U.S. patent application number 10/359435 was filed with the patent office on 2005-08-04 for expression of lipoproteins.
Invention is credited to Becker, Robert S., Erdile, Lorne F., Gray, Maryann B., Huebner, Robert C., Pyle, Derek J., Warakomski, Donald J. JR..
Application Number | 20050171343 10/359435 |
Document ID | / |
Family ID | 23889117 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050171343 |
Kind Code |
A1 |
Huebner, Robert C. ; et
al. |
August 4, 2005 |
Expression of lipoproteins
Abstract
Heterologous lipidated proteins formed recombinantly are
disclosed and claimed. The expression system can be E. coli. The
heterologous lipidated protein has a leader sequence which does not
naturally occur with the protein portion of the lipidated protein.
The lipidated protein can have the Borrelia OspA leader sequence.
The protein portion can be OspC, PspA, UreA, Ure B, or a fragment
thereof. Methods and compositions for forming and employing the
proteins are also disclosed and claimed.
Inventors: |
Huebner, Robert C.;
(Stroudsburg, PA) ; Erdile, Lorne F.;
(Stroudsburg, PA) ; Warakomski, Donald J. JR.;
(Tannersville, PA) ; Becker, Robert S.;
(Henryville, PA) ; Gray, Maryann B.;
(Bartonsville, PA) ; Pyle, Derek J.; (East
Stroudsburg, PA) |
Correspondence
Address: |
Patrick J. Halloran
Aventis Pasteur Inc.
Intellectual Property - Knerr Building
One Discovery Drive
Swiftwater
PA
18370
US
|
Family ID: |
23889117 |
Appl. No.: |
10/359435 |
Filed: |
February 6, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10359435 |
Feb 6, 2003 |
|
|
|
09067453 |
Apr 28, 1998 |
|
|
|
6538118 |
|
|
|
|
09067453 |
Apr 28, 1998 |
|
|
|
08475781 |
Jun 7, 1995 |
|
|
|
Current U.S.
Class: |
536/23.7 ;
424/190.1; 435/252.3; 435/320.1; 435/6.15; 435/69.3; 530/359 |
Current CPC
Class: |
Y02A 50/401 20180101;
C07K 2319/40 20130101; C07K 14/315 20130101; C12N 15/625 20130101;
Y02A 50/30 20180101; C07K 14/195 20130101; C07K 14/205 20130101;
A61K 38/00 20130101; C12P 21/02 20130101; C07K 2319/02
20130101 |
Class at
Publication: |
536/023.7 ;
424/190.1; 435/006; 435/069.3; 435/252.3; 435/320.1; 530/359 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 015/09; A61K 039/02; C07K 014/195; C12N 001/21 |
Claims
What is claimed is:
1. A hybrid nucleic acid molecule, comprising a first nucleic acid
sequence encoding a signal sequence of a lipoprotein and a second
nucleic acid sequence encoding a mature protein, or fragment
thereof, which is heterologous to the lipoprotein encoded by said
first-nucleic acid sequence, said first nucleic acid sequence being
contiguous with said second nucleic acid sequence when the mature
protein is naturally lipidated, or said first and second nucleic
acid sequences being separated by one codon coding for one amino
acid when the mature protein is not naturally lipidated.
2. The hybrid nucleic acid molecule of claim 1 wherein said signal
sequence is the signal sequence of an OspA protein of a Borrelia
species.
3. The hybrid nucleic acid molecule of claim 2 wherein said first
nucleic acid sequence and said second nucleic acid sequence are
coupled in a translational open reading frame relationship.
4. The hybrid nucleic acid molecule of claim 3 wherein said mature
protein is an OspC lipoprotein of a Borrelia species, or a fragment
thereof.
5. The hybrid molecule of claim 4 wherein said ospC lipoprotein is
that of a strain of B. burgdorferi.
6. The hybrid molecule of claim 5 wherein said strain of B.
burgdorferi is selected from the OspC sub-type families.
7. The hybrid molecule of claim 5 wherein said OspA protein is that
of a strain of B. burgdorferi.
8. The hybrid molecule of claim 7 wherein said strain of B.
burgdorferi is selected from the B31, ACA1 and Ip90 families of
strains.
9. The hybrid nucleic acid molecule of claim 3 wherein said mature
protein is a PspA protein of a strain of S. pneumoniae, or a
fragment thereof.
10. The hybrid molecule of claim 9 wherein said OspA protein is
that of a strain of B. burgdorferi.
11. The hybrid molecule of claim 10 wherein said strain of B.
burgdorferi is selected from the B31, ACA1 and Ip90 families of
strains.
12. The hybrid nucleic acid molecule of claim 3 wherein said mature
protein is a UreA protein of a strain of H. pylori, or a fragment
thereof.
13. The hybrid molecule of claim 12 wherein said ospA protein is
that of a strain of B. burgdorferi.
14. The hybrid molecule of claim 13 wherein said strain of B.
burgdorferi is selected from the B31, ACA1 and Ip90 families of
strains.
15. The hybrid nucleic acid molecule of claim 3 wherein said mature
protein is a UreB protein of a strain of H. pylori, or a fragment
thereof.
16. The hybrid molecule of claim 15 wherein said OspA protein is
that of a strain of B. burgdorferi.
17. The hybrid molecule of claim 16 wherein said strain of B.
burgdorferi is selected from the B31, ACA1 and Ip90 families of
strains.
18. A hybrid nucleic acid molecule, comprising a first nucleic acid
sequence encoding an OspC lipoprotein of a Borrelia species and a
second nucleic acid sequence encoding a signal sequence of an
expressed protein heterologous to OspC and coupled in translational
open reading frame relationship with said first nucleic acid
sequence.
19. The hybrid nucleic acid molecule of claim 18 wherein said OspC
lipoprotein is that of a strain of B. burgdorferi.
20. The hybrid nucleic acid molecule of claim 19 wherein said
strain of B. burgdorferi is selected from the OspC sub-type
families.
21. A hybrid nucleic acid molecule, comprising a first nucleic acid
sequence encoding a PspA protein of a strain of S. pneumoniae and a
second nucleic acid sequence encoding a signal sequence of an
expressed protein heterlogous to PspA and coupled in translational
open reading frame relationship with said first nucleic acid
sequence.
22. A hybrid nucleic acid molecule, comprising a first nucleic acid
sequence encoding a UreA protein of a strain of H. pylori and a
second nucleic acid sequence encoding a signal sequence of an
expressed protein heterlogous to UreA and coupled in translational
open reading frame relationship with said first nucleic acid
sequence.
23. A hybrid nucleic acid molecule, comprising a first nucleic acid
sequence encoding a UreB protein of a strain of H. pylori and a
second nucleic acid sequence encoding a signal sequence of an
expressed protein heterlogous to UreB and coupled in translational
open reading frame relationship with said first nucleic acid
sequence.
24. An expression vector containing the hybrid nucleic acid
molecule of claim 1 under control of a promoter for expression of
said mature protein.
25. The expression vector of claim 24 wherein said mature protein
is an OspC lipoprotein of a Borrelia species.
26. The expression vector of claim 24 wherein said mature protein
is a PspA lipoprotein of a strain of S. pneumoniae.
27. The expression vector of claim 24 wherein said mature protein
is a UreA protein of a strain of H. pylori.
28. The expression vector of claim 24 wherein said mature protein
is a UreB protein of a strain of H. pylori.
29. An expression vector containing the hybrid nucleic acid
molecule of claim 18 under control of a promoter for expression of
said OspC lipoprotein.
30. An expression vector containing the hybrid nucleic acid
molecule of claim 21 under control of a promoter for expression of
said PspA protein.
31. An expression vector containing the hybrid nucleic acid
molecule of claim 22 under control of a promoter for expression of
said UreA protein.
32. An expression vector containing the hybrid nucleic acid
molecule of claim 23 under control of a promoter for expression of
said UreB protein.
33. A method for forming a recombinant protein, which comprises:
incorporating the expression vector of claim 24 into a host
organism; and effecting expression of said mature protein from the
host organism.
34. The method of claim 33 wherein said mature protein is an OspC
lipoprotein of a Borrelia species.
35. The method of claim 34 wherein said host organism is E.
coli.
36. The method of claim 33 wherein said mature protein is a PspA
protein of a strain of S. pneumoniae.
37. The method of claim 36 wherein said host organism is E.
coli.
38. The method of claim 33 wherein said mature protein is a UreA
protein of a strain of H. pylori.
39. The method of claim 38, wherein said host organism is E.
coli.
40. The method of claim 33 wherein said mature protein is a UreB
protein of a strain of H. pylori.
41. The method of claim 40 wherein said host organism is E.
coli.
42. A method for forming recombinant ospC lipoprotein, which
comprises: incorporating the expression vector of claim 29 into a
host organism; and effecting expression of said OspC lipoprotein
from the host organism.
43. The method of claim 42 wherein said host organism is E.
coli.
44. A method for forming recombinant PspA lipoprotein, which
comprises: incorporating the expression vector of claim 30 into a
host organism; and effecting expression of said PspA lipoprotein
from the host organism.
45. The method of claim 44 wherein said host organism is E.
coli.
46. A method for forming recombinant UreA lipoprotein, which
comprises: incorporating the expression vector of claim 31 into a
host organism; and effecting expression of said UreA lipoprotein
from the host organism.
47. The method of claim 46 wherein said host organism is E.
coli.
48. A method for forming recombinant UreB lipoprotein, which
comprises: incorporating the expression vector of claim 32 into a
host organism; and effecting expression of said UreA lipoprotein
from the host organism.
49. The method of claim 48 wherein said host organism is E.
coli.
50. A process for the production of a recombinant lipoprotein,
which comprises: constructing a hybrid nucleic acid molecule
comprising a first nucleic acid sequence encoding a signal sequence
of a lipoprotein and a second nucleic acid sequence encoding a
mature protein, or fragment thereof, which is heterologous to the
lipoprotein encoded by said first nucleic acid, said second nucleic
acid sequence being contiguous with said first sequence; forming an
expression vector containing said hybrid nucleic acid molecule
under control of a promoter for expression of said mature protein;
incorporating said expression vector into a host organism;
effecting expression of said recombinant lipoprotein by said host
organism; lysing the cells of the host organism; treating the lysed
cells with a surfactant which selectively solubilizes said
recombinant lipoprotein in preference to bacterial and other
proteins and which is able to effect phase separation of a
detergent phase under mild conditions; effecting phase separation
at a detergent phase containing solubilized recombinant
lipoprotein, an aqueous phase containing bacterial and other
proteins and a solid phase containing cell residue; separating and
recovering said detergent phase from said solid phase and said
aqueous phase; contacting said detergent phase with a first
chromatographic column under conditions which result in binding of
protein other than said recombinant lipoprotein to said column to
provide a flow-through from said first chromatographic column
containing the recombinant lipoprotein and recovering said
flow-through from said first chromatographic column; contacting the
flow-through from said first chromatographic column with a second
chromatographic column under conditions which result in binding of
the recombinant lipoprotein to the second chromatographic column in
preference to contaminant proteins and lipopolysaccharides which
pass through said second chromatographic column; eluting said
recombinant lipoprotein from said second chromatographic column to
provide an eluant containing said recombinant lipoprotein
substantially free from lipopolysaccharide and contaminant
proteins; and recovering said eluant.
51. The process of claim 50 wherein said signal sequence is the
signal sequence of an OspA protein of a Borrelia species.
52. The process of claim 51 wherein said first nucleic acid
sequence and said second nucleic acid sequence are coupled in a
translational open reading frame relationship.
53. The process of claim 52 wherein said surfactant is TRITON.TM.
X-114.
54. The process of claim 53 wherein said treating of lysed cells is
effected at a temperature of about 0.degree. C. to about 10.degree.
C., the resulting mixture is treated to a mildly elevated
temperature of about 35.degree. C. to about 40.degree. C. to effect
separation of said detergent phase, and said detergent phase is
separated from said aqueous phase and said solid phase by
centrifugation.
55. The process of claim 52 wherein said mature protein is an OspC
lipoprotein of a Borrelia species.
56. The process of claim 55 wherein said first chromatographic
column is further contacted with a buffer medium at a pH to provide
liquid containing the recombinant ospC lipoprotein from the first
chromatographic column while the other proteins are retained on the
first chromatographic column and the flow-through from the further
contact is collected and combined with that from the first
contacting step on said first chromatographic column and the
combined flow-through is contacted with said second chromatographic
column.
57. The process of claim 52 wherein said mature protein is a PspA
protein of a strain of S. pneumoniae.
58. The process of claim 57, wherein said first chromatographic
column is further contacted with a buffer medium at a pH to provide
liquid containing the recombinant PspA lipoprotein from the first
chromatographic column while the other proteins are retained on the
first chromatographic column and the flow-through from the further
contact is collected and combined with that from the first
contacting step on said first chromatographic column and the
combined flow-through is contacted with said second chromatographic
column.
59. The process of claim 52 wherein said mature protein is a UreA
protein of a strain of H. pylori.
60. The process of claim 59 wherein said first chromatographic
column is further contacted with a buffer medium at a pH to provide
liquid containing the recombinant UreA lipoprotein from the first
chromatographic column while the other proteins are retained on the
first chromatographic column and the flow-through from the further
contact is collected and combined with that from the first
contacting step on said first chromatographic column and the
combined flow-through is contacted with said second chromatographic
column.
61. The process of claim 52 wherein said mature protein is a UreB
protein of a strain of H. pylori.
62. The process of claim 61 wherein said first chromatographic
column is further contacted with a buffer medium at a pH to provide
liquid containing the recombinant UreB lipoprotein from the first
chromatographic column while the other proteins are retained on the
first chromatographic column and the flow-through from the further
contact is collected and combined with that from the first
contacting step on said first chromatographic column and the
combined flow-through is contacted with said second chromatographic
column.
63. The process of claim 52 wherein said host organism lysis is
effected by freezing and thawing the host organism.
64. Recombinantly-produced, isolated and purified lipoprotein
produced by the process of claim 50.
65. Recombinantly-produced, isolated and purified ospC lipoprotein
of a Borrelia strain having a purity of at least about 80% and
substantially free from contaminant proteins and
lipopolysaccharides.
66. Recombinantly-produced, isolated and purified lipidated PspA
protein of a strain of S. pneumoniae having a purity of at least
about 50% and substantially free from contaminant proteins and
lipopblysaccharides.
67. Recombinantly-produced, isolated and purified lipidated UreA
protein of a strain of H. pylori having a purity of at least about
80% and substantially free from contaminant proteins and
lipopolysaccharides.
68. Recombinantly-produced, isolated and purified lipidated UreB
protein of a strain of H. pylori having a purity of at least about
80% and substantially free from contaminant proteins and
lipopolysaccharides.
69. A lipoprotein fusion vector PLF100 having ATCC Accession No.
69750.
70. A method for inducing an immunological response in a human or
animal comprising the step of administering to said human or animal
a composition comprising the lipoprotein of claim 64 or 65.
71. A composition for inducing an immunological response comprising
the lipoprotein of claim 64 or 65.
72. A method for inducing an immunological response in a human or
animal comprising the step of administering to said human or animal
a composition comprising the lipidated PspA protein of claim 64 or
66.
73. A composition for inducing an immunological response comprising
the lipidated PspA protein of claim 64 or 66.
74. A method for inducing an immunological response in a human or
animal comprising the step of administering to said human or animal
a composition comprising the lipidated UreA protein of claim
67.
75. A composition for inducing an immunological response comprising
the lipidated UreA protein of claim 67.
76. A method for inducing an immunological response in a human or
animal comprising the step of administering to said human or animal
a composition comprising the lipidated UreB protein of claim
68.
77. A composition for inducing an immunological response comprising
the lipidated UreB protein of claim 68.
78. A method for enhancing the immunogenicity of a protein,
comprising the steps of: forming a hybrid nucleic acid molecule
comprising a first nucleic acid sequence encoding a signal sequence
of a lipoprotein and a second nucleic acid sequence encoding a
mature protein, or fragment thereof, which is heterologous to the
lipoprotein encoded by said first nucleic acid, said first nucleic
acid sequence being contiguous with said second nucleic acid
sequence; incorporating said hybrid nucleic acid molecule into an
expression vector; effecting expression vector into a host
organism; and effecting expression of said mature protein from said
host organism.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] Reference, especially with respect to recombinant Borrelia
proteins, is made to each of applications Ser. No. 07/973,338;
filed Oct. 29, 1992; Ser. No. 08/373,455 (Rule 62 FWC of U.S. Ser.
No. 07/973,338), filed Jan. 17, 1995, Ser. No. 07/888,765, filed
May 27, 1992; Ser. No. 08/211,891; filed Oct. 16, 1992 (national
phase of PCT/US92/08697); and Ser. No. 07/779,048, filed Oct. 18,
1991.
[0002] Reference, especially with respect to structural genes of
pneumococcal proteins, epitopic regions thereof, and administration
of pneumococcal proteins, is made to each of applications Ser. Nos.
656,773, filed Feb. 15, 1991; Ser. No. 835,698, filed Feb. 12,
1992; Ser. No. 072,056, filed Jun. 3, 1993; Ser. No. 072,068, filed
Jun. 3, 1993; Ser. No. 214,222, filed Mar. 17, 1994; Ser. No.
214,164, filed Mar. 17, 1994; Ser. No. 247,491, filed May 23, 1994;
Ser. No. 048,896, filed Apr. 20, 1993; Ser. No. 246,636, filed May
20, 1994; ______ (continuation-in-part of application Ser. No.
246,636), filed October 7, 1994; ______, filed Jun. 2, 1995
(Attorney Docket No. 454312-2040, Briles et al., entitled, "ORAL
ADMINISTRATION OF PNEUMOCOCCAL ANTIGENS"); ______, filed May 19,
1995 (Attorney Docket No. 454312-2018); Ser. No. 08/312,949, filed
Sep. 30, 1994; and _____, filed Jun. 7, 1995 (Attorney Docket No.
454312-2051, Becker et al.,entitled, "IMMUNOGENIC COMBINATION
COMPOSITIONS AND METHODS").
[0003] Reference is also made to application Ser. No. ______, filed
Jun. 7, 1995 (Attorney Docket No. 454312-2052, Huebner et al.,
entitled, "EXPRESSION OF LIPOPROTEINS").
[0004] Each of the aforementioned applications is hereby
incorporated herein by reference.
FIELD OF INVENTION
[0005] The present invention is concerned with genetic engineering
to effect expression of lipoproteins from vectors containing
nucleic acid molecules encoding the lipoproteins. More
particularly, the present invention relates to expression of a
recombinant lipoprotein wherein the lipidation thereof is from
expression of a first nucleic acid sequence and the protein thereof
is from expression of a second nucleic acid sequence, the first and
second nucleic acid sequences, which do not naturally occur
together, being contiguous. The invention further relates to
expression of such lipoproteins wherein the first nucleic acid
sequence encodes a Borrelia lipoprotein leader sequence. The
invention also relates to recombinant lipidated proteins expressed
using the nucleic acid sequence encoding the OspA leader sequence,
methods of making and using the same compositions thereof and
methods of using the compositions. The invention additionally
relates to nucleic acid sequences encoding the recombinant
lipoproteins, vectors containing and/or expressing the sequences,
methods for expressing the lipoproteins and methods for making the
nucleic acid sequences and vectors; compositions employing the
lipoproteins, including immunogenic or vaccine compositions, such
compositions preferably having improved immunogenicity; and methods
of using such compositions to elicit an immunological or protective
response.
[0006] Throughout this specification, various documents are
referred to in order to more fully describe the state of the art to
which this invention pertains. These documents are each hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0007] Lyme borreliosis is the most prevalent tick-borne disease in
the United States as well as one of the most important tick-borne
infectious diseases worldwide. The spirochete Borrelia burgdorferi
is the causative agent for Lyme disease. Infection with B.
burgdorferi produces local and systemic manifestations. Local
symptoms that appear early after infection are a skin lesion at the
site of the tick bite, termed erythema migrans. Weeks to months
after infection, systemic manifestations that include rheumatic,
cardiac and neurological symptoms appear. The early local phase of
B. burgdorferi infection is easily treatable with antibiotics.
However, the later systemic phases have proved to be more
refractory to antibiotics.
[0008] Substantial effort has been directed toward the development
of a vaccine for Lyme disease. Two distinct approaches have been
used for vaccine development. One approach is to use a vaccine
composed of whole inactivated spirochetes, as described by Johnson
in U.S. Pat. No. 4,721,617. A whole inactivated vaccine has been
shown to protect hamsters from challenge and has been licensed for
use in dogs.
[0009] Due to the concerns about cross-reactive antigens within a
whole cell preparation, human vaccine research has focused on the
identification and development of non-cross-reactive protective
antigens expressed by B. burgdorferi. Several candidate antigens
have been identified to date. Much of this effort has focused on
the most abundant outer surface protein of B. burgdorferi, namely
outer surface protein A (OspA), as described in published PCT
patent application WO 92/14488, assigned to the assignee hereof.
Several versions of this protein have been shown to induce
protective immunity in mouse, hamster and dog challenge studies.
Clinical trials in humans have shown the formulations of OspA to be
safe and immunogenic in humans [Keller et al., JAMA (1994)
271:1764-1768]. Indeed, one formulation containing recombinant
lipidated OspA as described in the aforementioned WO 92/14488, is
now undergoing Phase III safety/efficacy trials in humans.
[0010] While OspA is expressed in the vast majority of clinical
isolates of B. burgdorferi from North America, a different picture
has emerged from examination of the clinical Borrelia isolates in
Europe. In Europe, Lyme disease is caused by three genospecies of
Borrelia, namely B. burgdorferi, B. garinii and B. afzelli. In
approximately half of the European isolates, OspA is not the most
abundant outer surface protein. A second outer surface protein C
((OspC) is the major surface antigen found on these spirochetes. In
fact, a number of European clinical isolates that do not express
OspA have been identified. Immunization of gerbils and mice with
purified recombinant OspC produces protective immunity to B.
burgdorferi strains expressing the homologous OspC protein [V.
Preac-Mursic et al., INFECTION (1992) 20:342-349; W. S. Probert et
al., INFECTION AND IMMUNITY (1994) 62:1920-1926]. The OspC protein
is currently being considered as a possible component of a second
generation Lyme vaccine formulation.
[0011] Recombinant proteins are promising vaccine or immunogenic
composition candidates, because they can be produced at high yield
and purity and manipulated to maximize desirable activities and
minimize undesirable ones. However, because they can be poorly
immunogenic, methods to enhance the immune response to recombinant
proteins are important in the development of vaccines or
immunogenic compositions.
[0012] A very promising immune stimulator is the lipid moiety
N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine,
abbreviated Pam.sub.3Cys. This moiety is found at the amino
terminus of the bacterial lipoproteins which are synthesized with a
signal sequence that specifies lipid attachment and cleavage by
signal peptidase II. Synthetic peptides that by themselves are not
immunogenic induce a strong antibody response when covalently
coupled to Pam.sub.3Cys [Bessler et al., Research Immunology (1992)
143:548-552].
[0013] In addition to an antibody response, one often needs to
induce a cellular immune response, particularly cytoxic T
lymphocytes (CTLB). Pam.sub.3Cys-coupled synthetic peptides are
extremely potent inducers of CTLs, but no one as yet reported CTL
induction by large recombinant lipoproteins.
[0014] The nucleic acid sequence and encoded amino acid sequence
for OspA are known for several B. burgdorferi clinical isolates and
is described, for example, in published PCT application WO 90/04411
(Symbicom AB) for B31 strain of B. burgdorferi and in Johnson et
al., Infect. Immun. 60:1845-1853 for a comparison of the ospA
operons of three B. burgdorferi isolates of different geographic
origins, namely B31, ACA1 and Ip90.
[0015] As described in WO 90/04411, an analysis of the DNA sequence
for the B31 strain shows that the OspA is encoded by an open
reading frame of 819 nucleotides starting at position 151 of the
DNA sequence and terminating at position 970 of the DNA sequence
(see FIG. 1 therein). The first sixteen amino acid residues of OspA
constitute a hydrophobic signal sequence of OspA. The primary
translation product of the full length B. burgdorferi gene contains
a hydrophobic N-terminal signal sequence which is a substrate for
the attachment of a diacyl glycerol to the sulfhydryl side chain of
the adjacent cysteine residue. Following this attachment, cleavage
by signal peptidase II and the attachment of a third fatty acid to
the N-terminus occurs. The complete lipid moiety is termed
Pam.sub.3Cys. It has been shown that lipidation of OspA is
necessary for immunogenicity, since OspA lipoprotein with an
N-terminal Pam.sub.3Cys moiety stimulated a strong antibody
response, while OspA lacking the attached lipid did not induce any
detectable antibodies [Erdile et al., Infect. Immun., (1993),
61:81-90].
[0016] Published international patent application WO 91/09870
(Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA
sequence of the ospC gene of B. burgdorferi strain Pko and the OspC
(termed pC in this reference) protein encoded thereby of 22 kDa
molecular weight. This sequence reveals that OspC is a lipoprotein
that employs a signal sequence similar to that used for OspA. Based
on the findings regarding OspA, one might expect that lipidation of
recombinant OspC would be useful to enhance its immunogenicity;
but, as discussed below, the applicants experienced difficulties in
obtaining detectable expression of recombinant OspC.
[0017] U.S. Pat. No. 4,624,926 to Inouye relates to plasmid cloning
vectors, including a DNA sequence coding for a desired polypeptide
linked with one or more functional fragments derived from an outer
membrane lipoprotein gene of a gram negative bacterium. The
polypeptide expressed by thee transformed E. coli host cells
comprises the signal peptide of the lipoprotein, followed by the
first eight amino acid residues of the lipoprotein, which in turn
are followed by the amino acid sequence of the desired protein. The
signal peptide may then be translocated naturally across the
cytoplasmic membrane and the first eight amino acids of the
lipoprotein may then be processed further and inserted into the
outer membrane of the cell in a manner analogous to the normal
insertion of the lipoprotein into the outer membrane.
Immunogenicity of the expressed proteins was not demonstrated.
Moreover, Inouye was not at all concerned with recombinant
lipidation, particularly to enhance immunogenicity.
[0018] Published international patent application WO91/09952
describes plasmids for expressing lipidated proteins. Such plasmids
involve a DNA sequence encloding a lipoprotein signal peptide
linked to the codons for one of the .beta.-turn tetrapeptides QANY
or IEGR, which in turn is linked to the DNA sequence encoding the
desired protein. Again, immunogenicity of the expressed proteins
was not demonstrated.
[0019] Streptoccus pneumoniae causes more fatal infections
world-wide than almost any other pathogen. In the U.S.A., deaths
caused by S. pneumoniae rival in numbers those caused by AIDS. Most
fatal pneumoccal infections in the U.S.A. occur in individuals over
65 years of age, in whom S. pneumoniae is the most common cause of
community-acquired pneumonia. In the developed world, most
pneumococcal deaths occur in the elderly, or in immunodeficient
patents including those with sickle cell disease. In the
less-developed areas of the world, pneumococcal infection is one of
the largest causes of death among children less than 5 years of
age. The increase in the frequency of multiple antibiotic
resistance among pneumococci and the prohibitive cost of drug
treatment in poor countries make the present prospect for control
of pneumococcal disease problematical.
[0020] The reservoir of pneumococci that infect man is maintained
primarily via nasopharyngeal human carriage. Humans acquire
pneumococci first through aerosols or by direct contact.
Pneumococci first colonize the upper airways and can remain in
nasal mucosa for weeks or months. As many as 50% or more of young
children and the elderly are colonized. In most cases, this
colonization results in no apparent infection. In some individuals,
however, the organism carried in the nasopharynx can give rise to
symptomatic sinusitis of middle ear infection. If pneumococci are
aspirated into the lung, especially with food particles or mucus,
they can cause pneumonia. Infections at these sites generally shed
some pneumococci into the blood where they can lead to sepsis,
especially if they continue to be shed in large numbers from the
original focus of infection. Pneumococci in the blood can reach the
brain where they can cause menigitis. Although pneumococcal
meningitis is less common than other infections caused by these
bacteria, it is particularly devastating; some 10% of patients die
and greater than 50% of the remainder have life-long neurological
sequelae.
[0021] In elderly adults, the present 23-valent capsular
polysaccharide vaccine is about 60% effective against invasive
pneumococcal disease with strains of the capsular types included in
the vaccine. The 23-valent vaccine is not effective in children
less than 2 years of age because of their inability to make
adequate responses to most polysaccharides. Improved vaccines that
can protect children and adults against invasive infections with
pneumococci would help reduce some of the most deleterious aspects
of this disease.
[0022] The S. pneumoniae cell surface protein PspA has been
demonstrated to be a virulence factor and a protective antigen. In
published international patent application WO 92/14488, there are
described the DNA sequences for the pspA gene from S. pneumoniae
R.times.1, the production of a truncated form of PspA by genetic
engineering, and the demonstration that such truncated form of PspA
confers protection in mice to challenge with live pneumococci.
[0023] In an effort to develop a vaccine or immunogenic composition
based on PspA, PspA has been recombinantly expressed in E. coli. It
has been found that in order to efficiently express PspA, it is
useful to truncate the mature PspA molecule of the R.times.1 strain
from its normal length of 589 amino acids to that of 314 amino
acids comprising amino acids 1 to 314. This region of the PspA
molecule contains most, if not all, of the protective epitopes of
PspA. However, immunogenicity and protection studies in mice have
demonstrated-that the truncated recombinant form of PspA is not
immunogenic in naive mice. Thus, it would be useful to improve the
immunogenicity of recombinant PspA and fragments thereof.
[0024] Many bacterial and viral pathogens, such as S. pneumoniae
and Helicobacter pylori, and HIV, herpes and papilloma viruses gain
entry through mucosal surfaces. The principal determinant of
specific immunity at mucosal surfaces is secretory IgA (S--IgA)
which is physiologically and functionally separate from the
components of the circulatory immune system. Mucosal S--IgA
responses are predominantly generated by the common mucosal immune
system (CMIS) [Mestecky, J. Clin. Immunol. (1987), 7:265-276], in
which immunogens are taken up by specialized lympho-epithelial
structures collectively referred to as mucosa-associated lymphoid
tissue (MALT). The term common mucosal immune system referes to the
fact that immunization at any mucosal site can elicit an immune
response at all other mucosal sites. Thus, immunization in the gut
can elicit mucosal immunity in the upper airways and vice versa.
Further, it is important to note that oral immunization can induce
an antigen-specific IgG response in the systemic compartment in
addition to mucosal IgA antibodies [McGhee, J. R. et al., (1993),
Infect. Agents and Disease 2:55-73].
[0025] Most soluble and non-replicating antigens are poor mucosal
immunogens, especially by the peroral route, probably because they
are degraded by digestive enzymes and have little or no tropism for
the gut associated lymphoid tissue (GALT). Thus, a method for
producing effective mucosal immunogens, and vaccines and
immunogenic compositions containing them, would be desirable.
[0026] Of particular interest is H. pylori, the spiral bacterium
which selectively colonizes human gastric mucin-secreting cells and
is the causative agent in most cases of nonerosive gastritis in
humans. Recent research indicates that H. pylori, which has a high
urease activity, is responsible for most peptic ulcers as well as
many gastric cancers. Many studies have suggested that urease, a
complex of the products of the ureA and ureB genes, may be a
protective antigen, However, until now it has not been known how to
produce a sufficient-mucosal immune response to urease.
[0027] Antigens, such as OspC, PspA, UreA, UreB or immunogenic
fragments thereof, stimulate an immune response when administered
to a host. Such antigens, especially when recombinantly produced,
may elicit a stronger response when administered in conjunction
with an adjuvant. Currently, alum is the only adjuvant licensed for
human use, although hundreds of experimental adjuvants such as
cholera toxin B are being tested. However, these adjuvants have
deficiencies. For instance, while cholera toxin B is not toxic in
the sense of causing cholera, there is general unease about
administering a toxin associated with a disease as harmful as
cholera, especially if there is even the most remote chance of
minor impurity.
[0028] Thus, it would be desirable to enhance the immunogenicity of
antigens, by methods other than the use of an adjuvant, especially
in monovalent preparations; and, in multivalent preparations, to
have the ability to employ such a means for enhanced immunogenicity
with an adjuvant, so as to obtain an even greater immunological
response.
[0029] As to expression of recombinant proteins, it is expected
that the skilled artisan is familiar with the various vector
systems available for such expression, e.g., bacteria such as E.
coli and the like.
[0030] It is believed that heretofore the art has not taught or
suggested: expression of a recombinant lipoprotein wherein the
lipidation thereof is from expression of a first nucleic acid
sequence, the protein thereof is from expression of a second
nucleic acid sequence, the first and second sequences, which do not
naturally occur together, being contiguous, especially such a
lipoprotein wherein the first sequence encodes a Borrelia
lipoprotein leader sequence, preferably an OspA leader sequence,
and even more preferably wherein the first sequence encodes an OspA
leader sequence and the second sequence encodes OspC, PspA, UreA,
UreB, or an immunogenic fragment thereof; or genes containing such
sequences; or vectors containing such sequences; or methods for
such expression; or such recombinant lipoproteins; or compositions
containing such recombinant lipoproteins; or methods for using such
compositions; or methods for enhancing the immunogenicity of a
protein by lipidation from a nucleic acid sequence not naturally
occurring with the nucleic acid sequence encoding the protein
portion of the lipoprotein.
OBJECTS AND SUMMARY OF THE INVENTION
[0031] It is an object of the invention to provide a recombinant
lipoprotein wherein the lipidation thereof is from expression of a
first nucleic acid sequence and the protein portion thereof is from
expression of a second nucleic acid sequence and the first and
second sequences do not naturally occur together; especially such a
lipoprotein wherein the first sequence encodes a Borrelia
lipoprotein leader sequence, preferably an OspA leader sequence,
and more preferably wherein the second sequence encodes a protein
portion comprising OspC, PspA, UreA, UreB, or an immunogenic
fragment thereof.
[0032] It is another object of the invention to provide expression
of genes and/or sequences encoding such a recombinant lipoprotein,
vectors therefor and methods for effecting such expression.
[0033] It is a further object of the invention to provide
immunogenic compositions, including vaccines, containing the
recombinant lipoproteins and/or vectors for expression thereof.
[0034] It has surprisingly been found that an immunogenic
recombinant lipidated protein, preferably OspC or a portion
thereof, can be expressed from a vector system, preferably E. coli,
without the toxicity to the vector system evident when the native
lipoprotein signal sequence encoding region is present. This result
has been achieved by replacing the nucleotide sequence encoding the
native leader or signal sequence of a lipoprotein with the
nucleotide sequence encoding a leader or signal of another
lipoprotein, preferably of a Borrelia lipoprotein, and more
preferably the OspA leader or signal sequence. Proteins not
naturally lipidated, such as PspA, UreA, and UreB, may be expressed
as recombinant lipidated proteins as well, by fusing the
lipoprotein signal sequence to the first amino acid of the desired
protein. These recombinant lipidated proteins have been shown to
elicit an immune response, including a mucosal immune response.
[0035] It is surprising that fusion of DNA encoding a lipoprotein
leader sequence, directly to the DNA encoding a protein, without
any intervening nucleotide sequences, can lead to expression of an
immunogenic recombinant lipoprotein in significant quantities
without the toxicity evident with the native leader sequence,
because previous attempts to express recombinant lipidated proteins
have been unsuccessful. For example, Fuchs et al. report that
recombinantly formed OspC (referred to as pC in this reference)
with its native leader protein was only weakly expressed in E. coli
[Mol. Microbiology (1992) 6(4):503-509]. Applicants, in addition to
Fuchs, attempted to obtain lipidated recombinant OspC by expression
of the OspC-encoding sequence in E. coil using the pET vector
system described in the aforementioned WO 92/14488 for the
expression of OspA and using the pDS12 plasmid systems described.
However, OspC was barely detectable by immunoblotting of cell
extracts using these systems to express OspC.
[0036] Further, as discussed supra, it was believed that an
additional nucleotide sequence, preferably one encoding a peptide
sequence forming a .beta.-turn, was necessary for expression of
recombinant lipoproteins, and the immunogenicity of recombinant
lipoproteins previously expressed had not been demonstrated.
[0037] The procedure of the present invention, therefore, enables
large quantities of pure recombinant, immunogenic lipidated
proteins, e.g., OspC, PspA, UreA, UreB and portions thereof, to be
obtained, which has not heretofore been possible. The
recombinantly-formed lipidated proteins provided herein are
significantly more immunogenic than a non-lipidated recombinant
analog.
[0038] The present invention, it is believed, represents the first
instance of effecting expression of a heterologous lipidated
protein using a non-native, preferably Borrelia and more preferably
the ospA leader sequence. The invention, therefore, includes the
use of non-native, preferably Borrelia and more preferably the OspA
leader sequence to express proteins heterologous to the leader
sequence.
[0039] Accordingly, in one embodiment, the present invention
provides an isolated hybrid nucleic acid molecule, preferably DNA,
comprising a first nucleic acid sequence encoding the signal
sequence preferably of an OspA protein of a Borrelia species,
coupled in translational open reading frame relationship with a
second nucleic acid sequence encoding a mature protein heterologous
to the signal sequence, preferably to OspC or PspA. More
preferably, the first and second sequences are contiguous when the
mature protein is naturally lipidated, and separated by one codon
coding for one amino acid, preferably cysteine, when the mature
protein is not naturally lipidated.
[0040] The mature protein encoded by the second nucleic acid
sequence generally is a lipoprotein, preferably an antigenic
lipoprotein, and more preferably is the mature OspC lipoprotein of
a Borrelia species, preferably a strain of B. burgdorferi, more
preferably a strain of B. burgdorferi selected from the OspC
sub-type families. In another preferred embodiment, the mature
protein is the mature PspA protein, or an immunogenic fragment
thereof, of a strain of S. pneumoniae. In yet another preferred
embodiment, the mature protein is UreA or UreB protein of a strain
of H. pylori. Similarly, the signal sequence of the OspA protein of
a Borrelia strain encoded by the first nucleic acid sequence
preferably is that of a strain of B. burgdorferi, more preferably a
strain of B. burgdorferi selected from the B31, ACA1 and Ip90
families of strains.
[0041] The hybrid gene provided herein may be assembled into an
expression vector, preferably under the control of a suitable
promoter for expression of the mature lipoprotein, in accordance
with a further aspect of the invention, which, in a suitable host
organism, such as E. coli, causes initial translation of a chimeric
molecule comprising the leader sequence and the desired
heterologous protein in lipidated form, followed by cleavage of the
chimeric molecule by signal peptidase II and attachment of lipid
moieties to the new terminus of the protein, whereby the mature
lipoprotein is expressed in the host organism.
[0042] The present invention provides, for the first time, a hybrid
nucleic acid molecule which permits the production of recombinant
lipidated protein, e.g., recombinant lipidated OspC of a Borrelia
species, recombinant lipidated PspA of a strain of S. pneumoniae or
recombinant lipidated UreA or UreB of a strain of H. pylori, to be
obtained. Accordingly, in a further aspect of this invention, there
is provided a hybrid nucleic acid molecule, comprising a first
nucleic acid sequence encoding a lipoprotein, preferably an OspC
lipoprotein of a Borrelia species, more preferably a strain of B.
burgdorferi, still more preferably a strain of B. burgdorferi
selected from the OspC sub-type families; or encoding a PspA
lipoprotein of a strain of S. pneumoniae or immunogenic fragment
thereof; or encoding a UreA or UreB lipoprotein of a strain of H.
pylori; and a second nucleic acid sequence encoding a signal
sequence of an expressed protein heterologous to the protein
encoded by the first nucleic acid sequence and coupled in
translational open reading frame relationship with said first
nucleic acid sequence, preferably encoding the signal sequence of
an OspA protein of a Borrelia species.
[0043] As described above, the hybrid gene can be assembled into an
expression vector under the control of a suitable promoter for
expression of the lipoprotein, which, in a suitable host organism,
such as E. coli, causes expression of the lipoprotein from the host
organism.
[0044] It has also surprisingly been found that enhanced
immunogenicity can be obtained by a recombinant lipoprotein when
the lipoprotein is expressed by a hybrid or chimeric gene
comprising a first nucleic acid sequence encoding a leader or
signal sequence and a second nucleic acid sequence encoding the
protein portion of the lipoprotein, wherein the first and second
sequences do not naturally occur together.
[0045] Accordingly, the present invention also provides a
recombinant lipoprotein expressed by a hybrid or chimeric gene
comprising a first nucleic acid sequence encoding a leader or
signal sequence contiguous with a second nucleic acid sequence
encoding a protein portion of the lipoprotein, and the first and
second sequences do not naturally occur together. The first and
second sequences are preferably coupled in a translational open
reading frame relationship. The first sequence can encode a leader
sequence of a Borrelia lipoprotein, preferably the leader sequence
of ospA; and the second sequence can encode a protein comprising an
antigen, preferably OspC, PspA, UreA, UreB or an immunogenic
fragment thereof. The first and second sequences can be present in
a gene; and the gene and/or the first and second sequences can be
in a suitable vector for expression.
[0046] The vector can be a nucleic acid in the form of, e.g.,
plasmids, bacteriophages and integrated DNA, in a bacteria, most
preferably one used for expression, e.g. E. coli, Bacillus
subtilis, Salmonella, Staphylocoocus, Streptococcus, etc., or one
used as a live vector, e.g. Lactobacillus, Mycobacterium,
Salmonella, Streptococcus, etc. When an expression host is used the
recombinant lipoprotein can be obtained by harvesting product
expressed in vitro; e.g., by isolating the recombinant lipoprotein
from a bacterial extract. The gene can preferably be under the
control of and therefore operably linked to a suitable promoter;
and the promoter can either be endogenous to the vector, or be
inserted into the vector with the gene.
[0047] The invention further provides vectors containing the
nucleic acid encoding the recombinant lipoprotein and methods for
obtaining the recombinant lipoproteins and methods for preparing
the vector.
[0048] As mentioned, the recombinant lipoprotein can have enhanced
immunogenicity. Thus, additional embodiments of the invention
provide immunogenic or vaccine compositions for inducing an
immunological response, comprising the isolated recombinant
lipoprotein, or a suitable vector for in vivo expression thereof,
or both, and a suitable carrier, as well as to methods for
eliciting an immunological or protective response comprising
administering to a host the isolated recombinant lipoprotein, the
vector expressing the recombinant lipoprotein, or a composition
containing the recombinant lipoprotein or vector, in an amount
sufficient to elicit the response.
[0049] Documents cited in this disclosure, including the
above-referenced applications, provide typical additional
ingredients for such compositions, such that undue experimentation
is not required by the skilled artisan to formulate a composition
from this disclosure. Such compositions should preferably contain a
quantity of the recombinant lipoprotein or vector expressing such
sufficient to elicit a suitable response. Such a quantity of
recombinant lipoproprotein or vector can be based upon known
amounts of antigens administered. For instance, if there is a known
amount for administration of an antigen corresponding to the second
sequence expressed for the inventive recombinant lipoprotein, the
quantity of recombinant lipoprotein can be scaled to about that
known amount, and the amount of vector can be such as to produce a
sufficient number of colony forming units (cfu) so as to result in
in vivo expression of the recombinant lipoprotein in about that
known amount. Likewise, the quantity of recombinant lipoprotein can
be based upon amounts of antigen administered to animals in the
examples below and in the documents cited herein, without undue
experimentation.
[0050] The present invention also includes, in other aspects,
processes for the production of a recombinant lipoprotein, by
assembly of an expression vector, expression of the lipoprotein
from a host organism containing the expression vector, and
optionally isolating and/or purifying the expressed lipoprotein.
The isolating/purifying can be so as to obtain recombinant
lipoprotein free from impurities such as lipopolysaccharides and
other bacterial proteins.
[0051] The present invention further includes immunogenic
compositions, such as vaccines, containing the recombinant
lipoprotein as well as methods for inducing an immunological
response.
BRIEF DESCRIPTION OF DRAWING
[0052] In the following detailed description, reference is made to
the accompanying drawings, wherein:
[0053] FIG. 1 is a schematic representation of a procedure for
assembling plasmid pLF100;
[0054] FIG. 2 is a schematic representation of a procedure for
assembling plasmid vectors pPko9a (strain Pko) and pB319a (strain
B31);
[0055] FIG. 3 is a schematic representation of the procedure
employed for the isolation and purification of lipidated OspC;
[0056] FIG. 4 is a schematic representation of the procedure
employed for the isolation and purification of non-lipidated OspC
for comparative purposes;
[0057] FIG. 5 shows an SDS-PAGE analysis of lipidated OspC produced
herein at various stages of the purification procedure illustrated
schematically in FIG. 3;
[0058] FIG. 6 shows an SDS-PAGE analysis of non-lipidated OspC
produced herein at various stages of the purification procedure as
described in WO 91/09870;
[0059] FIG. 7 is a graphical representation of the immune response
of mice immunized with OspC formulations containing antigen from
two OspC sub-types as measured in an anti-OspC ELISA assay;
[0060] FIG. 8 is a graphical representation of the immune response
of mice immunized with a two sub-type OspC formulation that
contains alum adjuvant as measured in an anti-OspC ELISA assay;
[0061] FIG. 9 is a schematic representation of a procedure for
assembling plasmid vector pPA321-L;
[0062] FIG. 10 is a schematic representation of a procedure for
assembling plasmid vector pPA321-NL;
[0063] FIG. 11 is a schematic representation of the procedure
employed for the isolation and purification of lipidated PspA;
[0064] FIG. 12 is a schematic representation of the procedure
employed for the isolation and purification of non-lipidated PspA
for comparative purposes;
[0065] FIG. 13 shows an SDS-PAGE analysis of lipidated PspA
produced herein at various states of the expression and host cell
fractionation procedure illustrated schematically in FIG. 11;
[0066] FIG. 14 shows an SDS-PAGE analysis of non-lipidated PspA
produced herein at various stages of the expression and host cell
fractionation procedure illustrated schematically in FIG. 12;
and
[0067] FIG. 15 shows an SDS-PAGE analysis of the PspA column
chromatography results illustrated schematically in FIGS. 11 and
12.
DETAILED DESCRIPTION OF INVENTION
[0068] As noted above, the present invention is concerned with the
use of a nucleic acid sequence encoding the OspA signal sequence to
express lipidated proteins heterologous to OspA protein, preferably
an OspC protein of a Borrelia species, a PspA protein or portion
thereof of a strain of S. pneumoniae, or a UreA or UreB protein of
a strain of H. pylori, and to the use of a nucleic acid sequence
encoding the signal sequence of a protein heterologous to the
protein to be expressed, to express the lipidated OspC protein of a
Borrelia species or the lipidated PspA protein of a strain of S.
pneumoniae.
[0069] The leader amino acid sequence and encoding DNA sequence for
the ACA strain of B. burgdorferi are as follows:
1 M K K Y L L G I G (SEQ ID NO: 1) L I L A L I A C ATG AAA AAA TAT
TTA TTG GGA ATA GGT (SEQ ID NO: 2) CTA ATA TTA GCC TTA ATA GCA
TGC
[0070] The corresponding leader amino acid sequences and encoding
DNA sequences for the ospA of other strains of B. burgdorferi are
known in the art and may be employed in the present invention from
this disclosure, without any undue experimentation.
[0071] A hybrid gene molecule is assembled comprising the OspA
leader encoding sequence and the gene encoding the heterologous
protein to be expressed, preferably the ospc or pspA gene, arranged
in translational reading-frame relationship with the ospA gene
fragment.
[0072] For production of the lipidated protein, the appropriate
hybrid gene molecule can be incorporated into a suitable expression
vector and the resulting plasmid incorporated into an expression
strain of E. coli or other suitable host organism. The vector can
also be a bacteriophage or integrated DNA.
[0073] The lipidated protein is expressed by the cells during
growth of the host organism. The lipidated protein may be recovered
from the host organism in purified form by any convenient procedure
which separates the lipidated protein in undenatured form. One
schematic of a procedure in accordance with this aspect of the
invention is shown in FIG. 3.
[0074] Following cell growth and induction of protein, the cells
are subjected to freeze-thaw lysis and DNase I treatment. The
lysate is treated with a detergent which is selective for
solubilization of the recombinant lipidated protein, in preference
to the other bacterial proteins in the lysate. While the present
invention preferably utilizes polyethylene glycol tert-octylphenyl
ether having the formula
t-Oct-C.sub.6H.sub.4-(OCH.sub.2CH.sub.2).sub.xOH wherein x=7-8 as
the detergent (commercially available as, and hereinafter referred
to as, TRITON.TM. X-114), other materials may be used exhibiting a
similar selective solubility for the lipidation protein as well as
the phase separation property under mild conditions, as discussed
below.
[0075] Following addition of the TRITON.TM. X-114, the mixture is
warmed to a mild temperature elevation of preferably about
35.degree. C. to 40.degree. C., at which time the solution becomes
cloudy as phase separation occurs. The purification procedure for
such phase separation should occur under conditions to avoid any
substantial denaturing or any other substantial impairment of the
immunological properties of the recombinant lipoprotein.
[0076] Centrifugation of the cloudy mixture results in separation
of the mixture into three phases, namely a detergent phase
containing about 50% or more of the recombinant lipidate protein
and a small amount (approximately 5 wt %) of other proteins, an
aqueous phase containing the balance of the other proteins, and a
solid pellet of cell residue. The detergent phase is separated from
the aqueous phase and the solid pellet for further processing.
[0077] Final purification of the protein preferably is effected by
processing of the detergent phase to provide a recombinant
lipidated protein having a purity of at least about 80 wt %, and
which is substantially free from other contaminants such as
bacterial proteins, and lipopolysaccharides (LPS), and which has
endotoxin levels compatible with human administration.
[0078] Such purification is conveniently effected by column
chromatography. Such chromatographic purification may include a
first chromatographic purification using a first chromatographic
column having the pH, ionic strength and hydrophobicity to bind
bacterial proteins, but not the recombinant lipidated protein.
[0079] Such first chromatographic purification may be effected by
loading the detergent phase onto the first chromatographic column
and the flow-through, which contains the purified lipidated
protein, is collected. The bound fraction contains substantially
all the bacterial protein impurities from the detergent phase. The
chromatography medium used for such first purification operation
may be a DEAE-Sephacel or DEAE-Sepharose column.
[0080] The flow-through from the first chromatographic purification
operation may be subjected to further purification on a second
chromatographic column. The flow-through is loaded onto the column
having the pH, ionic strength and hydrophobicity which will
selectively bind the recombinant lipidated protein to the second
chromatographic column, while bacterial contaminants and LPS pass
through the column. The chromatography medium for the second
chromatographic column may be S-Sepharose.
[0081] Preferably the recombinant lipoprotein is purified to 80%
purity or to greater than 80% purity, e.g., 85-90% or even 90-95%
or greater than 95% purity. The lipidated proteihaceous material
can then be formulated into immunogenic compositions, preferably
vaccines.
[0082] The vaccine or immunogenic composition elicits an immune
response in a host subject which produces an immunological
response, such as antibodies which may be opsonizing or
bactericidal. Should a subject immunized with a recombinant
lipoprotein of the invention then be challenged, such immunological
response can inactivate the challenge organism. Furthermore,
opsonizing or bactericidal antibodies may also provide protection
by alternative mechanisms.
[0083] Immunogenic compositions including vaccines can be prepared
as injectables, as liquid solutions or emulsions, or as formulating
for oral, nasal or other orifice administration e.g., vaginal,
rectal, etc. Oral formulations can be liquid solutions, emulsions
and the like, e.g., elixers, or solid preparations, e.g., tablets,
caplets, capsules, pills, liquid-filled-capsules, gelatin and the
like. Nasal preparations can be liquid and can be administered via
aerosol, squeeze spray or pump spray dispensers. Documents cited
herein provide exemplary formulation types and ingredients
therefor, including the applications cited above.
[0084] The immunogens can be mixed with pharmaceutically acceptable
excipients which are compatible with the immunogens. Such
excipients may include water, saline, dextrose, glycerol, ethanol,
and combinations thereof. The immunogenic compositions and vaccines
may further contain auxiliary substances, such as wetting or
emulsifying agents, pH buffering agents, or adjuvants to enhance
the effectiveness thereof. Immunogenic compositions and vaccines
may be administered parenterally, by injection subcutaneously or
intramuscularly. The immunogenic preparations and vaccines are
administered in a manner compatible with the dosage formulation,
and in such amount as will be therapeutically effective,
immunogenic or protective. The quantity to be administered depends
on the subject to be treated, including, for example, the capacity
of the immune system of the individual to synthesize antibodies,
and, if needed, to produce a cell-mediated immune response. Precise
amounts of active ingredient required to be administered depend on
the judgment of the practitioner, taking into account such factors
as the age, weight, sex, condition of the host or patient to whom
there is to be administration. However, suitable dosage ranges are
readily determinable by one skilled in the art and may be of the
order of micrograms of the immunogens. Suitable regimes for initial
administration and booster doses are also variable, but may include
an initial administration followed by subsequent administrations.
The dosage may also depend on the route of administration and will
vary according to the size of the host.
[0085] The concentration of the immunogens in an immunogenic
composition according to the invention is in general about 1 to
about 95%. A vaccine or immunogenic composition which contains
antigenic material of only one pathogen is a monovalent vaccine.
Vaccines or immunogenic compositions which are multivalent or which
contain antigenic material of several pathogens (also known as
combined vaccines or combined imunogenic compositions) also belong
to the present invention. Such combined vaccines or immunogenic
compositions contain, for example, material from various pathogens
or from various strains of the same pathogen, or from combinations
of various pathogens.
[0086] Immunostimulatory agents or adjuvants have been used for
many years to improve the host immune responses to, for example,
vaccines or immunogenic compositions. Intrinsic adjuvants, such as
lipopolysaccharides, normally are the components of the killed or
attenuated bacteria used as vaccines or immunogenic compositions.
Extrinsic adjuvants are immunomodulators which are typically
non-covalently linked to antigens and are formulated to enhance the
host immune responses. Some of these adjuvants are toxic, however,
and can cause undesirable side-effects, making them unsuitable for
use in humans and many animals. Indeed, only alum is routinely used
as an adjuvant in human and veterinary vaccines.
[0087] In view of the difficulties associated with the use of
adjuvants, it is thus an advantage of the present invention that
the recombinant lipidated proteins are the most immunogenic forms,
and are capable of eliciting immune responses both without any
adjuvant and with alum.
[0088] The following examples illustrate but do not limit the scope
of the invention disclosed in this specification.
EXAMPLES
Example 1
[0089] Construction of a Vector Containing a Gene Encoding the OspA
Leader Sequence
[0090] Plasmid pBluescript KS+ (Stratagene) was digested with XbaI
and BamHI and ligated with a 900 bp XbaI-BamHI DNA fragment
containing the complete coding region of B. burgdorferi strain ACA1
ospA gene, to form a lipoprotein fusion vector pLF100. This
procedure is shown schematically in FIG. 1.
[0091] The vector pLF100 has been deposited with the American Type
Culture Collection at Rockville, Md. on Feb. 2, 1995 under
Accession No. 69750. This deposit was made under the terms of the
Budapest Treaty.
Example 2
[0092] construction of a pET9a Expression Vector Containing a
Hybrid ospA-ospC Gene
[0093] Specifically designed oligonucleotide primers were used in a
polymerase chain reaction (PCR) to amplify the portion of the ospc
gene downstream from the cysteine-encoding codon terminating the
signal-peptide recognition-encoding sequence to the C-terminal end
of the coding region from the Pko and B31 strains of B.
burgdorferi.
[0094] The 5'-end primer had the nucleotide sequences respectively
for the Pko and B31 strains:
2 5'-GGC GCG CAT GCA ATA ATT (Pko) (SEQ ID NO: 3) CAG GGA AAG G-3'
5'-GGC GCG CAT GCA ATA ATT (B31) (SEQ ID NO: 4) CAG GGA AAG A-3'
while the 3'-end primer had the nucleotide sequence: 5'-CGC GGA TCC
TTA AGG TTT (B31 & (SEQ ID NO: 5) TTT TGG-3' Pko)
[0095] The PCR amplification was effected in a DNA Thermal Cycler
(Perkins-Elmer Cetus) for 25 cycles with denaturation for 30 secs
at 94.degree. C., annealing at 37.degree. C. for 1 minute and
extension at 72.degree. C. for 1 minute. A final extension was
effected at 72.degree. C. for 5 minutes at the completion of the
cycles. The product was purified using a Gene Clean II kit (B10
101) and the purified material was digested with SphI and BamHI.
This procedure introduced a silent mutation in the Pko ospC gene
which changes the codon for amino acid 60 of the mature protein
from ATT to ATA.
[0096] The materials produced for the Pko and B31 B. burgdorferi
strains were handled identically from this point on and hence only
the further handling of the Pko strain OspC material is
described.
[0097] The plasmid pLF100 (Example 1) was digested with SphI and
BamHI and the amplified PKo sequence was ligated into the plasmid
to form plasmid ppko 100 (pB31 100 for the B31 strain) containing a
hybrid ospA/ospC gene. The hybrid gene was excised from plasmid
pPko 100 by digestion with NdeI and BamHI and cloned into the NdeI
and BamHI sites of the plasmid vector pET9 to place the ospA/ospC
hybrid gene under control of a T7 promoter and efficient
translation initiation signals from bacteriophage T7, as seen in
FIG. 2. The resulting plasmid is designated pPko9a (pB319a for the
B31 strain).
Example 3
[0098] Expression and Purification of Lipidated OspC.
[0099] Plasmid pPko9a, prepared as described in Example 2, was used
to transform E. coli strains BL21(DE3) (pLysS) and
HMS174(DE3)(pLysS). The transformed E. coli was inoculated into LB
media with 30 .mu.g/ml kanamycin sulfate and 25 .mu.g/ml of
chloramphenicol at a rate of 12 ml of culture for every liter
prepped. The culture was grown overnight in a flask shaker at
37.degree. C.
[0100] The next morning, 10 ml of overnight culture medium was
transferred to 1 L of LB media containing 30 .mu.g/ml of kanamycin
sulfate and the culture was grown in a flask shaker at about
37.degree. C. to a level of OD.sub.600=0.6-1.0 (although growth up
to OD.sub.600=1.5 can be effected), in approximately 3-5 hours.
[0101] To the culture medium was added isopropylthiogalactoside
(IPTG) to a final concentration of 0.5 mM and the culture medium
was grown for a further two hours at about 30.degree. C. The
cultures were harvested and samples analyzed on Coomassie stained
SDS-PAGE gels (FIG. 5). The culture medium was cooled to about
4.degree. C. and centrifuged at 10,000.times.G for 10 minutes. The
supernatant was discarded while the cell pellet was collected.
Purified lipidated OspC was recovered from the pellet by effecting
the procedure shown schematically in FIG. 3 and described
below.
[0102] The cell pellet first was resuspended in {fraction (1/10)}
the volume of PBS. The cell suspension was frozen and stored at
-20.degree. C. or below, if desired. Following freezing of the cell
suspension, the cells were thawed to room temperature (about
20.degree. C. to 25.degree. C.) which causes the cells to lyse.
DNase I was added to the thawed material to a concentration of 1
.mu.g/ml and the mixture was incubated for 30 minutes at room
temperature, which resulted in a decrease in the viscosity of the
material.
[0103] The incubated material was chilled on ice to a temperature
below 10.degree. C. and Triton.TM. X-114 was added as a 10 wt %
stock solution, to a final concentration of 0.3 to 1 wt %. The
mixture was kept on ice for 20 minutes. The chilled mixture next
was heated to about 37.degree. C. and held at that temperature for
10 minutes.
[0104] The solution turned very cloudy as phase separation
occurred. The cloudy mixture then was centrifuged at about
20.degree. C. for 10 minutes at 12,000.times.G, which caused
separation of the mixture into a lower detergent phase, an upper
clear aqueous phase and a solid pellet. Analysis of the phases
fractionated by SDS-PAGE (FIG. 5) revealed that the OspC
partitioned into the detergent phase, showing that it is in
lipidated form. The detergent phase was separated from the other
two phases and cooled to 4.degree. C., without disturbing the
pellet.
[0105] Buffer A, namely 50 mM Tris pH 7.5, 2 mM EDTA and mM NaCl
and 0.3% polyethylene glycol tert-octylphenyl ether having the
formula t-Oct-C.sub.6H.sub.4--(OCH.sub.2CH.sub.2).sub.xOH wherein
x=9-10 as the detergent (commercially available as, and hereinafter
referred to as, Triton.TM. X-100), was added to the cooled
detergent phase to reconstitute back to 1/3 the original volume.
The resulting solution may be frozen and stored for later
processing as described below or may be immediately subjected to
such processing.
[0106] A DEAE-Sepharose CL-6B column was prepared in a volume of 1
ml/10 ml of detergent phase and was washed with 2 volumes of Buffer
C, namely 50 mM Tris pH 7.5, 2 mM EDTA, 1 M NaCl, 0.3 wt %
Triton.TM. X-100, and then with 4 volumes of Buffer B, namely 50 mM
Tris pH 7.5, 2 mM EDTA, 0.3 wt % Triton.TM. X-100.
[0107] The detergent phase then was loaded onto the column and the
flow-through containing the OspC, was collected. The column was
washed with 2 volumes of Buffer B and the flow-through again was
collected. The combined flow-through was an aqueous solution of
purified OspC, which may be frozen for storage.
[0108] The column may be freed from bacterial proteins for reuse by
eluting with 4 volumes of Buffer C.
[0109] Further and final purification of the flow-through from the
DEAE-Sepharose column was effected by chromatography on S-Sepharose
Fast Flow. The flow-through from the DEAE-Sepharose column first
was acidified to pH 4.3 by the addition of 1 M citric acid and the
acidified material was loaded onto the S-Sepharose column. The
S-Sepharose Fast Flow column had been washed with 3 column volumes
of Buffer A and then with 5 column volumes of Buffer A made up to
pH 4.3. The OspC binds to the column. The loaded column was washed
with 4 column volumes of pH 4.3 Buffer A followed by 4 column
volumes of pH 5.5 Buffer A.
[0110] Highly-purified OspC was eluted from the column using Buffer
A, adjusted to pH 6.0 with 1N HCl. A schematic of the purification
process described in this Example is shown in FIG. 3.
[0111] The aqueous solution of highly purified lipidated ospC
obtained by the chromatography procedures was analyzed by Coomassie
stained gels (FIG. 5), and confirmed to be OspC in highly purified
form by immunoblot analysis using rabbit anti-OspC polyclonal
antiserum. The purity of the product was estimated to be greater
than 80%.
[0112] By this procedure, about 2 to 4 mg of pure OspC was
recovered from a 1 liter culture of the BL21 host and about 1 to 2
mg of pure OspC was recovered from a 1 liter culture of the HMS 174
host.
Example 4
[0113] Expression and Purification of Non-Lipidated OspC.
[0114] E. coli JM 109 transformants containing plasmid vector
containing chromosomal gene fragment encoding non-lipidated OspC
were prepared and grown as described in WO 91/09870. The cultures
were harvested, the culture medium centrifuged at 10,000.times.G
for 10 minutes at 4.degree. C., the supernatant discarded and the
pellet collected.
[0115] The cell pellet was first resuspended in lysis buffer A,
namely 50 nM Tris-HCI pH 8.0, 2 mM EDTA, 0.1 mM DTT, 5% glycerol
and 0.4 mg/ml lysozyme, and the suspension stirred for 20 minutes
at room temperature. TRITON.TM. X-100 then was added to the cell
suspension to a concentration of 1 wt %, DNase I was added to a
concentration of 1 .mu.g/ml, and the suspension stirred at room
temperature for a further 20 minutes to effect cell lysis. Sodium
chloride next was added to the cell suspension to a concentration
of 1M and the suspension again stirred at 4.degree. C. for a
further 20 minutes. The suspension then was centrifuged at
20,000.times.G for 30 minutes, the resultant supernatant separated
from the pellet and the pellet was discarded.
[0116] The separated supernatant was dialyzed against a buffer
comprising 50 mM Tris pH 8, 2 mM EDTA. The supernatant next was
loaded onto a DEAE-Sepharose CL-6B column and the non-lipidated
OspC was collected in the column flow-through. The flow-through was
dialyzed against a 0.1 M phosphate buffer, pH 6.0.
[0117] The dialyzed flow-through next was bound to a S-Sepharose
fast flow column equilibrated with 0.1M phosphate buffer, pH 6.0.
Purified non-lipidated OspC then was eluted from the S-Sepharose
column using the dialysis buffer with 0.15 M NaCl added. A
schematic of the purification process is shown in FIG. 4.
[0118] The aqueous solution of highly purified non-lipidated OspC
was analyzed by Coomassie stained gels (FIG. 6). The purity of the
product was estimated to be greater than 80%.
Example 5
[0119] Immunogenicity of Recombinant Lipidated OspC.
[0120] Purified recombinant lipidated OspC, prepared as described
in Example 3, was tested for immunogenicity in mice and compared to
that from non-lipidated OspC prepared as described in Example 4.
For this study, 4 to 8 week old female C3H/He mice were immunized
on day 0 and boosted on day 21 and 42. All animals were given 1
.mu.g each of OspC expressed from the B31 and Pko genes per dose.
Both lipidated and non-lipidated forms of the antigen were tested.
Formulations were tested with and without alum as an adjuvant.
[0121] Representative animals were exsanguinated on days 21, 42, 63
and 91. ELISA testing was performed on these sera using purified
non-lipidated OspC as the coating antigen.
[0122] The test results from mice immunized with unadjuvanted
antigen (FIG. 7) show that only animals immunized with the
lipidated antigen make a detectable ELISA response. However, the
immune response of animals immunized antigens formulated on alum
(FIG. 8) shows that two types of antigen give comparible ELISA
responses and these responses develop more rapidly.
Example 6
[0123] Construction of a pET9a Expression Vector Containing a
Hybrid ospA/pspA Gene
[0124] Specifically designed oligonucleotide primers were used in a
PCR reaction to amplify the portion of the pspA gene of interest
(in this case from amino acid 1 to 321) from the S. pneumoniae
strain R.times.1.
[0125] The 5'-end primer had the nucleotide sequence:
3 5'-GGG ACA GCA TGC GAA GAA (PspN1). (SEQ ID NO: 6) TCT CCC GTA
GCC AGT-3'
[0126] The 3'-end primer had the nucleotide sequence:
4 5'-GAT GGA TCC TTT TGG (PspC370). (SEQ ID NO: 7) TGC AGG AGC TGG
TTT-3'
[0127] The PCR reaction was as follows: 94.degree. C. for 30
seconds to denature DNA; 42.degree. C. for one minute for annealing
DNA; and 72.degree. C. for one minute for extension of DNA. This
was carried out for 25 cycles, followed by a 5 minute extension at
72.degree. C. This procedure introduced a stop codon at amino acid
315. The PCR product was purified using the Gene Clean II method
(Bio101), and digested with SphI and BamHI.
[0128] The plasmid pLF100 (Example 1) was digested with SphI and
BamHI and the amplified pspA gene was ligated to this plasmid to
form the plasmid pLF321, which contained the hybrid ospA-pspA gene.
The hybrid gene was excised from pLF321 by digestion with NdeI and
BamHI and cloned into the NdeI and BamHI sites of the plasmid
vector pET9a to place the ospA-pspA hybrid gene under the control
of a T7 promoter. The resulting plasmid is called pPA321-L. This
process is shown schematically in FIG. 9.
Example 7
[0129] Construction of a pET9a Expression vector Containing the
pspA Gene
[0130] Specifically designed oligonucleotide primers were used in a
PCR reaction to amplify the portion of the pspA gene of interest
(in this case from amino acid 1 to 321) from the S. pneumoniae
strain R.times.1 using plasmid pPA321-L of Example 6.
[0131] The 5'-end primer had the nucleotide sequence:
5 5'-GCT CCT GCA TAT GGA AGA (PspNL-2) (SEQ ID NO: 8) ATC TCC CGT
AGC C-3'
[0132] The 3'-end primer had the nucleotide sequence:
6 5'-GAT GGA TCC TTT TGG (PspC370). (SEQ ID NO: 7) TGC AGG AGC TGG
TTT-3'
[0133] The PCR reaction was as follows: 94.degree. C. for 30
seconds to denature DNA; and 72.degree. C. for one minute for
annealing and extension of DNA. This was carried out for 25 cycles,
which was followed by a 5 minute extension at 72.degree. C. This
procedure introduced a stop codon at amino acid 315. The PCR
product was purified using the Gene Clean II method (Bio 101), and
digested with NdeI and BamHI. The digested PCR product was cloned
into the NdeI and BamHI sites of the plasmid vector pET9a to place
the pspA gene under the control of a T7 promoter. The resulting
plasmid is called pPA321-NL. This process is shown scematically in
FIG. 10.
Example 8
[0134] Expression and Purification of Lipidated PspA
[0135] Plasmid pPA321-L was used to transform E. coli strain
BL21(DE3)pLyS. The transformed E. coli was inoculated into LB media
containing 30 .mu.g/ml kanamycin sulfate and 25 .mu.g/ml
chloramphenicol. The culture was grown overnight in a flask shaker
at 37.degree. C.
[0136] The following morning 50 ml of overnight culture was
transferred to 1L LB media containing 30 .mu.g/ml kanamycin sulfate
and the culture was grown in a flask shaker at 37.degree. C. to a
level of OD 600 nm of 0.6-1.0, in approximately 3-5 hours. To the
culture medium was added IPTG to a final concentration of 0.5 mM
and the culture was grown for an additional two hours at 30.degree.
C. The cultures were harvested by centrifugation at 4.degree. C. at
10,000.times.G and the cell pellet collected. Lipidated PspA was
recovered from the cell pellet.
[0137] The cell pellet was resuspended in PBS at 30 g wet cell
paste per liter PBS. The cell suspension was frozen and stored at
-20.degree. C. The cells were thawed to room temperature to effect
lysis. DNaseI was added to the thawed material at a final
concentration of 1 .mu.g/ml and the mixture incubated for 30
minutes at room temperature, which resulted in a decrease in
viscosity of the material.
[0138] The material was then chilled in an ice bath to below
10.degree. C. and Triton.TM. X-114 was added as a 10% stock
solution to a final concentration of 0.3 to 1%. The mixture was
kept on ice for 20 minutes. The chilled mixture was then heated to
37.degree. C. and held at that temperature for 10 minutes. This
caused the solution to become very cloudy as phase separation
occurred. The mixture was then centrifuged at about 20.degree. C.
for 10 minutes at 12,000.times.G, which caused a separation of the
mixture into a lower detergent phase, an upper clear aqueous phase
and a pellet. The lipidated PspA partitioned into the detergent
phase. The detergent phase was separated from the other two phases,
diluted 1:10 with a buffer comprising 50 mM Tris, 2 mM EDTA, 10 mM
NaCl pH 7.5, and was stored at -20.degree. C.
[0139] A Q-Sepharose column was prepared in a volume of 1 ml per 5
ml diluted detergent phase. The column was washed with 2 column
volumes of a buffer comprising 50 mM Tris, 2 mM EDTA, 0.3%
Triton.TM. X-100, 1M NaCl pH 4.0, and then equilibrated with 5 to
10 column volumes 50 mM Tris, 2 mM EDTA, 0.3% Triton.TM. X-100, 10
mM NaCl pH 4.0. The pH of the diluted detergent phase material was
adjusted to 4.0, at which time a precipitation occurred. This
material was passed through a 0.2 .mu.M cellulose acetate filtering
unit to remove the precipitated material. The filtered diluted
detergent phase was applied to the Q-Sepharose column and the flow
through (containing PA321-L) was collected. SDS-PAGE analysis of
this step is shown in FIG. 15. The column was washed with 1-2
column volumes of 50 mM Tris, 2 mM EDTA, 0.3% Triton.TM. X-100, 10
mM NaCl pH 4.0, and the flow through was pooled with the previous
flow through fraction. The pH of the flow through pool was adjusted
to 7.5. The bound material, contaminating E. coli proteins, was
eluted from the Q-Sepharose with 2 column volumes of 50 mM Tris, 2
mM EDTA, 0.3% Triton.TM. X-100, 1M NaCl pH 4.0. A schematic of the
purification process described in this Example is shown in FIG.
11.
Example 9
[0140] Expression and Purfication of Non-Lipidated PspA
[0141] Plasmid pPA321-NL was used to transform E. coli strain
BL21(DE3)pLyS. The transformed E. coli was incolulated into LB
media containing 30 .mu.g/ml kanamycin sulfate and 25 .mu.g/ml
chloramphenicol. The culture was grown overnight in a flask shaker
at 37.degree. C.
[0142] The following morning 50 ml of overnight culture was
transferred to 1L LB media containing 30 .mu.g/ml kanamycin sulfate
and the culture was grown in a flask shaker at 37.degree. C. to a
level of OD 600 nm of 0.6-1.0, in approximately 3-5 hours. To the
culture medium was added IPTG to a final concentration of 0.5 mM
and the culture was grown for an additional two hours at 30.degree.
C. The cultures were harvested by centrifugaton at 4.degree. C. at
10,000.times.G and the cell pellet collected. Non-lipidated PspA
was recovered from the cell pellet.
[0143] The cell pellet was resuspended in PBS at 30 g wet cell
paste per liter PBS. The cell suspension was frozen and stored at
-20.degree. C. The cells were thawed to room temperature to effect
lysis. DNaseI was added to the thawed material at a final
concentration of 1 .mu.g/ml and the mixture incubated for 30
minutes at room temperature, which resulted in a decrease in
viscosity of the material. The mixture was centrifuged at 4.degree.
C. at 10,000.times.G, and the cell supernatant saved, which
contained non-lipidated PspA. The pellet was washed with PBS,
centrifuged at 4.degree. C. at 10,000.times.G and the cell
supernatant pooled with the previous cell supernatant.
[0144] A MonoQ column (Pharmacia) was prepared in a volume of 1 ml
per 2 ml cell supernatant. The column was washed with 2 column
volumes of a buffer comprising 50 mM Tris, 2 mM EDTA, 1M NaCl pH
7.5, and then equilibrated with 5 to 10 column volumes of a buffer
comprising 50 mM Tris, 2 mM EDTA, 10 mM NaCl pH 7.5. The cell
supernatant pool was applied to the Q-Sepharose column and the flow
through was collected. The column was washed with 2-5 column
volumes of 50 mM Tris, 2 mM EDTA, 10 mM NaCl pH 7.5, and the flow
through pooled with the previous flowthrough.
[0145] The elution of bound proteins began with the first step of a
5-10 column volume wash with 50 mM Tris, 2 mM EDTA, 100 mm NaCl pH
7.5. The second elution step was a 5-10 column volume wash with 50
mM Tris, 2 mM EDTA, 200 mM NaCl pH 7.5. The non-lipidated PspA was
contained in this fraction. SDS-PAGE analysis of this step is shown
in FIG. 15. The remaining bound contaminating proteins were removed
with 50 mM Tris and 2 mM EDTA pH 7.5 with 300 mM-1M NaCl.
[0146] A schematic of the purification process described in this
Example is shown in FIG. 12.
Example 10
[0147] Immunogenicity of Recombinant Lipidated PspA
[0148] Purified recombinant lipidated PspA, prepared as described
in Example 8, was tested for immunogenicity in mice and compared to
that from non-lipidated PspA prepared as described in Example 9.
For this study, CBA/N mice were immunized subcutaneously in the
back of the neck with 0.5 ml of the following formulations at the
indicated PspA antigen concentrations.
7 PspA Antigen Formulation Concentration Native PspA molecule of
the RX1 200 ng/ml strain (Native RX1) Non-Lipidated Recombinant
PspA 200 and 1000 ng/ml (pPA-321-NL) Alone in PBS* Non-Lipidated
Recombinant PspA 200 and 1000 ng/ml (pPA-321-NL) Adsorbed to Alum
Lipidated Recombinant PspA (pPA- 200 and 1000 ng/ml 321-L) Alone in
PBS Lipidated Recombinant PspA 200 and 1000 ng/ml (pPA0321-NL)
Adsorbed to Alum* Alum* 0 ng/ml PBS 0 ng/ml *Alum was Hydrogel at a
concentration of 200 .mu.g/ml
[0149] Four mice were immunized on days 0 and 21 for each dosage of
the formulations. The mice were then bled on day and subsequently
challenged with S. pneumoniae of A66 strain. The days of survival
after challenge for the mice were recorded and surviving mice were
bled on days 36, 37, 42 and 46. From these subsequent bleeds the
blood was assayed for the number of colony forming units (CFU) of
S. pneumoniae/ml. The sera taken on day 35 were assayed by ELISA
for antibodies against PspA using ELISA. The days to death for the
challenged mice are shown in the following table.
8 Survival in Immune and Non-Immune CBA/N Mice Immunization
Efficacy dose Days to P value time Alive: P value Group Antigen in
.mu.g Alum Death to death* Dead Survival* #1A pPA-321-L 1.0 - 4x
> 14 0.01 4:0 0.01 #1B PpA-321-L 0.2 - 4x > 14 0.01 4:0 0.01
#2A pPA-321-L 1.0 + 4x > 14 0.01 4:0 0.01 #2B pPA-321-L 0.2 + 4x
> 14 0.01 4:0 0.01 #3A pPA-321-NL 1.0 - 1, 1, 2, 2 n.s. 0:4 n.s.
#3B pPA-321-NL 0.2 - 1, 1, 2, .gtoreq.15 n.s. 1:3 n.s. #4A
pPA-321-NL 1.0 + 4x > 14 0.01 4:0 0.01 #4B pPA-321-NL 0.2 + 4x
> 14 0.01 4:0 0.01 #5 FL-Rx1 0.2 - 4x > 14 0.01 4:0 0.01 #6
none 0.0 + 1, 1, 3, 6 n.s. 0:4 n.s #7 none 0.0 - 1, 1, 1,
.gtoreq.15 n.s. 1:3 n.s. pooled 0.0 5 .times. 1, 3, 6, .gtoreq.15
-- 1:7 none Note: *indicates versus pooled controls; time to death,
by one tailed two sample rank test; survival, by one tailed Fisher
Exact test. Calculations have been done using "one tail" since we
have never observed anti-PspA immunity to consistently cause
susceptibility.
[0150] The number of CFU in the blood of the mice are shown in the
table below.
9 Bacteremia in Immune and Non-Immune CBA/N Mice Immunization
Cog.sub.10CFU dose in 2 6 7 Group Antigen .mu.g Alum 1 day day day
day #1A pPA-321-L 1.0 - .ltoreq.1.6, 1.9, 2.1, 4x .ltoreq. 1.6 4x
.ltoreq. 1.6 n.d. 2.5 #1B pPA-321-L 0.2 - 3x .ltoreq. 1.6, 1.7 4x
.ltoreq. 1.6 4x .ltoreq. 1.6 n.d. #2A pPA-321-L 1.0 + 2x .ltoreq.
1.6, 1.7, 2.9 3x .ltoreq. 1.6, 1.7 4x .ltoreq. 1.6 n.d. #2B
pPA-321-L 0.2 + 2x .ltoreq. 1.6, 1.7, 1.7 4x .ltoreq. 1.6 4x
.ltoreq. 1.6 n.d. #3A pPA-321-NL 1.0 - .ltoreq.1.6, 1.7, d, d d, d,
d, d d, d, d, d d, d, d, d #3B pPA-321-NL 0.2 - 2x > 7, d, d
.ltoreq.1.6, d, d, d .ltoreq.1.6, d, d, d n.d., d, d, d #4A
pPA-321-NL 1.0 + 2x .ltoreq. 1.6, 6.7, >7 3x .ltoreq. 1.6, 1.7
4x .ltoreq. 1.6 n.d. #4B pPA-321-NL 0.2 + .ltoreq.1.6, 1.7, 2.1, 4x
.ltoreq. 1.6 4x .ltoreq. 1.6 n.d. 2.4 #5 FL-Rx1 0.2 - 2x .ltoreq.
1.6, 2.6, 2.7 4x .ltoreq. 1.6 4x .ltoreq. 1.6 n.d. #6 none 0.0 +
.ltoreq.1.6, 4.1, >7, d .ltoreq.1.6, 5.1, d, d 6.1, d, d, d d,
d, d, d #7 none 0.0 - 1.7, >7, >7, d .ltoreq.1.6, d, d, d
.ltoreq.1.6, d, d, d n.d, d, d, d pooled none 0.0 .ltoreq.1.6, 4.1,
>7, 2x .ltoreq. 1.6, 5.1, .ltoreq.1.6, 6.1, n.d, d, >7, d d,
d, d, d, d d, d, d, d, d, d d, d, d, d, d Note: 1 colony at the
highest concentration of blood calculated out to 47 CFU or Log 1.7.
Thus ".ltoreq.1.6" indicates no colonies counted. >10.sup.7
indicates that the mouse was alive but the number of colonies at
the highest dilution was too high to count. "d" indicates the mice
had died prior to assay.
[0151] These results indicate that the recombinant protein was not
protective when injected alone. The recombinant antigen adjuvanted
with alum and/or PAM.sub.3Cys lipidation was immunogenic and
protective. The native full length PspA antigen did not need an
adjuvant to be protective. The CFU results indicate that mice
protected by immunization cleared the challenging S. pneumoniae
from the blood in two days.
[0152] ELISA analysis of sera taken on day 35 indicated that there
was a good correlation between protection of the mice from S.
pneumoniae challenge and the induction of measurable antibody
responses. No detectable antibody reponses were observed in the
sera of mice immunized with the non-lipidated antigen (pPA-321-NL)
in saline or to the negative controls that did not contain PspA
antigen, (as shown in the table below). Good antibody responses
were detected to the Native R.times.1 PspA antigen and to the
recombinant PspA when it was lipidated with PAM.sub.3cys and/or
adsorbed to alum.
10 ELISA Analysis of Day 35 Mouse Sera PspA Dose PspA Alum (.mu.g/
Resulting OD at Indicated Dilution of the Antisera* Antigen
Adsorption mouse 600 1200 2400 4800 9600 19200 pPA-321-L No 0.1
0.885 0.497 0.271 0.146 0.075 0.039 (0.082) (0.043) (0.025) (0.017)
(0.012) (0.009) pPA-321-L No 0.5 1.857 1.437 1.108 0.750 0.459
0.284 (0.060) (0.137) (0.150) (0.139) (0.092) (0.057) pPA-321-L Yes
0.1 1.373 1.048 0.745 0.490 0.288 0.171 (0.325) (0.376) (0.362)
(0.304) (0.197) (0.147) pPA-321-L Yes 0.5 1.202 0.787 0.472 0.296
0.162 0.087 (0.162) (0.184) (0.187) (0.102) (0.061) (0.035)
pPA-321-NL No 0.1 0.022 0.030 0.014 0.007 0.006 0.001 (0.035)
(0.060) (0.024) (0.018) (0.005) (0.001) pPA-321-NL No 0.5 0.029
0.014 0.008 0.003 0.002 0.002 (0.035) (0.014) (0.007) (0.004)
(0.002) (0.002) pPA-321-NL Yes 0.1 0.822 0.481 0.278 0.154 0.082
0.042 (0.181) (0.166) (0.085) (0.051) (0.029) (0.015) pPA-321-NL
Yes 0.5 1.017 0.709 0.447 0.253 0.141 0.075 (0.139) (0.128) (0.101)
(0.057) (0.034) (0.020) Native RX1 No 0.1 1.367 1.207 0.922 0.608
0.375 0.209 (0.084) (0.060) (0.070) (0.077) (0.048) (0.029) None No
0 0.018 0.012 0.009 0.005 0.005 0.005 (0.003) (0.008) (0.003)
(0.002) (0.002) (0.002) None Yes 0 0.013 0.009 0.004 0.004 0.001
0.000 (0.006) (0.008) (0.004) (0.003) (0.001) (0.000) *The OD is
the mean of the result of the four tested animals and the standard
deviation is in parentheses.
[0153] To determine whether protection was at least in part
mediated by the anti-PspA antibody responses, a passive experiment
was run. BALB/c mice were immunized with 0.5 .mu.g of recombinant
lipidated PspA alone or absorbed to alum, or with recombinant
non-lipidated PspA adsorbed to alum on days 0 and 21; and were bled
on day 35. The anti-sera were diluted 1:3 or 1:15 in saline and 0.1
ml of the dilution was injected i.p. into two mice for each
dilution. A 1/3 dilution of normal BALB/c mouse serum was used as a
negative control. Subsequently one hour after passive immunization,
the animals were challenged i.v. with the WU2 strain of S.
pneumoniae (15,000 CFU). Mice passively immunized with anti-PspA
sera were protected as compared-to those mice that received
dilutions of normal mouse sera as shown in the following table.
11 Passive Protection of BALB/c to WU2 Immunizing Formulation PspA
Dose Dilution Days to Death PspA Antigen Alum (.mu.g/animal) of
Serum Post Challenge pPA-321-L No 0.5 3 4, >7 15 2, 4 pPA-321-L
Yes 0.5 3 >7, >7 15 4, >7 pPA-321-NL Yes 0.5 3 2, 4 15
>7, >7 None No 0 3 2, 2
[0154] Having thus described in detail certain preferred
embodiments of the present invention, it is to be understood that
the invention defined by the appended claims is not to be limited
by particular details set forth in the above description, as many
apparent variations thereof are possible without departing from the
spirit or scope thereof.
Sequence CWU 1
1
8 1 17 PRT B. burgdorferi leader amino acid sequence for ACA strain
1 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1
5 10 15 Cys 2 51 DNA B. burgdorferi DNA encoding leader amino acid
sequence for ACA strain 2 atgaaaaaat atttattggg aataggtcta
atattagcct taatagcatg c 51 3 28 DNA Artificial Sequence Description
of Artificial Sequence 5'-end PCR primer for portion of ospC gene
of Pko strain of B. burgdorferi 3 ggcgcgcatg caataattca gggaaagg 28
4 28 DNA Artificial Sequence Description of Artificial Sequence
5'-end PCR primer for portion of ospC gene of B31 strain of B.
burgdorferi 4 ggcgcgcatg caataattca gggaaaga 28 5 24 DNA Artificial
Sequence Description of Artificial Sequence 3'-end PCR primer for
portion of ospC gene of B31 & Pko strains of B. burgdorferi 5
cgcggatcct taaggttttt ttgg 24 6 33 DNA Artificial Sequence
Description of Artificial Sequence 5'-end PCR primer for portion of
pspA gene of S. pneumoniae strain RX1 6 gggacagcat gcgaagaatc
tcccgtagcc agt 33 7 30 DNA Artificial Sequence Description of
Artificial Sequence 3'-end PCR primer for portion of pspA gene for
S. pneumoniae strain RX1 7 gatggatcct tttggtgcag gagctggttt 30 8 31
DNA Artificial Sequence Description of Artificial Sequence 5'-end
PCR primer for portion strain RX1 pspA gene encoding aa's 1-321 of
S. pneumoniae 8 gctcctgcat atggaagaat ctcccgtagc c 31
* * * * *