IE83340B1 - Vaccine against lyme disease - Google Patents

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-deUXfi£d\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.

Ham CH manwwmom Po: ma: wosmowwmosamossaefl an Hhmwaocmmzp maawhpom mo QOflP0®P®© d o mawofisaaonsm .mHm>mm + mmvmamcos I\+ I mmzon |_um3oHHcw mm UGPGOHUQH ma wmnmzo Hmoawoaonpmmopmwn as P we mmkwmn one ‘P I ‘x -H +I+| + + +I +l +1 + +l +I +I H pm A mm mm Ammmov H.>mu Ammmov a.mmu¢g A fsflaam ummamv Hmuaq Anflaaw ummamv oHu A m2H nmwufipmm mzH Hmmuflpqm Aaohpnoo m>Hpmmmqv mzz Aaoapqoo ®>H#mwmQV mmm I“'\C\J\ON\ P0 N\ P(\ ll w M Q Amommommnosawossafifi wsm WQHHSPHSOV flHmMHO@w%5Q .m mo QOfiPo®#m© mavwmrnmo mflpflpgpam \mfl»Hggpnm Ifiamn mmoao nsgagopmfiz mflpflggpga Hmofizflao mmzpnsm amH QPH3 pfimapmwhp Awowa mo hmpadzv uflom >H1m.o Sflmppm mwsofi M manna - 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)

CLAIMS:

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—