IE83320B1 - Vaccine against lyme disease - Google Patents

Description

PATENTS ACT, 1992 3377/90 VACCINE AGAINST LYME DISEASE MAX-PLANCK—GESELLSCHAF T ZUR F ORDERUNG DER WISSENSCHAFTEN E.V. and DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNG DES OFFENTLICHEN RECHTS Lymie borreliosis is the commonest infectious disease transmitted by ticks in the temperate regions.

It is caused by the Spirochaeta Borrelia burgdorferi which is transmitted to humans in particular by ticks of the genus lxodes. The disease is a chronic, progressive 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 dependable 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 B. burgdorferi. In the case of persons afflicted by lyme disease, there is admittedly ascertained a high titre of antibodies against B. burgdorferi which, however, does not bring about a protection 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 This would mean that a protection by antibodies is only possible be directly reached by the immune system. immediately after commencement of the infection, thus as long as the pathogen is still present in the blood circulation.

The fact that a natural infection with B. burgdorferi has been found in various kinds of animals has led to the attempt 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 the mouse a specific immune response for B. burgdorferi; it was found that the infection of inbred mouse strains with a long since cultured B. burgdorferi isolate 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 disease (Schaible (1988) Infect. of a serious aspect of the disease in animals was et al., Immun., 1, 41). The development presumably prevented either by the immune defence of the host and/or by the reduced virulence of Spiro- chaetes cultured in vitro for a comparatively long time (Johnson et al., (1984), J. Clin. Microbiol., 20, 747; Schwan et al., (1988), Infect. and Immun., 56, 1837).

Howe et al. (Infection and Immunity 54 (1986), 207-212) disclose that the genes coding for the outer membrane proteins OspA and OspB of Borrelia burgdorferi are localised in a single transcription unit.

Bergstrom et al. (Mol. Microbiol. 3 (1989), 479-486) describe a molecular analysis for the proteins OspA and OspB from the B. burgdorferi strain B31-coding DNA (Ann. N.Y. Acad. 539 (1988), -143) describe a variability of the surface antigens sequences. Wilske et al. in various B. burgdorferi strains.

The task forming the basis of the invention is to make available an effective vaccine against lyme disease. However, for this purpose, the development of a suitable animal laboratory model is first necessary.

It is now suggested that a mouse strain without functional T- and B-cells, the so-called Scid mouse (Bosma et al., (1983), Nature, 10, 52) can serve as experimental animal since scid mice, in the case of infection with a pathogenic B. burgdorferi isolate, develop a multi-systemic disease, namely, mainly poly- arthritis and carditis. By means of this animal model, it is possible for the first time to test the action of vaccines against lyme disease.

A subject of the invention is a passive vaccine against lyme disease which contains one or more specific monoclonal antibodies for the 31kD antigen (0spA) and/or the 34 kD antigen (OspB) of B.burgdorferi of the strains B3l (ATCC 35210) and/or ZS7 (DSM 5527).

A vaccine is preferred which contains one of the antibodies of the class lgG according to the invention, especially preferably of the subclass lgG2b or lgGl.

Surprisingly, in contradistiuction to the administ- ration of another antibody, e.g. against the 41 kD surface antigen of B. burgdorferi (flagellin), the administration of the antibody according to the invention brings it about, in the case of immune- deficient experimental animals, preferably of scid mice which have been infected with viable pathogenic B. burgdorferi, preferably B. burgdorferi ZS7, that the development of arthritis, carditis and hepatitis is completely or at least substantially prevented.

The vaccine according to the invention with the antibody as active material can possibly also contain usual carrier, filling and adjuvant materials.

Furthermore, the invention also comprises a process for the obtaining of a passive vaccine against lyme disease from lymphocytes or spleen cells of an experimental animal, preferably of a mouse, which is immunised with B. burgdorferi organisms or parts thereof, preferably with complete B. burgdorferi B31 and/or ZS7 organisms, whereby, from the lymphocytes or spleen cells of the immunised animals, one obtains, by cell fusion, a hybridoma which produces a monoclonal antibody according to the invention.

A hybridoma cell line (ECACC 89091302) which produces an antibody LA-2 against OspA (IgG2b) %ccord- ing to the invention is also described. Furthermore, the antibody LA-26.1 against OspA (IgGl)-producing hybridoma cell line ECACC 90050406, as well as anti— bodies LA—25.l and LA-27.1, respectively, against 0spB (IgG2b and IgGl, respective1y)~producing hybridoma cell lines ECACC 90050405 and ECACC 90050407 are also described.

Furthermore, there is given the pathogenic B. burgdorferi strain ZS7 (DSM 5527).

Furthermore, there is described an antigen which immune—reacts with a monoclonal antibody according to the invention. Thereunder is to be understood an antigen which contains the whole amino acid sequence of OspA or OspB, respectively, or also only an immunogenically-acting part sequence (immunogenic epitope) of OspA or OspB, respectively. An expert can determine potentially immunogenic epitopes of these proteins without difficulty by a structural analysis of the OspA protein (e.g. Chou-Fassmann analysis) and then test experimentally for their effectiveness.

In particular, there is given a recombinant antigen which immune—reacts with the antibody according to the invention whereby the DNA sequence coding for the antigen is present on a recombinant vector, prefer- ably a prokaryotic vector, which is suitable for the protein expression.

In particular, there is given an antigen from B. burgdorferi ZS7 which specifically immune—reacts with the antibody according to the invention and which contains the amino acid sequence shown in Fig. l or an immunogenic epitope of this sequence. Consequently, a recombinant DNA is also described 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 (3) one hybridis~ ing under stringent conditions with a sequence from (1) and/or (2), which sequence codes for the 31 kD antigen of B. burgdorferi ZS7 or an immunogenic epitope thereof. The term “stringent hybridising conditions“ is thereby to be understood as in Maniatis et al., Molecular Cloning, A Laboratory Manual (1982), Cold Spring Harbor Laboratory, New York.

Especially preferred is an antigen which is a recombinant non-fusion protein or 5-galactosidase fusion protein.

Furthermore, there is described a recombinant vector which contains one or more copies of a recombinant DNA according to the invention. The vector according to the invention can be a prokaryotic and/or eukaryotic vector, it is preferably a prokaryotic vector. The recombinant vector can be present extrachromosomally in the host cell (e.g. plasmid) or it can also be integrated in the genome of the host cell (e.g. bacteriophage lambda). The vector according to the invention is preferably a plasmid.

The recombinant vector pZS—7/31-2 (DSM 5528) is especially preferred.

Furthermore, there is given a process for the obtaining of antigens according to the invention by investigation of a B. burgdorferi gene bank with one or more antibodies according to the invention, whereby one isolates the clones which show a positive immune reaction with the antibodies used.

Furthermore, there is described a process for the obtaining of a passive vaccine against lyme disease, whereby one immunises experimental animals, preferably mice, with the antigen and obtains protective monoclonal antibodies in the usual way from the immunised experimental animals.

Finally, a process is also described for the isolation and reculturing of pathogenic B. burgdorferi organisms which is characterised in that, from immune- deficient experimental animals, preferably mice, which have previously been infected with the pathogen, one obtains the pathogen, whereby the pathogeneity of the pathogen is retained. Especially preferred is a process in which one obtains pathogenic B. burgdorferi ZS7 (DSM 5527) organisms from blood and/or joints of infected scid mice.

The clarification of the invention takes place by the following Examples and Figures 1 and 2. There are shown: Fig. l the DNA and amino acid sequence of the 31 kD antigen (OspA) from B. burgdorferi ZS7, Fig. 2 the immunological characterisation of the recombinant protein rZS7/31-2.

Example 1 Induction of arthritis, carditis and hepatitis in scid mice by infection with B. burgdorferi strain ZS7.

Treatment of the mice with B. burgdorferi Adult mice of the strains C.B-17 scid (homozygous for the scid mutation) and C.B~17 were injected sub- cutaneously into the tail root with 1 x 105, 5 X 105, x 106 or 1 X 108 B. burgdorferi organisms. of viable or killed (UV irradiation) Isolation of B. burgdorferi from ticks and mice The investigations were carried out with the already long+since cultured B. burgdorferi strain B31 (ATCC 35210) and the fresh isolate B. burgdorferi ZS7 (DSM 5527) which had been isolated from a female lxodes rizinus tick. All B. burgdorferi strains were cultured in modified Kelly's medium (Barbour et al., (1983) Switzerland Curr. Microbiol., 8, 123). B. burgdorferi organisms which had been obtained from the middle gut of ticks sterilised with ethanol or from blood of infected mice were initially cultured in Kelly's medium with the addition of 8 pg/ml of kanamycin and 230 pg/ml fluorouracil (Johnson et al., (1984) J. Clin. Microbiol., 1, 81).

Serological tests The detection of B. burgdorferi—specific anti- bodies was carried out in a conventional ELISA process (1988) Wehrmed. Mschr., 32, 263).

The standard curve for the content of immunoglobulin (Justus et al., (lg) was obtained by coating a dish with anti-mouse Ig (l:5OO dilution of the serum solution of Paesel, Frankfurt, FRG) and titration of the total mouse IgG or IgM content (Calbiochem, La Jolla, USA). Total serum IgM and IgG were measured similarly. The concentration of B. burgdorferi-specific IgM or IgG antibodies is given in pg lg/ml of serum. lmmunofluorescence and Giemsa staining pl of blood were pipetted into a haemocrit test tube (Becton and Dickinson, Heidelberg, FRG) and centrifuged at 5000 g in a haemocrit centrifugse (ECCO, FRG). between serum and erythrocytes and 5 pl of the serum The test tubes were cut up on the interphase were applied to microscope slides (Superior, Bad Mergentheim, FRG). The microscope slides loaded with the serum samples were dried in the air and fixed in lOOZ ethanol for one minute at -20°C. After one hour incubation with rabbit anti—B. burgdorferi hyperimmune serum (l:lOO dilution) at room temperature, the micro- scope slides were washed five times in PBS and then stained for one hour with FITC-conjugated goat anti- rabbit antiserum (1:20 dilution, Jackson Lab., West Grove, USA). embedded in Kaiser's glycerol gelatine (Merck, The microscope slides were washed and Darmstadt, FRG) and immediately investigated fluor- escence microscopically. Untreated blood droplets were dried in the air, fixed in methanol, stained with Giemsa (0.1%, Merck, Darmstadt, FRG), decolorised in PBS and embedded in Entellan (Merck, Darmstadt, FRG).

Histological preparations and staining processes Various internal organs (brain, heart, lungs, liver, kidneys, spleen and joints) were removed from mice previously infected with B. burgdorferi at different times after the infection and stored either in liquid nitrogen for the preparation of frozen sections or in 5% formaldehyde (in PBS) for the embedding in paraffin or methacrylate. Sections of A to 7 pm thickness were prepared, stained with haemoroXylin—eosin and embedded in Entellan (Merck, Darmstadt, FRG). with the use of the streptavidin—biotin—peroxidase The immunohistology was carried out system (Kramer et al., (1989) Eur. J. 151). lmmuno1., 19, Table 1 shows that B. burgdorferi organisms of the isolates ZS7 and B31 were detected during the whole of the experimental period in the blood of scid mice which had previously been inoculated with viable organisms. However, only Spirochaeta of the strain ZS7 but not of the strain B31 could be recultured in vitro.

In the case of comparison of the recultured organisms with the primary B. 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 B. burgdorferi during the whole observation period. No IgM or IgG antibodies specific for B. burgdorferi could be found in these animals (Table 1). which had been infected with B. burgdorferi, expressed On the other hand, all C.B—l7 control mice, large amounts of total lg and increased titres of IgM and IgG antibodies specific for B. burgdorferi.

Between 7 and 20 days after the infection with B. burgdorferi, the scid mice showed the first clinical symptoms of arthritis (reddening and swelling of both tibiotarsal joints), which increased with time. On the other hand, no symptoms of arthritis were found in scid mice which had been infected either with UV- irradiated B. burgdorferi ZS7 or with viable B. burgdorferi B31 organisms and in C.B-l7 control mice which had been infected with viable B. burgdorferi ZS7 organisms.

Arthritic joint changes were also detected histopathologically in scid mice which had been infected with viable B. burgdorferi ZS7 (Table l).

Severe joint damages were ascertained, characterised by the presence of hyperplastically inflamed synovial covering cells, combined with erosion and destruction of cartilaginous tissue and/or bone. Furthermore, there was ascertained pancarditis with infiltration of mononuclear cells in the endocardium, myocardium and pericardium. There was also ascertained a progressive inflammation of the liver, whereby 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 was observed. In addition, smaller damages in the kidneys, the lungs, the brain and the striated musculature were ascertained. mAWV mucaom .Amv UOOHD.:5Hw COHUNHOWH %« I Nmm.H cam I ¢om.m «om mom c~m.o mq~.~ wmq mom.m mHm.w I qom ¢m I I I mm mm U: Us US Us I I%+V I I I I wofi I I won I I I I .I II I 1 > 1 uwwaummw wa Hmuou ooAHE\w1V xwonwufim mo cowumaowwmw EDHWW He\w1 m.m v wucflom ummwumuownflu msu mo wcflaawzm Ucm wcwcmvvwu U +0 m®UC®Um®HO3HMOC5FFH HO wcwcflmum $Ew®H© xn % I I I I ¢N I I I I ma I I I I mm I I I I mm I I I + am I I I + mm I I I I ofi + + + + om w: + U: vs I w: + U: vs I + + w\m+ + mm + + W+ + 0N + + I + mm + + m+ + mm + + I + A + + m+ + mm + + m+ + me + + m+ + om Hmowwoa %wOOHn cofluummcw Io£umm oamoa %¢coHum OLD CH Hmuwm Iouwwfi Icwao Iaomw coauumumw mxmv mfluauzuum Huwmuovwufln .m WHUHHSUHM mo COwumEHOw wcm OHUHU Afioflwucw mHMmmuOUwu5£ .m £uH3 woHE Howucou mHIm.U Ucm OUHE Uwom mHIm.D MO COHuUwwCH H mqm mofi wofi wofi oofi mofi X X X K x X H H m H ~ mofi X m cowuuwwcw mo wwmo mzu cw HwnE5c Hmm pu>:NwN cwmuum Huwuuowwufln .m An I :v ~HIm.o A.~cu fiwum ~HIm.o EHNHUW wmDOE mmsmwau wzu Eoww mmuwmfiuouamm Example 2 Action of a monoclonal antibody specific for the B. burgdorferi 31 kD antigen on the course of lyme borreliosis in scid mice Preparation of the monoclonal antibody In the case of immunisation of a mouse which possesses an intact immune system with B. burgdorferi organisms, polyclonal antibodies are expressed which are specific for B. burgdorferi (see Table l).

Ten week old female mice of the inbred strain BALB/c were immunised with Borrelia (B. burgdorferi, strain B31; ATCC 35210) homogenised by acoustic irradiation.

Immunisation protocol: day 0: 200 pg Borrelia antigen in complete Freund's adjuvant subcutaneously day 21, 35, 49, 63: antigen in phosphate-buffered saline i.p. challenge with 100 pg Borrelia day 66: removal of the spleen and preparation of an individual cell suspension.

The immune spleen cells were fusioned with the Ag8-PAT myeloma cell line according to standard methods with use of polyethylene glycol (J.H. Peters, H. Baumgarten, M. Schulze, “Monoklonale Antikorper“, Springer Verlag, Heidelberg).

The fusion products were seeded out into 96 well tissue culture plates. After 8 days, the cell culture supernatants were investigated for the presence of E. burgdorferi-specific monoclonal antibodies with the help of a solid—phase ELISA (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 IgG2b is produced by a monoclonal hybridoma line and secreted and reacts in the Western blot with the 3lkDa structure (OspA) of the isolates ZS7 and B3l) in the case of contact with B. burgdorferi proteins separated electrophoretically via an SDS gel and transferred by means of Western blot The monoclonal antibodies LA—26.lC (anti—OspA IgGl), LA 25.l (anti-OspB (34kDA antigen); lgG2b) and LA 27.1 (anti-OspB (34 kDa antigen) lgGl) were prepared and characterised in an analogous manner. to a membrane.

Infection of mice with B. burgdorferi ZS7 C.B—l7 scid mice were infected subcutaneously in the tail root with 1 x 108 viable B. burgdorferi ZS7 organisms.

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 31 kD antigen of B. burgdorferi). The dosage of the administered antisera amounted to lOO pl or lOO pg in the first week in the case of LA-2, to 200 pl or 200 pg in the second week in the case of LA-2 and to 300 pl or 300 pg in the third week in the case of LA-2.

Table 2 shows that scid mice, untreated or treated with NMS, developed clinical and histopethological indications of arthritis or carditis and hepatitis after 12 days. monoclonal antibody LA-2 brings about a distinct On the other hand, the administration of the 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 IMS showed no clinical arthritis findings.

A detection of B. burgdorferi pathogen by in Vitro culturing only succeeded in the case of mice which were either untreated or treated with NMS. In the case of mice treated with LA-2 or IMS, B. burgdorferi could not be detected (Table 2).

TABLE 2 Treatment of C.B—17 scid mice with B. burgdorferi and antisera mouse treatment arthritis CarditiS/ B- burg- strain with (after 12 days) h?Patiti5 dorfefi c_B-17 antiserum C1iD_ hiStO_ hist0— detection Scid ical pathol- pathO_ (culture) Ogical logical n = 3 — + + + + n = 3 NMS + + + + n=Z ms — — — — n = 3 LA-2 —° —°° — — ° = slight reddening of the joint °° = only marginal change Example 3 Expression cloning of the 3lkD antigen (OspA) of B. burgdorferi ZS7 DNA preparation High molecular DNA from the B. burgdorferi strain ZS7 was purified after culturing in modified Kelly's medium. The spirochaetes were pelleted by centrifug- ing at l0,000 g and washed three times in PBS buffer.

The dry pellet was resuspended in l0 ml TE (10 mmol/1 Tris, 1 mmol/l EDTA, pH 7.4), treated with lysozyme (5 mg/ml) for 15 minutes at 30°C and the DNA liberated by addition of l ml 20% SDS. After addition of 1.5 ml NaCl (5 mol/l), the solution was extracted with an equal volume of phenol, followed by an extraction with chloroform. The DNA was then precipitated by addition of 2 volumes of absolute ethanol and incubation at -20°C overnight. After centrifuging, the residue was dissolved in 0.5 ml TE and incubated with DNAse—free RNAse A (20 ug/ml) for 45 minutes at 55°C, followed by a one hour treatment with proteinase K (0.1 pg/ml) at 37°C. and extracted with phenol-chloroform as described above.

The solution was adjusted to 0.3 mol/l NaOAc After precipitation with ethanol, the DNA was again taken up in TE.

Preparation of the gene bank High molecular DNA was statistically comminuted by 3 second long ultrasonic treatment. T4—DNA polymerase (30 minutes at 37°C) and Klenow enzyme (5 minutes at °C) were used in order to smooth the ends of the DNA fragments produced. into the BamHI position of an expression vector pUEXl DNA with smooth ends was ligated with the use of an adaptor cloning strategy (Bresan and Stanley (1987) Nucl. Acid Res., p. 1056). selection step by a molecular sieve chromatography over After a size Sephacryl S-l0OO and transformation of competent host cells E. coli (MC lO6l), the proportion of recombinant plaque—forming units (pfu) was determined as follows: randomly selected colonies were picked and cultured up to saturation in 2 ml of selection medium (LB with pg/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, on average, b 1.5 kb-long DNA insertions.

Plating out and screening of the B. burgdorferi ZS7 gene bank The cells were plated out on 24 x 24 cm plates at a density of 7000 pfu per plate and incubated overnight at 30°C. cellulose filters (NC), the expression of B-galactosid- After transfer of the colonies to nitro- ase fusion proteins was induced by two hours incubation at 42°C. paper which had been treated with 5% SDS and incubated The filters were transferred to a Whatman 3MM for about 25 minutes at 95°C. The proteins were then electro-blotted with the use of a usual apparatus to the half-dried Western blotting. 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 positions on the NC filters were saturated by four hours incubation with PBS containing 0.2% (weight per volume) of gelatine and 3 mmol/l NaN3 at room temperature.

Subsequently, the filters were incubated for 18 hours with continuous shaking with culture supernatants of the anti-3l kD monoclonal antibody clone LA—2. After thorough washing (PBS + l% volume/volume Triton X—lO0; PBS + 0.5 mol/l sodium chloride; PBS + 1 mol/l sodium chloride; each step 10 minutes), the filters were incubated with the l:l000O dilution of a peroxidase- labelled F(ab)2 preparation of rabbit—anti-mouse—lgG antibodies for l.5 hours at room temperature with continuous shaking. The filters were again washed as above described and then incubated with diamino- Of 104 PFU‘s, 20 clones reacted with the monoclonal antibody LA—2. benzidine as peroxidase substrate. recombinant Seouence analysis of the 31 kD antigen (OSDA) The inserted DNA of a recombinant E. coli clone with positive antibody reaction with LA—2 was isolated in the usual manner. The DNA insertion of this clone contained the OspA gene coding for the B. burgdorferi RD antigen in complete length. The plasmid which contains the insertion was designated pZS-7/3l~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 rZS7/31-2. The cloning DNA sequence of the OspA gene was determined.

It is shown in Fig. 1, together with the amino acid sequence of the OspA protein derived therefrom.

From Fig. 1, it can also be seen that the 31 kD antigen from B. burgdorferi corresponds to a protein with 273 amino acids.

Preparation of non-fusion proteins a) The clone which expresses the immune-reactive protein rZS7/31-2 was cultured overnight at 30°C in 10 ml LB with ampicillin. 1 ml of the culture was introduced into l00 ml of selection medium and cultured at 30°C with good aeration up to a density of 8 x 107 ml (A600 = 0.2). protein was achieved by a transfer to the host cells at 42°C. After cooling and centrifuging, the cells were washed in STE buffer (10 mmol/l Tris, 100 mmol/l sodium chloride, 1 mmol/l EDTA, pH 8.0) and the residue resuspended in 0.6 ml of lysis buffer (25% sucrose, mmol/l Tris, pH 8.0). After addition of 150 pl of lysozyme (10 mg/ml), the mixture was incubated for cells per The expression of the recombinant minutes on ice, followed by a further incubation (15 minutes on ice) with 18 pl DNAse l (10 mg/ml) in the presence of 5 pl of 1 mol/l magnesium chloride.

Finally, 250 pl 4x detergent mix (lZ Triton X100, .5% deoxycholate, 0.1 mol/l NaCl, 10 mmol/1 Tris, pH 7.4) were added thereto and incubated on ice for minutes. After centrifuging, the residue was washed twice with buffer A (50 mmol/l Tris, 50 mmol/1 NaCl, mmol/l EDTA, pH 8.0) and resuspended in 9 volumes of buffer A additionally containing 8M urea and incubated for one hour at room temperature. The sample was diluted with 9 parts of buffer B (50 mmol/1 KHZPOA/K2HP04, 50 mol/1 NaCl, 1 mmol/l EDTA, pH 10.7) and stirred for 30 minutes at room temperature, whereby the pH value was maintained at 10.7 by addition of KOH.

After adjustment of a pH of the solution to 7.0 by addition of HCl, the sample was dialysed overnight against buffer A (at 4°C) and centrifuged for 10 minutes at 4°C and 10,000 rotations per minute (r.p.m.) in an SS34 rotor. recombinant protein, was stored at —20°C.

The supernatant, which contains the b) Since the clone also secretes the immune~reactive protein rZS7/31-2 into the culture medium, a purific- ation (affinity chromatography) directly from the culture supernatant is possible.

Preparation of recombinant OspA (non-fusion) protein and affinity chromatographic purification The recombinant proteins were subsequently purified affinity chromatographically.

For this purpose, purified monoclonal antibodies LA-2 were covalently bound to activated Sepharose CL 4B.

The dialysed urea extract with the recombinant protein was adsorbed on mouse lgG—Sepharose CL 4B and subse- quently passed over the LA—2—Sepharose CL AB column.

After intensive washing, the bound recombinant protein was eluted with 0.1 mol/1 glycine/HCl — 0.1 mol/1 NaCl, pH 2.5. neutralised by immediate addition of 1/10 vol. of 0.5 mol/1 K HPO . were concentrated and dialysed.

The pH value of the collected fractions was The protein-containing fractions The degree of the purification was determined with the help of SD8- polyacrylamide gel electrophoresis.

Immunological characterisation of the recombinant protein rZS7/31-2 The recombinant protein rZS7/31-2 was investigated immunologically. For comparison, there was used the recombinant protein rB3l/41-9 (B. burgdorferi 41 kD surface antigen).

Flat-bottomed microtitre plates were coated with urea extracts of the recombinant proteins rZS7/31-2 and rB3l/41-9 or with a urea extract of the E. coli strain MC 1061 used for the gene expression. Non- specific binding positions were blocked with 0.2% gelatine in phosphate—buffered common salt solution.

Cups of the so-prepared microtitre plates were mixed with the given monoclonal antibodies LA-2 (anti-31 kD, OspA), LA-l (anti-41 kD, flagellin) and ACHT—2 (anti- Nl-antichymotrypsin), respectively.

The bound monoclonal antibodies were brought to reaction with peroxidase-labelled, species-specific anti-mouse immunoglobulins. Bound peroxidase-labelled antibodies were quantified with the use of the peroxidase substrate ortho-phenylenediamine. The adsorption at 492 nm (A492) was determined directly in the microtitre plates with the help of an automated plate photometer. The strength of the adsorption is a measure for the amount of_bound monoclonal antibodies.

The monoclonal antibody LA-2 reacts in a specific manner with rZS7/31-2 but not with MC 1061 or rB3l/41-9, respectively. The control reaction of the monoclonal antibody LA-l is specific for rB3l/41-9. The mono- clonal control antibody ACHT-2 (negative control) does not show a significant reaction on any of the proteins.

Fig. 2 shows that the antigenic epitope recognised in specific manner by the monoclonal antibody LA—2 is expressed on the recombinant protein rZS7/31-2 which was cloned from the genome of B. burgdorferi ZS7: Example 4 Comparison of antibodies specific for the 31 kD (OspA) and 34 kD antigen (OspB) with antibodies which are specific for the 41 kD antigen (flagellin) The monoclonal antibodies LA-2 and LA-26.1 recognise the 31 kD antigen OspA and are of the isotype IgG2b and IgGl, respectively. The monoclonal antibodies LA—25.1 and LA—27.1 recognise the 34 kD antigen OspB and are of the isotype IgG2B and IgGl, respectively.

The monoclonal antibodies LA-10 and LA-21 are specific for the flagella-associated 41 kD periplasmatic protein of B. burgdorferi and are of the isotype IgG2a and IgG1, respectively. All above-mentioned antibodies were obtained according to the process described in Example 2. In this experiment, it is to be ascertained whether monoclonal antibodies against another B. burgdorferi antigen in scid mice also bring about a protection against the clinical symptoms of lyme borreliosis.

The polyclonal anti-B31 immune serum (IMS) was taken from C57BL/6 mice 91 days after a subcutaneous inocculation with 1 x 108 B. burgdorferi B31 organisms.

The polyclonal anti-ZS7 IMS was taken from C57BL/6 mice 68 days after a subcutaneous inoculation with 1 x 108 B. burgdorferi ZS7. Both sera contained 60 pg/ml of specific antibodies, as was determined in an ELISA system (Schaible et al., J. Exp. Med., 170 (1989), 1427-1432). The normal mouse serum (NMS) was taken from non-infected C57BL/6 mice.

At the time point 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 3: 100 pl day 7 and day 10: 200 pl day 13 and day 17: 300 pl.

Scid mice which had been treated either with anti~ ZS7lMS, anti~B3lIMS or with 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 to be 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 OspA-specific monoclonal antibodies LA—26.l of the isotype lgGl, as well as the OspB- specific antibodies LA-25.1 and LA-27.1, were also able to mitigate the clinical symptoms of arthritis, carditis and hepatitis. Slight pathological changes in the investigated organs were here shown.

In contradistinction thereto, scid mice to which had been administered either PBS buffer, NMS or mono- clonal antibodies against flagellin (LA-lO or LA—2l) showed clinical signs of arthritis, the pathological changes typical for untreated scid mice (Table 3).

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 whole observation period. No Spirochaetes could be isolated from scid mice which had previously been treated either with anti—ZS7IMS or with the antibody LA-2. In contra- distinction thereto, the detection was possible of Spirochaetes by immunofluorescence and by culturing from the blood of scid mice which had been treated with PBS buffer, NMS or the monoclonal antibodies LA—25.l,

Claims (12)

Patent Claims

1. Vaccine against lyme disease, characterised in that it contains one or more monoclonal antibodies which are specific for the 31 RD antigen (0spA) or the 34 kD antigen (OspB) of B. burgdorferi.

2. Vaccine according to claim 1, characterised in that the antibody is specific for the 31 kD or the 34 kD antigen of B. burgdorferi of the strains ZS7 (DSM 5525) and/or B31 (ATCC 35210).

3. Vaccine according to claim 1 or 2, characterised in that it contains as active material a monoclonal antibody of the class IgG, preferably of the subclass IgG2b or lgG1.

4. Vaccine according to one of claims 1 to 3, characterised in that it prevents the formation of arthritis, carditis and hepatitis in immune—deficient experimental animals which have been infected with viable, pathogenic B. burgdorferi organisms.

5. Vaccine according to one of claims 1 to 4, characterised in that it substantially prevents the formation of arthritis, carditis and hepatitis in immune—deficient scid mice which have been infected with viable B. burgdorferi organisms, strain ZS7.

6 Vaccine according to one of claims 1 to 5, characterised in that it contains the monoclonal anti- body LA-2 against OspA, secreted by the hybridoma cell line ECACC 89091302.

7. Vaccine according to one of claims 1 to 5, characterised in that it contains the monoclonal anti- body LA—26.l against 0spA secreted by the hybridoma cell line ECACC 90050406.

8. Vaccine according to one of claims 1 to 5, characterised in that it contains the monoclonal anti- body LA-25.1 against OspB secreted by the hybridoma -1'1 1- r.‘f‘A("f‘ onnzn/.r\I: 24

9. Vaccine according to one of claims 1 to 5, characterised in that it contains the monoclonal antibody LA—27.1 against OspB secreted by the hybridoma call line ECACC 90050407.

10. Process for producing a vaccine against Lyme disease, wherein experimental animals are immunized with the 31 kD antigen (OspA) or/and with the 34 kD antigen (OspB) of B. burgdorferi and protective, polyclonal or monoclonal antibodies against OspA or/and OspB are isolated in the usual manner from the immunized experimental animal.

11. Process as claimed in claim]O, wherein mice are used as experimental animals.

12. Process as claimed in clain1lO or ll,wherein the OspA antigen or/and the OspB antigen from B. Burgdorferi ZS7 (DSM 5527) is used. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS