Note: Descriptions are shown in the official language in which they were submitted.
2~ 2112
7~,IT~,E OF INVENTION
PREPARATION OF RECOb~INANT BORRBLIA ~Q,$OTBINS
FIELD OF 3~,N~ION
The present invention relates to the preparation of
recombinant Borrelia lipoproteins, particularly the outer
surface protein A (OspA) of ~orrelia burcdorferi, the
spirochete responsible for Lyme disease.
BACKGROUND TO THE INVENTION
Lyme disease is a zoonosis caused by the tick-borne
spirochete Borrelia ~xradorferi. The spirochete can
cause
serious dermatological, arthritic, neurological and
other
pathological disorders in an infected host. Recently
Lyme
disease has become a serious epidemiological concern,
particularly in North America, but also elsewhere in
the
world.
Attempts are underway to develop a vaccine against
the disease and immunodiagnostic reagents useful in
the
detection of antibodies against the spirochete. Much
2o attention has focused on the major outer surface protein
OspA, antibodies against which have been demonstrated
to
.. provide protection against challenge with the spirochete
,, in mice (refs. 1, 2 - identification of the literature
references appears at the end of the disclosure).
Previous attempts have bean made to isolate purified,
soluble $orrelia lipoproteins through the growth and
subsequent purification of ~orrp~:~ia cell cultures.
However, the growth and subsequent purification of
the
Proteins from crude cell extracts of ~orrelia is very
time
consuming and expansive.
2121121
Recombinant techniques have been suggested for
producing OspA and derivatives thereof, in view of the
potential for production of large quantities of pure
protein material. Such techniques would involve
expression of B~crrelia genes in a suitable host/vector
expression system, such as E.E. coll.
Published International (PCT) Patent application No.
WO 90/04411 describes a DNA fragment encoding the OspA
protein of B. burgdorferi of the New York strain B31 (ATCC
35210) and containing the osQA gene. The nucleotide
sequence of the osoA gene, coding for the full-length
wild-type OspA, is described in the published PCT patent
application, along with the derived amino-acid sequence.
Dune et al have described the preparation of a
modified form of OspA protein which is soluble, yet
retains specific reactivity to antibodies against wild
type B. Burgdorferi OspA (ref. 3). The paper describes
the preparation of two plasmids, pET9-OspA and pET9
preOspA, the former containing a polymerase chain reaction
(PCR)-amplified DNA sequence coding for a recombinant
truncated version of OspA, and the latter containing a
PCR-amplified DNA sequence coding for the full-length,
wild-type OspA. Both sequences are expressed from the
bacteriophage T7 ~ 10 promoter.
The primary translation product of the full-length
gene contains a hydrophobic N-terminal leader sequence,
which is a substrate for the attachment of lipid moiety to
the sulfhydryl side chain of the adjacent cysteine
residue. Following this attachment, cleavage by signal
peptidase II and the attachment of lipid moieties to the
new N-terminus occurs. On the other hand, the protein
translated from the truncated gene is not lipidated, and
is soluble in aqueous solution. Expression of the
soluble, truncated derivative of OspA is said to overcome
certain problems said to be associated with expressing
3 2121121
recombinant versions of the full-length wild-type Bo~elia
~uradorferi lipoproteins using E. o ', namely that
the
protein~ has poor solubility properties, due to the
association of the protein with the outer cell membrane
of
the host during expression, requiring the use of
detergents to effect separation of the protein and
difficult purification procedures.
The plasmids were transferred to an expression strain
of E. coli, namely BL21(DE3)(pLysS) (a host strain
l0 containing a chromosomal copy of the gene for T7 RNA
;..,, polymerase under control of the inducible ac promoter
and a pACYC184 based plasmid pLysS, which produces
low
levels of T7 lysozyme). Upon induction, plasmid pET9-
preOspA was found to produce relatively smaller amounts
of
inducible protein than plasmid pET9-OspA. The latter
product was found to be soluble in the absence. of
detergent while the former required treatment with
the
detergent Triton* X-100 to solubilize.. The Dunn et
al
paper contains no description of any subsequent isolation
and purification of the detergent-solubilized OspA
protein.
Although production of recombinant full-length OspA
may be achieved in lower quantities than the soluble
truncated variation of OspA and the use of detergents
is
necessary for recovery of the full-length protein
(treatment of lipoproteins with detergent$ often impairs
reactivity), nevertheless production of the recombinant
wild-type protein is considered desirable, since the
lipidated wild-type protein is thought to produce a
greater immune response than the truncated protein,
due to
the stimulation of both B lymphocytes (refs. 4, 5)
and
cytotoxic T lymphocytes (ref. 6) by lipid covalently
attached to the N-terminus of a peptide in an identical
fashion to that in which lipid moiety is attached to
$~"
buradorferi OspA (ref. 7).
* Trade-mark
vA
212~~21
Long-lasting protective immunity to spirochetal
challenge has been produced in mice by vaccination with
OspA (refs. 1, 8). The antigen used in these studies was
a fusion protein containing a large domain of glutathione-
S-transferase at the amino terminus of OspA. This fusion
protein is unlikely to be a substrate for a post-
translational lipid attachment. The ability of this
antigen to induce a protective response in these studies
may be due to the fact that the antigen was delivered in
Freund's complete adjuvant, with multiple secondary doses
in Freund' s incomplete adj uvant ( ref s . 1, 8 ) , or in the
form of live or killed whole E. coli expressing the fusion
protein (ref. 9). With respect to this prior activity,
reference also is made to published International
application WO 92/00055.
As will be seen herein, it has now been shown that
the recombinant protein encoded by the full-length os_pA
gene, in unadjuvanted or alum-absorbed form, exhibits an
immunogenicity which is not shown by the corresponding
recombinant OapA protein lacking the attached lipid. This
difference in immunogenicity has been shown not to be due
to any difference between the antigenicity of the two
proteins. The present invention represents the first
disclosure of which the inventors are aware of enhancement
of the immunogenicity of a large protein antigen by
expression of a bacterial lipoprotein.
SUMMARY OF INVENTION
The present invention provides, in one aspect, a
novel, simple and effective manner of producing a highly
purified immunologically-effective recombinant protein
encoded by the full-length wild-type B. buradorferi osnA
gene and other full-length Borrelia lipoproteins encoded
by the respective genes. The recombinant protein exhibits
an immunogenicity which is equal to that of the native
protein.
2121121
The present invention employs selective separation of
the recombinant lipoprotein from other cellular
constituents using particular detergents. The procedure
of the present invention enables the perceived prior art
5 problems associated with production of the full-length
OspA protein to be overcome, while providing a product
having enhanced immunogenicity.
For the first time, there is provided by the present
invention a highly-purified immunologically-effective
recombinant protein which is encoded by a full-length
wild-type Bor~elia lipoprotein gene, particularly the
~.bur_cdorferi ,pspA gene, and which is formed recombinantly
from a host organism transformed by a plasmid containing
the gene. Such novel protein forms a further aspect of
the present invention. Certain novel plasmids, useful in
such transformation, also are provided herein.
The novel protein product provided herein is useful
in vaccines against infection by Borrelia organisms,
particularly those causing Lyme disease, and such
vaccines, comprising an immunologically-effective amount
of the protein, particularly that encoded by the full-
length wild-type B. burgdorferi osoA gene, constitute an
additional aspect of the invention.
In a yet further aspect of the invention, there is
provided a method of immunization of a mammal against
infection by Borrelia organisms, particularly those
causing Lyme disease, by administering an immunologically
effective amount of the protein, particularly that encoded
by the full-length wild type B. buradorferi osnA gene, in
the absence of adjuvant.
The novel proteins also may be used as
immunodiagnostic agents for the detection of antibodies
against infection of a host by a Borrelia organism,
particularly that causing Lyme disease, and hence the
presence of such infection.
2I~1~.21
6
There have been produced herein, as described below,
purified immunologically-effective recombinant protein
encoded by the full-length osnA gene from several
different specific strains of B. burcrdorferi, specifically
the B31, ACAl and Ip90 strains. There also exist full-
length os~A genes which differ in nucleotide sequence from
the sequences of these specific genes by at the most a few
nucleic acids, resulting in gene products which differ at
most by a few amino acids from those produced by the
actual B31, ACA1 and Ip90 strain os~A genes. Such genes
and gene products are encomposed herein within the 831,
ACA1 and Ip90 families of strains.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the PCR oligonucleotides used in
cloning the B-31, ACA1 and Ip90 full-length osDA gene of
B. burgdorferi,into the pET9a expression vector;
Figure 2 illustrates the cloning strategy for
inserting the full-length ospA gene into the pET9a
expression vector so as place the ospA gene under control
of the T7 ~ 10 promoter, to form pOAl from the B31 gene,
pOA9 from the ACA1 gene and pOAlO from the Ip90 gene;
Figure 3 is a predicted restriction map of plasmid
pOAl;
Figure 4 shows the results of various restriction
digests of plasmid pOAl, demonstrating that all predicted
sites are present (M= markers (~X 174 DNA digested with
Hae III); B= Bam HI; E= Eco RI; H= Hind III; N= Nde I);
Figure 5 shows the PCR nucleatides used in cloning
the B-31, ACA1 and Ip90 full-length os gene of B.
burgdorferi into the pCMBl expression vector;
Figure 6 illustrates the cloning strategy for
inserting the full-length os~A gene into the pCMBi
expression vector, so as to place the ospA gene under the
control of the Trc promoter, to form pOA5 from the B31
gene, pOA7 from the ACA1 gene and pOAB from the Ip90 gene;
7 2121121
Figures 7A, 7B and 7C show a time course of induction
with ITPG of OspA in two host strains containing pOA1
(Figure 7A), and a host strain containing pOAS (Figure
7B)
and pOA6 (Figure 7C);
Figures 8A, SB and SC are flow charts showing.
respectively, the cell growth and lysis, the detergent
extraction and the purification steps involved in the
production and purification of recombinant full-length
OspA from E. coli in accordance with one embodiment
of the
invention;
.., Figure 9 shows the solubilization of proteins in
whole cell lysate with various detergents (Deo= sodium
deoxycholate; Emp= Empigen; Sarc= sodium lauryl
sarcosinate; SDS= sodium dodecyl sulfate; TX-114 Triton*
X-114), with the lanes being (M= markers (Bio-Rad Low
Molecular Weight Standards); W= whole lysate; S= soluble
fraction; P= pellet; A= aqueous phase; D= detergent);
Figure 10 is an immunoblot with the anti-OspA
monoclonal antibody H5332, showing that the 31 Kilodalton
species in the detergent phase is OspA, Wh= whole cell
lysate, Aq= aqueous phase after Triton X-114 extraction,
Dt= detergent phase, BX= aqueous phase back extracted
with
Triton X-114, Pl= insoluble pellet;
Figure 11 illustrates Triton X-114 phase partitioning
of lipoprotein and non-lipoprotein for pOAl and pOA2,
in
which bacteria containing p0A1 expressing OspA lipoprotein
or pOA2 expressing OspA non-lipoprotein were grown
to rnid-
log phase and induced with ITPG for 2 hours for pOAl
and
4 hours for pOA2. After centrifugation, cells were
resuspended and lysis was effected by freezing and
thawing. Triton X-114 was added and phase partitioning
was produced by heating to 37C. An equal aliquot of
each
fraction was electrophoresed on 4 to 20% SDS-PAGE gel,
and
the gel was stained with Coomassie Brilliant Blue.
The
fractions are molecular weight markers (M), whole lysate
* Trade-mark
=A
2121121
(W), aqueous phase (A), detergent phase (D), and insoluble
pellet (P) for p0A1 and p0A2 as indicated;
Figures 12A, 12B and 12C illustrate purification of
OspA lipoprotein produced from pOAl (Figure 12A), from
pOA9 and pOAlO (Figure 12B) and from p0A5, pOA7 and pOA8
(Figure 12 C), where an aliquot of each fraction was
electrophoresed on a 4 to 20% SDS-PAGE gel and the gel
was
stained with Coomassie Brilliant Blue. Figure 12A : lane
1, low molecular weight markers; lane 2, whole lysate;
lane 3, detergent phase; lane 4, DEAF-Sepharose*; and
lane
5, S-Sepharose* elution; Figure 12B . P= prestained low
molecular weight markers (106 kDa, 80kDa, 49.5kDa,
32.5kDa, 27.5kDa, 18.5kDa), CL= whole cell lysate,
D=Triton X114 detergent phase, De= DEAF Sepharose flow
through fraction. S= Sepharose, pH5.7 elution (purified
OspA-2); Figure 12C . P=prestained low molecular weight
markers (106kDa, 80kDa, 49.5kDa, 32.5kDa, 27.5kDa,
18.5kDa), O= B31 OapA-L S-Sepharose purified, derived
from
pOAl (Figure 12A), CLa whole cell lysate, D= Triton X114
detergent phase, De=DEAF Sepharose flow through fraction.
Figure 13 is a silver-stained gel of three test
.. vaccines used in a study comparing the immunogenicity
of
,,
,,
the purified full-length OspA to that of the protein
from
Borrelia burcdorferi (M= markers; OA= purified recombinant
OspA; E= B. buradorferi fraction E; SA= bovine serum
albumin);
Figure 14 shows the IgG serum titres of mice given a
boost four weeks after primary injection with 10 ~g of
the
purified full-length OspA and B. burgdorferi fraction
of
Figure 13, with an ELISA assay being performed with 100
ng
purified OspA in the solid phase, mouse serum as the
firs
antibody and goat anti-mouse IgG conjugated to alkaline
phoaphatase as the second antibody;
Figures 15A to 15E contain a graphical depiction of
the dose response of mice to lipoprotein. Figure 15A
* Trade-marks
A
," 2I2~1~1
9
shows the dose response of C3H/He mice to unadsorbed
lipoprotein; Figure 15B shows the dose response of BALB/c
mice to unadsorbed lipoprotein; Figure 15C shows the dose
response of C3H/He mice to lipoprotein adsorbed to alum;
Figure 15D shows the dose response of CH3/He mice to
unadsorbed lipoprotein; Figure 15E shows the dose response
CH3/He mice to non-lipidated protein. Mice were
vaccinated at week 0 with the indicated amount of protein,
in PBS except where noted, and boosted with the same
vaccine at week 3. Serum IgG titre at week 4 was
determined by ELISA using OspA lipoprotein as antigen;
Figure 16 contains a graphical depiction of the
antigenicity of the lipoprotein and non-lipoprotein; and
Figure 17 illustrates the production of plasmids
pCMBl and pCMB2.
IDENTIFICATION OF osbA GENE-CQNTAINING PLASMIDS
Plasmid ~. burgdorferi Expression
I.D. Strain Vector
pOA1 B31 pET9a
pOA2* B31 pET9a
pOA5 B3I pCMBl
pOA6 B31 pCMB2
pOA7 ACA1 pCM81
pOA8 Ip90 pCMBl
pOA9 ACA1 pET9a
pOAlO Ip90 pET9a
* This plasmid contains truncated ospA of B31 strain.
All other plasmids contain full-length Q~pA of various
strains.
30' GENERAL DESCRIPTION OF INVENTION
The cloned os~A gene of B. buradorferi strain B31 (as
described in the above-mentioned WO 90/04411) (N-terminal
region : SEQ ID No: 1, C-terminal region . SEQ ID No: 2.
The remainder of the sequence is shown in WO 90/04411) was
used as a template (pTRH44) and specially-designed
l0 2'1~~.I~1
oligonucleotide primers (PET-IN [C01] (SEQ ID No: 3) and
PET-273C [C03] (SEQ ID No: 4)) were used in a polymerase
chain reaction (PCR) to amplify the whole of the wild-type
osDA gene, as shown in Figure 1.
Similarly, the cloned os~A gene of B. buradorferi
strains ACA1 and Ip90 (as described in reference 10 - N-
terminal region of ACA1 and Ip90 is: SEQ ID No: 1; C-
terminal region of ACA1: SEQ ID No: 5; C-terminal region
of Ip90: SEQ ID No: 6) was used in a PCR reaction with
oligonucleotide primer pairs (a) OspN2 (SEQ ID No: 7) and
BZ1 (SEQ ID No: 8) and (b) OspN 2 (SEQ ID No: 7) and pK4
(SEQ ID No: 9), respectively at the N- and C-terminal ends
to form the appropriate amplified fragments, as shown in
Figure 1.
The basic methods for amplifying a desired target
nucleic acid sequence using oligonucleotide primers are
generally known in the art and are described in U.S.
Patents No. 4,683,202 and 4,800,159. Reference may be had
to such patents for description of the techniques to be
employed.
The resulting fragments were cloned into the NdeI and
Bam HI sites of the plasmid vector pET9 to place the ospA
gene under control of a T7 promoter and efficient
translation initiation signals from bacteriophage T7, as
seen in Figure 2. The pET9 and pLysS plasmids, the
bacterial hosts for cloning, growth media and the methods
used to direct expression of cloned genes by T7 RNA
polymerase have previously been described in U.S. Patent
No. 4,952,496 and reference may be had thereto for such
description. While a T7 promoter system is one preferred
expression system in the present invention, expression of
the full-length OspA gene may be achieved utilizing other
expression systems compatible with the host organism, as
described below.
,....,
~I2112.~
11
The pET9 expression vector was used since it has a
kan gene as its selective marker rather than a gene.
Consequently, ampicillin is not used during cell growth
and hence there is no possibility that an immunogenic
ampicilloyl/OspA target protein conjugate can be formed.
Such conjugates are believed to be major antigenic
determinants in penicillin allergy and may complicate
immunological studies.
The resulting plasmids have been designated pOAl,
pOA9 and pOAlO, containing the osDA genes from B31, ACA1
and Ip90 strains of B. bu~g;g~orferi respectively. The pOA1
plasmid is nearly identical to the pET9-preOspA plasmid
described by Dunn et al (supra), except that the
oligonucleotides used for the PCR reaction were different
in the two cases. A predicted restriction map for the
plasmid pOAl is shown in Figure 3, while Figure 4 contains
the results of various restriction digests of plasmid
pOAl, demonstrating that all the predicted sites are
present.
For protein production, the plasmids pOAl, pOA9 and
pOAlO were transformed into the expression strain of E.
o i or other suitable host organism. Preferably, the E.
coli strain is the T7 expression strain of E. coli, as
described in the aforementioned U.S. Patent No. 4,952,496.
Specifically, the strain may be the expression strain
BL21(DE3)(pLysS) of E.E. coli, as described above, or
o i strain HI~tS174(DE3)(pLysS). The transformed host was
grown and protein was induced with isopropyl-i3-D-
thiogalactoside (IPTG). A time course of induction of
OspA from plasmid pOAl, following IPTG addition, is shown
in Figure 7A. Identical results to those for pOAl were
obtained using pOA9 and pOAlO. Synthesis of OspA protein
from plasmid pOAl ceased approximately one hour after
induction, implying some toxicity of the protein to E.
~'I2~I2I
12
coli. Nevertheless, the protein production was at an
acceptable level of approximately 10 mg/L of cell culture.
In addition to the provision of plasmids pOAl, pOA9
and pOAlO and expression of OspA lipoprotein in E. coli
using the T7 promoter, further plasmids have been
constructed containing the full-length B31, ACA1 and Ip90
os~A gene under a different promoter and expression of
lipoprotein has been achieved. In this regard, plasmids
pOA5 and pOA6 were prepared by cloning a PCR-amplified
fragment of ospA from B31 strain into the Ncol and Bam HI
sites of plasmid expression vectors pCMBl and pCMB2 while
plasmids pOA7 and pOAB were prepared by cloning a PCR-
amplified fragment of OspA from ACA1 strain (pOA7) and
from Ip90 strain (p0A8) into the Ncol and Bam FiI sites of
expression vector pCMBl (see Figure 6 for pOA5, pOA7 and
pOAB ) .
As seen in Figure 5, the cloned oanA genes of
burgdorferi strains B31, ACA1 and Ip90 were amplified by
PCR reaction using oligonucleotide primer pairs (a) PK3
(SEQ ID No: 10) and C03 (SEQ ID No: 3), (b) PK3 and (SEQ
ID No: 10) and BZ1 (SEQ ID No: 8), and (c) PK3 (SEQ ID No:
10) and PK4 (SEQ ID No: 9), respectively at the N- and C-
terminal ends of the respective Qenes to form the
appropriate amplified fragments.
Plasmids pCMBl and pCMB2 were constructed by
digesting plasmid pTrc99a (Pharmacia Catalog No. 27-5007-
01) and a Kanamycin resistance gene (Pharmacia Catalog No.
27-4897-O1), isolating the resulting fragments by the
Geneclean procedure, and ligating the fragments together
(Figure 17). Plasmid pTrc99a, 4197 bp, contains a strong
promoter adjacent to a multiple cloning site, followed by
a strong transcription termination signal (rrnB).
Expression of the target gene uses the host cell RNA
polymerase, allowing its use in a wide variety of E. coli
strains. Expression is tightly controlled by the lactose
13
suppressor gene (lacIq) included on the vector. The
lactose repressor protein prevents transcription of the
target gene in the absence of the inducer IPTG. The
kanamycin resistance gene is a linear double stranded 1282
by DNA fragment containing the gene from the transposon
Tn903 flanked by restriction enzyme sites and encodes the
enzyme aminoglycoside 3'-phosphotransferase, which confers
resistance to kanamycin and neomycin.
pCMBl, 5.5 kb, contains the kanamycin resistance gene
oriented such that its transcription is in the same
direction as that originating from the Trc promoter while
pCMB2 contains the kanamycin resistance gene oriented in
the opposite direction, such that transcription of the
resistance gene and the gene of interest under the control
of the Trc prompter result in converging transcripts.
Restriction enzyme digests of pCMBl and pCMB2 using SmaI,
HindIII and BamHI+NcoI showed the exact size fragments
predicted.
The plasmids pOAS and pOA6 were transformed into the
expression strain of E. coli or other suitable organism,
preferably the DHSa competent cells. The transformed
hosts were Brawn and protein was induced with IPTG. The
time course of induction with pOAS (Figure 7B) was similar
to that for pOAl (Figure 7A) while the levels of OspA
produced by pOA6 (Figure 7C) was several times lower.
Identical results to those for pOAS were obtained using
pOA7 and pOA8.
The steps involved in the production and purification
of the recombinant full-length OspA are shown
schematically in Figures 8A to 8C. Specific process
conditions are recited therein as set forth in the
Examples below.
Following the cell growth step and induction of
protein (Figure 8A), the cells are subjected to freeze
thaw lysis. The lysate is treated with a detergent which
-~- ~ ~. ~ f I 2 I
14
is selective for OspA protein solubilization, in
preference to other bacterial proteins present in the
lysate. A series of experiments was conducted employing
different detergents to determine which detergent was
selective for OspA protein. Of those tested Triton X-114
was found to selectively solubilize a 31 kilodalton
protein (Figure 9), which was shown to be OspA by
immunoblotting (Figure l0).
The invention is not limited to the employment of
Triton X-114 but clearly also includes other materials
exhibiting a similar selective solubility for OspA as well
as the phase separation property under mild conditions
referred to below.
Following addition of the selective detergent, the
mixture is warmed to a mild temperature elevation of about
35° to 40°C at which time the solution becomes cloudy as
phase separation occurs. It is essential to the
purification procedure of the present invention that such
phase separation occurs under mild conditions to avoid any
denaturing or other impairment of the immunological
properties of the protein. (For example, if Triton X-100,
mentioned by Dunn et al (ref. 3), is used as the
detergent, while selective separation of OspA protein is
effected, much higher temperatures, about 60 to 65°C, are
required to effect phase separation, which is highly
disadvantageous with respect to the utility of the
product).
Centrifugation of the cloudy mixture results in
separation of the mixture into three phases, namely a
detergent phase containing 50% or more of the OspA protein
and a small amount (approximately 5 wt%) of the bacterial
proteins, an aqueous phase containing the balance of the
bacterial proteins and a solid pellet of cell residue.
The detergent phase is separated from the aqueous phase
and solid pellet.
2~ 2> > z~
8y the steps of treatment with a selective detergent for
OspA and subsequent phase separation of the detergent phase,
substantially complete separation of ospA from bacterial
proteins is achieved (Figure 12). There remains the final
5 purification of OspA from residual bacterial protein present
in the detergent phase.
Final purification of the protein is effected on a
chromatography column selective for binding bacterial proteins
but no OspA, specifically DEAE-Sepharose*, DEAE-Sepharose, or
10 other equivalent chromatography material. The detergent phase
is loaded onto the column and the flow-through, which contains
all the purified OspA protein is collected. The bound
fraction contains all the bacterial proteins in the detergent
phase. Following further purification using S-sepharose or
15 equivalent chromatographic column (Figure 12), in addition to
being free from contaminating proteins, the flow-through
fraction is substantially free from contaminating proteins,
the flow-through fraction is substantially free from
lipopolysaccharide (LPs) as indicated by lack of pyrogenicity,
as determined by limulus amebocyte lysate (LAL). The highly
purified solution of OspA may be freeze dried or otherwise
processed. ' .
Although the procedure described above has been
specifically directed to production of highly-purified
recombinant OspA protein, the procedures and techniques
described are readily adaptable to the production of other
highly-purified recombinant ~iorrelia lipoproteins, by suitable
cloning of the appropriate gene, construction of a suitable
plasmid vector containing the gene, transformation of a
suitable host by the plasmid vector, and production and
purification of the lipoprotein, by suitable choice of
selective surfactant.
* Trade-mark
~,A
~z2zz2z
16
EXAMPLES
Example 1
Plasmid pOAl was prepared as described above and used
to transform E~cg~i strains BL21 (DE3) (pLysS) (pOAl) and
HMS174(DE3)(pLysS)(pOA1). The transformed E. coli was
inoculated into LB media with 25 ~.g/ml of kanamycin
sulfate and 25 ~,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 about 37°C.
l0 The next morning, 10 ml of overnight culture medium
was transferred to 1L of LB media containing 25 ~.g/ml of
kanamycin sulfate and the culture was grown in a flask
shaker at about 37°C to a level of OD=0.6 (although growth
up to OD=1.5 can be effected), in approximately 3 hours.
To the culture medium was added
isopropylthiogalactoaide (IPTG) to a final concentration
of 0.5mM and the culture medium was grown for a further
two hours at about 37°C. At the end of this period, the
culture medium was cooled to about 4°C and centrifuged at
10000 xg for 10 minutes. The supernatant was discarded
while the cell pellet was resuspended in 1/10 the volume
of PBS. The cell suspension was frozen in liquid nitrogen
and may be stored indefinitely at -70°C, if desired.
Following freezing of the cell suspension, the cells
Were thawed to room temperature (about 20° to 25°C) which
causes the cells to lyre . DNase I was added to the thawed
material to a concentration of 1 ~,g/ml and the mixture was
incubated for 30 minutes at room temperature, which
resulted in a decrease in the viscosity of the material.
The incubated material was chilled on ice to a
temperature below 10°C and Triton 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°C and held at
that temperature for 10 minutes.
17 2121121
The solution turned very cloudy as phase separation
occurred. The cloudy mixture then was centrifuged
at
about 20C for 10 minutes at 12,000 xg, which caused
separation of the mixture into a lower detergent phase,
an
upper clear aqueous phase and a solid pellet. The
detergent phase was separated from the other two phases
and cooled to 4C, without disturbing the pellet. Buffer
A, namely 50 mM Tris, pH 7.5, 2 mM EDTA and 10 mM
NaCl,
was added to the cooled detergent phase to reconstitute
back to 1/3rd 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.
A DEAE-Sepharose CL-6B*column was prepared in a
volume of 1 m1/10 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 X-100, and then with
4
volumes of Buffer B, namely 50 mM Tris pH 7.5, 2 mM
EDTA,
0.3 wt % Triton X-100.
The detergent phase then was loaded onto the column
and the flow-through containing the OspA, was collected.
The column was washed with 1 volume of Buffer 8 and
the
flow-through again was collected. The combined flow-
through was an aqueous solution of purified OspA,
which
may be frozen for storage.
The column may be freed from bacterial proteins for
reuse by eluting with 2 volumes of Buffer C.
Further and final purification of the flow-through
from the DEAE-Sepharose column by chromatography on
S-
Sepharose Fast Flow. The flow-through from the DEAE-
Sepharose column first was acidified to pH 4.2 by
the
addition of 0.1 M citric acid. The S-Sepharose Fast
Flow
column was washed extensively with Buffer C, adjusted
to
pH 4.2 with citric acid.
Highly-purified OapA was eluted from the column using
Buffer C, adjusted to pH 5.7 with citric acid. The
eluate
* Trade-mark
'~'A
18~~.21121 ,
was immediately adjusted back to pH 7.5 by the addition of
2 M Tris base.
The aqueous solution of highly purified OspA obtained
by both chromatography procedures was analyzed by
Coomassie stained gels (Figure 12A) and confirmed to
contain OspA in highly purified form. The purity of the
product produced by the latter chromatography procedure
was greater than that formed by the former chromatography
procedure, exhibiting very low levels of endotoxin.
Examp~~e 2
The procedure of Example 1 was repeated, except that
other surfactants were used as well as Triton X-114 to
form the detergent phase. The results which were obtained
are shown in Figure 9. As may be seen from Figure 9, only
Triton X-114 of the detergents tested was able to provide
a sufficiently selective solubilization of OspA to permit
ready purification by column chromatography.
Exam 1p a 3
Plasmids pOA5 and pOA6 were prepared as described
above and used to transform E. coli strain DHSa. The
protein expression procedure employed in Example 1 was
repeated and the time course expression of the OspA
lipoprotein by p0A5 was identical to that observed for
pOAl while OspA expression by pOA6 was found to be several
times lower than by pOAS (Figures 78 and 7C).
Purification of the OspA protein was continued in pOAS-
expressing cells only.
The B31 OspA lipoprotein produced by the Trc
expression system,in this way was purified following the
identical procedure to that described in Example 1, with
the exception that 0.1 mg/ml of lysozyme was added to the
cell pellet after harvesting and the cells were suspended
for 30 minutes at room temperature prior to freezing.
The DEAF-Sepharose column flow through contained OspA
lipoprotein in a highly purified form (Figure 9C).
19 2121121
Example 4
The procedures for production of plasmid pOAI (pET
promoter) and pOA5 (TRC promoter) were repeated, as described
above, with the cloned CsoA gene of Asian OspA strain (Ip90)
of B. burg~dorferi, to form plamids pOAB (Trc promoter) and
pOAlO (pET promoter) containing the gene. A restriction
digest was performed on these plasmids, which shoed all
predicted sites to be present.
The procedures also were repeated with the cloned csaA
gene of the European strain (ACA1) of B. buradorferi, to form
plasmids pOA7 (Trc promoter) and p0A9 (pET promoter)
containing the gene. A restriction digest was performed on
these plasmids, which showed all predicted sites to be
present.
Growth and induction of the pOA7 and pOA8 expression
strains proceeded identically to the pOA5 strain (Example 3)
while growth and induction of the p0A9 and pOAlO expression
strains proceeded identically to the p0A1 strain (Example 1).
2o The ACAl and Ip90 OspA lipoproteins obtained by these
operations were purified identically to the B31 (pOAl) OspA
lipoprotein as described in Example 1. The DEAE-Sepharose
column flow through contained OspA lipoprotein in a highly .
purified form (Figures 128, 12C).
Examgle 5
The full-length recombinant OspA purified from E, coli in
accordance with the procedure described in Example 1 was
compared with the B. burq~ior eri fraction E, which is enriched
for OspA (prepared as described in the aforementioned PCT No.
WO 90/04411), and with bovine serum,albumin (BSA) for
immunogenicity in mice. C3H/He mice were injected with l0 ~g
of each vaccine (Figure 13) in unadjuvanted form. Four weeks
after primary rejection, the mice were given a boost of the
same antigen as used in the primary injection. The serum
titres were assayed by
~A
2~ 2I21 i 21
an ELISA against recombinant, purified OspA lipoprotein
(Figure 14).
As may be seen, both immunogens elicited a very
similar response, that is a weak but detectable primary
serum IgG response, with as much as a 100-fold increase in
serum titres following the boost. For both immunogens,
serum IgG titres remained stable for at least 6 weeks. In
neither case was any significant serum IgM response
observed.
These experiments indicate that the recombinant OspA
is equally immunogenic as the protein purified from B.
burcrdorf eri .
The immunological integrity of the recombinant OspA
lipoprotein was further demonstrated by the fact that sera
from mice immunized with the recombinant lipoprotein were
fully capable of inhibiting the growth of B. buradorfer~
strain B31 spirochetes in vitro, as seen from the
following Table 1:
21
c~
Ea
~ '~ ~ tn
G
.. ~ ~ b ~ oD N ~ N aD CO OD a0 d, 10 ~ op o0 CD
V ('~1 N ~ V V V V r1 ~ V V V
r-1 ~~
'
fI~ err ~ 13
3
'
b~
,1 umn .mn u~ m un ~ umn
o o .
O O N O O N O O N O O
N
al A
~
L',
.~
N ~ U
-rl 1.7 r1 J.~
O ~ O
1~ ~1 1J
~1 O S-1 O
O v O
~i
r1 ' r1 '
ri G r-I
O Gl' O R'
O O
t",
(C
S-iN U
x w
w
N M
W U G1
O
r~
22
As may be seen from this Table, the inhibitory
activity of the serum was found to be proportional to the
dose of the lipoprotein administered to mice and
correlated very well with the results obtained by the
ELISA assay for anti-OspA IgG. Serum from mice vaccinated
with OspA non-lipoprotein, which contained no detectable
anti-OspA antibodies, had no effect on spirochete growth.
The growth inhibitory titres achieved in mice
immunized with 2.5 ~.g of OspA lipoprotein in PBS were
equal to or greater than those obtained in mice vaccinated
with 20 ~g of total B. buradorferi protein in Freund's
complete adjuvant and boosted with total protein in PBS.
The ability of the recombinant OspA lipoprotien to elicit
a serum response capable of inhibiting spirochete growth
demonstrates that the critical protective epitopes on the
protein are conserved during expression in E. coli and
subsequent purification.
Exam In a 6
Plasmid pOA2 was prepared in similar manner to that
described above for plasmid pOAl, except that primer PET
18N, having the sequence (SEQ ID No. 11):
5' CAG CAT ATG GCT AAG CAA AAT PTT ABC 3'
was used together with the C-terminal primer PET-273C, in
the PCR reaction to amplify the truncated form of the osnA
gene lacking the lipoprotein signal peptide. The
underlined region in PET-18N is identical to nucleotides
51 to 67 of the coding strand of the OspA gene.
The plasmid pOA2 was used to transform E. coli
strains BL21 (DE3) (pLysS) (pOA2) and HMS174 (DE3) (pLysS)
(pOA2) and the truncated OspA protein was expressed from
the transformed E.E. coli, as described in Example 1. Upon
performing the cell lysis and detergent extraction steps
of Example 1, the non-lipidated protein was found to be
present in the aqueous phase, as would be expected.
~~~~mz
Examsle 7
The effect of lipid attachment to the OspA protein on
immunogenicity was investigated.
C3H/He and BALB/c mice were vaccinated with either
the lipoprotein form of OspA formed and isolated as
described in Example 1 or the non-lipoprotein form of OspA
prepared by Dunn et al (ref. 3) and boosted three weeks
later. Sera were collected 1 to 2 weeks following
boosting, and assayed by an ELISA using purified OspA
lipoprotein as the antigen.
Figures 15A and 15B show that OspA lipoprotein in PBS
induced a strong dose-dependent, secondary IgG response in
both C3H/He (Figure 15A) and BALB/c (Figure 15B) mice.
Little, if any, increase in immunogenicity of the
lipoprotein was observed when the protein was absorbed to
aluminum hydroxide at a ratio of protein: aluminum of l:5
by weight prior to injection, as seen from Figure 15C.
In constrast to the response seen for the OspA
lipoprotein, the non-lipoprotein was unable to induce any
detectable anti-OspA antibody, even at the highest dose of
2.5 ~,g/mouse, as seen in Figure 15E. Even alum-absorbed
non-lipoprotein appeared incapable of producing an immune
response.
It should be observed that the lipoprotein and non-
lipoprotein versions of OspA are indentical in deduced
amino acid sequence except for the amino terminal residue
which is modified cysteine for the lipoprotein and
methionine-alanine for the non-lipoprotein, which strongly
suggests that the observed difference in immunogenicity is
due to the presence of the lipid moiety.
Example 8
The inherent antigenicity of the lipidated and non-
lipidated OspA also was investigated, to determine whether
the difference in immunogenicity observed in Example 7 was
due to differences in their inherent antigenicity or to
zm~~z~
24
differences in how the immune system recognizes and
responds to the two proteins.
Sera from mice immunized with the lipoprotein gave a
positive signal in an ELISA using either version of OspA
as the test antigen, as seen in Figure 16. Similarily,
sera from mice immunized with the non-lipoprotein failed
to react with either antigen. Accordingly, while both
versions of OspA are equally antigenic, the lipoprotein is
recognized well by the immune system while the non
lipoprotein is not.
In addition, these results strongly suggest that,
while the lipid group is essential for immunogenicity, the
antibodies made against the lipoprotein are directed
principally against the peptide portion of the molecule,
rather than the lipid moiety.
Example 9
The effect of strain-source for OspA protein on
immunogenicity was observed.
Mice were vaccinated with purified OspA lipoproteins
B31, ACA1 and Ip90 derived from pOAl, pOA9, pOAlO
respectively. The mice were vaccinated with 2.5 ~,g of
protein at week 0, boosted with the same dose at week 3,
and bled out at week 4. Serum IgG titres at week 4 were
determined by an ELISA assay using purified B31, ACA1 or
Ip90 OspA lipoprotein as the antigen. The titre was
determined using the secondary antibody goat anti-mouse
IgG.
The results obtained in these experiments are shown
in Figure 15D. As may be seen from this data, recombinant
OspA for all the strains tested is immunogenic in mice and
polyclonal sera show a partial cross-reactivity.
2~z~~z~
SUMMARY OF DISCLOSURE
In summary of this disclosure, the present invention
provides a novel and simple procedure to produce a highly-
purified immunologically-effective recombinant protein
5 encoded by a full-length wild-type Borrelia lipoprotein
gene, particularly the osr~A gene of B.burg~dorferi, by the
use of a detergent to selectively extract the protein from
the host strain and subsequent purification of the
detergent solution by column chromatography.
10 Modifications are possible within the scope of this
invention.
26 2I211~I.
R
1. Fikrig, E., S.W. Barthold, F.S. Kantor, and R.A.
Flavell (1990). Protection of Mice Against the Lyme
Disease Agent by Immunizing with Recombinant OspA.
Science, 250: 553-556
2. Simon, M.M., U.E. Schaible, M.D. Kramer, C.
Eckerskorn, C. Museteanu, H.K. Muller-Hermelink, and
R. Wallich (1991). Recombinant Outer Surface Protein
A from Borrelia burgdorferi Induces Antibodies
Protective Against Spirochetal Infection in Mice.
Infect. Dis. 164: 123-132.
3. Dunn, J.J., B.N. Lade, and A.G. Barbour (1990).
Outer Surface Protein A (OspA) from the Lyme Disease
Spirochete, Borrelia burgdorferi: High Level
Expression and Purification of Soluble Recombinant
Form of OspA. ,protein Expression and Purification 1:
159-168.
4. Bessler, W.G., B. Suhr, H.-J. Buhring, C.P. Muller,
K.-H. Wiesmuller, G. Becker, and C. Jung (1985).
Specific Antibodies Elicited by Antigen Covalently
Linked to a Synthetic Adjuvant. Immunobiolociv 170:
239-244.
5. Biesert, L., W. Scheuer, and Bessler, W.G. (1987).
Interaction of Mitogenic Bacterial Lipoprotein and a
Synthetic Analogue with Mouse Lymphocytes. Eur. J.
Biochem. 162: 651-657.
6. Deres, K., H. Schild, K.H. Wiesmuller, G. Jung, and
H.G. Rammensee (1989). In vivo Priming of Virus-
Specific Cytotoxic T Lymphocytes with Synthetic
Lipopeptide Vaccine. Nature 342: 561-564.
7. Brandt, M.E., B.S. Riley, J.D. Radolf, and M.V.
Norgard (1990). Immunogenic Integral Membrane
Proteins of Borrelia burgdorferi Are Lipoproteins.
Infection and Immunity 58: 983-991.
27
8. Fikrig, E., S.W. Barthold, F.S. Kantor, and R.A.
Flavell (1992). Long-term protection of mice from
Lyme disease by vaccination with OspA. Infect. and
Immun. 60: 773-777.
9. Fikrig, E., S.W. Barthold, F.S. Kantor, and R.A.
Flavell (1991). Protection of mice from Lyme
borreliosis by oral vaccination with Escherichia coli
expressing OspA. J. Infect. Dis. 164:1224-1227.
10. Johnsson, M., L. Noppa, A.G. Barbour, and S.
Bergstram 1992. Heterogenicity of Outer Membrane
Proteins in Borrelia burgdorferi: Comparison of osp
operons of three isolates of different geographic
origins. Infect. Immun. 60:1845-1853.
28
SEQUENCE IDENTIFICATION
SEO ID NO. DESCRIPTION OF SEOUEN~~, FIGURE
1 Coding strand for N-terminal 1,5
region of osnA from B31 strain,
ACA1 and 1p90 strain
2 Coding strand for C-terminal 1,5
region of ose~A from B31 strain
I
3 Oligonucleotide primer PET-IN 1
I (CO1]
I
4 Oligonucleotide primer PET-273C 1,5
(co3]
Coding strand for C-Terminal 1,5
region of osnA from ACA1
6 Coding strand for C-terminal 1,5
region of ospA from Ip90
7 Olignucleotide primer OspN2 1
8 Olignucleotide primer BZ1 1,5
9 Olignucleotide primer PK4 1,5
Olignucleotide primer PK3 5
11 Olignucleotide primer pET-18N -
29
SEQtTENCE LISTING
(1) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
ATATATTATG AAAAAATATT TATTGGG
27
{2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
GAGGAATAAA ATTTCGCAAA AATTAAA
27
(3) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
212112
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCACATATGA AAAAATATTT ATTGGG
26
(4) INFORMATION FOR SEQ ID N0:4:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CGGGGATCCC TCCTTATTTT AAAGCG
26
(5) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GAGGAATAAA TAAAGTTTCG CAi'~AAATTCA A
31
{6) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
''' 2I21I2~
31
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GAGGGATAAA ATTTCGTAGA AATTCAA
27
(7) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGCCGCACAT ATGAA1:1AAAT ATTTATTGGG
(B) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic}
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
CGGGGATCCC CTTATTTATT TCAAAGCG
28
(9) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
2I~ZI~~
32
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CGGGGATCCC TATTTTAAAG CATC
24
(10) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CGGCCATGGA AAAATATTTA TTGGG
(11) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CAGCATATGG CTAAGCAAAA TGTTAGC
27
