface.
The humoral immune response involves helper T lymphocytes produced
by the T cell maturation pathway TH-2. Cells of this pathway secrete cytokines
such as Interleukin 4 (IL-4}, IL-S, IL-6, IL-9, IL-10 and tumor necrosis
factor
(TNF). These cytokines inactivate macrophage proliferation, contributing to a
down-regulation of the Ta-1 response. TNF causes tissue inflammation and
necrosis when released at high levels, which are the indications of failure of
the
overall immune system in many diseases. CD4+ T lymphocytes become
activated through contact with antigens displayed in association with MHC
class II molecules (MHC II), at the surface of macrophages and antigen
presenting cells. Antibodies are produced by B cells when they interact with
these activated CD4+ T lymphocytes. The MHC II molecules reside in the
vesicles that engulf and destroy extracellular materials. Thus, their location
within the cell gives them their specific function in monitoring the content
of
these vesicles. They specifically bind to antigens that have been
enzymatically
2~ processed in the lysosomes of the immune cells after phagocytosis. The
humoral immune response is required to protect the extracellular environment
against extracellular antigens and parasites through antibodies which can be
effective in neutralizing infectious agents. However, the humoral immune
response cannot eliminate whole cells that become diseased, it causes tissue
destruction and necrosis, and it is not effective in fighting intracellular
diseases.


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Consequently, the body relies on t:he cellular immune response for
protection from pathologies that start in the intracellular environment.
Cellular
immune response is carried out through cytotoxic immune cells which are
capable of killing diseased cells. The cellullar immune response involves
helper
T lymphocytes produced by the T cell maturation pathway TH-1. Cells of this
pathway secrete cytokines such as IL-2, IL; 12, IL-15, gamma Interferon (IFN)
lymphotoxin, and Granylocyte Macrophage Colony Stimulating Factor
(GMCSF). These cytokines activate macrophages. The cytotoxic T
lymphocytes are CD8+ T cells that becomf; activated through contact with
antigens associated with MHC class I molecules (MHC I). MHC I molecules
reside around the protein factories such as the endoplasmic reticulum. Thus,
their location within the cell gives them their specific function of
monitoring the
output and transport of materials producedl inside the cell. They specifically
bind to antigens that have been synthesized in the intracellular environment
like
in the case of cancer or intracellular diseases. The cellular immune response
protects against chronic intracellular diseases such as intracellular
infection,
parasitism and cancer, by activating the macrophages and facilitating the
detection and lysis of diseased cells. The result is the formation of a
granuloma
which is the paradigm of protective immunity in intracellular diseases.
Although the immune system has evolved to be efficient in selecting the
target antigens against which an immune reaponse is delivered, it does not
always succeed in selecting the appropriate: combination of the humoral and
cellular immune components necessary to <;ontain or eliminate the disease. For
example, intracellular diseases resulting from genetic disorders, cancer,
infections, allergies and autoimmune reactions are particuiariy difficult to
treat
and continue to be life threatening illnesses despite the advances in
detection,
diagnosis and treatment. Many of these diseases are able to circumvent the
immune system and progress without challenge. For others with a long latency
period, diagnosis is often made too late. Some display multi-resistance
profiles
against drug treatment or have their disease processes originating in
environments accessible only to high doses of existing drugs. Many of these


CA 02284736 1999-09-27
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-- 4
drug treatments have high toxic side effects. Treating with chemotherapy is
expensive and may be implemented only after significant expansion of the
pathological process, or if there is transmission of infection and damage to
the
host. Although these diseases may elicit an immune response, they usually
compromise its effectiveness by suppressing or mimicking the MHC molecules.
In this type of illness, a TH-1 immune response favors protection, while down-
regulation of this pathway, conversion to TH-2 during the chronic course of
the
disease, or up-regulation of the pathway TH-2 is detrimental to the host.
Accordingly, a shift to TH-1 response or up-regulation of the TH-1 pathway
should be beneficial on its own, and when associated with appropriate
chemotherapy, would mount an effective response to resistance, chronicity, and
disease. Therefore, treatment methods for intracellular diseases are needed
which favor a TH-1 immune response rather than a TH-2 response.
Cancers are caused by genetic alterations that disrupt the metabolic
activities of the cell. These genetic changes can result from hereditary
and/or
environmental factors including infections by pathogenic viruses. Like in
other
intracellular diseases, cellular immunity plays a major role in the host
defense
against cancer. Traditionally, cancer immunotherapies were designed to boost
the cellular immune response by using specific and non-specific stimuli,
including: 1 ) passive cancer immunotherapies where antibodies have been
administered to patients, showing success only in rare cases; 2) active cancer
immunotherapies where materials expressing cancer antigens have been
administered to patients (e.g., the injection of whole or fractions of cancer
cells
that have been irradiated, modified chemically, or genetically) showing little
impact in experimental tumor models; and 3) the combination of adoptive
lymphocytes and IL-2, wMch caused regression of tumors in mice and metastic
melanoma in humans. Tumor infiltrating lymphocytes (TIL) capable of
mediating tumor regression are lymphoid cells that can be grown from single
cell suspensions of the tumor incubated with IL-2. Thus, antigens recognized
by TIL are more likely to be involved in vivo in anticancer immune response,
and the cDNA and the amino-acid sequences of several of these antigens have


CA 02284736 1999-09-27
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.. 5
been identified. While these findings have opened new opportunities for the
development of cancer specific immunotherapies, treatment methods based on
mixing cancer antigens or the cloning and expression of the genes encoding
these antigens into a delivery system that favors a TH-1 response rather than
a
TH-2 response to these antigens are needed.
Intracellular infections are caused by bacteria, viruses, parasites, and
fungi. These infectious agents are either present free in the environment or
carried by untreated hosts. Humans, animals and plants can serve as hosts, and
if not treated, they can act as reservoirs facilitating the further spreading
of
such agents to others. Intracellular pathogens such as M. tuberculosis, M.
leprae, and tumor viruses cause disease worldwide in millions of people each
year. It is estimated that M. tuberculosis infects at least thirty million
people/yeau- and will cause an average of tluee million deaths/year during
this
decade, making tuberculosis (TB) the number one cause of death from a single
infectious agent (World Health Organization, 1996). TB occurs most
commonly in developing countries, but the. prevalence of TB has increased
recently in the U. S., as well as in developing countries, due to an increase
in the
number of immune compromised individua~is with HIV infection. The risk of
TB infection has also increased in individuals with diabetes, hemophilia,
lymphomas, leukemias, and other malignant neoplasms, because these
individuals have compromised immune systems. Leprosy and viruses which
cause neoplasia are also important intracellular pathogens worldwide. Leprosy
presently causes disease in more than twelve million people, and at least 15%
of human cancers are thought to be caused by neoplastic transformation of
cells
2~ by viruses.
Intracellular infections with highly virulent strains are quickly resolved
resulting in death or cure of the patient. Hlowever, organisms of lower
virulence can persist in the host and develop chronic diseases. Mycobacterium
infections develop through a spectrum thavt ranges from a state of high
resistance associated with cellular immunity to an opposite extreme of low
resistance associated with humoral immunity. For example, leprosy is caused


CA 02284736 1999-09-27
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-. 6
by Mycobacterium leprae which remains uncultivable. The disease manifests
an immuno-histological spectrum with six groups. At one end of the spectnam,
there is the polar tuberculous leprosy (TT), a paucibacillary form of the
disease
which is characterized by a strong TH-1 immune response and a bacteriolytic
effect that lead to granuloma formation and restrict the growth ofM. leprae,
respectively. At the opposite end of the spectrum there is the polar
lepromatous leprosy (LL), a multibacillary form of the disease which is
characterized by a strong but inefficient TH-2 immune response and a down-
regulation of the TH-1 pathway. During the chronic course of the disease the
I 0 levels of IL-2 and cells with IL-2 receptors diminish, the T cells become
defective in their functions, and M. leprae proliferates unrestricted within
the
macrophages and the schwann cells. With this immune failure the clearance of
the bacteria is markedly retarded, and the patient continues to harbor bacilli
in
the tissues even after prolonged drug therapy. The antibodies react with
circulating antigens forming immune complexes that lead to tissue damage,
necrosis and organ failure. Between these two extremes there are four
borderline forms of leprosy reflecting the different balances achieved by the
body between TH-1 and TH-2 immune responses. Likewise, tuberculosis
caused by Mycobacterium tuberculosis also manifests an immuno-clinical
spectrum with multiple (four) groups. The reactive polar group (RR) is
associated with a Tx-1 immune response while the opposite pole (UU) is
unreactive and is associated with a TH-2 immune response. Therefore, there
are clear indications that the TH-1 immune response is the main defense
mechanism in leprosy and tuberculosis. Thus, treatment and immuno-
prophylaxis agai~t these diseases should be aimed at enhancing the TH-1
pathway.
Allergic diseases are characterized by the sustained production of Ig E
molecules against common environmental antigens. This production is
dependent of IL,-4 and is inhibited by gamma interferon. Thus, the allergic
reactions involve a TH-2 immune response which requires a low level of
stimulation by allergens. Therefore, preferable treatment for allergies would


CA 02284736 1999-09-27
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. . 7
include the following: switching to a TH-1 immune response, which requires a
high level of stimulation; activating CDS+ T cells and the production of gamma
interferon; reducing the production of Ig l3 and recruitment of eosinophils
and
mast cells; and increasing the threshold concentration of the allergen to
trigger
a reaction.
Mycobacterium gene products, esopecially heat shock proteins, show
homologies with bacterial, viral, parasitic, mycotic, and tumor antigens
suggesting that these similarities may reflect regions in Mycobacterium
antigens
which can serve as potential inducers of cross immunity to different diseases.
Heat shock proteins are overexpressed by stressed cells in many pathologies
including infections, cancer, and autoimmune diseases. Thus, vaccinated
individuals would have circulating cytoto~;ic T lymphocytes (CTL) that can
interact and lyse the stressed cells, while the expression of putative
autoimmunity antigenic domains in a susceptible host may lead to the
1 S suppression of the immune response and the chronicity of the disease.
(Labidi,
et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum
var. fortuitum plasmid, pAL 5000," Plasmid 27:130-140).
The available methods for prophylaxis and treatment of intracellular
diseases include antibiotics, chemotherapy, and vaccines. Antibiotics have not
been effective in treating diseases caused by M. tuberculosis or M. leprae
because the lipid-rich cell wall of a mycobacteria is impermeable to
antibiotics.
Likewise, antibiotics have no effect on viral pathogenesis. Chemotherapy as a
means of prophylaxis for high-risk individuals can be effective against M.
tuberculosis or M. leprae, but it has disadvantages. Chemotherapeutic agents
have undesirable side-effects in the patient, are costly, and lead to the
potential
existence of mufti-drug resistant Mycobacterium strains. In addition to these
disadvantages, chemotherapy as a means of treating active TB, leprosy, and
virus-induced neoplasms has minimal effect since it is used only after
significant
disease progression. Consequently, vaccination is the therapy of choice
because it provides the best protection at the lowest cost with the least
number
of undesirable side-effects.


CA 02284736 1999-09-27
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g
Early vaccines administered as protection against acute infections were
developed using antigens to initiate an immune response regardless of its
nature
or its mechanism. The aim was to protect against acute infections where a TH-
2 immune response may be efficient. These vaccines were made of a variety of
S crude antigens including killed or attentuated whole cells, toxins, and
other
structural components derived from the pathogen. Bacterial products such as
peptidoglycan, lipoproteins, lipopolysaccharides, and mycolic acids were used
as therapeutic and prophylactic agents in several diseases. The administration
of non-specific stimulants derived from Corynebacterium parvum,
Streptococci, Serratia marcescens, and Mycobacterium, to cancer patients
showed some efficacy and concomitantly enhanced the immune response
against the disease. Adjuvants were developed to stimulate the immune
response to antigenic material. One such adjuvant was complete Freund's
adjuvant, which consisted of killed Mycobacterium tuberculosis suspended in
oil and emulsified with aqueous antigen solution. This preparation was found
to be too toxic for human use. (Riott, et al., Immunology, 5th ed., Mosby,
Philadelphia, pp. 332, 370 (1998).
Following these first steps, efforts have been made to isolate and to
develop single antigens and even single epitopes into vaccines. Molecular
techniques have been used for the last two decades to clone the genes and map
the domains of the corresponding proteins. However, individual antigens or
cytokines did not reproduce the same physiological effects like a whole
bacterial adjuvant. For example, antigen development for M. tuberculosis, M.
leprae, and other intracellular parasites were fruitless because the dogma of
the
specific protective antigens or epitopes could not accurately define a
protective antigen for these diseases. The dogma, fizrthermore, has ignored
the fact that the immune response to a pathogen is a coherent response to a
mosaic complex of epitopes displayed by the pathogen with some epitopes
conferring protection and other epitopes mediating virulence and
immunopathology. These vaccines have been unsuccessful in establishing the
favored TH-1 response over the TH-2 response.


CA 02284736 1999-09-27
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. . 9
Early vaccines were also not potent against intracellular diseases. The
vaccines were inefficient, short-lived, or triggered inappropriate immune
responses similar to hypersensitivity reactions in allergic diseases that
result in
necrosis, which worsens the outcome of the pathological process in many
chronic infections such as tuberculosis and leprosy. For example, BCG
(Bacille-Calmette Guerin) is a vaccine that has been used for TB and leprosy
prophylaxis, but has questionable efficacy. BCG is an attenuated live vaccine
derived from M. bovis, a Mycobacterium strain that is closely related to M.
tuberculosis. BCG has been only marginally effective against leprosy and is
not
currently recommended for leprosy prophylaxis. Results from controlled
studies to determine the efficacy of BCG vaccines for TB prophylaxis have
been conflicting. Estimates of BCG efficacy from placebo-controlled studies
range from no efficacy to 80% efficacy. A large scale BCG trial in India
(n=360,000 people) showed that BCG failed to provide a protective effect
against the onset of pulmonary TB. Other studies have shown that BCG
produces an inconsistent, fluctuating immunity. Because no effective vaccine
has been developed to protect against leprosy or virus-induced cancers, and
because BCG is unreliable for TB prophylaxis, a more effective vaccine is
needed. An example of such new vaccines would combine selective antigens
with potent adjuvants and stimulate the cellular immune response to deliver a
lasting protective immunogen.
In U.S. Patent No. 3,956,481, Joll~es et al. discloses a hydrosoluble
extract of mycobacteria suitable as an adjuvant, wherein delipidated bacterial
residues are subjected either to a mild extraction process or treatment with
2~ pyridine followed by treatment with ethanol or water. These extracts were
found to be toxic in humans, discouraging their use as a vaccine.
In U.S. Patent No. 4,036,953, Adam et al. discloses an adjuvant for
enhancing the effects of a vaccine, wherein the adjuvant is prepared by
disrupting mycobacteria or Nocardia cells:, separating and removing waxes,
free
lipids, proteins, and nucleic acids; digesting delipidated material from the
cell


CA 02284736 1999-09-27
10365/05602
. . ~ ~ ~ , _ ..
..
. _. __.. ...
;; 10 ' , . ,
~~ n, . n ~v f . ..
wall with a murolytic enzyme; and collecting the soluble portion. Adjuvants
of this type were also noted to be toxic in hunnans.
In U.S. Patent No. 4,724,144, Rook, ea al. discloses an
immuntherapeutic agent comprising antigenic; material from killed
~ Mycobacterium vaccae cells useful for the treatment of diseases such as
tuberculosis and leprosy. The vaccine has been shown to be effective against
persistent microorganisms which survived long exposure to chemotherapeutic
agents. Although the vaccine shows improved immune response, it is limited
only to antigens endogenous to Mycobacterium vaccae.
In U.S. Patent No. 5,599,545, Stanford, et al. discloses an ,
immunotherapeutic agent comprising killed ~t~Iycobacterium vaccae cells in
combination with an antigen exogenous to mycobacteria which promotes a TH i
1 response. The exogenous antigen may be combined with the killed
i
Mycobacterium vaccae by admixture, chemical conjugation or absorption, or
alternatively produced by expression of an exogenous gene in Mycobacterium
vaccae via plasmid, cosmid, viral or other expression vector, or inserted into
.
the genome. While these compositions promote the TH-1 immune response,
.;
they were limited only to killed Mycobacterium vaccae cells. Further, the ~i
patent provides no guidance as to how to make Mycobacterium expression
vectors, or how to incorporate the expression vectors into either a plasmid,
cosmid, or viral expression vector, or how to integrate the expression vector
into the genome.
In U.S. Patent No. 5,583,038, Stover disclosed an expression vector for
expressing a protein or polypeptide in a bacterium which comprises a first
DNA sequence encoding at least a secretion signal of a lipoprotein and a
second DNA sequence encoding a desired protein, protein fragment,
polypeptide, or peptide heterologous to the bacterium which expresses the .
desired protein, etc. Stover demonstrated~use of an origin of replication _'
recognized in Mycobacterium and the desirability of eliminating sequences not
necessary for plasmid replication, e.g., reducing a pAL5000 plasmid fragment
containing such an origin of replication to 1910 base pairs. Stover also
discloses use of an attP-integrase gene fragment from mycobacteriophage L5
a
to transform M. smegmatis and BCG.
rn ~E~
PMFNn


CA 02284736 1999-09-27
1036,5/0560,2
.. .~,, ~~ H_
.. - o . ., " ~ r.
, n a n ... o O 3
.. -~ ., _, ,~ . , r, n n.~. n .. n a w w w w n
i ~~ _ o a a v s
" 11 -,, ,nn, ~oo~ oe o w w
WO 92/01783 disclosed a DNA which includes a first DNA sequence
containing a phage integration gene and a second DNA sequence encoding a
protein or polypeptide heterologous to the mycobacterium in which the DNA
is to be integrated for integrating DNA into a :mycobacterium chromosome and
then administering the mycobacteria as a vaccine and/or therapeutic agent.
WO 92/01783 also disclosed use of an origin of replication recognized in
Mycobacterium and the desirability of eliminating sequences not necessary for
plasmid replication, e.g., reducing a pAL5000 plasmid fragment containing
such an origin of replication to 1910 base pales; and the use of an attP-
integrase gene fragment from mycobacteriophage LS to transform M.
smegmatis and BCG.
David, et al. i(David, et al. 1992. Plas.mid 28:267-271) discloses a
plasmid shuttle vector for E coli and mycoba.eteria constructed from an E.
coli
plasmid containing the ColEl origin, a 2.6 kb PstI fragment from
bacteriophage D29, and kanamycin resistancE; gene, which successfully
transformed Mycobacterium smegmatis. Mistakenly reporting that
transformation was achieved due to an origin of replication from the D29
fragment, David, et~al. did not teach the use of a minimal functional
component of D29 comprising an attachment site and and integrase gene.
With respect ~to Mycobacterium diseases, advances made in the area of
genetic tools and vaccine strategy included: the isolation, characterization
and
sequencing of the Mycobacterium plasmid pAI, 5000; the identification of the
kanamycin resistance gene as a selection ma~~ker for Mycobacterium; the
development of the first Escherichia coli (E. coli)lMycobacterium shuttle
vectors; the construction of M. tuberculosis amd M. leprae genomic libraries;
and the expression of Mycobacterium DNA i.n E. coli. (Labidi, et al. 1984.
"Plasmid profiles of Mycobacterium fortuinsm complex isolates," Curr.
Microbiol. 11, 235-240; I,abidi, et al.;1.9°85. "Cloning and
expression of
mycobacterial plasmid DNA in Escherichia coli, " FEMS Microbiol Lett. 30,
221-225; Labidi, et al. 1985. "Restriction endonuclease mapping and cloning
of Mycobacterium fortuitum var. fortuitum hlasmid pAL 5000," Ann. Insti.
PasteurlMicrobiol. 136B, 209-215; Labidi, et al. May 8-13, 1988.
"Nucleotide sequence analysis of a 5.0 kilobase plasmid from
AMENO~ SN~Ef


CA 02284736 1999-09-27
10365/056Q2
..
. __ ,
~~ a oas aso
.,, .~, .. ..
12 ~ ' . . ° y
Mycobacterium fortuitum," Abstract U6 of the 88th Annual Meeting of the
American Society for Microbiology, Miami, Florida, USA; Labidi, et al.
1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var.
fortuitum plasmid, pAL 5000," Plasmid 27, 130-140; Labidi, A. January,
1986. "Contribution to a plan. of action for research in molecular biology and
immunology of mycobacteria," Ph.D. Thesis. University of Paris and
Pasteur Institute, Paris, France). Such adva~lcements have opened the way
for the application of recombinant DNA technology to Mycobacterium.
Lazraq, et al. 1990. Conjugative transfer of a shuttle plasmid from
Escherichia
coli to Mycobacterium smegmatis. FEMS lLl'icrobiol. Lett. 69, 135-138;
Konicek, et al. 1991. Gene manipulation in rnycobacteria. Folia Microbiol.
36(5), 411-422; and Falkinham, III, J.O. and J.T. Crawford. 1994. Plasmids,
p. 185-198. In Barry Bloom (ed.), Tubercul.osis: Pathogenesis, protection and
control. American Society for Microbiology, Washington, D.C.).
The Mycobacterium expression vectors resulting from such
advancements are not suitable for vaccine development because: 1) the
expression vectors are large so tl-ie vectors hame limited cloning capacity
and
low transformation efficiency (calculated as 'the number of transformants
obtained per microgram of vector DNA), 2) she vectors lack multiple-cloning
sites, 3) the protocols for transfo I~mation of mycobacteria with these
expression
plasmids result in inefficient transformation, 4) the spectrum of mycobacteria
- transformed by the vectors is restricted because transformation is host-
dependent, and 5) the current expression plasmids do not stably transform
mycobacteria. Therefore; suitable Mycobacterium expression vectors are
needed which can provide efficient transformation and stable expression of
multiple protective immunogens in mycoba<aeria.
Suitable antigen delivery.systems using nonpathogenic Mycobacterium
strains, cloning vectors, and Mycobacterium expression vectors have now been
found which contain protective immunogens that specifically stimulate a cell-
mediated immune response by the induction. of TH-1 cells, or cytotoxic T
lymphocytes, and provide a consistent, prolonged immunity to intracellular
pathogens.
AMEI'!DFD SHEET


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 _
. . 13
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 depicts a sequence of the origin of replication in E. coli (695
bp). The underlined base indicates the replication point.
Fig. 2 depicts a sequence for the k:anamycin gene (932 bp). The
underlined sequences are in the 5' to 3' order: the (-35) region for the gene,
the (-10) region for the gene, the ribosomal binding site region for the gene,
the starting codon (ATG), and the stop codon (TAA~.
Fig. 3A depicts a sequence of the ;pAL 5000 origin of replication (1463
bp) obtained by restriction enzymes analysis. The numbers in superscript
indicate the position of the nucleotides in the published sequence of pAL 5000
(Labidi, et al. 1992. "Cloning and DNA sf;quencing of the
Mycobacteriumfortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27:130-
140). The underlined sequences indicate i~.n the S' to 3' order: the position
of
the forward (F~, F,, FZ, and F3), and the reverse (R4, R3, R2, R,, and R~)
primers used in PCR analysis, respectively.
Fig. 3B depicts a sequence of the pAL 5000 origin of replication ( 1382
bp) obtained after PCR analysis. The numbers in superscript indicate the
position of the nucleotides in the published sequence of pAL 5000 (Labidi, et
al. 1992. Plasmid 27:130-140). The underlined sequences indicate in the 5' to
3' order: the position of the forward ( F,, FZ and F3), and the reverse (R4,
R3,
RZ and R,) primers used in PCR analysis, respectively.
Fig. 4A depicts a sequence of the attachment site (attP) and the
integase gene (int) of the Mycobacterioplhage D29, obtained by restriction
enzymes analysis ( 1631 bp). The numbers in superscript indicate the position
of the nucleotides in the sequence. The underlined sequences delimited by
numbered nucleotides indicate in the 5' to 3' order: the position of the
forward (F~, F,, F2, F3, and F4) and the reverse (R4, R3, R2, R,, and ~)
primers
used in PCR analysis, respectively. The underlined sequences not delimited by
numbered nucleotides indicate in the 5' to 3' order: the attachment site
(attP),
the (-35) region for the gene (int), the (-11)) region for the integrase gene
(int),
the ribosomal binding site region for the integrase gene (int), and the
starting


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 _
14
codon (ATG) for the integrase gene (int). The stop codon for the integrase
gene (int) is the TGA1531.
Fig. 4B depicts a sequence of the attachment site (attP) and the
integrase gene (int) of the Mycobacteriophage D29, obtained after PCR analysis
( 1413 bp). The numbers in superscript indicate the position of the
nucleotides
in the sequence. The underlined sequences delimited by numbered nucleotides
indicate in the 5' to 3' order: the position of the forward (F3, and F4) and
the
reverse (R4, R3, and Rz) primers used in PCR analysis, respectively. The
underlined sequences not delimited by numbered nucleotides indicate in the S'
to 3' order: the attachment site (attP), the (-35) region for the gene (int),
the
(-10) region for the integrase gene (int), the ribosomal binding site region
for
the integrase gene (int), and the starting codon (ATG) for the integrase gene
(int). The stop codon for the integrase gene (int) is the TGA'S3y
Fig. 4C depicts a sequence of the attachment site (attP) and the
integrase gene (int) of the Mycobacteriophage Dz9, obtained after PCR analysis
( 13 74 bp). The numbers in superscript indicate the position of the
nucleotides
in the sequence. The underlined sequences delimited by numbered nucleotides
indicate in the 5' to 3' order: the position of the forward (F4) and the
reverse
(R4, R3, and Rz) primers used in PCR analysis, respectively. The underlined
sequences not delimited by numbered nucleotides indicate in the 5' to 3'
order:
the attachment site (attP), the (-35) region for the gene (int), the (-10)
region
for the integrase gene (int), the ribosomal binding site region for the
integrase
gene (int), and the starting codon (ATG) for the integrase gene (int). The
stop
codon for the integrase gene (int) is the TGA'ssy
Fig. 5 depicts a sequence for the kanamycin gene promoter( I 02 bp)
and the first ATG codon. The underlined sequences are in the 5' to 3' order:
the (-35) region for the gene, the (-10) region for the gene, the ribosomal
binding site region for the gene, and the starting codon (ATG).
Fig. 6 depicts a sequence of the pAL 5000 fragment containing the
open reading frame ORF 2 (2096 bp). The numbers in superscript indicate
the position of the nucleotides in the published sequence of pAL 5000 (Labidi,


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
et al. 1992. Plasmid 27:130-140). The underlined sequence (GGATCC) is
the unique Bam HI site which is spanned by the ORF 2 promoter.
Fig. 7 is a gene map of a representative genetic transfer system, wherein
"C-terlanch/seq." = C terminal anchoring sequence; "MCS/express." = multiple
5 cloning site for expression; "N-ter/lead/seq." = N terminal leading
sequence;
"MycolProm." =Mycobacterium promoter'; "Repllnteg/Myco" _
Mycobacterium origin of replication or phage attachment site and integrase
gene (either one or the other but not both is present in a given vector);
"MCS/gen/clon." = multiple cloning site for general cloning;
10 "univ/selectlmark." = universai selection marker;
and "ori/E. coli" = E. coli origin of replication.


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
16
DETAILED DESCRIPTION OF TIIE INVENTION
The therapeutic or prophylactic vaccines of the present invention
combine a protective immunogen with one or more Mycobacterium strains
acting as a delivery system and an adjuvant, preferably in addition to
cytokines
and appropriate chemotherapy. The rationale is that the Mycobacterium cells
will be ingested by macrophages and remain within the macrophage, blocking
the killing mechanism of the macrophage while synthesizing the protective
immunogen. The immunogen will be processed and presented on the
macrophage cell surface to T cells, resulting in TH-1 cell activation and a
cell-
mediated immune response that is protective against the intracellular disease.
One aspect of the present invention uses an antigen delivery system in
the form of a nonpathogenic Mycobacterium strain to provide products
combining nontoxic immuno-regulating Mycobacterium adjuvants, nontoxic
immuno-stimulating protective immunogens specific for a variety of diseases,
and nontoxic amounts of cytokines that boost the TH-1 pathway. Preferably,
the present invention uses a protective immunogen delivery system in the form
of a nonpathogenic Mycobacterium strain, a genetic transfer system in the form
of cloning vectors, and expression vectors to carry and express selected genes
in the delivery system.
Protective immunogen deliver~r_~ystem
The protective immunogens of the present invention form pure non-
necrotizing complete granuloma. Such immunogens can be protein antigens
or other immunogenic products produced by culturing and killing the diseased
cell or infectious microorganism, by separating and purifying the immunogens
from natural or recombinant sources, or by the cloning and expression into a
Mycobacterium delivery system of the genes encoding these protein antigens or
the enzymes necessary to modify an endogenous lipid to a stage where it is
immunogenic and specific. The protective immunogens of the present
invention include antigens associated with: 1 ) cancer including but not
limited
to lung, colorectum, breast, stomach, prostate, pancreas, bladder, liver,
ovary,
esophagus, oral and pharynx, kidney, non-Hodgkin's, brain, cervix, larynx,


CA 02284736 1999-09-27
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17
myeloma, corpus uteri, melanoma, thyroid., Hodgkin's, and testis; 2) bacterial
infections including but not limited to mycobacteriosis (e.g., tuberculosis
and
leprosy), Neisseria infections (e.g., gonorrhea and meningitis), brucellosis,
plague, spirochetosis (e.g., trypanosomiasiis, Lyme disease and tularemia),
rickettsiosis (e.g., typhus, rickettsialpox, and anaplasmosis), chlamydiosis
(e.g.,
trachoma, pneumonia, atherosclerosis, anal urethritis}, and Whipple's disease;
3)
parasitic diseases including but not limited to malaria, leishmania,
trypanosomiasis, and toxoplasmosis; 4) viral diseases including but not
limited
to measles, hepatitis, T-cell leukemia, denl;ue, AIDS, lymphomas, herpes, and
warts; 5) autoimmune diseases including but not limited to rheumatoid
arthritis,
ankylosing spondylitis, and Reiter's syndrome; 6) allergy diseases including
but
not limited to asthma, hay fever, atopic eczema, and food allergies; 7)
veterinary diseases including but not limited to feline immunodeficiency,
equine
infectious anemia, avian flue, heartworm, and canine flea allergy; and 8)
other
diseases including but not limited to leukemia, multiple sclerosis, bovine
spongiform (BSE), and myoencephalitis (11~IE). These antigens can be used
singly or in combination in one vaccine. V~~hen a combination of antigens is
used, they can be administered together at one time or they can be
administered
separately at different times.
Preferred endogenous lipid protectiive immunogens for the treatment of
tuberculosis, leprosy, and other mycobacterioses include but are not limited
to
complex lipid heteropolymers such as the phenolic glycolipids PGL I and PGL
Tbl, the sulfolipid SL I, the diacyl-trehalos;e DAT and the lipo-
oligosaccharide
LOS. These lipid immunogens are not synthesized, or modified to their final
2~ forms by all Mycobacterium species. Therefore, the host strain must provide
the necessary precursors to synthesize the desired final immunogenic products.
When using an expression vector, the expression system must provide the
necessary genes that encode the necessary enzymes to modify the lipid to a
stage where it is immunogenic.
The mycobacterial adjuvant of the present invention is one that boosts
the TH-1 immune response, and preferably down-regulates the Ta-2 response.


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
. 18
The Mycobacterium strains are characterized by their lack of pathogenicity to
mammals and their capacity to be ingested mammalian macrophages. The
Mycobacterium strains of the present invention may be live or dead upon
administration. When the vaccines of the present invention are administered to
immunocompromised patients, only dead Mycobacterium strains are used.
Preferable Mycobacterium strains can be obtained from the American Type
Culture Collection (Rockville, MD). One or more types ofMycobacterium
species may be utilized in the preparation of a vaccine. Examples include but
are not limited to nonpathogenic Mycobacterium vaccae, Mycobacterium
gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium
thermoresistible, Mycobacterium chitae, Mycobacterium duvalii,
Mycobacterium flavescens, Mycobacterium norrchromogenicum,
Mycobacterium neoaurum, and Mycobacterium bovis BCG. M. bovis BCG
and M. gastri are the only known Mycobacterium species that have precursors
for producing M. tuberculosis and M. leprae lipids; therefore, M. gastri must
be used if the precursors of exogenous lipids are to be expressed in a vaccine
for TB or leprosy. M. gastri and M. triviale can be found in the
gastrointestinal tract, and are, thus, important for use in oral vaccines. The
Mycobacterium adjuvants of the present invention can utilize either one
Mycobacterium strain or multiple strains; however, when killed Mycobacterium
vaccae is used, it is preferably administered in combination with other
Mycobacterium species.
Preferably, the vaccine of the present invention also comprises
cytokines that associate with the TH-1 pathway. Examples of such cytokines
include but are not limited to gamma interferon (IF-N), interleukin(IL)-2, IL-
12,
IL-15 and granulocyte macrophage colony stimulating factor (GMCSF).
Additionally, the vaccine of the present invention may also be
administered in combination with appropriate chemotherapy for treatment of
patients with active diseases. If a live Mycobacterium strain is used as an
adjuvant, appropriate chemotherapy must be selected that does not interfere
with the adjuvant function of the live Mycobacterium. Examples of


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
- 19
appropriate concommitant chemotherapy :is Taxol-R for the treatment of cancer
or protein inhibitors for AIDS treatment.
The protective immunogens, cytokines, and concommitant
chemotherapy may be produced separately in a synthetic or in a recombinant
S form, purified by any conventional technique. They may be used in parallel
with, mixed with, or conjugated to live or dead Mycobacterium cells of
interest.
Genetic transfer s, s~tem_
The genetic transfer system of the :present invention comprises cloning
vectors where the genes of interest are cloned and the transformation
technique
is used to introduce and express the recombinant molecules into the delivery
system. Previous cloning vectors which have been used in
Mycobacterium species include the extracllromosomal M. fortuitum plasmid
pAL 5000 (Labidi, et al. 1992. "Cloning and DNA sequencing of the
Mycobacterium fortuitum var. fortuitum p:lasmid, pAL 5000," Plasmid 27:130-
140) which replicate extrachromosomally and the mycobacteriophage Dz9.
(Forman, et al. 1954. "Bacteriophage active against virulent Mycobacterium
tuberculosis: isolation and activity," Am J.Public Health 44:1326-1333)
Mycobacteriophage D29 is a large spectrum virulent phage able to infect and
efficiently reproduce itself in cultivated Mycobacterium species and attach
itself
to uncultivated M. leprae.
New cloning vectors have now been developed which are generally
made of either origins) of replication or integration system(s), selection
marker(s), and multiple cloning sites) (MC;S). The cloning vectors are
comprised of the minimum functional sizes of various components including the
following components: the E toll replicor~, the kanamycin selection marker,
the pAL 5000 origin of replication, and the Dz9 attachment site (attP) and
integrase gene (int). Using conventional dE:letion techniques, the coding
region
for each component have been reduced to the point that further loss of base
pairs resulted in loss of function, hence the designation of minimum
functional
size. The sequences for each minimum functional component are given as


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
follows: origin of replication in E. toll (695 bp) as SEQ ID NO: l and Fig. 1;
kanamycin gene (932 bp) as SEQ ID N0:2 and Fig. 2; origin of replication in
pAL 5000 ( 1463 bp) obtained by restriction enzyme analysis as SEQ ID N0:3
and Fig. 3A; origin of replication in pAL 5000 (1382 bp) obtained after PCR
5 analysis as SEQ ID N0:4 and Fig. 3B; Mycobacteriophage D29 attachment site
and integrase gene ( 1631 bp) obtained by restriction enzyme analysis as SEQ
ID NO:S and Fig. 4A; Mycobacteriophage D29 attachment site and integrase
gene (1413 bp) obtained after PCR analysis as SEQ ID N0:6 and Fig. 4B; and
Mycobacteriophage Dz9 attachment site and integrase gene {1374 bp) obtained
10 after PCR analysis as SEQ ID NO:? and Fig. 4C. It is well understood in the
art of deletion techniques that while the above-identified sequences provide
the
coding regions for each minimum functional component, an additional loss of a
few base pairs from the minimum functional component could still result in a
functional component of the present invention.
1 S Numerous E. toll origins of replication are commercially available and
can be utilized in the present invention. For example, the E. toll origin of
replication CoIE 1 is found in most commercially available plasmid vectors
designed for E toll. Although the replication point is usually indicated for
these vectors, the smallest fragment that can support an efficient replication
in
20 E. toll has not heretofore been specified. Using the commercially available
plasmid vector pNEB 193 ( Guan C., New England Biolabs Inc., USA, 1993)
as starting material, it has now been determined through restriction
endonuclease deletions, cloning, and transformation analysis that the smallest
DNA fragment that can support an efficient CoIE 1 replication in E. toll is
limited to a 695 ~ sequence given in SEQ ID NO:1 and Fig. 1. This E. toll
origin of replication of minimum functional size has been successfully
utilized in
the construction of E. toll cloning vectors and E. toll Mycobacterium shuttle
vectors of the present invention.
While a variety of selection markers are available for the selection of
transformed cells and can be used in the present invention, the Streptococcus
faecalis 1489 by gene coding for resistance to kanamycin has been selected as


CA 02284736 1999-09-27
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21
a representative selection marker for Mycobacterium (Labidi, et al. 1992.
"Cloning and DNA sequencing of the Mycobacterium fortuitum var. fortuitum
plasmid, pAL 5000," Plasmid 27:130-140; Labidi, et al. 1985. "Restriction
endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum
plasmid pAL 5000," Ann. Insti. Pasteurl'hlicrobiol. 136B, 209-215). While
this gene is well established as the selection marker for Mycobacterium
(Konicek, et al. 1991. FoliaMicrobiol. :36(5), 411-422), the smallest
fragment capable of supporting kanamycin selection in Mycobacterium has not
heretofore been established. It has now been found that the minimal functional
sequence for this gene is about 932 by as shown in SEQ ID:N02 and Fig. 2.
The kanamycin gene of minimum functional size described herein has been
successfully utilized in the construction of E. coli cloning vectors and E.
coli-
Mycobacterium shuttle vectors of the present invention.
Vectors containing a plasmid origin of replication do not usually
integrate in the chromosome of the host strain. Thus, they are extra-
chromosomal vectors. The replication and maintenance in Mycobacterium
strains of the extra-chromosomal vectors developed in this study, are
supported
by the origin of replication of the Mycobacterium fortuitum plasmid pAL 5000.
Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum complex
isolates," Curr. Microbiol. 11, 235-240. The pAL 5000 plasmid is the most
thoroughly studied Mycobacterium plasm:id and has been used worldwide to
develop vectors for genetic transfer in Mycobacterium (Falkinham, III, J.O.
and J.T. Crawford. 1994. Plasmids, p. 185-198. In Barry Bloom (ed.),
Tuberculosis: Pathogenesis, protection and control. American Society for
Microbiology, Washington, D.C.). Functional analysis ofthe pAL 5000
plasmid has indicated the location of two open reading frames coding for a 20
KDa and a 65 KDa protein, respectively, and a 2579 by fragment containing its
origin of replication (Labidi, et al. 1992. F'lasmid 27:130-140). In the
present
invention, the 2579 by fragment was reduced through deletions with restriction
enzymes to a 1463 by fragment extending from nucleotide 4439 to nucleotide
1079 without loosing its function (SEQ II) N0:3 and Fig. 3A). It has been


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
22
found that the 1247 by fragments extending from nucleotide 4439 to nucleotide
863, and the 1315 by fragment extending from nucleotide 4587 to nucleotide
1079 do not support replication in Mycobacterium (SEQ 1D NO: 3 and Fig.
3A). Thus, the role of the sequences extending from nucleotide 4439 to
S nucleotide 4587, and from nucleotide 863 to nucleotide 1079 have now been
investigated. In the absence of usable restrictions sites in these two areas
of the
pAL 5000 sequence, sets of forward and reverse primers that span the two
areas have been designed. PCR is then used to amplify the different fragments
which are subsequently cloned into an E. toll replicon containing the
kanamycin gene. Using PCR analysis technique, the minimal functional pAL
5000 origin of replication has been reduced to a 1382 by fragment extending
from nucleotide 4468 to nucleotide 1027 as given in SEQ ID N0:4 and Fig.
3B. Although it has been determined that the 1383 by fragment extending
from nucleotide 4519 to nucleotide 1079, and the 1356 by fragment extending
IS from nucleotide 4439 to nucleotide 972 did not support replication in
Mycobacterium, it is further believed that some of the 51 by sequence
extending from nucleotide 4468 to nucleotide 4518 and the 55 by sequence
extending from nucleotide 973 to nucleotide 1027 also might not be needed for
replication. This pAL 5000 origin of replication of minimum functional size
described herein has been successfully utilized in the Mycobacterium cloning
vectors and construction of E. toll Mycobacterium shuttle vectors of the
present invention.
Vectors can also include a phage attachment site (attP) and its
accompanying integrase gene. A preferred embodiment of the present
invention comprises the attachment site (attP) and the integrase gene ant) of
the Mycobacteriophage Dz9 (Forman, et al. 1954. Am JPublic Health
44:1326-1333). The phage D29 is a large spectrum virulent phage able to infect
cultivated Mycobacterium species and efficiently reproduce itself. To develop
integrative vectors, a map of its attachment site (att P) and integrase gene
(int)
has been determined by constructing a set of hybrid plasmids containing
overlapping fragments of Dz9 genome. The recombinant plasmids were then


CA 02284736 1999-09-27
-- WO 98/44096 PCT/US98106056
23
electroporated into the Mycobacterium strains and plated on LB medium
containing 50 ug/ml kanamycin. A plasmiid containing a 2589 by fragment
generated Mycobacterium transformants. The 2589 by fragment was isolated
and further analyzed. After establishing its restriction map, another set of
S hybrid plasmids were constructed containing overlapping segments of the 2589
by fragment. These recombinant plasmids were electroporated into the
Mycobacterium strains then plated on selective media. The smallest fragment
still able to generate kanamycin resistant Mycobacterium transformants were
isolated and sequenced using a double strand plasmid template and sequenase
version 2.0 (USB, Cleveland, Ohio, USA). The sequence analysis indicated
that the fragment size was 1631 bp, which comprised from 5' to 3' the phage
attachment site (attP), the integrase gene promoter and the integrase gene
(int)
(SEQ ID NO:S and Fig. 4A). Subsequent deletions studies regarding the 1631
by were performed. A 1413 by originating from base pair 119 to 1531 ,
illustrated in Fig. 4B afforded a high transformation efficiency. Additional
deletion studies resulted in a 1374 by fragment originating from base pair 158
to 1531, illustrated in Fig. 4C. The 1374 by fragment generated
Mycobacterium transforlnants, but the transformation efficiency was 100 times
lower and the incubation time becomes much longer, probably due to low
efficiency of integration and stability. It is believed that some of the 39 by
sequence extending from nucleotide 119 to nucleotide 157 might not be needed
for integration. These Dz9 (AttP), (int) and the preceding sequence as
described above are the smallest phage Dl'JA fragment so far used in the
construction ofMycobacterium integrative expression vector and E . cold
Mycobacterium integrative shuttle vectors.
The MCS is a synthetic fragment o~f DNA containing the recognition
sites for certain restriction enzymes that do not cut in the vector sequence.
The
choice of enzymes to be included in the MCS is based on their frequent use in
cloning and their availability. Representative enzymes include BamH I, EcoR
V, and Pst I.


CA 02284736 1999-09-27
WO 98/44096 PCT/ITS98/06056
24
From these minimal functional components, cloning vectors have been
developed which maximize the capacity for multiple cloning sites. Preferably,
the cloning vectors comprise each component at its minimal functional size.
For example, extra-chromosomal cloning vectors have been constructed by
assembling the minimum functional fragments for the E. toll origin of
replication, the pAL 5000 origin of replication, the kanamycin gene, and the
MCS. Exemplary integrative cloning vectors have the same structure except
the origin of pAL 5000 is replaced by the attP and the integrase gene of Dz9
When each component of the cloning vector is reduced to its smallest
functional size, the vectors have a size of about 3 Kb and a transformation
efficiency about 108. Each vector has a theoretically unlimited cloning
capacity
and is capable of transforming Mycobacterium species. Each cloning vector is
presented in Table I.
Fig.7 presents a genetic map of an exemplary cloning and expression
vector. The present invention does not require any particular ordering of the
functional components within the cloning vector.
Further, the cloning vectors of the present invention, do not require that
each component contained in the vector be reduced to its minimum functional
size. The degree to which the minimal functional components are utilized in
each cloning vector is dictated ultimately by the application of the vaccine
and
the maximum transformation size. For example, an integrative cloning vector
may contain the minimal functional component for the attachment site and
integrase gene while the selection marker is larger than its minimal
functional
size. Such an arrangement can arise because the cloning vector contains only
one site for cloning a protective immunogen, thereby allowing other
components of the vector to range in size as long as the vector is of a small
enough size to allow for efficient transformation into Mycobacterium cells.
Preferably, the present invention uses an E. toll Mycobacterium shuttle
vector constructed by applying various recombinant DNA techniques. The
constructed vector can be efficiently transformed into either an E toll or
Mycobacterium host, allowing selected mycobacterial genes to be exponentially


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 _
cloned and expressed. Preferably, the E. c:oli Mycobacterium shuttle vector
uses a selection marker that can be expressed in both genera. One shuttle
vector is comprised of a kanamycin selectiion marker, an origin of replication
for E. coli, and an origin of replication for the Mycobacterium plasmid pAL
5 5000. Another shuttle vector is comprised of a kanamycin selection marker,
an
origin of replication for Is. coli, and an attachment site and integrase gene
of
the Bacteriophage D29. Each component of the constructed shuttle vector has
been reduced to its smallest functional size: thereby enhancing its cloning
and
transformation efficiency.
10 By reducing the vector components to their minimum functional size,
the cloning vectors have the capacity for a multiple cloning site with a large
number of restriction sites. Therefore, the genetic transfer system of the
present invention preferably comprises cloning vectors for more than one
protective immunogen. When more than one Mycobacterium strain is used in a
15 vaccine, the genetic transfer system of each Mycobacterium strain comprises
cloning vectors for one or more protective immunogens.
Transformation
Mycobacterium strains have been successfirlly transformed through
electroporation. (Labidi, et al. 1992. "Cloning and DNA sequencing of the
20 Mycobacteriumfortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27:130-
140) It is understood that other transformation techniques developed for
Mycobacterium would be usefi~l in the present invention. The electroporation
techniques of the present invention are described in Example 3, and the
results
are given in Table 1. The vector designs, culture medium, and the
25 transformation technique described have improved significantly the
transformation efficiency for Mycobacterium species and brought it for the
first
time to a level comparable to that obtained with E. coli.


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
- 26
The integrative vectors containing the attachment site (attP) and the
integrase gene (int) of the phage Dz9 have been found to integrate into the
chromosomes of their hosts at a region complementary of the region (attP).
This region is the bacterial attachment site (attB) and is located between the
genes encoding the Proline transfer RNA (tRNA~'°) and the Glycine
transfer
RNA (tRNAG~').


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
27



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CA 02284736 1999-09-27
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WO 98/44096 PCT/US98/06056
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CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
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CA 02284736 1999-09-27
WO 98/44096 PC1'/LTS98/06056
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CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
58
Expression vectors
The expression vectors of the present invention are made by inserting
functional promoters from plasmid or chromosomal origin into the cloning
vectors which serve as backbones. The expression vectors are tailored to carry
and express selected genes in the delivery system. They contain in their
structures the genetic information necessary for their auto-replication in the
cytoplasm, or their integration into the chromosome of the host. They provide
the promoter and the regulatory sequences necessary for 1 ) gene expression,
and if necessary, 2) the secretion of the gene product out of the cytoplasm to
the cell membrane structure or to the extracellular environment.
While the kanamycin gene is a preferred selection marker for the
present invention, it is also well expressed in a wide range of hosts
including
Mycobacterium and E. coli species, and therefore, vectors containing the
promoter of this gene can express foreign genes in E. coli and Mycobacterium
strains, respectively. Using conventional PCR techniques, the minimum
functional component of this promoter was determined and is given in SEQ 1D
N0:8 and Fig. 5. The use of a kanamycin promoter to construct E coli-
Mycobacterium expression shuttle vectors is reported for the first time.
Another preferred expression vector in the present invention used the
promoter of pAL 5000 open reading frame (ORF) 2. An open reading frame
(ORF 2) encoding a 60 - 65 KDa protein in E coli minicells was identified in
the plasmid pAL 5000. To map the promoter region of this ORF, the 2096 by
fragment containing this open reading frame (SEQ ID N0:9 and Fig. 6) has
been isolated. Through restriction endonuclease deletions, cloning, and
transformation analysis, a set of hybrid plasmids containing overlapping
segments of the 2096 by fragment were constructed. These recombinant
plasmids were electroporated into E. coli DS410. Minicells were prepared
from transformants and plasmid encoded proteins were analyzed as indicated in


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
59
Example 4. The promoter of the ORF was found in the sequence spanning the
unique Bam HI site in the fragment indicated in Fig. 6.
The products of the invention are administered by injection given
intradermal or via other routes (e.g., oral, nasal, subcutaneous,
intraperitoneal,
intramuscular) in a volume of about 100 nnicroIiters containing 10' to 10"
live
or killed cells of recombinant Mycobacterium, or the same amount of
nonrecombinant Mycobacterium cells mixed with, or conjugated to
predetermined amounts of the exogenous antigens, the cytokines, and/or the
drugs. If the products are being used with patients with active diseases, they
should be associated with drug treatments. that do not interfere with the live
form of the vaccine if it is being used. If the products of the invention are
being used separately, they can be administered in any order, at the same or
at
different sites, and using the same or different routes. The invention takes
in
consideration that the products are designed to be used in humans or in
animals
and therefore they must be effective and safe with or without any further
pharmaceutical formulation that may add other ingredients.
In summary, the preferred cloning and expression vectors of the present
invention comprise an E. coli Mycobacterium shuttle vector which contains the
following: an origin of replication for both E. coli (E. coli replicon) and
Mycobacterium (pAL 5000 origin of replication), a kanamycin resistance
marker, multiple cloning sites, promoters and regulatory sequences for
secretion of gene products out of the bacteria and for insertion into the cell
membrane, and the attachment site (att P) and integrase gene (int) of phage
Dz9. Another type of preferred cloning and expression vectors contain all of
these elements listed above except the pha.ge DZ9 attachment site and
integrase
gene. The multiple cloning sites allow cloning of a variety of DNA fragments.
The E. coli replicon, the pAL 5000 origin of replication, the kanamycin
resistance marker, and the D29 attP site and int genes have been mapped and
reduced to their minimum functional sizes to maximize the cloning capacity of
the vector and to increase the transformation efficiency. A new transformation
protocol was developed so that the efficiency with which these vectors


CA 02284736 1999-09-27
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- 60
transform Mycobacterium strains ( 1 Og Mycobacterium transformants/,ug
DNA) approaches the transformation efficiency for E. codi.
The vaccine system of the present invention has a number of advantages
over current vaccines. The major advantage of such a system over current
vaccines is the ability to specifically express immunogens that elicit a
consistent, protective immune response, i.e., a prolonged activation of Tx-1
cells with concomitant activation of macrophages. Additional advantages
include: 1 ) protective immunogens for more than one intracellular disease can
be incorporated into one vaccine, 2) such a genetically engineered vaccine is
flexible in that new technology can be easily incorporated to improve the
vaccine, and 3) Large amounts of immunogen can be synthesized by using a
genetically engineered expression vector to induce protective immunity, 4) the
Mycobacterium itself acts as an adjuvant injected along with the immunogen to
induce immunity, 5) the vaccine is naturally targeted to macrophages because
the Mycobacterium infect these cells, 6) and prolonged immunity will result
since a Mycobacterium strain remains live within by the macrophages for a long
time.
Methodologies for performing various aspects of the present invention
are presented below.
DNA, RNA and o,~~gonucleotide np 'mers
DNA and RNA were extracted and purified at Cytoclonal
Pharmaceutics, Inc., Dallas, Texas. The oligonucleotide primers were
purchased from National Biosciences Inc., Plymouth, MN., or from Integrated
DNA Technologies Inc., Coralville, IA.
En~;rmes.
Restriction endonucleases were purchased from United States
Biochemical Inc., Cleveland, OH.; New England Biolabs Inc., Beverly, MA.;
Promega Inc., Madison, WL; Stratagene Inc., La Jolla, CA.; MBI Fermantas
Inc., Lithuania.; and TaKaRa Biomedicals Inc., Kyoto, Japan. DNA ligase was
purchased from Boehringer Mannheim Biochemica Inc., Indianapolis, IN.;
Gibco-BRL Inc., Gaithersburg, MD., and New England Biolabs. RNase was


CA 02284736 1999-09-27
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- - bl
purchased from 5 Prime --------->3 Prime :(nc., Boulder, CO.
Deoxyribonucleotides and DNA polymera.se I (Klenow fragment) were
purchased from New England Biolabs. A,Ikaline phosphatase was purchased
from Boehringer Mannheim Biochemica and New England Biolabs. Taq
polymerase was purchased from Qiagen Inc., Chatsworth, CA. AMV reverse
transcriptase was purchased from Promega Inc. DNase-free RNase and
RNase-free DNase were purchased from A.mbion Inc., Austin, TX.
Computer software
The computer software Oligo (Natiional Biosciences Inc, Plymouth,
MN} and MacVector (Oxford Molecular Group Inc., Campbell, CA) were
used to design primers and to analyze nucleic acid and protein sequences.
Prgparation of Microorganisms
Bacterial strains and bacteriophages were used from the collection of
the Vaccine Program at Cytoclonal Pharmaceutics Inc., Dallas, TX.
I S Antibiotics ampicillin, kanamycin and tetracycline were purchased from
Sigma Chemical Co., Inc. (Saint Louis, MO).
The requirements for Mycobacterizrm species to grow are usually more
complex and more diversified than those fir E coli strains. Consequently, a
general culture medium, hereinafter designated Labidi's medium, has been
developed which can support the growth of all Mycobacterium species and
which contributes to the increased transformation rate of the present
invention.
The composition of the Labidi's medium pe;r liter contains: about 0.25%
proteose peptone No 3; about 0.2% nutrient broth, about 0.075% pyruvic acid,
about 0.05% sodium glutamate, about 0.5°,io albumin fraction V, about
0.?%
dextrose, about 0.0004% catalase, about 0.005% oleic acid, Lc_~ amino-acid
complex (about 0.126% alanine, about 0.0!7% leucine, about 0.089% glycine,
about 0.086% valine, about 0.074% arginine, about 0.06% threonine, about
0.059% aspartic acid, about 0.057% serine, about 0.056% proline, about
0.05% glutamic acid, about 0.044% isoleuc:ine, about 0.033% glutamine, about
0.029% phenylalanine, about 0.025% asparagine, about 0.024% lysine, about
0.023% histidine, about 0.021% tyrosine, about 0.02% methionine, about


CA 02284736 1999-09-27
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62
0.014% tryptophan, and about 0.01% cysteine), about 0.306% NazHP04,
about 0.055% KHzP04, about 0.05% NH4Cl, about 0.335% NaCI, about
0.0001% ZnS04, about 0.0001% CuS04, about 0.0001% FeCl3, about 0.012%
MgS04, about 0.05% Tween 80, and about 0.8% Glycerol (except for M.
bovis), pH 7Ø A solid form of this medium can be obtained by adding 2.0%
agar. Whenever it is necessary, this medium can be supplemented with
preferred selection markers and/or with special factors required for the
growth
of certain species such as mycobactin for M paratuberculosis and hemin X
factor for M. haemophilium.
For transformation, cultures were grown on Labidi's medium. The
cultures were incubated at the appropriate temperature for each strain.
Cultures in liquid media were shaken at 150 rpm in a rotatory shaker Gyromax
703 (Amerex Instruments Inc., Hercules, CA).
In growing Mycobacterium cells for the vaccine, cultures were grown
on protein-free media: [per liter: 6.0% glycerol, 0.75%glucose, 0.4%
asparagine, 0.25% NaZHP04, 0.2% citric acid, 0.1% KHZP04, 0.05% ferric
ammonium citrate, 0.05% MgS04, 0.02% Tween 80, 0.0005% CaClz,
0.0001% ZnS04, and 0.0001% CuS04, at a final pH of 7 ]. Whenever it is
necessary, this medium can be supplemented with the required selection
markers and/or the growth factors.
For routine culture of E. coli strains, the bacteria were cultivated on
Luria Broth (LB) medium [per liter of medium: 1 % tryptone, 1 % NaCI, and
0.5% yeast extract in distilled or deioninzed water]. The solid form of the LB
medium was obtained by adding 2.0% agar to the previous formula. When
necessary, the met~um was supplemented with selection markers. The cultures
were incubated at 37°C except if the culture required otherwise.
Cultures in
liquid media were shaken at 280 rpm in a rotatory shaker Gyromax 703
(Amerex Instruments Inc., Hercules, CA).
Spheroplasts were prepared from Mycobacterium cultures as
previously described (Labidi, et al. 1984. Curr. Microbiol. 11, 235-240).
Briefly, the spheroplast solution [for every ml of Mycobacterium culture ( 14


CA 02284736 1999-09-27
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63
mg of glycine, 60 beg of D-cycloserine, 1 m;g of lithium chloride, 200 ~g of
lysizyme, and 2 mg of EDTA)) was added to the Mycobacterium cultures in
exponential growth phase, and the incubation was continued for three
generations to induce spheroplast formation. The spheroplasts were
subsequently collected by centrifugation for 20 min, at 3000 rpm, at 4
° C,
washed and resuspended in the spheroplast storage solution [per liter, (6.05
gm
of tris, 18.5 gm of EDTA, 250 gm of sucrose, and pH adjusted to 7)).
Culturing MXcobacterium for Adjuvants
The adjuvants are made of Mycobacterium cells harvested after
preferably growing the corresponding Mycobacterium strains in a liquid protein
free medium. The medium is inoculated and incubated at the appropriate
temperature. The culture is shaken at 150 rpm for appropriate aeration. The
ODD of the culture is monitored daily to determine when the culture reaches
stationary phase. At the stationary phase, the number of cells per milliliter
is
determined through serial dilutions and plating each dilution in triplicate.
The
culture is sterilely centrifuged for 30 minutes, at 5000 rpm, at 4 ° C.
The
pelleted cells are washed twice with ice cold. sterile distilled water and
pelleted
as indicated above. The pellet is re-suspended into pyrogen-free saline (for
injection only), to form a suspension of cells ranging from 1 Og - 10'Z cells
per
ml. The Mycobacterium cell suspension is dlispensed into suitable mufti-dose
vials and used alive, or dead. Preferred methods for killing the mycobacterium
cells include the use of chemicals, radiation, or intense heat (autoclaving
for 30
min, at 15 - 18 psig ( 104 - 124 kPa) at 120 - 122 ° C).
DNA and RNA Preparations
Plasmid DNA was prepared from E. coli strains, as described in prior
text (Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum
complex isolates," Curr. Microbiol. 11, 235-240). 300 ul of spheroplasts
were microcentrifuged in another preferred method of the invention. The pellet
was resuspended in 360 ~1 of freshly prepare;d SI solution [250 mM tris (pH7),
50mM EDTA (pH8), 50 mM glucose, and 2.5 ~cg/ml losozyme]. 240 gel of S II
[ 10% SDS (pH7)) was added and the pellet :incubated at 65 ° C for 15
minutes.


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- - 64
Subsequently, 300 ~cl of S III [7.5 ammonium acetate (pH 7.5), or 5 M NaCl,
or 3 M potassium acetate (pH 5.2), or 3 M sodium acetate (pH 5.2)J was
added and the pellet was incubated on ice for 15 minutes and microcentrifuged
for 15 minutes at 0 ° C at 14 Krpm. 2. 5 /.cl of proteinase K (20
mg/ml) was
added and incubated at 37° C for 15 minutes. The aqueous phase is
extracted
three times by adding 250 ,ul of buffered phenol and 250 ~cl of chloroform/iso-

amyl-alcohol (24:1, v/v) each time. The pellet is vortexed, microcentrifuged
for 15 minutes at 14 Krpm at room temperature and the aqueous phase
recovered. To the last aqueous phase is added 1 ml of isopropanol, vortex
briefly and microcentrifuge for 10 minutes at 14 Krpm at room temperature.
The pellet is dried at 37 ° C for 5 minutes and the DNA is dissolved in
50 ~cl of
sterile distilled water.
Total DNA was prepared from Mycobacterium strains as described
before (Labidi, A., 1986). Another preferred method is to add sterile glass
beads to the pellet obtained from 20 ml of spheroplasts. The pellet is
vortexed
vigorously to have a homogeneous suspension. The suspension is treated with
ml of SI, 8 ml of SII, and 14 ml of SIII. The aqueous phase is extracted
several times, each time with 10.5 ml of a buffered phenoUchloroform/iso-
amyl-alcohol solution. The total DNA is precipitated with 0.6 volume of
20 isopropanol, then dissolved in a cesium chloride gradient and ethidium
bromide. The gradient is centrifuged and treated according to techniques that
are well established in the art. The plasmid DNA then be separated from the
chromosomal DNA.
Total RNA was prepared from E. coli strains containing the appropriate
plasmids and application of a preferred two step protocol. A crude preparation
of total RNA was made using the protocol provided with the kit "Ultraspec
RNA Isolation System" (Biotex Laboratories Inc., Houston, TX). Since the
latter was always contaminated with plasmid DNA, the total RNA was further
purified using the protocol provided with the kit "Qiagen Total RNA Isolation"


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
(Qiagen Inc., Chatsworth, CA). The combination of the two systems
efI'lciently separated total RNA from other contaminating nucleic acids.
Prgt~ration of Electro-competent Cells
Mycobacterium strains can be transi:ormed only through electroporation
(Labidi, A., 1986). Therefore, the bacterial cells must be made electro-
competent before being subject to this procedure. E. coli strains were made
electro-competent following the protocol provided with the BRL Cell Porator
apparatus ( BRL Life Technologies, Gaithersburg, MD).
For Mycobacterium strains, a single colony of Mycobacterium culture
10 was inoculated into 25 ml of Labidi's medium in a 250 ml screw capped
flask.
The culture was shaken at 150 rpm at appropriate temperature until the ODD
reached 0.7. The culture was checked for contamination by staining. If there
was no contamination, a second culture was started by inoculating 50 ,ul of
the
first culture into 200 ml of Labidi's medium iin a 2000 ml screw capped flask.
15 The culture was shaken at 150 rpm at appropriate temperature until the ODD
reached 0.7. The culture was cooled on ice/water for 2 hours, and then the
bacterial cells were harvested by centrifugation (7.5 Krpm) for 10 minutes at
4°C. The first pellet was suspended into 31 ml of3.5% sterile cold
glycerol
and centrifuged (5 Krpm) for 10 minutes at ~4°C. The second pellet was
20 suspended into I2 ml of 7% sterile cold glycerol and centrifuged (3 Krpm)
for
10 minutes at 4 ° C. The third pellet was suspended into 6 ml of 10%
sterile
cold glycerol and centrifuged (3 Krpm) for 1 ~0 minutes at 4 ° C. The
fourth
pellet was suspended in a minimum volume o~f about 2.0 ml of 10.0% sterile
cold glycerol, aliquoted into 25.0 ~cl fractions. then used immediately or
stored
25 at minus 80°C.
Transformation
The technique of electroporation was applied to E. coli and
Mycobacterium strains. E. coli or Mycobacterium electro-competent cells (25
~cl) were mixed with vector DNA ( I 0 ng in 1 E.cl), incubated on ice/water
for 1
30 minute then transferred to an electroporation cuvette (0.15 cm gap). The
electroporation was conducted with a BRL Cell Porator apparatus Cat. series


CA 02284736 1999-09-27
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66
1600 equipped with a Voltage Booster Unit Cat. series 1612 (BRL Life
Technologies, Gaithersburg, MD). The Voltage Booster Unit was set at a
resistance of 4 kiloohms and the Power Supply Unit was set at a capacitance
of 330 microfarad, a fast charging speed rate and a iow Ohm mode to
S eliminate extra-resistance. Once the cuvettes were in the safety chamber,
the
"charge/arm button" was set to "charge", the "up button" was held down until
the capacitors voltage displayed in the Power Supply Unit reached 410 volts
forE coli and 330 volts for Mycobacterium strains. The "charge/arm button"
was set to "arm" and the capacitors voltage was allowed to fall down to 400
volts for E coli and to 316 volts for Mycobacterium strains. The "trigger
button" was pushed to deliver about 2.5 kilovolts for E. coli and
Mycobacterium strains, respectively. These voltage values were displayed on
the Voltage Booster Unit. Each voltage value corresponds to 2.5 kilovolts
divided by 0.15 cm equals 16.66 kilovolts/cm across the cuvette gap for E coli
strains and 1.9 kilovolts divided by 0.15 cm equals 12.66 kilovolts/cm across
the cuvette gap for Mycobacterium strains. The electroporated cells of each
sample were immediately collected with 1 m1 of Labidi's medium, transferred to
a 15 ml falcon tube with a round bottom (Becton Dickenson Inc., Lincoln Park,
NJ) and incubated for one generation time under appropriate temperature and
shaking conditions. The cultures were diluted 1:102 to 1:105 into sterile
distilled water. The diluted cultures were plated ( 100 ~l) in triplicates on
Kanamycin-containing LB and Labidi's media, respectively. The plates were
incubated at appropriate temperatures until colonies were visible and easy to
count. The numbers counted were averaged and used to calculate
transformation erTiciencies. A negative and a positive control were included
for
each species and each experiment.
DNA Sequencing
The DNA was sequenced using a double strand plasmid template and
the protocol provided with the kit "Sequenase Version 2.0" (LJSB, Cleveland,
Ohio, USA). The sequence was computer analyzed using MacVector program
(Oxford Molecular Group Inc., Campbell, CA).


CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
67
In Vitro Analysis of Vector's Stabilit,X,
Single Mycobacterium transformant colonies were grown to saturation
on Labidi's medium containing kanamycin (50 ~cg/ml). The number of
generations required to reach saturation is significantly different between
slow
and rapidly growing mycobacteria. The saturated cultures were diluted to
1:102 and to 1:106 into antibiotic-free Labidi's medium. The dilution 1:106
was
immediately plated (0.1 ml per plate) on antibiotic containing Labidi's medium
to determine the number of Kanamycin-resistant colonies per ml of culture at
the start of the experiment. For calculation purposes, the number of
Kanamycin-resistant colonies per ml of this culture was considered to be 100%.
Fractions of 0.1 ml of the dilution 1:102 were used to inoculate fresh
antibiotic-free Labidi's medium and allowed to grow to saturation. This
procedure was repeated for six months. >=:ach time the number of Kanamycin-
resistant colonies was determined. The proportion of antibiotic-resistant
colonies in the culture after the six month period was found to be 96%.
DNA and RNA transactions.
DNA and RNA were treated with the appropriate enzymes respectively,
as recommended by the manufacturers.
Integration analysis
The integration of vectors containing the attachment site (attP) and the
integrase gene (int) of the Mycobacteriophage Dz9 into the chromosomes of
the Mycobacterium host strains was analyzed by plasmid DNA preparation and
by hybridization using the cloned fragment :From the Dz9 genome as a probe.
M~nicells analysis
Minicells analysis was performed using the E. coli DS410, which is a
mutant strain of E. coli (MinA and MinB). This mutant divides asymmetrically
and produces normal cells and small anucleated cells called minicells. The
minicells are easily separated from normal cells by their differential
sedimentation on a sucrose gradient. If the mirucells producing strain
contains
a mufti-copy plasmid, each of its minicells vrill not have a chromosome but
will


CA 02284736 1999-09-27
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68
carry at least one copy of the plasmid. Since minicells are capable of
supporting DNA, RNA and protein synthesis for several hours, they are used
as an in vivo gene expression system for prokaryotes. The expression product
is labeled with S35-methionine and analyzed by protein gel electrophoresis.
S Nutrient Broth is the medium used in this technique.
Preparation of minicells originated with the preparation of
electrocompetent cells of E. toll DS410 with the appropriate recombinant
plasmids. Each plasmid containing clone is grown overnight in 400 ml NB
having the appropriate selection markers. One clone of the non transformed
DS410 was grown on 400 ml NB alone to serve as a control.
Three 3 S ml sucrose gradients ( 10-30% w/v) were prepared per clone
using M9-mm-S[per liter of medium: 200 gm of sucrose, 100 ml of sterile
l OX I- M9-mm, 10 ml of sterile 10 mM CaCl2, and 10 ml of sterile 100 mM
Mg S04]. The gradients are then placed at minus 70 ° C for at least
one hour
or until the gradients are completely frozen. The gradients are then placed at
4 ° C overnight to allow the gradient to thaw and to be established.
The
bacterial cultures are centrifuged for 5 minutes at 2 Krpm at 4° C. The
supernatants are then centrifuged for 15 minutes at 8 Krpm at 4° C.
Each
pellet is subsequently resuspended in 6 ml of M9-mm [per l OX liter of medium:
400 mM NaHZP04, 200 mM KHZP04, 80 mM NaCI, and 200 mM NH4C1)].
Each 3 ml of cell suspension is layered on top of a sucrose gradient. The
gradients are then centrifuged for 18 minutes at 5 Krpm at 4° C. The
top one-
third of the white transparent minicells band are recovered from each
gradient.
An equal volume of M9-mm is added to each tube and centrifuged for 10
2~ minutes at 10 Krpm at 4° C. Each pellet is subsequently resuspended
in 3 ml
of M9-mm and the suspension is layered on top of the last gradient and
centrifuged for 18 minutes at 5 Krpm at 4 ° C. The top one-third of the
white
transparent minicells band is recovered and the optical density is read at 600
nm. The number of cells in the minicells preparation is calculated using the
equation of 2 ODD, which equals 10'° minicells per ml. Preferably, the
level of
whole cell contamination is determined in the minicells' preparation. The


CA 02284736 1999-09-27
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69
minicell suspension is centrifuged for 10 minutes at 10 Krpm at 4 ° C
and
resuspended in M9-mm-G [per 100 ml of medium: 30 mi of sterile ( 100%)
glycerol, 1 ml of sterile 10 mM CaCI2, 1 rr~l of sterile 100 mM MgS04, and 10
ml of sterile lOX I-M9-mm).
The labeling of the plasmid encoded proteins with 535 methionine is
achieved by placing 100 ,~l of minicells in tlhe microcentriuge for 3 minutes
at
4 ° C. The pellet is resuspended in 200 ~cl of M9-mm and 3 ~cl of MAM [
10.5
gm of methionine assay medium per 100 m:l of medium]. The pellet is
incubated at 37° C for 90 minutes and 25 ~,cCi of S35-methionine is
added. The
pellet is incubated at 37° C for 60 minutes. 10 ~l of unlabeled MS (0.8
gm of
L(-) methionine in 100 ml of distilled water] is added and incubated at
37° C
for S minutes and microcentrifuged for 3 minutes at room temperature. The
pellet is resuspended in SO ~l of BB (per 100 ml of solution, (0.71 gm of
NazHP04, 0.27 gm of KHzP04, 0.41 gm of NaC 1, and 400 ,ul of sterile 100
1 S mM MgS04)] and 50 ~cl of SDS-SB [per 10 ml of solution, ( 12.5 ml of
sterile 1
M tris (pH 6.8), 20 ml of sterile (100%) glycerol, 10 ml of 20% SDS (pH 7.2),
Sml of mercaptoethanol, and 250 ~cl of 0.4°.% bromophenol blue)]. The
pellet is
boiled for 3 minutes, centrifuged, and the top 25 ~cl of the sample is applied
to
a 12% SDS-polyacrylamide slab gel.
Primer extension analv
Primer extension analysis was conducted according to the protocol
provided with the kit "Primer Extension Sy:>tem" (Promega Inc., Madison, WI).
Ribonuclease protection as av anahr
Ribonuclease protection assay (RPA) was conducted according the
protocol provided with the "Ambion HypSpeed RPA Kit" (Ambion Inc.
Austin, TX).
DNA amplification b~~y~erace chain reaction
DNA fragments from the Mycobacteriophage Dz9 genome and
Mycobacterium plasmid and chromosomal l7NA were amplified by polymerise
chain reaction using a Progene Programmat~le Dri-Block Cycler (Techne Inc.,
Princeton, NJ). The reaction mixture was subject to denaturation
(94°C for 3


CA 02284736 1999-09-27
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_ 70
minutes), followed by 10 cycles of amplification (94 ° C for 2 minutes,
55 ° C for
2 minutes, 72°C for 2 minutes), followed by 30 cycles of amplification
(94 ° C for 2 minutes, 63 ° C for 2 minutes, 72 ° C for 2
minutes). The
programming described above is disclosed for the first time in this report.
Examples 1-3 demonstrate the present invention in terms of use of
specific antigens in the treatment of various diseases. These examples are
illustrative and are not meant to be limiting with regard to the selected
antigen
and Mycobacterium strain nor the application of the E. coli Mycobacterium
shuttle.
Example 1: Exem l~ary AIDS Vaccine
If the product is being used to vaccinate against AIDS, E. coli-
Mycobacterium expression vectors containing genes encoding HIV env, rev,
and gag/pol proteins (National Institutes of Health, Bethtesda MD), and genes
encoding IL-2, gamma INF and GMCSF (Cytoclonal Pharmaceutics, Inc.,
Dallas, Texas) are electroporated into a recipient strain M. aurum. The
transformants are checked for their plasmid content. A clone containing the
expected hybrid plasmid is grown in the protein-free liquid medium. The
inoculated medium is incubated at a temperature of 35 to 37°C. The
culture is
shaken at 150 rpm for appropriate aeration. The ODD of the culture is
measured daily, and a growth curve featuring optical densities versus time is
established. At the stationary phase, the number of cells per milliliter is
determined through serial dilutions ( 1:10 to 1:10'° ), and plating in
triplicates of
each dilution on Labidi's medium. The culture is sterilely centrifuged for 30
minutes, at 5000 rpm, at 4°C. The pelleted cells are washed twice with
ice
cold sterile distilled water and pelleted as indicated above. The pellet is re-

suspended into pyrogen-free saline for injection only, to have a suspension of
108 to 10'z cells per ml. The Mycobacterium cell suspension is dispensed into
suitable mufti-dose vials. The product is administered by injection given
intradermaI in a volume of about 100 ul containing 10' to I 0" cells of
recombinant Mycobacterium. If a killed form of the vaccine is preferred, the
cells can be killed either chemically, by radiation, or by autoclaving for 30
min,


CA 02284736 1999-09-27
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71
at 15 - 18 psig ( 104 - 124 kPa) at 120 - 12 2 ° C. If a killed form of
the vaccine
is used, those antigens or cytokines that may be inactivated during the
process
are added to the product separately, or they recombinant cells are killed by
radiation.
example 2~ Exem~lanr Cancer Vaccine
If the product is being used to vaccinate against cancer such as prostate
cancer, the gene encoding the cancer antigen such as the prostate cancer
antigen PSA (National Institutes of Health., Bethesda, MD), is cloned
according
to the procedure given in Example 1. The product is prepared and adminstered
according to the procedure given in Example 1.
Example 3 ~ Exemplary Aller~v Vaccine
If the product is being used for vaccination against allergies such as
reactions to the major allergen of birch pollen, the gene encoding the
allergen
such as the birch pollen allergen BetVla (Llniveristy of Vienna, Austria) is
cloned according to the procedure given in Example 1. The product is
prepared and adminstered according to the procedure given in Example 1.


CA 02284736 1999-09-27
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72
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Cytoclonal Pharmaceutics, Inc.
(B) STREET: 9000 Harry Hines Blvd, Suite 330
(C) CITY: Dallas
(D) STATE: Texas
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 75235
(G) TELEPHONE: (214) 353-2923
(H) TELEFAX: (214) 350-9514
(I) TELEX:
(ii) TITLE OF INVENTION: Mycobacterium Recombinant Vaccines
(iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sidley & Austin
(B) STREET: 717 N. Harwood, Suite 3400
(C) CITY: Dallas
(D) STATE: Texas
(E) COUNTRY: United States
(F) ZIP: 75201
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/042849
(B) FILING DATE: 28-MAR-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hansen, Eugenia S.
(B) REGISTRATION NUMBER: 31,966
(C) REFERENCE/DOCKET NUMBER: 10365/05602
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 214-981-3300
(B) TELEFAX: 214-981-3400

CA 02284736 1999-09-27
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73.
(2) INFORMATION FOR SEQ ID NO:1:


(i} SEQUENCE CHARACTERISTICS:


(A) LENGTH: 695 base pairs


(B) TYPE: nucleic acid


(C) STRANDEDNESS: double


(D) TOPOLOGY: linear


(ii) MOLECULE TYPE: DNA (genomic)


(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:


GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCAC'AAAAATCGACGCT CAAGTCAGAG~
60


GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTTCCCCCTGGAA GCTCCCTCGT120


GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATAC'CTGTCCGCCTTTC TCCCTTCGGG180


AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTAT'CTCAGTTCGGTGT AGGTCGTTCG240


CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCCGACCGCTGCG CCTTATCCGG300


TAACTATCGT CTTGAGTCC'.A ACCCGGTAAG ACACGAC'TTATCGCCACTGG CAGCAGCCAC360


TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGT'GCTACAGAGTTCT TGAAGTGGTG420


GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGT'ATCTGCGCTCTGC TGAAGCCAGT480


TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGC'AAACAAACCACCG CTGGTAGCGG540


TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGp,AAAAAAGGATCTC AAGAAGATCC600


TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAAC'.GAAAACTCACGTT AAGGGATTTT660


GGTC'ATGAGA TTATCAAAAA GGATCTTCAC CTAGA 695


(2) INFORMATION FOR SEQ ID N0:2:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 932 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOL9~Y: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NC1:2:
GTTGTGTCTC AAAATCTCTG ATGTTAC.ATT GCACAAGiATA AAAATATATC ATCATGAACA 60

CA 02284736 1999-09-27
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74
ATAAAACTGTCTGCTTACAT ACAAGGGGTGTTATGAGCCA 120
AAACAGTAAT TATTCAACGG


GAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTAT 180


AAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAG 240


CCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACA 300


GATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCAT 360


TTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCA 420


TTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTG 480


TTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTA 540


TTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTT 600


GATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTG 660


CCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTT 720


GACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATAC 780


CAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGG 840


CTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATG 900


CTCGATGAGTTTTTCTAATCAGAATTGGTTAA 932


(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1463 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TGTTCCTCCT GGTTGGTACA GGTGGTTGGG GGTGCTCGGC TGTCGCGGTT GTTCCACCAC 60
CAGGGCTCGA CGGGAGAGCG GGGGAGTGTG CAGTTGTGGG GTGGCCCCTC AGCGAAATAT 120
CTGACTTGGA GCTCGTGTCG GACCATACAC CGGTGATTAA TCGTGGTCTA CTACCAAGCG 180
TGAGCCACGT CGCCGACGAA TTTGAGCAGC TCTGGCTGCC GTACTGGCCG CTGGCAAGCG 240

CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
ACGATCTGCT CGAGGGGATCTACCGCCAAA GGCCCTAGGCCGCCGGTACA300
GCCGCGCCiTC


TCGAGGCGAA CCCAACAGCGCTGGCAAACCTGCTGGTCGTGGACGTAGACCATCCAGACG360


CAGCGCTCCG AGCGCTCAGCGCCCGGGGGTCCCATCCGCTGCCC'.AACGCGATCGTGGGCA420


ATCGCGCCAA CGGCCACGCAC'ACGCAGTGTGGGCACTC:AACGCCCCTGTTCCACGCACCG480


AATACGCGCG GCGTAAGCCGCTCGCATAC:ATGGCGGCCDTGCGCCGAAGGCCTTCGGCGCG540


CCGTCGACGG CGACCGCAGTTACTCAGGCCTCATGACC'AAAAACCCCGGCCACATCGCCT600


GGGAAACGGA ATGGCTCCACTCAGATCTCTACACACTC'AGCCACATCGAGGCCGAGCTCG660


GCGCGAACAT GCC.'ACCGCCGCGCTGGCGTCAGC.AGACC'ACGTACAAAGCGGCTCCGACGC720


CGCTAGGGCG GAATTGCGCACTGTTCGATTCCGTCAGGTTGTGGGCCTATCGTCCCGCCC780


TCATGCGGAT CTACCTGCCGACCCGGAACGTGGACGG~1,CTCGGCCGCGCGATCTATGCCG840


AGTGCCACGC GCGAAACGCCGAATTCCCGTGCAACGAC'GTGTGTCCCGGACCGCTACCGG900


ACAGCGAGGT CCGCGCCATCGCCAACAGCATTTGGCGT'TGGATCACAACCAAGTCGCGCA960


TTTGGGCGGA CGGGATCGTGGTCTACGAGGCCAC:ACTC'AGTGCGCGCCAGTCGGCCATCT1020


CGCGGAAGGG CGCAGCAGCGCGCACGGCGGCGAGCACA.GTTGCGCGGCGCGCAAAGTCCG1080


CGTCAGCCAT GGAGGC,AT'TGCTATGAGCGACGGCTACA.GCGACGGCTACAGCGACGGCTA1140


CAACCGGCAG CCGACTGTCCGCAAAAAGCCGTGACGCGCCGAAGGCGCTCGAATCACCGG1200


ACTATCCGAA CGCCACGTCGTCCGGCTCGTGGCGCAGGAACGCAGCGAGTGGCTCGCCGA1260


GCAGGCTGCA CGCGCGCGAAGC'ATCCGCGCCTATCACGACGACGAGGGCCACTCTTGGCC1320


GC.AAACGGCC AAAC.ATTTCGGGCTGCATCTGGACACCGTTAAGCGACTCGGCTATCGGGC1380


GAGGAAAGAG CGTGCGGCAGAACAGGAAGCC,GCTC.'AAAAGGCCCACAACGAAGCCGAC'AA1440


TCC:ACCGCTG T'TCTAACGCAATT 1463


(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1382 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear

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76
(ii) MOLECULE
TYPE:
DNA (genomic)


(xi) SEQUENCE CRIPTION:
DES SEQ ID
N0:4:


GGGTGCTCGGCTGTCGCGGTTGTTCCACCACCAGGGCTCGACGGGAGAGCGGGGGAGTGT 60


GCAGTTGTGGGGTGGCCCCTCAGCGAAATATCTGACTTGGAGCTCGTGTCGGACCATACA 120


CCGGTGATTAATCGTGGTCTACTACCAAGCGTGAGCCACGTCGCCGACGAATTTGAGCAG 180


CTCTGGCTGCCGTACTGGCCGCTGGCAAGCGACGATCTGCTCGAGGGGATCTACCGCCAA 240


AGCCGCGCGTCGGCCCTAGGCCGCCGGTACATCGAGGCGAACCCAACAGCGCTGGCAAAC 300


CTGCTGGTCGTGGACGTAGACCATCCAGACGCAGCGCTCCGAGCGCTCAGCGCCCGGGGG 360


TCCCATCCGCTGCCCAACGCGATCGTGGGCAATCGCGCCAACGGCCACGCACACGCAGTG 420


TGGGCACTCAACGCCCCTGTTCCACGCACCGAATACGCGCGGCGTAAGCCGCTCGCATAC 480


ATGGCGGCGTGCGCCGAAGGCCTTCGGCGCGCCGTCGACGGCGACCGCAGTTACTCAGGC 540


CTCATGACCAAAAACCCCGGCCACATCGCCTGGGAAACGGAATGGCTCCACTCAGATCTC 600


TACACACTCAGCCACATCGAGGCCGAGCTCGGCGCGAACATGCCACCGCCGCGCTGGCGT 660


CAGCAGACCACGTACAAAGCGGCTCCGACGCCGCTAGGGCGGAATTGCGCACTGTTCGAT 720


TCCGTCAGGTTGTGGGCCTATCGTCCCGCCCTCATGCGGATCTACCTGCCGACCCGGAAC 780


GTGGACGGACTCGGCCGCGCGATCTATGCCGAGTGCCACGCGCGAAACGCCGAATTCCCG 840


TGCAACGACGTGTGTCCCGGACCGCTACCGGACAGCGAGGTCCGCGCCATCGCCA~1CAGC900


ATTTGGCGTTGGATCACAACCAAGTCGCGCATTTGGGCGGACGGGATCGTGGTCTACGAG 960


GCCACACTCAGTGCGCGCCAGTCGGCCATCTCGCGGAAGGGCGCAGCAGCGCGCACGGCG 1020


GCGAGCACAGTTGCGCGGCGCGCAAAGTCCGCGTCAGCCATGGAGGCATTGCTATGAGCG 1080


ACGGCTACAGCGACGGCTACAGCGACGGCTACAACCGGCAGCCGACTGTCCGCAAAAAGC 1140


CGTGACGCGCCGAAGGCGCTCGAATCACCGGACTATCCGAACGCCACGTCGTCCGGCTCG 1200



TGGCGCAGGAACGCAGCGAGTGGCTCGCCGAGCAGGCTGCACGCGCGCGAAGCATCCGCG 1260


CCTATCACGACGACGAGGGCCACTCTTGGCCGCAAACGGCCAAACATTTCGGGCTGCATC 1320


TGGACACCGTTAAGCGACTCGGCTATCGGGCGAGGAAAGAGCGTGCGGCAGAACAGGAAG 1380


CG
1382



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77
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE
CHARACTERISTICS:


(A) LENGTH:1631 base
pairs


(B) TYPE:
nucleic
acid


(C) STRANDEDNESS:
double


(D) TOPOLOGY:
linear


(ii)
MOLECULE
TYPE:
DNA
(genomic)


(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:5:


GTGAGAGAATCTTCACTGCACC'AGCTCCGATCTGGTGTACCGCCCCTCGT CTGTTGCAGC60


AGGCGGGGGGCTTTCTTCGTCTGTCC~GAGGTCGAAGGTAGCAGATGTGTC GCTGTATCCG120


GGCAGCATAAATGCAGGTCATTAGTGTCGCTCTAAGGTCGCGGCCCCCTC TCGGGGATCC180


GGTCCTCGGGCTAAAAACCACCTCTGACCTGTGGAGCGiGGCGACGGGAAT CGAACCCGCG240


TAGCTAGTTTGGAAGTAAGGGGGTCGGCGTGTCACAT9'CTCCCAGCTCAG ACCCTGTTTT300


TAGCTCTGACCCTGTGCGACCTTGAAGTGGACAAAAATGCCTGTTCACGG ACACGCAAAG360


ACGTCTGAAGGTCGCAATAAGGTCGCATTCCGGTAGCC'.TGTTTCGCATGG CAGCAAGACG420


GAGAGGATGGGGATCGCTGCGGACCCAGCGCAGCGGTC'.GAGTGCAAGCGT CGTACGTC.'AG480


CCCGATCGACGGGCAGCGGTACTTCGGGCCGAGGAACTACGACAACCGGA TGGACGCCGA540


AGCGTGGCTCGCGTCTGAGAAGCGGCTGATCGACAACCiAGGAGTGGACCC CGCCGGCCGA600


GCGCGAGAAGAAGGCTGCGGCGAGTGCCATCACGGTCCiAGGAGTACACCA AGAAGTGGAT660


CGCCGAGCGAGACCTCGCTGGCGGCACCAAGGATCTCTACAGCACGCACG CTCGCAAGCG720


GATCTACCCGGTGTTGGGCGACACCCCGGTCGCCGAGATGACCCCCGCCC TTGTCCGGGC780


GTGGTGGGCCC~GGATGGGTAAGCAGTACCCGACGGCAC:GGCGGCACGCCT ACAACGTACT840


CCGGGCGGTCATGAATACCGCTGTAGAGGACAAGCTGGTGTCGGAGAACC CGTGCCGGAT900


CGAGCAGAAGGCACCCGCTGAGCGCGACGTGGAAGCCC:TCACACCGGAGG AGCTGGACGT960


AGTGGCCGGGGAGGTGTTCGAGC:ACTACCGCGTGGCCC:TCTACATCCTGG CGTGGACCAG1020


CCTGCGGTTCGGTGAGCTGATCGAGATCCGCCGCAAGCsACATCGTGGATG ACGGCGAGAC1080


GATGAAGCTCCGCGTGCGCCGGGGCGCGGCCCGCGTCCxGCGAGAAGATCG TCGTCGGCAA1140


CACCAAGACCGTCAGGTCCAAGCGGCCGGTGACCGTGCCGCCTCACGTCG CGGCGATGAT1200



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CCGCGAGCACATGGCTGACCGGACGAAGATGAACAAGGGGCCGGAAGCTCTCCTGGTGAC 1260


CACCACGCGGGGGCAGCGGCTGTCGAAGTCTGCGTTCACTCGCTCGCTGAAGAAGGGCTA 1320


CGCCAAGATCGGTCGACCGGACCTCCGCATCCACGACCTCCGGGCCGTGGGAGCCACGCT 1380


GGCGGCTCAGGCCGGTGCGACGACCAAGGAGCTGATGGTGCGCCTCGGGCACACGACTCC 1440


GCGCATGGCGATGAAGTACCAGATGGCCTCAGCAGCCCGTGACGAGGAGATAGCGAGGCG 1500


AATGTCGGAGCTGGCAGGGATTACCCCCTGAAACGCAAAAAGCCCCCCTCCCAAGGCCAT 1560


ACAGCCTCAAGAGGGGGGTTTCTTGTCACTCAGTCCACACGGTCCATTGGATCTTGGGCG 1620


TGTAGACGATC 1631


(2) INFORMATION
FOR SEQ
ID N0:6:


(i) SEQUENCE
CHARACTERISTICS:


(A) LENGTH: 1413 base pairs


(B) TYPE: nucleic acid


(C) STRANDEDNESS: double


(D) TOPOLOGY: linear


(ii) MOLECULE
TYPE:
DNA (genomic)


(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:6:


CGGGCAGCATAAATGCAGGT CATTAGTGTC GCTCTAAGGTCGCGGCCCCC TCTCGGGGAT60


CCGGTCCTCGGGCTAAAAAC CACCTCTGAC CTGTGGAGCGGGCGACGGGA ATCGAACCCG120


CGTAGCTAGTTTGGAAGTAA GGGGGTCGGC GTGTCACATTCTCCCAGCTC AGACCCTGTT180


TTTAGCTCTGACCCTGTGCG ACCTTGAAGT GGACAAAAATGCCTGTTCAC GGACACGCAA240


AGACGTCTGAAGGTCGCAAT AAGGTCGCAT TCCGGTAGCCTGTTTCGCAT GGCAGCAAGA300


CGGAGAGGATGGGGATCGCT GCGGACCCAG CGCAGCGGTCGAGTGCAAGC GTCGTACGTC360


AGCCCGATCGACGGGCAGCG GTACTTCGGG CCGAGGAACTACGACAACCG GATGGACGCC420


GAAGCGTGGCTCGCGTCTGA GAAGCGGCTG ATCGACAACGAGGAGTGGAC CCCGCCGGCC480


GAGCGCGAGAAGAAGGCTGC GGCGAGTGCC ATCACGGTCGAGGAGTACAC CAAGAAGTGG540


ATCGCCGAGCGAGACCTCGC TGGCGGCACC AAGGATCTCTACAGCACGCA CGCTCGCAAG600


CGGATCTACCCGGTGTTGGG CGACACCCCG GTCGCCGAGATGACCCCCGC CCTTGTCCGG660



CA 02284736 1999-09-27
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GCGTGGTGGGCCGGGATGGGTAAGCAGTACCCGACGGC:ACGGCGGCACGCCTACAACGTA 720


CTCCGGGCGGTCATGAATACCGCTGTAGAGGACAAGC7:GGTGTCGGAC,AACCCGTGCCGG 780


ATCGAGCAGAAGGCACCCGCTGAGCGCGACGTGGAAGC:CCTCACACCGGAGGAGCTGGAC 840


GTAGTGGCCGGGGAGGTGTTCGAGCACTACCGCGTGGC:CGTCTACATCCTGGCGTGGACC 900


AGCCTGCGGTTCGGTGAGCTGATCGAGATCCGCCGCAAGGACATCGTGGATGACGGCGAG 960


ACGATGAAGCTCCGCGTGCGCCGGGGCGCGGCCCGCG7:CGGCGAGAAGATCGTCGTCGGC 1020


AACACCAAGACCGTCAGGTCCAAGCGGCCGGTGACCG7CGCCGCCTCACGTCGCGGCGATG 1080


ATCCGCGAGCACATGGCTGACCGGACGAAGATGAACAAGGGGCCGGAAGCTCTCCTGGTG 1140


ACCACCACGCGGGGGCAGCGGCTGTCGAAGTCTGCGT7CCACTCGCTCGCTGAAGAAGGGC 1200


TACGCCAAGATCGGTCGACCGGACCTCCGCATCCACGACCTCCGGGCCGTGGGAGCCACG 1260


CTGGCGGCTCAGGCCGGTGCGACGACCAAGGAGCTGA".CGGTGCGCCTCGGGCACACGACT 1320


CCGCGCATGGCGATGAAGTACC:AGATGGCCTCAGCAGCCCGTGACGAGGAGATAGCGAGG 1380


CGAATGTCGGAGCTGGCAGGGATTACCCCCTGA 1413


(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:


(A) LENGTH: 1374 base pairs


(B) TYPE: nucleic acid


(C) STRANDEDNESS: double


(D) TOPOLOGY: linear


(ii) MOLECULE TYPE: DNA (genomic)


(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:


TCGCGGCCCC CTCTCGGGGA TCCGGTCCTC GGGCTAAAAPa CCACCTCTGA 60
CCTGTGGAGC


GGGCGACGGG AATCGAACCC GCGTAGCTAG TTTGGAAGTA AGGGGGTCGG 120
CGTGTCACAT


TCTCCCAGCT CAGACCCTGT TTTTAGCTCT GACCCTG'TGC GACCTTGAAG 180
TGGACAAAAA


TGCCTGTTCA CGGACACGCA AAGACGTCTG AAGGTCGCAA TAAGGTCGCA 240
TTCCGGTAGC


CTGTTTCGCA TGGCAGCAAG ACGGAGAGGA TGGGGATCGC TGCGGACCCA 300
GCGCAGCGGT


CGAGTGCAAG CGTCGTACGT CAGCCCGATC GACGGGC.AGC GGTACTTCGG 360
GCCGAGGAAC



CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
TACGACAACCGGATGGACGC CGAAGCGTGGCTCGCGTCTGAGAAGCGGCT GATCGACAAC420


GAGGAGTGGACCCCGCCGGC CGAGCGCGAGAAGAAGGCTGCGGCGAGTGC CATCACGGTC480


GAGGAGTACACCAAGAAGTG GATCGCCGAGCGAGACCTCGCTGGCGGCAC CAAGGATCTC540


TACAGCACGCACGCTCGCAA GCGGATCTACCCGGTGTTGGGCGACACCCC GGTCGCCGAG600


ATGACCCCCGCCCTTGTCCG GGCGTGGTGGGCCGGGATGGGTAAGCAGTA CCCGACGGCA660


CGGCGGCACGCCTACAACGT ACTCCGGGCGGTCATGAATACCGCTGTAGA GGACAAGCTG720


GTGTCGGAGAACCCGTGCCG GATCGAGCAGAAGGCACCCGCTGAGCGCGA CGTGGAAGCC780


CTCACACCGGAGGAGCTGGA CGTAGTGGCCGGGGAGGTGTTCGAGCACTA CCGCGTGGCC840


GTCTACATCCTGGCGTGGAC CAGCCTGCGGTTCGGTGAGCTGATCGAGAT CCGCCGCAAG900


GACATCGTGGATGACGGCGA GACGATGAAGCTCCGCGTGCGCCGGGGCGC GGCCCGCGTC960


GGCGAGAAGATCGTCGTCGG CAACACCAAGACCGTCAGGTCCAAGCGGCC GGTGACCGTG1020


CCGCCTCACGTCGCGGCGAT GATCCGCGAGCACATGGCTGACCGGACGAA GATGAACAAG1080


GGGCCGGAAGCTCTCCTGGT GACCACCACGCGGGGGCAGCGGCTGTCGAA GTCTGCGTTC1140


ACTCGCTCGCTGAAGAAGGG CTACGCCAAGATCGGTCGACCGGACCTCCG CATCCACGAC1200


CTCCGGGCCGTGGGAGCCAC GCTGGCGGCTCAGGCCGGTGCGACGACCAA GGAGCTGATG1260


GTGCGCCTCGGGCACACGAC TCCGCGCATGGCGATGAAGTACCAGATGGC CTCAGCAGCC1320


CGTGACGAGGAGATAGCGAG GCGAATGTCGGAGCTGGCAGGGATTACCCC CTGA 1374


(2) INFORMATION
FOR SEQ
ID N0:8:


(i) SEQUENCE
CHARACTERISTICS:


(A) LENGTH: 105
base pairs


(B) TYPE: nucleic
acid


(C) STRANDEDNESS:
double


(D) TOPOLOGY: linear


(ii) MOLECULE
TYPE:
DNA (genomic)


(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:8:


GTTGTGTCTCAAAATCTCTG ATGTTACATTGCACAAGATAAAAATATATC ATCATGAACA60


ATAAAACTGTCTGCTTACAT AAACAGTAATACAAGGGGTGTTATG 105



CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056
81
(2) INFORMATION
FOR
SEQ
ID N0:9:


(i) SEQUENCE
CHARACTERISTICS:


(A) LENGTH:2096 base
pairs


(B) TYPE:
nucleic
acid


(C) STRANDEDNESS:
double


(D) TOPOLOGY:
linear


(ii)
MOLECULE
TYPE:
DNA
(genomic)


(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:9:


GGTCACCTGCGATCACACCGAGCGTGCAGGTAGCGAAGTCCTCATCACCA CCAGGACGGG60


CCTGGGCGATACCAGCGCCGGGGGCGATCCCGCCAGGF~AATGCCGTCCAA TCGGTGTCCG120


CGACTGCGGCGGAGCGGACACTCCGACCAACACAACAP~CCAACGTCGTCA TAGCGACGAC180


GAACCACGATCGGATGATCCGAATCACTGCGCTGTCCF~TACAGGCGGCCA CCCCTCGAAC240


TCACCAGCTTCAATGCGCGTCTGCAAAGACTGCCATGGAGCGCTACTCGG GCCGGTCTCA300


ACGCACTGCTCGAAGAAATCGACAGCGGCCAGTGCACC:GAACTCCTTGTG CTGCTCGGCT360


TGCAGCTCGGCGCTCCACGTCTTCACCTCGGGCGCGGPvCAATTCGACGAC CTTGTTAGCG420


ATCGACGCATTGGTCGCCGCAGCAATGCCCGCCACATC:CCAGTCCCCTGG ATCGAGGTCG480


GCGCGGCACAAC.AGCTCCGCGATCCGACCCCGATCCAGCGCCTGCCTCAC CACTTTTCGT540


CGTCGCGGGGCTCACCCGGGTACTGAACCGGATCGCCACTATCGAAACGG CTACGCGCGG600


CGGCAGCGGCGGCGCTGGCGGCGGCACGTTCATCACCF~CCGGACCGGGAA CCAGCGTCGA660


TTCATCGATGGCCGGCTGAATCGGCCGGCGTTCGTCGCDGCAGCAGGTCCG CGAGCTCGTC720


GGCATCGATGTACTGCCGGCCGGCGGATCGTCGTCACCiCAGAATGTGGGA CACCAGCGCC780


TTGTCGCGGGCCTCTTCGCCGGTGAGGATCCGCTCGGAGGCGCGGTCGCG GCGCGGCTGT840


GGCATGTCGGGGCGTGCCGCTCCCCCGGCGCCGCCCATCGGCCCGCCCAT TGGCATTCCG900


CCCATGCCGCCCATCATTCCTGTGGAGCCAGCTGGCCC:GGTCTTCAATGG AGGCAGGCCC960


GCTGACGGCGACGTC~GAGGCGGTGCGCCCCGAAATCTC:GGCCGGATCAAC TCGGCC.ACCG1020


GTCACGGTCGGATTGGCGGCCGGTGTTGTCGGTGCGAC:AACACCGCCGAC AACGCCGCGC1080


CCCGCCATCGCCGAACCACGGGGTGGTC~GGTGCGTCCC~ACCTGCCAGAAT CGTCCCGGCG1140


TCGCGGCTGCTGCTGAACACCGCCGAGCCCGCCGCCAGTCGGGAAAGCGC TGGGCATCAT1200



CA 02284736 1999-09-27
WO 98/44096 PCT/US98/06056 -
82


GGTCGGGCCGGGGGCCATCGGAGCGGGTGCACCTGTCGGGGCTGGTGGCGGCGTCAGCGC 1260


CGTCGCCTGCACCATCGGCCGTGGGCCGCCGACACCTCCGTGGTCGCACCGCCGCCGCCG 1320


ACGATCGTGTCGTCAGCGCCGCCGCCGACGATGGTGTCGTCCCAACCGTCGCGCGGCTGG 1380


AGGTCGCGGGGCGACCGGAAAATGCCTTTATCGTGGCCGGACACCTTGGAATCGGTGTCC 1440


GGCTCGTCGGGCAGGCCTTCCGTCGCTGACGTGCACGCGCGCTCCAATCGCTCCAGCGCC 1500


GCCTGGACCTCGGGATCGGCAGCCGTCCCGCCCCGAATGACCGGGCGGCCGCGGCCGGCC 1560


TCTCCCACCGCACGCAGGGCCGTCGGCGATTTTCAGCAGGTCGCCGCCCATTTCCGACAT 1620


CTTTTCCTCGGCGGCCGATCGCCGCACCGGACCCAATGTCGTCCGGAAACGGCTCGGCCG 1680


CGATCGACTCCAGCAACGCGGCCATGTCGATGCGCTCCTGAAACTCGGCCTCGTTGGTCA 1740


GCGAATCGCCGTCATAACGGATGGCGCCCGGGCCGCCGCGCGATATCGAGCCGAGAACGT 1800


TATCGAAGTTGGTCATGTGTAATCCCCTCGTTTGAACTTTGGATTAAGCGTAGATACACC 1860


CTTGGACAAGCCAGTTGGATTCGGAGACAAGCAAATTCAGCCTTAAAAAGGGCGAGGCCC 1920


TGCGGTGGTGGAACACCGCAGGGCCTCTAACCGCTCGACGCGCTGCACCAACCAG



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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 1998-03-27
(87) PCT Publication Date 1998-10-08
(85) National Entry 1999-09-27
Dead Application 2004-03-29

Abandonment History

Abandonment Date Reason Reinstatement Date
2003-03-27 FAILURE TO REQUEST EXAMINATION
2003-03-27 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 1999-09-27
Application Fee $150.00 1999-09-27
Maintenance Fee - Application - New Act 2 2000-03-27 $50.00 2000-02-17
Maintenance Fee - Application - New Act 3 2001-03-27 $50.00 2001-02-28
Maintenance Fee - Application - New Act 4 2002-03-27 $50.00 2002-02-18
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
CYTOCLONAL PHARMACEUTICS, INC.
Past Owners on Record
LABIDI, ABDEL HAKIM
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Cover Page 1999-11-22 1 42
Description 1999-09-27 82 3,536
Claims 1999-09-27 28 1,225
Description 2000-03-23 84 3,513
Claims 2000-03-23 28 1,059
Abstract 1999-09-27 1 51
Drawings 1999-09-27 10 518
Correspondence 1999-11-02 1 3
Assignment 1999-09-27 4 121
PCT 1999-09-27 46 1,865
Prosecution-Amendment 1999-11-01 1 47
Correspondence 2000-03-23 43 1,535
Assignment 2000-09-28 3 139

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