Immunobiol., vol. 181, pp. 357-366 (1990)
Universitats-Hautklinik, Heidelberg, Max-Planck-Institut fur Immunbiologie, Freiburg, and 3 Institut fur Radiologie und Pathophysiologie, Abt. fUr Angewandte Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, FRG
Characterization of Borrelia burgdorferi Associated Antigens by Monoclonal Antibodies MICHAELD. KRAMERl, ULLRICH
E. SCHAIBLE2, REINHARD WALLICH 3,
l SABINE E. MOTER l, DETLEF PETZOLDT ,
and MARKUS M.
Received May 5, 1990· Accepted in Revised Form July 10, 1990
Abstract In this paper, we present a series of murine mAb recognizing B. burgdorferi antigens. The antibodies were characterized by immuno-blotting and immuno-fluorescence studies using isolates of B. burgdorferi from North America and Europe, respectively. Moreover, reactivity of the antibodies with recombinant B. burgdorferi flagellin and OspA was studied. The results suggest these anti-B. burgdorferi mAb as valuable tools for the serological analysis of B. burgdorferi isolates and for affinity-purification of the respective proteins. Moreover, these mAb appear suitable to classify antigenic variants of B. burgdorferi and to study the protective capacity of antibodies in a murine model for B. burgdorferi infection.
Introduction Lyme disease in humans is a complex multisystem illness, caused by the tick-transmitted spirochete Borrelia burgdorferi (B. burgdorferi). The earliest feature of B. burgdorferi infection following the tick bite is a local infection of the skin known as erythema (chronicum) migrans. If not treated with antibiotics, hematogenous spread of B. burgdorferi may occur, and the local infection may be followed by organ manifestation and development of a systemic disease involving the joints, heart, nervous system and liver. A chronic progressive disease with persistent organ involvement may develop. Antibiotic treatment can interfere with all stages of the infection, but may fail in some cases, in particular during later stages of the disease (for a recent review see (1)). Abbreviations: ATCC=American type culture collection; B 31=American prototype B. burgdorferi isolate (ATCC 35210); B=Borrelia; mAb = monoclonal antibody(ies); OspA = B. burgdorferi outer surface protein A (31 kDa); OspB = B burgdorferi outer surface protein B (34 kDa); PBS=phosphate buffered saline; ZS7=German B. burgdorferi isolate (Freiburg area); ZQl = German B. burgdorferi isolate (Freiburg area).
358 . MICHAEL D. KRAMER et al.
All stages of Lyme disease are associated with specific humoral and cellular immune responses (2-6). The analysis of the humoral immune response by immuno-blotting experiments using sera from infected humans revealed several immuno-dominant proteins of B. burgdorferi in the molecular weight range of 20-65 kDa (7, 8). These include: the common antigen (= 60 kDa) (9), the «flagellin» (41 kDa) (10, 11), and the Outer surface proteins (Osp) A (31 kDa) (12) and B (34 kDa) (13), and a = 20 kDa protein (14). In the past, several monoclonal mouse antibodies with specificities for B. burgdorferi antigens, such as flagellin and OspA have been described (11, 15). The antibodies had been raised against the prototype B. burgdorferi isolate B31 from North-America. As tools to study antigenic epitopes recognized by B cells on immunodominant proteins and to reveal antigenic variation between B. burgdorferi isolates derived from different areas, we have now generated a panel of new mAb including those against isolates from Europe. The serological characterization of these mAb and their application are described.
Materials and Methods Bacterial strains, culture conditions and preparation of soluble antigen(s) The prototype strain of B. burgdorferi, named B31 (ATCC 35210), from North America and two isolates of B. burgdorferi from Europe recovered from ticks collected in the Freiburg (FRG) area (ZS7, ZQl) (17) were used in this study. Spirochetes were grown in modified Kelly'S medium as recently described in detail (18). The preparation of soluble antigen(s) was performed as described previously (18). Aliquots of the bacterial suspensions and soluble antigens were stored at -20°C until further use. Immunization of mice, production of hybridomas and screening and selection of clones secreting anti-B. burgdorferi monoclonal antibodies Female BALB/c mice of 6-8 wks of age were immunized as follows: on day 1, 100 [lg of soluble antigen emulsified in Antibody Multiplier (Dunn Labortechnik; Asbach, FRG) subcutaneously and on days 14, 28 and 42, 100 [lg of the same preparation intraperitoneally. Three days prior to fusion the animals received 100 [lg of soluble antigen( s) dissolved in phosphate-buffered saline i.p. Fusion was carried out using Ag8/PAI myeloma cells (kindly provided by Dr. T. STAEHELIN, Ciba Geigy, Basel, Switzerland) according to standard protocols (19, 20). Fusion products were seeded into 96-well flat bottom tissue culture plates (Falcon; Oxnard, CA, USA). After 7-10 days the culture supernatants of individual microwells were assayed for antiB. burgdorferi antibodies using a standard solid-phase enzyme-linked immunosorbent assay (ELISA) system: 1 [lg/well of soluble B. burgdorferi antigens were coated onto flat bottom microtiter plates (Nunc GmbH; Wiesbaden, FRG). Remaining non-specific binding sites were then blocked by incubation with 0.2 % gelatine in PBS. Plates were then incubated with antibody-containing culture supernatants. Bound antibodies were detected via peroxidaselabelled species-specific anti-IgG antibody (dianova GmbH; Hamburg, FRG) and orthophenylenediamine as peroxidase substrate. Hybridoma cells from antibody positive cultures were then cloned by limiting dilution and retested similarly. The presented hybridomas secreting anti-B. burgdorferi antibodies were derived from three independent fusions.
Anti-Borrelia burgdorferi monoclonal antibodies . 359
The IgG isotypes were determined by using commercially available isotype specific peroxidase-labelled rabbit antisera. Activated flat-bottom microtiter plates were coated with nonlabelled anti-mouse Ig immunoglobulins (goat anti-mouse-Ig (7S); Cat. no. 15785; Nordic, Tilburg, The Netherlands) diluted 1:500. Afterwards, culture supernatants of the respective mAbs were added and incubated at room temperature for 1 h under permanent shaking. After several washings isotype-specific peroxidase-labelled immunoglobulin preparations (goat antimouse-IgM (Fc), -IgG (Fc), -IgG h -IgG 2 a> -IgG 2b , -IgG 3 ; all obtained from Nordic, Tilburg, The Netherlands) diluted 1:5000 (v/v) were added. Bound antibodies were detected by using ortho-phenylenediamine as peroxidase substrate. Immunoblotting
Whole cells of B. burgdorferi strain B31, ZS7, and ZQ1 (4 x 108 cells) were lysed in SDS buffer under non-reducing conditions, separated by SDS polyacrylamide gel electrophoresis (12 %) and transferred onto nitrocellulose. After blocking with 0.1 % bovine serum albumin in PBS, the membrane was incubated overnight with the respective mAb (diluted 1:2000 in PBS), washed with PBS and incubated with alkaline phosphatase labelled goat anti-mouse Ig (Southern Biotechnology; Birmingham, AL, USA) diluted 1:1500 in PBS. After intensive washings with PBS, bound labelled antibodies were visualized by incubation in 1 M diethanolamine buffer (pH 9.8) containing Nitrobluetetrazolium (70 [lg/ml) (Sigma; Deisenhofen, FRG) and Bromo-chloro-indoyl-phosphate (36 [lg/ml) (Sigma) as substrate. As molecular weight standards, a commercially available prestained marker mixture was used (BRL; Gaithersburg, USA). Immunofluorescence B. burgdorferi organisms were taken directly from exponentially growing cultures and washed twice in PBS, transferred onto adhesion slides (Superior, Bad Mergentheim, FRG) (1 x 105 spirochetes/reaction field) and air dried. Slides were fixed in absolute ethanol (2 min, -20°C) and air dried. Afterwards the fixed spirochetes were incubated with the respective mAbs diluted in PBS in a moist chamber for 30 min at room temperature. After three subsequent washings in PBS, the fixed spirochetes were incubated with a fluoresceine isothiocyanate-Iabelled goat anti-mouse Ig antiserum (Medac; Hamburg, FRG) at room temperature in a dark moist chamber for 30 min. After three additional washings in PBS, the preparations were embedded in Kaiser's Glyzerin-Gelatine (Merck AG; Darmstadt, FRG), immediately examined using a fluorescence microscope, and documented using a 400 ASA black and white film (HP5; Ilford, UK). Preparation of recombinant B. burgdorferi proteins B. burgdorferi flagellin (derived from strain B31; ATCC 35210) and outer surface protein A (derived from strain ZS7; 17) were cloned and expressed using the bacterial expression vector pUEX1, an adaptor cloning strategy and competent host cells of E. coli strain MC 1061 as described previously (21, 23, 24). Bacterial clones expressing recombinant flagellin (rB31/41-9) or OspA (rZS7/31-2) were identified by immuno-blotting using a mixture of the respective monoclonal antibodies (see Table 1). Reactivity of the monoclonal antibodies with enriched recombinant proteins was assessed by using a standard solid-phase enzyme-linked immunosorbent assay (ELISA) system: 1 [lg/well of urea extracted proteins was coated onto flat-bottom micro titer plates (Nunc GmbH; Wiesbaden, FRG) and non-specific binding sites were subsequently blocked by incubation with 0.2 % gelatine in PBS. Plates were then incubated with antibody-containing culture supernatants that were adjusted to 100 [lg/ml with dialysed urea extracts of E. coli clone MC 1061 (stock solution 1 mg/ml) to compete for reactivity with E. coli antigens. Antibodies bound to the recombinant antigens were detected via peroxidase-labelled speciesspecific anti- IgG antibody (dianova GmbH; Hamburg, FRG) diluted 1:5000 in PBS/0.05 % Tween 20 containing 100 [lg/ml MC 1061 extract.
Results and Discussion
Characterization of monoclonal antibodies recognizing B. burgdorferi antigens
Fusion of spleen cells from BALB/c mice previously immunized with sonic extracts of whole spirochetes from either strain B31 (isolate from North-America) or ZS7 (isolate from Europe) yielded several hybridoma clones secreting mAb specific for B. burgdorferi antigens. Those mAb which are reactive in immuno-blot analysis define the following structures as listed in Table 1: one mAb is specific for a = 20 kDa antigen (LA-7), 7 mAb are specific for outer surface protein A (OspA, 31 kDa; LA-2, -4, -5, Table 1. Reactivity of monoclonal antibodies with whole celllysates of B. burgdorferi strains Z57, ZQ1 and B31 in immuno-blotting, and with recombinant B. burgdorferi flagellin (rB311 41-9) and Outer surface protein A (rZ5/31-2) in ELISA B. burgdorferi Isolate B31
+ + + + + + +
31 kDa (OspA)
LA-2" LA-4'" LA-S" LA-ll'" LA-26'''' LA-2S""" LA-31"'"
IgG 2b IgG2a IgG2a IgG2a IgG 1 IgG 1 IgG 1
LA-2S'f'f LA-27""f LA-32'f'f
IgG 2b IgG 1 IgG 1
+ + +
LA-1" LA-lO" LA-21'f LA-22" LA-23'f LA-24"'" LA-3Y'" LA-34""f
IgG 2a IgG2a IgG 2b IgG 1 IgG2a IgG 1 IgG 1 IgG 1
+ + +
+ + +
+ + +
34 kDa (OspB)
41 kDa (flagellin)
+ + +
+ + +
+ + +
+ + +
+ + + + + + + +
+ + + + + + + +
'f raised against B. burgdorferi strain B31 ""f raised against B. burgdorferi strain Z57 nt = not tested Design. = designation
nt nt nt
Anti-Borrelia burgdorferi monoclonal antibodies . 361
-11, -26, -28, -31), 3 mAb are specific for outer surface protein B (OspB, 34 kDa; LA-25, -27, -32), 8 mAb are specific for flagellin (41 kDa; LA-I, -10, 21, -22, -23, -24, -33, -34), one mAb is specific for a ::::: 60 kDa antigen (LA -18), and one mAb is specific for a ::::: 70 kDa antigen. The immuno-chemical analysis of one B. burgdorferi strain from North America (B31) and two strains from Europe (ZS7, ZQ1) revealed differences in the expression of antigenic epitopes. MAb LA-31 recognizes an antigenic determinant expressed on OspA of all three strains (B31, ZS7, ZQ1), mAbs LA-2, -26 and -28 react with an epitope on OspA of strains ZS7 and B31 but not on ZQ1, and mAb LA-4 and LA-5 recognize a determinant expressed on OspA from strain B31 only. Furthermore, mAbs LA-25, -27, -32 specific for OspB react with the corresponding structure of ZS7 and B31 but not with that of ZQ 1. In contrast, no variations were observed between the three strains with respect to their binding of antiflagellin mAb (LA-I, -10, -21, -22, -23, -24, -33, -34). This is in line with the high degree of conservation of the nucleotide and deduced amino acid sequences of flagellins of two B. burgdorferi isolates from either Europe (GeHo) or North-America (B31) (21). MAb specific for either the::::: 60 kDa antigen (LA-18) or the::::: 70 kDa antigen (LA-3) reacted with all three strains of B. burgdorferi. MAb LA-32 reacted in addition to OspB (34 kDa) with a further::::: 20 kDa antigen when tested on B31 and ZS7 borreliae (Fig. 1; Lane H). The same mAb did not stain the corresponding antigens of strain ZQ1. Whether mAb LA-32 recognizes the same epitope on OspB and a putative cleavage product thereof or whether it binds to an immunogenic epitope common to two independent proteins - i.e. OspB and the ::::: 20 kDa antigen - is at present not clear and needs further experimentation. Yet, this finding is reminiscent of recently published data by JIANG et al. showing that their mAb 184.1 recognizes an antigenic epitope expressed on OspA and on a ::::: 22 kDa protein (22). Most notably, polyclonal anti-B. burgdorferi B31 antiserum (Fig. 1; lanes «AS») and anti-B. burgdorferi ZS7 antiserum (data not shown) did not recognize OspB of ZQ1 borreliae (Fig. I.e; lane «AS»). Moreover, a polyclonal antiserum raised against strain ZQ1 organisms did not reveal OspB or a related 34 kDa antigen in western-blotting studies using lysates of any of the three B. burgdorferi strains (B31, ZQ1, ZS7). This indicates either the absence of the respective molecule or a lack of immunogenicity in strain ZQ1 borreliae.
Reactivity of monoclonal antibodies with recombinant B. burgdorferi proteins MAb specific for B. burgdorferi flagellin or OspA were further analyzed for their binding to recombinant flagellin (rB31/41-9) cloned from strain B31 (21,24) and to recombinant OspA (rZS7/31-2) (23) cloned from strain ZS7. As expected from the reaction patterns on natural flagellin, all respec-
B ( 0 E
I K t1L
; -70 -60 ~-41
B( 0 E
...I K L
-70 -60 -41 -34 -31
B( 0 E
I K L t1
, ~ -70kO.
Anti-Borrelia burgdorferi monoclonal antibodies . 363
tive mAb reacted with the recombinant protein rB31/41-9 (recombinant flagellin). Anti-OspA mAb, on the other hand, displayed differential reactivity on recombinant OspA (rZS7/31-2): i.e. mAb LA-2, -26, -28, and -31 reacted with this protein, whereas mAb LA-4, -5, and -11 did not. Thus the reactivity pattern of these mAb is similar on native and recombinant OspA from strain ZS7 borreliae.
Immunofluorescence studies on intact B. burgdorferi orgamsms usmg monoclonal antibodies To explore the expression of the individual antigenic epitopes recognized by the different mAb on the outer surface of B. burgdorferi, we performed indirect immuno-fluorescence studies on intact spirochetes. As exemplified
Figure 2. Reactivity o f m onoclonal anti-B. burgdorferi antibodi es with whole o rganisms of B. burgdorferi strain B31 in indirect immunoflu orescence. Whole spirochetes were mounted onto adhesio n slides and fixed by using absolute ethanol. Afterwards the slides were incubated with a 1:100 dilution of the fo llowing antibody preparations: p olyclonal anti-3 1 k Da (OspA) of strain B31 ; LA-7 (anti- '" 20 kDa antigen); LA-2 (anti-O spA); LA-5 (anti-OspA); LA-25 (anti-OspB); LA-26 (anti-OspA); LA-lO and LA-21 (anti-flagellin); LA-1 8 (anti - '" 60 kDa antigen); LA-3 (anti- ""' 70 kDa antigen). Bound antibodies were visualized afterwards by using FITC-conjugated anti-mouse I g antibodies. Figure 1. Reactivity o f mo noclonal anti-B. burgdorferi antibodies with electrophoretic ally separated proteins of B. burgdorferi strains B31, ZS7 and ZQ1. Whole cell lysates of B. burgdorferi strains R31 (A; the American prototype isolate; ATCC 35210), ZS7 (B) and ZQl (C) (European i solates of the Freiburg area) were separated by SDS-PAGE and blotted onto nitrocellulose a s described in Materials and Methods. Individual nitrocellulose slices were incubated with 1:1 00 dilutions o f uclture supernatants containing the following mAbs: A=LA-7, B=LA-2, C=LA-5, D=LA-28.1, E=LA-31.1, F=LA-25.1 , G=LA-27.1 , H=LA-32.1, I=LA-lO, K =LA 21, L=LA 24.1, M=LA-34.1, N=LA-1 8, O=LA-3. ~ AS = m ouse anti-serum to whole B. burgdorferi strain B31 .
364 . MICHAEL D. KRAMER et a!.
for a selected range of mAb in Figure 2, mAb to OspA, in particular mAb LA-2, gave bright fluorescence staining which was comparable to the staining pattern observed with a monospecific polyclonal antiserum raised against enriched native OspA (anti 31 kD (B31); Fig. 2). On the other hand, mAb to OspB yielded much weaker staining. The finding is in agreement with previous studies showing that both antigens are differentially expressed at the outer surface of B. burgdorferi (12, 25). Weak but significant staining was seen with anti-flagellin mAb. This may be due to alteration of the outer membrane of B. burgdorferi during preparation for immunofluorescence analysis resulting in artificial exposure of flagellin epitopes. On the other hand, it is known that, although the flagella are localized in the periplasmic space, certain structures of the flagellin molecule may be exposed on the cell surface (26). No significant immunofluorescence staining was observed with mAb specific for either the = 20 kDa (LA-7), the = 60 kDa (LA-1S) or the = 70 kDa (LA-3) antigen. In conclusion, the presented data emphasize the importance of mAb in the analysis of the antigenic variability of B. burgdorferi associated structures. In addition, these antibodies can be readily applied for purification of individual B. burgdorferi proteins by affinity chromatography (data not shown). Moreover, we have recently used B. burgdorferi specific mAb of the LA series in passive transfer experiments in order to analyze their protective capacity in B. burgdorferi infection of mice (16). It was found that experimentally infected mice could be protected against the development of the infection by mAb to OspA or OspB and that the efficiency of protection was dependent on the isotype of the individual mAb, in that IgG2 mAb were more effective than IgG 1 mAb. Acknowledgements MDK is indebted to Dr. A. VOGT (Freiburg), Dr. B. WILSKE (Miinchen) and Dr. G. GASSMANN (Freiburg) for helpful discussions. The authors want to thank Ms. T. TRAN and Ms. U. SCHIRMER for expert technical assistance and Ms. S. PREUSSMANN and Ms. S. KAUTE for help with the photographic work. This work was supported in part by the Deutsche Forschungsgemeinschaft (Kr 93112-1).
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Anti-Borrelia burgdorferi monoclonal antibodies' 365 6. MOFFAT, C. M., L. H. SIGAL, A. C. STEERE, D. H. FREEMAN, and J. M. DWYER. 1984. Cellular immune findings in Lyme disease: Correlation with serum IgM and disease activity. Am. J. Med. 77: 625. 7. GRODZICKI, R. L., and A. C. STEERE. 1988. Comparison of immunoblotting and indirect enzyme-linked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease. J. Infect. Dis. 157: 790. 8. CRAFT, J. E., D. K. FISCHER, G. T. SHIMAMOTO, and A. C. STEERE. 1986. Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearence of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J. Clin. Invest. 78: 934. 9. HANSEN, K., J. M. BANGSBORG, H. FJORDVANG, N. STRANDBERG PEDERSEN, and P. HINDERSSON. 1988. Immunochemical characterization of and isolation of the gene for a Borrelia burgdorferi immunodominant 60-kilodalton antigen common to a wide range of bacteria. Infect. Immun. 56: 2047. 10. HANSEN, K., P. HINDERSSON, and N. S. PEDERSEN. 1988. Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease. J. Clin. Microbiol. 26: 338. 11. BARBOUR, A. G., S. F. HAYES, R. A. HEILAND, M. E. SCHRUMPF, and S. L. TESSIER. 1986. A Borrelia-specific monoclonal antibody binds to a flagellar epitope. Infect. Immun. 52: 549. 12. BARBOUR, A. G., S. L. TESSIER, and S. F. HAYES. 1984. Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45: 94. 13. BUNDOC, V. G., and A. G. BARBOUR. 1989. Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi. Infect. Immun. 57: 2733. 14. WILSKE, B., V. PREAC-MuRSIC, G. SCHIERZ, R. KOHBECK, A. G. BARBOUR, and M. KRAMER. 1988. Antigenic variability of Borrelia burgdorferi. Ann. NY Acad. Sci 539: 126. 15. BARBOUR, A. G., R. A. HEILAND, and T. R. HOWE. 1985. Heterogeneity of major proteins of Lyme disease borreliae. A molecular analysis of North-american and European isolates. J. Infect. Dis. 152: 478. 16. SCHAIBLE, U. E., M. D. KRAMER, K. EICHMANN, M. MODOLELL, C. MUSETEANU, and M. M. SIMON. 1990. Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci. USA 87: 3768. 17. SCHAIBLE, U. E., M. D. KRAMER, C. MUSETEANU, G. ZIMMER, H. MOSSMANN, and M. M. SIMON. 1989. The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis. J. Exp. Med. 170: 1427. 18. SCHAIBLE, U. E., M. D. KRAMER, C. W. E. JUSTUS, C. MUSETEANU, and M. M. SIMON. 1989. Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi. Infect. Immun. 57: 41. 19. PETERS, J. H., H. BAUMGARTEN, and M. SCHULZE. 1985. Monoklonale Antikorper. 1st Edn. Springer-Verlag: Berlin, Heidelberg, New York. 20. JUSTUS, C. W. E., S. MOLLER, and M. D. KRAMER. 1988. Application of novel monoclonal antibodies in the purification, quantification, and immunohistological localization of the proteinase inhibitor urmacroglobulin. Enzyme Microb. Technol. 10: 524. 21. GASSMANN, G. S., M. KRAMER, U. B. GOBEL, and R. WALLICH. 1989. Nucleotide sequence of a gene encoding the Borrelia burgdorferi flagellin. Nucl. Acid Res. 17: 3590. 22. JIANG, W., B. J. LUfT, P. MUNOZ, R. J. DATTWYLER, and P. D. GOREVIC. 1990. Crossantigenicity between the major surface proteins (OspA and OspB) and other proteins of Borrelia burgdorferi. J. Immunol. 144: 284. 23. WALLICH, R., U. E. SCHAIBLE, M. M. SIMON, A. HEIBERGER, and M. D. KRAMER. 1989. Cloning and sequencing of the gene encoding the outer surface protein A (OspA) of an European Borrelia burgdorferi isolate. Nucl. Acid Res. 21: 8864. 24. WALLICH, R., S. E. MOTER, M. M. SIMON, K. EBNET, A. HEIBERGER, and M. D. KRAMER. 1990. The Borrelia burgdorferi Flagellum-associated 41 kDa antigen «