IE83340B1 - Vaccine against lyme disease - Google Patents
Vaccine against lyme diseaseInfo
- Publication number
- IE83340B1 IE83340B1 IE1999/0173A IE990173A IE83340B1 IE 83340 B1 IE83340 B1 IE 83340B1 IE 1999/0173 A IE1999/0173 A IE 1999/0173A IE 990173 A IE990173 A IE 990173A IE 83340 B1 IE83340 B1 IE 83340B1
- Authority
- IE
- Ireland
- Prior art keywords
- antigen
- borrelia burgdorferi
- mice
- recombinant
- ospa
- Prior art date
Links
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Description
VACCINE AGAINST LYME DISEASE
Max—PIanck—Gesellschaft Zur Férderung Der Wissenschaften e.v.
and
Deutsches Krebsforschungszentrum Stiftung Des éffentlichen Rechts
The present invention is concerned.with a vaccine
against Lyme disease, with a process for pbtaining said
vaccine, with new monoclonal antibodies, with new
antigens and with new recombinant DNA's and vectors.
Lyme borreliosis is the most common infectidus «V
disease transmitted by ticks in the temperate regions.
It is caused by the spirochete ‘fiorrelia bgrgdorferi
which is transmitted to humans in particular by ticks
of the genus Ixodes, The disease is a chronic, pro-
gressive infection which attacks many organs, such as
the skin, the central and peripheral nervous system,
the heart, the liver, the kidneys and musculoskeletal
system. Since a reliable \treatment of this disease
by therapy with antibiotics is difficult, at the
moment great efforts are being made to investigate the
pathogen itself and the immune response of the host to
infection with Borrelia burgdorferi. In the case of
persons afflicted by Lyme disease, there is admittedly
ascertained a high titre of antibodies against Borrelia
burgdorferi which, however,_do not provide any pro-
tection against the infection. It is assumed that the
pathogen passes over very quickly from the blood
circulation into the tissues and can there no longer
be directly reached by the immune system. This would
mean that a protection by antibodies is only possible
immediately after commencement of the infection, i.e.
as long as the pathogen is still present in the blood
circulation.
The fact that a natural infection with Borrelia
burgdorferi has been found in various kinds of animals
has led to attempts to establish laboratory models for
Lyme disease, This also took place with limited success.
Thus, in the case of experiments which had the object
of inducing in mice a specific immune response for ‘
Borrelia burgdorferi , it was found that the infection
of inbred mouse strains with a proflonged cultured
isolate of Borrelia burgdorferi led to moderate but
significant pathomorphological changes in various organs,
such as the brain, the heart, the lungs and the kidneys,
which were comparable to those which are to be observed
in patients with Lyme disase (see Schaible et al,,
T Infect. Immun., l, 41/1988). The development of a
It is an object of the present invention to provide
an effective vaccine against Lyme disease. However, for
this purpose, it is first necessary to develop-an.
appropriate animal laboratory model. It is now suggested
that a mouse strain without functionable T- and B—cells,
the so—called scid mouse (see Bosma et al,, Nature, lg,
/1985) can serve as experimental animal since scid
_
mice, in the case of infection with a pathogenic
Borrelia burgdorferi isolate, develop a. multi-
systemic disease, namely, mainly polyarthritis and
carditis. By means of this animal model,it is possible
for the first time to test the action of vaccines
against nyme disease.
One subject of the present invention is a passive
vaccine against Lyme disease which contains one or more
specific monoclonal antibodies for the 5lkD antigen
(OspA) and/or the 54 kD antigen (OspB) of Borrelia
burgdorferi and especially OspA and/or OspB of
Borrelia burgdorferi of the strain B51 (ATCC 55210)
and/or ZS7 (DSM 5527). A vaccine is preferred which
contains one of the antibodies of the class IgG
according to the present invention and especially
preferably of the subclass IgG2b or IgGl. Surprisingly,
in contradistinction to the administration of another
antibody, for example against the 41 kD surface antigen
of Borrelia burgdorferi (flagellin), the administration
of the antibody according to the present invention has
the result,in.the case of immune-deficient experimental
animals and preferably of scid mice which have been
infected with viable pathogenic Borrelha burgdorferi
and preferably with Borrelia burgdorferi ZS7,that the
development of arthritis, carditis and hepatitis is
completely or at least substantially prevented.
The vaccine according to the present invention with
the antibody as active material can possibly also contain
-
conventional carrier, filling and adjuvant materials.
Furthermore, the present invention provides a process
for obtaining a passive vaccine against Lyme disease
from lymphocytes or spleen cells of an experimental
animal, preferably of a mouse, which has been immunised
with Borrelia burgdorferi organisms or parts thereof,
preferably with complete Borrelia burgdorferi B51 and/or
ZS7 organisms, in which, from the lymphocytes or spleen
cells of the immunised animals, there is obtained, by
cell fusion, a hybridoma which produces a monoclonal
antibody according to the present invention.
Thus, a subject of the present invention is also a
ahybridoma cell line (ECACC 89091502) which produces an
antibody LA—2 against OspA (IgG2b) according to the
present invention. Furthermore, the subject of the
present invention is also the hybridoma cell line
ECACC 90050406 producing the antibody LA-26.l against
OspA,(IgGl), as well as the hybridoma cell lines
ECACC 90050405 and ECACC 90050407 producing antibodies
LA_25,l and LA—27_l, respectively,against OspB (IgG2b
and IgGl, respectively). I
Furthermore, the present invention provides the
pathogenic Borrelia burgdorferi strain ZS7 (DSM 5527).
In addition, the subject of the present invention
is an antigen which immune—reacts with a monoclonal
antibody according to the present invention. By this is
to be understood an antigen which contains the whole
amino acid sequence of OspA or OspB or also only an
_5_ _
immunogenically—acting part sequence (immunogenic
epitope) of OspA or OspB, respectively. Potentially‘
immunogenic epitopes of these proteins can be determined
without difficulty by a structural analysis of the
OspA protein, for example a Chou-Fagmm ianalysis, and
then tested experimentally for their effectiveness.
Yet another-subject of the present invention is
also, in particular, a recombinant antigen which immune-
reacts with the antibody according to the present
invention in which the DNA sequence-coding for the
antigen is present on a recombinant vector, preferably
a prokaryotic vector, which is suitable for the protein
expression.
In particular, a subject of the present invention
is an antigen from Borrelia burgdorferi ZS7 which
specifically immune—reacts with the antibody according
to the present invention and which contains the amino
acid sequence shown in Fig. l of the accompanying
drawings or an immunogenic epitope of this sequence,
Consequently, the present invention also concerns a
recombinant DNA.which contains (1) the sequence shown in
Fig, l, (2) a nucleic acid sequence corresponding to it
in the scope of the degeneration of the genetic code or
(5) one hybridising under stringent conditidns with a
sequence from (1) and/or (2), which sequence codes for
the 5l.kD antigen of Borrelia burgdorferi gm3h3Zg7or;m
immunogenic epitope thereof, The term stringent hybrid-
ising conditions is thereby to be understood as in
_
Maniatis et al., Molecular Cloning, A Laboratory Manual
(lo82), Cold Spring Harbor Laboratory, New York.
Especially preferred is an antigen according to the
present invention which is a recombinant non-fusion
protein or B—galactosidase fusion protein. V ‘
Furthermore, the present invention is concerned
with a recombinant vector which contains one or more
copies of_a recombinant DNA according to the present
invention, The vector according to the present invention
can be a prokaryotic and/or eukaryotic vector,but is
preferably a prokaryotic vector. The recombinant vector
can be present extrachromosomally in the host cell
(for example plasmid) or it can also be integrated in the
genome of the host cell (fon'example bacteriophage
lambda), The vector according to the present invention
is preferably a plasmid, the recombinant vector
pZS—7/51-2 (DSM 5528) being especially preferred.
The present invention also provides a process for
obtaining antigens according to the present invention
by investigation of a Borrelia burgdorferi gene bank
with one or more antibodies according to the present
invention in which the clones are isolated which show a
positive immune reaction with the antibodies used,
Since the antigen according to the present invention
itself also can be used for active immunisation, i.e.
for the induction of antibody formation in the
organism, the present invention also provides an active
vaccine against Lyme disease which, as active material,
-8. -
contains an antigen according to the present invention,
optionally together with conventional carrier, filling
and adjuvant materials. A preferred embodiment is when
the antigen according to the present invention is obtained
gene-technologically. ‘
Indeed, it could be shown that the administration of
native or recombinant OspA to normal mice induces the
formation of protective antibodies which, after passive
transfer into soid mice, protect these against Lyme L
borreliosis. In particular, it is found that recombinant
OspA induces a protective immune response comparable
with native OspA and, therefore, represents a highly
promising condidate for a vaccine against Lyme
borreliosis in humans,
The present invention also provides a process for
obtaining a passive vaccine against Lyme disease in
which experimental animals, preferably mice, are
immunised with an antigen according to the present
invention and protective, polyclonal or monoclonal
antibodies are obtained in the usual way from the
immunised experimental animals.
Finally, the present invention also provides a
process for the isolation and reculturing of pathogenic
Borrelia burgdorferi organisms, wherein, from immune~
deficient experimental animals, preferably mice, which
have previously been infected with the pathogen, there
is obtained the pathogen, whereby the pathogeneity of
the pathogen is retained. Especially preferred is a
_
process in which pathogenic Borrelia burgdorferi gga31zs7
(DSM 5527) organisms are obtained from the blood and/or
joints of infected scid mice. A T
The following examples are given for the purpose of
illustrating the present, reference being made to the
accompanying drawings, in which:
Fig. 1 shows the DNA and amino acid sequence of the 51 kD
antigen (OspA) from Borrelia burgdorferi stran1Zs7and
Fig. 2 shows the immunological characterisation of the
recombinant protein rZS7/51-2.
Examplell.
Induction of arthritis, carditis and hepatitis in send
mice by infection with Borrelia burgdorferi strain ZS7,
Treatment of the mice with Borrelia burgdorferi.
Adult mice of the strains C,B-l7 scid (homozygous
for the scid mutation) and G;B—l7 were injected sub-
cutaneously into the root of the tail with l x 105,
x 105, 1 x 105 or l.x 108 viable or killed (ultra-
violet irradiation) Borrelia burgdorferi organisms.
Isolation of Borrelia burgdorferi from ticks and mice;
The investigations were carried out with the A
Borrelia burgdorferi strain B51 (ATCC 55210), already
cultured for a long time, and the fresh isolate Borrelia
burgdorferi strain zs7 (DSM5527) which had been isolated from
l985). Borrelia hmrmknferi organisms which had been
_
obtained from the midgut of ticks sterilised with
ethanol or from the blood of infected mice were
initially cultured in Kelly's medium with the addition
of 8 ;»g./ml. of kanamycin and 250 }»g./ml. fluorouracil
(see Johnson et al., J, Olin. Microbiol., l, 81/1984).
Serolagical tests;
The detection of Borrelia burgdorferi-specific
antibodies was carried out in a conventional ELISA
process (see Justus et al., wehrmed. Mschr,, gg, 263/
l988). The standard curve for the content of immune
globulin (lg) was obtained by coating a dish with anti-
mouse lg (l:5OO dilution of the serum solution of
Paesel, Frankfurt, Federal Republic of Germany) and
tfi:atkx1 of the total mouse IgG or IgM content
(Calbiochem, La Jolla, U.S.A,), Total serum IgM and
IgG were measured in a similar manner, The concentration
of Borrelia burgdorferi-specific IgM or IgG antibodies
is given in‘ }»g. Ig/ml. of serum.
Immunofluorescence and Giemsa staining,
‘#1. of blood were pipetted into a haemocrit
testtube (Becton and Dickinson, Heidelberg, Federal
Republic of Germany) and centrifugedat 5000 g in a
haemocrit centrifuge (ECCO, Federal Republic of Germany).
The test tubes were cut up on the interphase between
serum and erythrocytes and 5 )rl. of the serum were
applied to microscope slides (Superior, Bad Mergentheim,
Federal Republic of Germany). The microscope slides
loaded with the serum samples were dried in the air
-
and fixed in 100% ethanol for 1 minute at —2d°C, After
incubation for l hour with rabbit anti—Borrelia ~
burgdorferi hyperimmune serum (l:lOO dilution) at
ambient temperature, with microscope slides were washed
five times in PBS and then stained for 1 hour with Emc-
conjugated goat anti—rabbit antiserum (1:20 dilution,
Jackson Lab,, West Grove, U.S.A,). The microscope slides
were washed and embedded in Kaiser’s glycerol gelatine
(Merck, Darmstadt, Federal Republic of Germany) and
immediately investigated by fluorescence microscopy.
Untreated blood droplets were dried in the air, fixed
in methanol, stained with Giemsa's stain (0.1%, Merck,
Darmstadt, Federal Republic of Germany), decolorised
in PBS and embedded in Entellan (Merck, Darmstadt,
Federal Republic of Germany).
Histological preparations and staining processesL
Various internal organs (brain, heart, Lungs, liver,
kidneys, spleen and joints) were removed from mice
previously infected with Borrelia burgdorferi at different
times after the infection and stored either in liquid
nitrogen for the preparation of frozen sections or in
% formaldehyde (in PBS) for embedding in paraffin or
methacrylate. Sections of 4 to 7 fmm. thickness were
prepared, stained with haemotoxylin-eosin and embedded
in Entellan (Merck, Darmstadt, Federal Republic of
Germany). The immunohistology was carried out with the
use of streptavidin—biotin—peroxidase system (see Kramer
et al., Eur. J. Immunol., ig, l5l/1989)}
*
Table l shows that Borrelia burgdorferi organisms
of the isolates ZS7 and B51 were detected during the
whole of the experimental period in the blood of scid
mice which had previously been inoculated with viable
‘strain ZS7 *
organisms. However, only spirochetesof
but not of strain B51 could be recultured in vitro.
In the case of comparison of the recultured organisms
with the primary Borrelia burgdorferi ZS7 isolate, no
changes in the protein content or in the plasmid profile
could be ascertained. No or only extremely small titres
of irrelevant antibodies were detected in scid mice
infected with Borrelia burgdorferi during the entire
period of dbservation. No IgM or IgG antibodies specific
for Borrelia burgdorferi could be found in these animals
(seeTable 1). On the other hand, all C.B-17 control
mice, which had been infected with Borrelia burgdorferi,
expressed large amounts of total lg and increased titres
of IgM and IgG antibodies specific for Borrelia
burgdorferi. Between 7 and 20 days after infection with
Borrelia burgdorferi, scid mice showed the first _
clinical symptoms of arthritis (reddudng and swelling of
both tibiotarsal joints), which increased in the course
of time. On the other hand, no symptoms of arthritis V
were found in scid mice which had been infected either
with ultra-violet irradiated Borrelia burgdorferi anfimiams(ZSfl
or with viable horrelia burgdorferi organisms (Bin
‘in C,B—17 control mice which had been infected with
viable Borrelia burgdorferi ZS7 organisms.
-15..
Rrthritic joint changes were also detected histo-
pathologically in scid mice which had been infected,
with viable Borrelia burgdoriferi orqanisms (zs7-) (seeflfable 1).
Severe joint damages were ascertained, characterised by
the presence of hyperplastically inflamed synovial ‘
lining cells, combined with erosion and destruction
of cartilaginous tissue and/or bone. Furthermore, there
was ascertained panoarditis with infiltration of mono-
nuclear cells in the endocardium, myocardium and peri-
cardium. There was also ascertained a progressive
inflammation of the liver in which there was observed
an infiltration of mononuclear cells, which was limited
to the portal artery region and the central veins,
granulomatous reactions and, finally, the appearance of
liver fibrosis, In addition, smaller damage in the
kidneys, the lungs, the brain and the striated musculature
were ascertained.
mflPwH£PHN mo zowpmapow can mH#H# huonwpsm mmdwmav one EOHM mmvmm:oopHmm mo
:oHpmHomHon mwhompowwmsn waamumom spas mofis HOH#QoO wHIm.o cam QOHE ufiom >HIm.o mo QOHPOQMQH
H mHnma
+ = by Giemsa staining or immunofluorescence
++ = isolation from blood (B), joint (J)
o+ = neddening and swelling of the tibiotarsal joint
oo- = 4 7.5 fpg./ml. serum
Action of a monoclonal antibody specific for the
Borrelia burgdorferi 5lkD antigen on the course of
lyme borreliosis in scid mice;
Preparation of the monoclonal. antibody.
In the case of immunisation of a mouse which has
an intact immune system with Borrelia burgdorferi
organisms, polyclonal antibodies are expressed which
are specific for Borrelia burgdorferi (see Table 1).
Ten week old female mice of the inbred strain
BALB/c were immunised with Borreliaormyjsm(fimaelfiabumk
dorferi, strain B31; ATCC 55210) homogenised bye
sonication .
Immunisation protocol:
day 0: 200 }»g. Borrelia antigen in complete Freund‘s
adjuvant subcutaneously
day 21, 35, 49, 63: challenge with 100 fLg. Borrelia
antigen in phosphate—buffered saline (PBS)
intraperitoneally V
day 66: removal of the spleen and preparation of a
suspension of individual cells
The immune spleen cells were fusioned with the
Ag8—PAl myeloma cell line by standard methods with
the use of polyethylene glycol (see J,H_ Peters,
H. Baumgarten, M. Schulze, "Monoklonale Antikdrper”,
pub. Springer Verlag, Heidelberg),
The fusion products were seeded out into 96 well
tissue culture plates. Acter 8 days, the cell culture
supernatants were investigated for the presence of
Borrelia burgdorferi—specific monoclonal antibodies
with the help of a solid—phase ELISA (see J.H. Peters
et al., loc. cit.).
The hybridoma cells from antibody-producing cultures
were cloned according to the marginal dilution method.
The culture supernatants of individual clones were
subsequently again characterised in the solid-phase
ELISA, as well as by Western blot analysis and by
immunofluorescence investigations. The monoclonal
antibody LA-2 of the subclass LgG2b is produced by a
monoclonal hybridoma line and secreted and reacts in
the Western blot with the 5lkDa .structure (OspA) of
all investigated Borrelia burgdorferi strains (inter
alia the isolates ZS7 and B51) in the case of contact
with Borrelia burgdorferi proteins separated electro-
phoretically via an SDS gel and transferred by neans of
Western blot to a membrane. The monoclonal antibodies
LA~26,l (anti—OspA.LgGl), LA 25.1 (anti—OspB (54kDa
antigen); IgG2b) and LA 27;l (anti-bsm (54 kDa antigen)
IgGl) were prepared and characterised in an analogous
manner.
infection of mice with Borrelia burgdorferi ZS7,
-
lC_B-17 scid mice were infected subcutaneously in
the root of the tail with l x 108 viable Borrelia
burgdorferi ‘ organisms.(zsfl;‘
Treatment of the mice with antisera.
The infected scid mice were treated twice a week‘
with various antisera. One group was treated with NMS
(normal mouse serum), the second group with IMS (immune
mouse serum) and the third.group with the monoclonal
antibody LA—2 (against the 51 kD antigen of Borrelia
bergdorferi). The dosage of the administered antisera
was 100 /~l, or lOO )»g. in the first week in the case
of LA-2, 200 ,»l. or 200 ,,g. in the second week in the
case of LA-2 and 500 }»l, or 500 ,»g. in the third week
in the case of LA—2.
The following Table 2 shows that scid mice, untreated
or treated with NMS, develop clinical and histopatho—
logical indications of arthritis or carditis and hepat-
itis after l2 days. On the other hand, the administ-
ration of the monoclonal antibody LA—2 brings about a
distinct reduction of the symptoms in the case of scid
mice, Clinically, there were only ascertained slight
reddenings of the joints and histopathologically only
marginal changes. Mice treated with INS showed no-
clinical findings of arthritis. A
A detection of Borrelia burgdorfer}. Qfifimifimibyin
vitro culturing only succeeded in the case of mice which
were either untreated or treated with NHS, I“ the case
of mice treated with LA-2 or IMS, Borrelia burgdorferi
onfirnsm;
could not be detected (Table 2).
n = 5 - + + + +
n = 3 NMS + + + +
n = 2‘ IMS - — ~ —
n = 5 LA_2 _o _oo _ _
o = slight reddening of the joint
oo = only marginal change
Example 3,
Expression cloning of the 5lkD antigen (0spA) of
Borrelia burgdorferi ZS7,
High molecular weight DNA from the Borrelia burg-
dorferi strain ZS7 was purified after culturing in
modified Kelly's medium. The spirochetes \were pelleted
by centrifuging at 10,000 g and washed three times with
PBS buffer, The dry pellet was resuspended in 10 ml. TE
(10 mmole/litre Tris, l mmole/litre EDTA, pH 7.4),
treated with lysozyme (5 mg./ml.) for 15 minutes at
°C. and the DNA released. by the addition of 1 ml. 20%
SDS, After the addition of l.5 ml, sodium chloride
solution (5 mole/litre), the solution was extracted with
an equal Volume of phenol, followed by an extraction
with chloroform. The DNA was then precipitated by the
«
addition of 2 volumes of absolute ethanol and incubation
at —209C_ overnight, After centrifugatflxnthe peuet ‘ was
dissolved in 0.5 ml. rm: and incubated with DNAse—free
RNAse A (20 by. g,/ml.) for 45 minutes at 55°c,, followed
by treatment for 1 hour with proteinase K (0.1 F.g./ml.)
at 57°C. The solution was adjusted to 0.3 mole/litre
sodium acetate and extracted with phenol-chloroform as
described above. After precipitation with ethanol, the
DNA was dissolved in TE,
Preparation of the gene bank.
High molecular weight DNA was randomly sheared
by sonication for 5 seconds. T4-DNA
polymerase (50 minutes at 57°C.) and Klenow enzyme
(5 minutes at 20°C,) were used in orderfilljn am shglestnambd
ends of the generated DNA fragments. Blunt ended DNA
was ligated into the BamHI site of an expression
vector pUEXl by using an adaptor cloning strategy
(see Bresan and Stanley, Nucl, Acid Res., 1987, p, 1056).
After size selection by molecular sieve chromato-
graphy over Sephacryl S—lOOO and transformation of
competent host cells Escherichia coli (MC lO61), the
percentage of recombinant clones ' ‘ was
determined as follows: randomly selected colonies were
picked and cultured to saturation in 2 ml. of selection
medium (LB with 25 lig,/ml. of ampicillin), The plasmid
DNA was isolated according to the usual alkaline lysis
method and subsequently cleaved with BamHI, More than
_
50% of the analysed plasmids contained,tk¢e fimHfi’u3cm“Ein
UNA—inserts with an average size of}1.5 kb.
Plating and expression screenino ofiuthe Rorrel ia bljrgidnrferi
ZS2 gene bank.
The cells were plated on 24 X 24 cm,plates at a
density of7Mmcohxnesgerplate and incubated overnight
at 50°C, After transfer of thficolonies to nitrocerlulose
filtens(NC), the expression of B—galactosidase fusion
proteins was induced by incubation for 2 hours at 42°C,
The filters were transferred to a Whatman SMM paper»
which had been treated with 5% SDS and incubated for
about 25 minutes at 95°C, The proteins were then electro-
blotted with the use of a conventional Western blotting
fl%EHflfiS- _ After DNAse treatment of
the NC filters, immune-reactive clones were identified
by an expression screening with the use of monoclonal
antibodies. Non-specific binding guns on the NC
filters were blaflmfl gby incubation for 4 hours with
‘PBS containing 0.2 % w/v of gelatine and 3 mmole/litre
sodium azide at roan . temperature. Subsequently, the
filters were incubated for 18 hours with continuous
shaking with culture supernatants of the anti—5l kD
monoclonal antibody’ EA-2g After exuamive washing
(PBS + 1% v/V Triton X~lOO; PBS + 0,5 mole/litre sodium
chloride; P38 + 1 mole/litre sodium chloride; each step
minutes), the filters were incubated with a 1:1oooo
dilution of a peroxidase-labelled F(ab)2 preparation
of rabbit-anti-mouse-IgG antibodies for 1.5 hours at
nmmx _ temperature with penmment shaking, The filters
were again washed as
with diaminobenzidine as peroxidase substrate. Of 104
recombinant dunes, 20 clones reacted with the monocloanl
antibody LA—2. ' ’" ‘
Sequence analysis of the 3l.kD antigen (OspA),
The insert DNR of a recmbinant Escherichia coli clone with
posithm antflxfiy naxxim1wdthIAr2vrs muflysedanfl.sejnaxrn axnrdnryto
standard protocols (Maniatis et al., (1982) Molecular Cloning; A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor).
The DA insert of this clone cotained the OspA gene coding for the
Borrelia bgdorferi 31 kD antigen‘ in full length. The plasmid
which contains ‘die OspA gaie was designated pZS—7/ 31-2 and was deposited
according to the Budapest Convention at the DSM under the number DSM 5528.
The recombinant protein produced by this immune-
positive clone was designated as rZS7/51-2. The
DNA sequence of the OspA gene was determined. It is
shown in Fig, l.of the accompanying drawings, together
with the amino acid sequence of the OspA protein
deduced from the DNA sequence.
From Fig..l, it can also be seen that the 51 kD
antigen from Borrelia burgdorferi corresponds tdia
protein of 375 amino acids.
‘Preparation of non-fusion proteins,
a) The clone which expresses the immune-reactive protein
rzs7/31-2 was cultured overnight at 50°C, in 10 ml, LB
with ampicillin, 1 ml. of the culture was introduced
into the selection medium and cultured at 30°C, with
good aeration up to saturation.
After cooling and centrifugatmxythe cells were washed
in STE buffer (10 mmole/litre Tris, lOO mmole/litre «
sodium chloride, 1 mmole/litre EDTA, pH 8.0) and the
peuet wresuspended in 0.6 ml. of lysis buffer (25%
sucrose, 50 mmole/litre Tris, pH 8.0), After the addition
of 150 P1, of lysozyme (10 mg./ml.), the mixture was
incubated for 15 minutes on ice, followed by a further
incubation (15 minutes on ice) with 18 fpl. DNAse 1
(10 mg./ml.) in the presence of 5 }Ll, of 1 mole/litre
magnesium chloride. Finally, 250 3.1. 4x detergent
mixture (1% Triton X100” 0.5% deoxychoflate, O,T.mo1e/
litre sodium chloride, 10 mmole/litre Tris, pH 7.4)
were added, foxkmedky-an incubation on ice for
minutes. After centrifugatkm,the peuet was washed
twice with buffer-A (50 mmole/litre Tris, 50 mmole/litre
sodium chloride, 1 mmole/litre EDTA, pH 8.0) and
resuspended in 9 volumes of buffer~A
contained 8M urea and incubated for 1 hour at nxm
temperature. The sample was diluted with 9 parts of
buffer B (50 mmole/litre monopotassium dihydrogen.
phosphate/dipotassium monohydrogen phosphate, 50 mole/
litre sodium chloride, 1 mmole/litre EDTA, pH 10.7) and
stirred for 30 minutes at nxmx temperature, the pH
being maintained at 10.7 by the addition of pytassium
hydroxide solution, After adjustment of the miof Hm aflntum
-
to 7.0 by the addition of hydrochloric acid, the sample
was dialysed overnight against buffer A at 4°C, and
centrifuged for 10 minutes at #°C, and 10,000 r.p.m. in
an 8854 rotor. The supernatant, which contains the
recombinant protein, was stored at —20°C, «
b) Since the clone also secretes the immune—reactive
protein rZS7/51-2 into the culture medium, a purific-
ation (affinity chromatography) directly from the
culture supernatant was performed.
Preparation of recombinant OspA (non—fusion) protein
and purification by affinity chromatography.
The recombinant proteins were subsequently purified
by affinity chromatography. For this purpose, purified
monodonal antibodies LA-2 were covalently bound to
activated Sepharose CnJ4Bm The dialysed urea extract crime
mfltunasmxxnaumtvnth flK2recmmnnmn:pKfien1wasexboflxflcxxmmse
IgG-Sepharose CL.4B and subsequently passed over the
LA-2—Sepharose CL 4B column. After intensive washing,
the bound recombinant prdtein was eluted with 0,1 mole/
litre glycine/hydrochloric acid - O.l mole/litre srdium
chloride, pH 2.5. The pH value of the collected fractions
was immediately adjusted to neutral by the addition of
1/10 volume of 0.5 mole/litre dipotassium monohydrogen
phosphate. The prdtein—containing fractions were
concentrated and dialysed, The degree of purification
was determined, by SDS_polyacrylamide gel
electrophoresis.
,
Immunological characterisation of the recombinant
protein rZS7/31-2.
The recombinant protein rZS7/31-2 was investigated
immunologically, For comparison,
recombinant protein rB5l/4l~9 (Borrelia burgdcrferi ‘
-41 kD surface antigen) wasxxed.
Flat-bottomed microtitre plates were coated with
urea extracts of the recombinant proteins rZS7/31-2
and rB5l/41-9 or with a urea extract of the Escherichia
coli strain MC 1061 used for the gene expression.
Non-specific binding sius were blocked byjnmfl2mnm1wdth0.2%
gelatine in phosphate-buffered sodium chloride solution.
Microtitre plates were flxflhflfid
with the given monoclonal antibodies LA—2 (anti—5l kD,
OspA), LA-l (anti-41 kD, flagellin) or ACET_2 (anti-
al—antichymotrypsin). ’
Bound monoclonal antibodies were-deUXޣd\da
peroxidase-labelled, species—specific anti-mouse immuno-
globulins. Boufid peroxidase-labelled antibodies were
\the peroxidase substrate
quantified by using
g—phenylenediamine, The adsorption at 492 nm (Angz).
was determined directly in the microitire plates with
the help of an automated plate photometer, The
adsorption is pn$Dfijofl.to the amount of bound
monoclonal antibodies.
The monoclonal antibody LA—2 reacts in a specific
manner with rZS"7/51-2 but not with MC 1051 or real/41-9.
Ebr control reaction the monoclonal antibody LA—l wnchjs
_
specific for rB5l/41-9 is used. The monoclonal antiboay
ACHT_2 (negative control) does not show a significant
reaction on any of the proteins;
Fig. 2 of the accompanying drawings shows that the
antigenic epitope recognized in a specific manner by?the
monoclonal antibody LA—2 is expressed on the recombinant
protein rZS7/51-2 which was cloned from the genome of
anxeua burgdorferi ZS7.
Example«4.
Comparison of antibodies specific for the 51 kD (Ospa) or
the 54 kD antigen (OspB) and antibodies which are
specific for the 41 kD antigen (flagellin),
The monoclonal antibodies LA-2 and LA—26.l recognize
the El kD antigen OspA and are of the isotype IgG2b and
IgGl, respectively. The monoclonal.antibodies LA_25.l
and LA—27,l.recognize the 54 kD antigen OspB and are of
the isotype IgG2B and IgGl, respectively. The monoclonal
antibodies LA—lO and LAr2l are specific for. the
flagella—associated 41 kD perqiasmatic protein caageuim of
Borrelia burgdorferi and are of the isotype IgG2a and
IgG1, respectively? All the above-mentioned antibodies
were obtained according to theprocess described in
Example 2. In this experiment, it is to be ascertained
whether monoclonal antibodies against another Borrelia
burgdorferi antigen in scid mice also axfer
protection against the clinical symptoms of lyme
borreliosis.
‘
The polyclonal anti—B5l immune serum (INS) was
taken from C57BL/6 mice 91 days after a subcutaneous
inocculation with l x 108 Borrelia burgdorferi B51
organisms. The polyclonal anti—ZS7 IMS was taken from
C57BL/6 mice 68 days after a subcutaneous inoculation
with 1 x 108 Borrelia burgdorferi zs7, Both sera contained
60 ,Lg,/ml. of specific antibodies, as was determined
in an ELISA system (see Schaible et a1..,. J. Exp. Med,,
izg, 1427-1452/1989), The normal mouse serum (NMS) was
taken.from non-infected C57BL/6 mice.
At the point of time of the inoculation and there-
after in 4 day intervals, the given antibodies, the IMS,
the NMS or PBS buffer were passively transferred intra-
peritoneally into scid mice according to the following
protocol:
day 0 and day 5: 100 ;»l.
day 7 and day 10: 200 )Ll.
day 13 and day 17: 300 F,l.
Scid mice which had been treated either'with anti-
ZS7IMS, anti-B5lIMS or with the the monoclonal antibody
LA—2 showed no visible clinical symptoms of arthritis,
i.e. no reddening and swelling of tibiotarsal joints
occurred during the 21 days of observation. Also, no
symptoms of carditis and hepatitis were ascertained.
Histopathological investigations showed no changes in
the joints, the heart and the liver of scid mice which
had been treated either with anti-ZS7-IMS, anti-B31 IMS
_
or with the monoclonal antibody LA—2,
The other OspArspecific monoclonal antibodies
LA-26.1 of the isotype IgGl, as well as the OspB-specific
antibodies LA—25.l and LA-27.1, were able to mitigate the
clinical symptoms of arthritis, carditis and hepatitis.
Slight pathological changes in the investigated organs
were here shown-
which had
In cmfizasq ‘said mice
been tr&flxfl.eidmn‘wifl1 PBS buffer, NMS or monoclonal
antibodies against flagellin (LA-LO or LA-21) showed
clinical signs of arthritis, the pathological changes
typical for untreated scid mice (see-the following
Table 5). The severity of the symptoms in the last-
mentioned animals increased with increasing period of
time after the inoculation and did not weaken during
' the period of observation,
No spinxmeum
could be isolated from scid mice which had previously
been treated either with anti—ZS7IMS or with the anti-
contrast .de t ec t iOn
LA--2r In
of spirochaetes by immunofluorescence and by culturing
of blood from scid mouse was possible in those mice
which had been treated with PBS buffer, NMS 0? the
or LA—2l.
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-
Example.5.
Action of antiserum from mice immunised with 0spA on
the course of Lyme borreliosis in scid mice,
In this experiment, it couId be shown that an
administration of native 0spA (isolated from Borreliabunh
dotfixi $133,253 or of recombinant OspA (isolated from
Escherichia 99;; bacteria which had been transformed with
the recombinant plasmid pZS7/3l—2.(DSM 5528)) into normal
mice (mice strain C57BL/6) induced the formation of
protective polyélonal antibodies. If these antibodies
are administered to said mice, there is brought about a
protection against Lyme borreliosis, It is thereby
ascertained that recombinant OspA induces a protective
immune response comparable to that induced with native OspA. The
results and the details of carrying out of this experiment are
given in the following Table 4.
The obtaining of recombinant OspA is described in
Example 5,
The obtaining of native OspA, as well as the immun-
ising of mice with OspA, took place as follows:
Enrichment of native 5lkDa OspA,
,2 x 1010
Lspuxnmxes are stirred with a magnetic
stirrer for 2 hours at 4°C, in'5 ml. of PBS/7.5 mi,
n—butanol.in the presence of protease inhibitor
(5 mmole/litre EDTA, 5 mmole/litre benzamidine and
.5 mmole/litre PMSF). Thereafter, the mixture is
centrifuged for 90 minutes at 10,000 r,p,m, in a
Sorvall centrifuge (fixed-angle rotor), The aqueousphage
_5Q_ K
which contains the surface proteins is removed and
washed three times with chloroform. The protein content
is determined via the extinction at 280 nm or with the
BCA test,
In the silver gel or Western blot with anti-
Borrelia burgdorferi rabbit serum, there was found for
strain ZS? a main band in the molecular weight range of
51 kDa, as well as weak bands at 20, 54, and 65-68 kDa.
The butanol/water preparation of Borellia burgdorferi
strain B51 gave a main band at 51 kDa, as well as weak
bands at 20 and 54 kDa.
Immunfisation of mice with native and recombinant Ospn,
To CSWHJ6 and C,B-l7 mice were given three times
subcutaneously into the root of the tail at an interval
of 7 to 10 days 5 fig. (native OspA of strain B51) or
lO fhg, (native OspR of strain ZS7, recombinant OspA
of ZS7) in 100 ;»l. of adjuvant (ABM3; firm Sebak,
Aidenbach, Federal Republic of Germany). At the earliest
weeks after the last immunisation, serum could be
taken for 5 to 4 months. The content of specific
antibodies is determined in the ELISA system.
-51. _
Table 4
Effect of Borrelia burgdorferi—specific monoclonal‘and
Dolyclonal antibodies on soirochaetosis and development
of arthritis in scid mice infected with Borrelia
burgdorferi .
(number 1 2 5
of mice
n = 6 PBS (negative 1 + + 6/5
control)
n = 6 LA—2 - - — 0/;
n = 2 anti—0s A — — — O/2
(native? IMS
n = 5 anti—0spA — — — 0/3
(recomb,) IMS
First antibody transfer i.p, (100 ;rl,) day 0 (day of
the inoculation with QL burvdorferi gxau1zsL 1308
organisms, s.c. into th%root of the tail).
Further antibody transfers day 4 (100 fLl,), day 7
(200 ,».1,), day 11 (200 ,..1,), day 14 (300 #1,),
day 18 (300 ;v1.3,(i.D.).
Claims (8)
1. Antigen, wherein it reacts immunogenically with an antibody against the 31 kD antigen (OspA) of E; burgdorferi and contains the following amino acid sequence or an immunogenic epitope from this sequence:
2. _ Antigen as claimed in claim 1, wherein the DNA sequéfice that codes for it is located on a vector, preferably a prokaryotic vector, which is a suitable expression.
Antigen as claimed in claim 1 or 2, wherein the antigen is a B-galactosidase fusion protein or a non—fusion protein.
4. Recombinant DNA, wherein it codes for an antigen as claimed in one of the claims 1, 2 or 3 and contains (1) the nucleic acid sequence shown in the following or (2) a sequence corresponding to it within the scope of the
5. Recombinant vector, wherein it contains one or several copies of a recombinant DNA as claimed in claim 4.
6. Recombinant vector as claimed in claim 5, wherein the vector is a prokaryotic vector.
7. Recombinant vector as claimed in claim 6, wherein it is a plasmid.
8. Recombinant vector pzS—
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DEGERMANY19/09/1989P3931236.4 | |||
DE3931236 | 1989-09-19 | ||
DE4015911A DE4015911A1 (en) | 1989-09-19 | 1990-05-17 | VACCINE AGAINST LYME DISEASE |
Publications (2)
Publication Number | Publication Date |
---|---|
IE990173A1 IE990173A1 (en) | 2000-11-01 |
IE83340B1 true IE83340B1 (en) | 2004-03-10 |
Family
ID=
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