Vol. 60, No. 8
INFECTION AND IMMUNITY, Aug. 1992, p. 3098-3104
0019-9567/92/083098-07$02.00/0 Copyright X 1992, American Society for Microbiology
Selection of an Escape Variant of Borrelia burgdorferi by Use of Bactericidal Monoclonal Antibodies to OspB JAMES L. COLEMAN,' RENE C. ROGERS,' AND JORGE L. BENACHl 2* State of New York Department of Health1 and Department ofPathology,2 State University of New York at Stony Brook, Stony Brook; New York 11794-8692 Received 28 January 1992/Accepted 7 May 1992
Two immunoglobulin G (IgG) monoclonal antibodies (MAbs) to outer surface protein B (CB2 and CB6), affinity purified from mouse ascitic fluid, exhibited concentration-dependent inhibitory and bactericidal properties against Borrelia burgdorferi after a 24-h incubation period in spirochete medium. Fab fragments derived from these MAbs showed the same effects, indicating that they were not caused by agglutination of the organisms by the intact MAbs. The inhibition of spirochete growth in cultures containing MAbs was also detected by spectrophotometric analysis of the media. CB2 did not inhibit the growth of Borrelia hermsii or the BEP4 strain of B. burgdorferi, neither of which is recognized by the MAb. Affinity-purified IgG from hybridoma supernatants had similar effects on B. burgdorferi as the ascitic-fluid-derived IgG did, indicating that the inhibitory and bactericidal properties were not due to nonspecific toxic contaminants. The bactericidal properties of the MAbs were not complement dependent as there was none in the serum-free system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of B. burgdorfieri organisms surviving after exposure to CB2 revealed an escape variant which failed to express OspB. The continued presence of OspA in these escape variants indicates that the lack of OspB was not due to the loss of the plasmid which contains the genes for both of these proteins.
Borrelia burgdorferi, the etiologic agent of Lyme disease (5, 13, 32), has two known outer surface proteins, OspA and OspB (3, 4, 7). Although these two lipoproteins (9, 10) are very abundant in this organism, patients with Lyme disease do not produce antibodies to them until the later stages of the illness (6, 16, 19). OspA has been proposed as a suitable vaccine candidate since these outer surface proteins are recognized readily by the immune system in rodent models (20, 29). Protection conferred by OspA vaccines has been correlated with the development of antibodies and measured by resistance to challenge in vaccinated animals. The role of antibodies in these rodent vaccine trials is important since their appearance is linked to protection against infection. In the human patient, this is not the case, since antibodies coexist with infection and the human antibody repertoire often does not include those produced against OspA or OspB. Why rodents and humans respond to these lipoproteins in different ways remains a paradox and is one whose answer is likely to be complex since borrelial strain variation and antigenic variation within strains could account for the differences in response. Because of their external location, the outer surface proteins could also have a functional role as ligands of spirochetal adhesion to eukaryotic cells. B. burgdorferi adheres to a variety of cells (21, 26, 33) and molecules (22). Because of our interest in the molecular basis of adhesion, we produced murine monoclonal antibodies (MAbs) to OspA and OspB to attempt selective inhibition of the binding process. Through the course of these investigations, we noted that some of these MAbs had bactericidal effects which were unique in that the system in use was serum free and thus devoid of complement. In this report, we document the bactericidal effects of two MAbs to OspB and the
*
Corresponding author.
selection of an escape variant which does not express this antigen.
MATERIALS AND METHODS Spirochetes. The B31 strain of B. burgdorfieri from Shelter Island, N.Y. (13), was used for all experiments with the exception that B. burgdorferi BEP4 (18) and Borrelia hermsii were used as controls. Media and growth conditions for spirochetes. B. burgdorferi organisms were grown at 33°C in a serum-free medium that has been described previously (8). B. hernsii was grown at 33°C in BSK II medium (1). MAb production and purification. Whole, washed B. burgdorferi B31 spirochetes were used to immunize BALB/c mice. The immunization regimen, fusion, screening, subcloning, and isotyping of clones have been described previously (7, 17, 18). MAbs were purified from both ascitic fluid and culture supernatant. The protein G affinity purification procedure also has been described previously (18). Preparation of Fab fragments was done by papain digestion, followed by removal of the Fc fragment by protein A affinity chromatography (ImmunoPure Fab Preparation Kit; Pierce, Rockford, Ill.). MAbs of the immunoglobulin M (IgM) class were partially purified by precipitation in 40% ammonium sulfate. The MAbs were characterized for their antigenic specificities by Western blots (immunoblots) against wholecell lysates of B. burgdorferi. SDS-PAGE and Western blotting. B. burgdorferi organisms were harvested from the medium by centrifugation at 7,000 x g for 20 min (25°C) and washed two times in phosphate-buffered saline containing 5 mM magnesium chloride (pH 7.4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by using a Laemmli buffer system (27) in gels of 10% acrylamide. All other conditions have been described previously (7, 17, 18). West3098
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em blotting was also done as described previously (7, 17, 18,
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34).
RESULTS MAbs. The MAbs used in this study were all directed against either OspA or OspB of B. burgdorfen. During the
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Growth of B. burgdorferi in the presence of MAbs. B. burgdorferi organisms (5 x 106) were added to sterile tubes (Sarstedt, Newton, N.C.) containing 1 ml of serum-free BSK medium with a range of affinity-purified MAb concentrations (0.02, 0.1, 1.0, 1.5, and 2.0 ,ug/ml) and incubated at 33°C.
Protein determinations of MAbs were made by using a kit (BCA protein reagent; Pierce). Normal mouse IgG (Organon Teknika, Durham, N.C.) in the same concentrations and medium without antibody were used as controls. For some experiments, the MAb and normal mouse IgG concentrations were held at 2.0 ,ug/ml and the size of the B. burgdorferi inoculum was varied (5 x 105, 1 x 106, 5 x 106, and 1 x 107 spirochetes per tube). The growth of spirochetes was monitored by direct enumeration (by dark-field microscopy at x675 magnification) at 24-h intervals for a total of 72 h. Experiments involving B. hermsii were done in exactly the same manner, with the exception that BSK II medium (1) was used. For some experiments, the BSK medium containing the MAbs or normal mouse IgG was heated at 56°C for 30 min to inactivate any possible complement contaminant from the bovine serum albumin present in the medium or from the ascitic fluid from which the MAbs were purified. Indirect analysis of B. burgdorferi growth. In addition to direct enumeration, the growth of B. burgdorferi was measured spectrophotometrically by the change in color of the pH indicator in the medium as the cultures grew (the phenol red in the medium changes from red to yellow as spirochete density and metabolic activity increase, causing the medium to become increasingly more acidic). A scan of fresh BSK medium and BSK medium from a fully grown culture of B. burgdorferi was conducted to determine the optimum wavelength for measuring color change in the medium. A wavelength of 570 nm was chosen because it gave the greatest separation of absorbance values between the fresh and spent medium. For the assay, the spirochetes were removed by centrifugation at 7,000 x g for 10 min and the A570 for each sample was determined. At this wavelength, the absorbance readings were directly proportional to the amount of red in the medium, with the result that medium from cultures with the highest spirochete densities (and increased yellow color) actually had lower absorbance readings. To correct for this, the absorbance value for each sample was subtracted from the absorbance value of fresh BSK medium. Electron microscopy. For the ultrastructural analysis of damage to B. burgdorferi exposed to MAbs, B31 cultures were centrifuged and resuspended in BSK medium to a density of 5 x 107/ml. Aliquots of 100 ,ul received MAbs or normal mouse IgG to a concentration of 2.0 ,ug/ml. After incubation at 33°C for 1 h, the spirochetes were centrifuged and the pellets were fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate (pH 7.4) at 4°C. This was followed by fixation in 1% OS04 and dehydration through a graded ethanol series. The specimens were then embedded in Epon. Staining of thin sections was done with uranyl acetate and lead citrate. Specimens were observed in a transmission electron microscope (Carl Zeiss Instruments, Thornwood, N.Y.).
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FIG. 1. Western blot reactivities of MAbs to the OspB antigen of B. burgdorferi. Lanes: 1, CB2; 2, CB2 Fab fragment; 3, CB6; 4, CB6 Fab fragment. Approximately 10 ,ug of B. bwgdorferi was loaded per gel lane. All MAbs were used at 10 pg/ml. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG, used at 1 p,g/ml. SDS-PAGE was run under reducing conditions. Molecular mass is expressed in kilodaltons.
course of adhesion studies with these MAbs, we found evidence that some of them exhibited bactericidal properties. In a series of preliminary experiments to investigate this phenomenon, B. burgdorferi B31 was cultured in the presence of a range of MAb concentrations (from 0.02 to 10.0 Fg/ml) to assess their effects on the in vitro growth of the spirochete. In these experiments, the three MAbs to OspA (two of which were of the IgM class and one of which was an IgG3, all with kappa light chains) had no effect on the growth of B. burgdorferi over a 72-h period. The growth curve of B. burgdorferi in the presence of these MAbs was similar to that obtained for B. burgdorferi grown in the presence of identical levels of normal mouse IgG (data not shown). For this reason, these MAbs were not considered further in this study. CB2 and CB6 (IgGl and IgG3, respectively, with kappa light chains), both of which are directed to OspB, showed inhibitory and bactericidal effects on the growth of B. burgdorferi cultures at all MAb levels tested. These two MAbs were therefore selected and used for all experiments described hereafter. Since the results of these experiments were dependent upon direct enumeration of spirochetes under dark-field microscopy, microagglutination of spirochetes in the presence of outer surface-specific CB2 and CB6 could result in inaccurate counts. To avoid this, we prepared Fab fragments from CB2 and CB6. Figure 1 shows the Western blots of each MAb and its corresponding Fab fragment against B.
burgdorferi. Growth of B.
bugdorferi
in the presence of MAbs. (i)
Effect(s) of MAb concentration. The effects of increasing concentrations of CB2, CB6, and their Fab fragments on the in vitro growth of B. burgdorferi B31 were investigated. One-milliliter cultures received MAbs or normal mouse IgG at concentrations ranging from 0.02 to 2.0 p,g/ml and an inoculum of 5 x 106 B. burgdorferi organisms. Growth was measured by dark-field microscopy over a period of 72 h. All
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TIME (hours) in the presence of CB2-W (W, whole immunoglobulin molecule), CB2 Fab fragment, CB6-W, and CB6 FIG. 2. Growth of B. burgdorfeC Fab fragment: effect of MAb concentration. One-milliliter cultures received a range of MAb, Fab fragment, or normal mouse IgG concentrations (0.02 to 2.0 p.g/ml) and then were inoculated with 5 x 106 B. burgdorfern organisms. Symbols: A\, control with no antibody; 0, control with normal mouse IgG at 2.0 xg/ml; 0, MAb at 0.02 ,ug/ml; *, MAb at 0.1 Fg/ml; *, MAb at 1.0 ,uLg/ml; 0, MAb at 1.5 ,ug/ml; U, MAb at 2.0 ,ug/ml. measurements were compared with the normal growth rate of this strain in medium alone. The results of a representative experiment are summarized in Fig. 2. This experiment was repeated three times as described above (and numerous times with minor variations) and in duplicate for each experiment with high reproducibility. With the exception of the culture exposed to CB6 Fab at 0.02 ,uglml, B.
burgdorferi densities in cultures exposed to all MAbs and
Fab fragnents were below those of the controls. This inhibitory effect occurred in a concentration-dependent manner over the full 72 h. In addition, we noted a bactericidal effect at the 24-h time period with CB2 and CB2 Fab at
concentrations of 1.0, 1.5, and 2.0 jig/ml. With CB6 and CB6 Fab, this bactericidal effect was apparent at concentrations of 2.0 ,ug/ml for CB6 and 1.5 and 2.0 p,g/ml for CB6 Fab. The 50% lethal dose concentrations of CB2 (0.7 jig/ml), CB2 Fab (1.0 ,ug/ml), CB6 (1.0 p.g/ml), and CB6 Fab (1.2 ,ug/ml) for B. burgdorferi for the first 24 h of incubation were calculated by probit analysis from the data in Fig. 2. To test for the possibility that the inhibitory and bactericidal activity of these MAbs could be due to nonspecific toxic substances derived from the ascitic fluid, we investigated the effect of CB2 on the in vitro growth of a related spirochete, B. hennsii, which is not recognized by this MAb.
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CB2 had no effect on the growth of B. hermsii cultures. In another experiment, CB2 also failed to inhibit the growth of B. burgdorfei BEP4, which is not recognized by this MAb. In addition, affinity-purified MAb from CB2 hybridoma supernatant had the same effect on B. burgdorferi B31 that the affinity-purified IgG from ascitic fluid did (data not
shown). To investigate the possibility that complement could be present as a contaminant of bovine serum albumin in BSK medium or in the ascitic fluid-derived MAbs and thus be responsible for the bactericidal properties noted in the assay, we heat treated the medium and the antibodies (MAbs and normal mouse IgG at 2.0 ,ug/ml) for 30 min at 56°C. After monitoring by dark-field microscopy for 72 h, we found that CB2 and CB6 retained their bactericidal properties (data not shown). (ii) Effect(s) of inoculum size. We investigated the effects of initial inoculum size (B. burgdorferi B31) on the inhibitory and bactericidal properties of CB2 and CB6. One-milliliter culture tubes containing BSK medium and CB2 and CB6 at a fixed concentration of 2.0 ,ug/ml were prepared. Control antibody tubes were also made with normal mouse IgG at 2.0 p,g/ml. To these tubes were added increasing amounts of B. burgdorferi (5 x 105, 1 x 106, 5 x 106, and 1 x 107 spirochetes per 1-ml tube). A representative experiment is summarized in Fig. 3. At all four inoculum sizes, the growth of B. burgdorferi was depressed in comparison with that of the controls. As an alternative to direct enumeration, we also assessed spirochete growth indirectly by measuring the loss of the red color of the phenol red indicator in the medium by spectrophotometry. Metabolic activity of the spirochetes exposed to CB2 and CB6 was depressed in comparison with that of the control which received normal mouse IgG. This pattern held for inocula of 5 x 105, 1 x 106, 5 x 106, and 1 x 107 organisms (Fig. 4). Ultrastructural evidence of damage to B. burgdorfeni exposed to MAbs. Because of the bactericidal activity of the MAbs noted earlier, we sought to obtain direct evidence of spirochete damage after exposure to MAbs. In this experi-
ment, 5 x 107 B. burgdorferi organisms were exposed to 2.0-,ug/ml levels of CB2 for 1 h at 330C. After being fixed, sectioned, and stained, the spirochetes were examined in a transmission electron microscope and showed evidence of damage to the spirochetal outer envelope (Fig. 5A). The control, exposed to normal mouse IgG (2.0 p,g/ml), remained unaffected (Fig. SB). Selection of OspB- escape variants. Despite the marked bactericidal and/or inhibitory effects described by the data in Fig. 2, 3, and 4, a small number of surviving organisms was often seen in the B. burgdorfen-MAb assays. To investigate the possibility that these survivors were escape variants, B. burgdorfien (B31, uncloned) was exposed to CB2 at 2.0 p,g/ml. After 72 h in culture, the surviving organisms were analyzed by SDS-PAGE and Western blotting. If survival was the result of a mutation in the gene for the epitope recognized by CB2 in OspB, the entire molecule would still be detected by SDS-PAGE, but CB2 would no longer react in Western blots. Alternatively, if the plasmid which codes for OspA and OspB was lost, this loss would be apparent in SDS-PAGE by the absence of both proteins. The survivors failed to express OspB. A Coomassie brilliant blue-stained gel of survivors which are OspA+ and OspB- is shown in Fig. 6A. Stock B. burgdorfen, but not survivors, reacted with CB2 in Western blots (Fig. 6B). Both survivors and stock organisms reacted to MAb llGl (7), confirming the presence of OspA in both populations (Fig. 6B). While the survivors failed to express OspB, they did, however, express another protein with an apparent molecular mass of 21.5 kDa not present in the parent B31 population (Fig. 6A) but which did not react with the MAbs (Fig. 6B). Survivors have grown normally in medium containing up to 10 ,ug of CB2 per ml for several passages without expression of OspB. DISCUSSION We have selected an escape variant of B. burgdorferi which does not express OspB after treatment with bacteri-
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cidal MAbs directed against antigenic determinants present in this protein. The mechanism of action of these MAbs is not known at present. However, the bactericidal properties of these antibodies are not due to agglutination since Fab fragments of the affinity-purified IgG had similar effects on the organisms (Fig. 2). Furthermore, had agglutination been the reason for the decreased numbers of organisms detected after exposure to the MAbs, the indirect measurement of spirochete viability by spectrophotometric analysis of the color change of the phenol red indicator in the medium would have demonstrated metabolic activity similar to that of controls exposed to normal mouse IgG (Fig. 4). Agreement between the enumeration of the organisms and the lack of metabolic activity in the MAb-treated cultures strengthens the conclusion that the growth of the organisms was inhibited at the lower concentrations of MAbs and that the organisms were killed at the higher concentrations. The bactericidal effects of the MAbs did not appear to be due to a possible presence of nonspecific toxic compounds in the ascites. CB2 did not have an effect on the growth of B. hermsii, the agent of American relapsing fever, nor on the growth of the BEP4 strain of B. burgdorferi, which is not recognized by this MAb. Affinity-purified IgG from hybridoma supernatants of both CB2 and CB6 was found to have the same inhibitory and bactericidal action on B. burgdorferi that the affinity-purified IgG from ascitic fluid did. Lysis of B. burgdorferi by immune serum and complement
(25, 28) and immobilization of the organisms (14) have been demonstrated. The assay system used in our studies did not contain complement because the MAbs were used in the form of affinity-purified IgG from ascitic fluid and the spirochete medium was serum free. Nonetheless, medium containing MAbs was heat treated at 56°C for 30 min to inactivate any possible contaminating complement (possibly derived from the MAbs or from the bovine serum albumin in the medium). Heat treatment did not result in a decrease in inhibitory or bactericidal effects by the MAbs in the assay. Murine IgGl does not bind complement, and CB2 belongs to that subclass. From these observations, we can conclude that the inhibitory and bactericidal effects of CB2 and CB6 may be due to a unique action of these IgG antibodies on a still unknown epitope of OspB, which in turn has a disruptive effect on the outer envelope. Transmission electron microscopy of spirochetes treated with CB2 showed such a disruption of the outer envelope as compared with the intact organisms seen after treatment with normal mouse IgG (Fig. 5). Yet, the ultrastructural observations are of limited value in determining the mechanism of action of these MAbs, since some of the organisms were not bound by CB2 and others were only at one stage in the process of destruction. In this regard, recent evidence has shown that both OspA and OspB can be found in the periplasmic space, the protoplasmic cylinder, and the cytoplasmic membranes, as well as the outer enve-
ESCAPE VARIANTS SELECTED BY MAbs TO B. BURGDORFERI
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FIG. 6. (A) SDS-PAGE analysis. Lanes: 1, molecular mass standards; 2, stock B. burgdorferi B31; 3, OspB- escape variant. Arrowhead, absence of OspB; *, 21.5-kDa protein. Approximately 70 pg of antigen was loaded per gel lane. Gel contained 10% acrylamide and was run under reducing conditions. Staining was by Coomassie brilliant blue. Molecular mass is in kilodaltons. (B) Western blots confirming absence of OspB and presence of OspA in OspB- escape variant. Lanes: 1, lack of reactivity of CB2 to OspBescape variant; 2, reactivity of CB2 to stock B. burgdorferi; 3, reactivity of MAb llGl to OspB- escape variant; 4, reactivity of llGl to stock B. burgdorferi. llGl is directed against B. burgdorferi OspA and has been described previously (7). Reactivity of llGl to stock B. burgdorferi and OspB- variant confirms the presence of OspA in both strains. Approximately 10 ,ug of B. burgdorferi was loaded per gel lane. SDS-PAGE was run under reducing conditions in a gel of 10% acrylamide. MAbs were used at 10 Rg/ml. Secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG used at 1 gg/ml.
FIG. 5. (A) Thin sections of B. burgdorfeni B31 after exposure to CB2 (2.0 iLg/ml) for 1 h at 33°C; (B) control thin section after exposure of B. burgdorferi to normal mouse IgG (2.0 Fg/ml) for 1 h at 33°C. Bars, 0.2 ,um.
lope (11). This finding is relevant because if MAbs to OspB could gain entrance to the organism beyond the outer envelope, and bind internal OspB, the presence of such a complex may be sufficient to destroy the organism. It has been shown that immunogold electron microscopy of B. burgdorfen fixed in formalin after exposure to primary (MAb to OspA) and secondary antibodies showed aggregations of the gold particles. This phenomenon, not noted if the organisms were fixed before antibody treatment, was defined as patching or as a passive two-dimensional aggregation of
molecules in a fluid membrane (4). Leptospira spp. have been shown to permit longitudinal movement of antigens in the outer envelope (15), and this phenomenon may also occur in Borrelia spp. The escape variants selected by treatment with CB2 and CB6 failed to express OspB (Fig. 6). The variants also expressed a protein with a molecular mass of 21.5 kDa which was not present in the parent B31 population. Similar lower-molecular-mass proteins (21 and 18.5 kDa) in cloned strains which do not express or have altered OspB have been shown before (12). Unlike the reactivity to anti-OspB MAbs shown in this study (12), we did not find reactivity of the selecting MAbs for the 21.5-kDa polypeptide. Since both OspA and OspB are encoded by genes located in a 49-kb linear duplex plasmid with covalently closed ends (2, 23, 24), the fact that OspA was still expressed (Fig. 6) indicates that the entire plasmid was not lost and that an OspB- variant was present in the original heterogeneous B31 population. OspB- variants have been demonstrated before (3, 12, 35) as naturally occurring in various strains of B. burgdorferi. Loss of plasmids as a result of continuous in vitro cultivation has been documented and linked to a loss of infectivity of the strain for rodents (29). In fact, phenotypic expression of OspB has been lost in a heterogeneous strain of a human blood isolate after 11 to 15 in vitro passages (30, 31) and by experimentally transposable elements inserted in the OspA gene (23). If selection of OspB- variants could occur in vivo as a result of antibodies such as that which occurred in this
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experimental system, the organisms could avoid a potentially destructive antibody effect and prolong the illness. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI 27044 from the National Institutes of Health and by a grant from the G. Harold and Leila Y. Mathers Charitable Foundation to J.L.B. We appreciate the critical reviews of Jorge Galan of the Department of Microbiology and Gail S. Habicht of the Department of Pathology, State University of New York at Stony Brook. REFERENCES 1. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. 2. Barbour, A. G., and C. F. Garon. 1987. Linear plasmids of the bacterium Borrelia burgdorfeni have covalently closed ends. Science 237:409-411. 3. Barbour, A. G., S. L. Tessier, and S. F. Hayes. 1984. Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45:94-100. 4. Barbour, A. G., S. L. Tessier, and W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41:795-804. 5. Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G. Barbour, W. Burgdorfer, R. Edelman, and R. A. Kaslow. 1983. Spirochetes isolated from the blood of two patients with Lyme disease. N. Engl. J. Med. 308:740-742. 6. Benach, J. L., J. L. Coleman, J. C. Garcia-Monco, and P. C. Deponte. 1988. Biological activity of Borrelia burgdorferi antigens. Ann. N.Y. Acad. Sci. 539:115-125. 7. Benach, J. L., J. L. Coleman, and M. G. Golightly. 1988. A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete. J. Immunol. 140:265272. 8. Benach, J. L., H. B. Fleit, G. S. Habicht, J. L. Coleman, E. M. Bosler, and B. P. Lane. 1984. Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor. J. Infect. Dis. 150:497-507. 9. Bergstrom, S., V. G. Bundoc, and A. G. Barbour. 1989. Molecular analysis of linear plasmid-encoded major surface proteins, Osp A and Osp B, of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol. 3:479-486. 10. Brandt, M. E., B. S. Riley, J. D. Radolf, and M. V. Norgard. 1990. Immunogenic integral membrane proteins of Borrelia burgdorfen are lipoproteins. Infect. Immun. 58:983-991. 11. Brusca, J. S., A. W. McDowall, M. V. Norgard, and J. D. Radolf. 1991. Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorfeni. J. Bacteriol. 173:8004-8008. 12. Bundoc, V. G., and A. G. Barbour. 1989. Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi. Infect. Immun. 57:2733-2741. 13. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease-a tick-borne spirochetosis? Science 216:1317-1319. 14. Callister, S. M., R. F. Schell, and S. D. Lovrich. 1991. Lyme disease assay which detects killed Borrelia burgdorferi. J. Clin. Microbiol. 29:1773-1776. 15. Charon, N. W., C. W. Lawrence, and S. O'Brien. 1981. Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans. Proc. Natl. Acad. Sci. USA 78:71667170. 16. Coleman, J. L., and J. L. Benach. 1987. Isolation of antigenic components from the Lyme disease spirochete: their role in early diagnosis. J. Infect. Dis. 155:756-765.
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17. Coleman, J. L., and J. L. Benach. 1989. Identification and characterization of an endoflagellar antigen of Borrelia burgdorferi. J. Clin. Invest. 84:322-330. 18. Coleman, J. L., and J. L. Benach. 1992. Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria. J. Infect. Dis. 165:658-666. 19. Craft, J. E., D. K. Fischer, G. T. Shimamoto, and A. C. Steere. 1986. Antigens of Borrelia burgdorfen recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J. Clin. Invest. 78:934-939. 20. Fikrig, E., S. W. Barthold, F. S. Kantor, and R. A. Flavell. 1990. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 250:553-556. 21. Garcia-Monco, J. C., B. Fernandez-Vldlar, and J. L. Benach. 1989. Adherence of the Lyme disease spirochete to glial cells and cells of glial origin. J. Infect. Dis. 160:497-506. 22. Garcia-Monco, J. C., B. Fernandez-Villar, R. C. Rogers, A. Szczepanski, C. M. Wheeler, and J. L. Benach. Borrelia burgdorfen and other related spirochetes bind to galactocerebroside. Neurology, in press. 23. Howe, T. R, F. W. LaQuier, and A. G. Barbour. 1986. Organization of genes encoding two outer membrane proteins of the Lyme disease agent Borrelia burgdorferi within a single transcriptional unit. Infect. Immun. 54:207-212. 24. Howe, T. R., L. W. Mayer, and A. G. Barbour. 1985. A single recombinant plasmid expressing two outer surface proteins of the Lyme disease spirochete. Science 227:645-646. 25. Kochi, S. K., and R. C. Johnson. 1988. Role of immunoglobulin G in killing of Borrelia burgdorferi by the classical complement pathway. Infect. Immun. 56:314-321. 26. Kurtti, T. J., U. G. Munderloh, G. G. Ahlstrand, and R C. Johnson. 1988. Borrelia burgdorfen in tick cell culture: growth and cellular adherence. J. Med. Entomol. 25:256-261. 27. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packing events. J. Mol. Biol. 80:575599. 28. Lovrich, S. D., S. M. Callister, J. L. Schmitz, J. D. Alder, and R. F. Schell. 1991. Borreliacidal activity of sera from hamsters infected with the Lyme disease spirochete. Infect. Immun. 59:2522-2528. 29. Schaible, U. E., M. D. Kramer, K. Eichmann, M. Modoleli, C. Museteanu, and M. M. Simon. 1990. Monoclonal antibodies specific for the outer surface protein A (Osp A) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci. USA 87: 3768-3772. 30. Schwan, T., and W. Burgdorfer. 1987. Antigenic changes of Borrelia burgdorferi as a result of in vitro cultivation. J. Infect. Dis. 156:852-853. 31. Schwan, T., W. Burgdorfer, and C. F. Garon. 1988. Changes in the infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect. Immun. 56:1831-1836. 32. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista. 1983. The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308:733-740. 33. Szczepanski, A., M. B. Furie, J. L. Benach, B. P. Lane, and H. B. Fleit. 1990. Interaction between Borrelia burgdorferi and endothelium in vitro. J. Clin. Invest. 85:1637-1647. 34. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 35. Wilske, B., V. Preac-Mursic, G. Schierz, and K. Busch. 1986. Immunochemical and immunological analysis of European Borrelia burgdorferi strains. Zentralbl. Bakteriol. Hyg. A 263:92102.
INFECTION AND IMMUNITY, Aug. 1992, p. 3098-3104
0019-9567/92/083098-07$02.00/0 Copyright X 1992, American Society for Microbiology
Selection of an Escape Variant of Borrelia burgdorferi by Use of Bactericidal Monoclonal Antibodies to OspB JAMES L. COLEMAN,' RENE C. ROGERS,' AND JORGE L. BENACHl 2* State of New York Department of Health1 and Department ofPathology,2 State University of New York at Stony Brook, Stony Brook; New York 11794-8692 Received 28 January 1992/Accepted 7 May 1992
Two immunoglobulin G (IgG) monoclonal antibodies (MAbs) to outer surface protein B (CB2 and CB6), affinity purified from mouse ascitic fluid, exhibited concentration-dependent inhibitory and bactericidal properties against Borrelia burgdorferi after a 24-h incubation period in spirochete medium. Fab fragments derived from these MAbs showed the same effects, indicating that they were not caused by agglutination of the organisms by the intact MAbs. The inhibition of spirochete growth in cultures containing MAbs was also detected by spectrophotometric analysis of the media. CB2 did not inhibit the growth of Borrelia hermsii or the BEP4 strain of B. burgdorferi, neither of which is recognized by the MAb. Affinity-purified IgG from hybridoma supernatants had similar effects on B. burgdorferi as the ascitic-fluid-derived IgG did, indicating that the inhibitory and bactericidal properties were not due to nonspecific toxic contaminants. The bactericidal properties of the MAbs were not complement dependent as there was none in the serum-free system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of B. burgdorfieri organisms surviving after exposure to CB2 revealed an escape variant which failed to express OspB. The continued presence of OspA in these escape variants indicates that the lack of OspB was not due to the loss of the plasmid which contains the genes for both of these proteins.
Borrelia burgdorferi, the etiologic agent of Lyme disease (5, 13, 32), has two known outer surface proteins, OspA and OspB (3, 4, 7). Although these two lipoproteins (9, 10) are very abundant in this organism, patients with Lyme disease do not produce antibodies to them until the later stages of the illness (6, 16, 19). OspA has been proposed as a suitable vaccine candidate since these outer surface proteins are recognized readily by the immune system in rodent models (20, 29). Protection conferred by OspA vaccines has been correlated with the development of antibodies and measured by resistance to challenge in vaccinated animals. The role of antibodies in these rodent vaccine trials is important since their appearance is linked to protection against infection. In the human patient, this is not the case, since antibodies coexist with infection and the human antibody repertoire often does not include those produced against OspA or OspB. Why rodents and humans respond to these lipoproteins in different ways remains a paradox and is one whose answer is likely to be complex since borrelial strain variation and antigenic variation within strains could account for the differences in response. Because of their external location, the outer surface proteins could also have a functional role as ligands of spirochetal adhesion to eukaryotic cells. B. burgdorferi adheres to a variety of cells (21, 26, 33) and molecules (22). Because of our interest in the molecular basis of adhesion, we produced murine monoclonal antibodies (MAbs) to OspA and OspB to attempt selective inhibition of the binding process. Through the course of these investigations, we noted that some of these MAbs had bactericidal effects which were unique in that the system in use was serum free and thus devoid of complement. In this report, we document the bactericidal effects of two MAbs to OspB and the
*
Corresponding author.
selection of an escape variant which does not express this antigen.
MATERIALS AND METHODS Spirochetes. The B31 strain of B. burgdorfieri from Shelter Island, N.Y. (13), was used for all experiments with the exception that B. burgdorferi BEP4 (18) and Borrelia hermsii were used as controls. Media and growth conditions for spirochetes. B. burgdorferi organisms were grown at 33°C in a serum-free medium that has been described previously (8). B. hernsii was grown at 33°C in BSK II medium (1). MAb production and purification. Whole, washed B. burgdorferi B31 spirochetes were used to immunize BALB/c mice. The immunization regimen, fusion, screening, subcloning, and isotyping of clones have been described previously (7, 17, 18). MAbs were purified from both ascitic fluid and culture supernatant. The protein G affinity purification procedure also has been described previously (18). Preparation of Fab fragments was done by papain digestion, followed by removal of the Fc fragment by protein A affinity chromatography (ImmunoPure Fab Preparation Kit; Pierce, Rockford, Ill.). MAbs of the immunoglobulin M (IgM) class were partially purified by precipitation in 40% ammonium sulfate. The MAbs were characterized for their antigenic specificities by Western blots (immunoblots) against wholecell lysates of B. burgdorferi. SDS-PAGE and Western blotting. B. burgdorferi organisms were harvested from the medium by centrifugation at 7,000 x g for 20 min (25°C) and washed two times in phosphate-buffered saline containing 5 mM magnesium chloride (pH 7.4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by using a Laemmli buffer system (27) in gels of 10% acrylamide. All other conditions have been described previously (7, 17, 18). West3098
VOL. 60, 1992
ESCAPE VARIANTS SELECTED BY MAbs TO B. BURGDORFER
em blotting was also done as described previously (7, 17, 18,
1 2
34).
RESULTS MAbs. The MAbs used in this study were all directed against either OspA or OspB of B. burgdorfen. During the
4
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Growth of B. burgdorferi in the presence of MAbs. B. burgdorferi organisms (5 x 106) were added to sterile tubes (Sarstedt, Newton, N.C.) containing 1 ml of serum-free BSK medium with a range of affinity-purified MAb concentrations (0.02, 0.1, 1.0, 1.5, and 2.0 ,ug/ml) and incubated at 33°C.
Protein determinations of MAbs were made by using a kit (BCA protein reagent; Pierce). Normal mouse IgG (Organon Teknika, Durham, N.C.) in the same concentrations and medium without antibody were used as controls. For some experiments, the MAb and normal mouse IgG concentrations were held at 2.0 ,ug/ml and the size of the B. burgdorferi inoculum was varied (5 x 105, 1 x 106, 5 x 106, and 1 x 107 spirochetes per tube). The growth of spirochetes was monitored by direct enumeration (by dark-field microscopy at x675 magnification) at 24-h intervals for a total of 72 h. Experiments involving B. hermsii were done in exactly the same manner, with the exception that BSK II medium (1) was used. For some experiments, the BSK medium containing the MAbs or normal mouse IgG was heated at 56°C for 30 min to inactivate any possible complement contaminant from the bovine serum albumin present in the medium or from the ascitic fluid from which the MAbs were purified. Indirect analysis of B. burgdorferi growth. In addition to direct enumeration, the growth of B. burgdorferi was measured spectrophotometrically by the change in color of the pH indicator in the medium as the cultures grew (the phenol red in the medium changes from red to yellow as spirochete density and metabolic activity increase, causing the medium to become increasingly more acidic). A scan of fresh BSK medium and BSK medium from a fully grown culture of B. burgdorferi was conducted to determine the optimum wavelength for measuring color change in the medium. A wavelength of 570 nm was chosen because it gave the greatest separation of absorbance values between the fresh and spent medium. For the assay, the spirochetes were removed by centrifugation at 7,000 x g for 10 min and the A570 for each sample was determined. At this wavelength, the absorbance readings were directly proportional to the amount of red in the medium, with the result that medium from cultures with the highest spirochete densities (and increased yellow color) actually had lower absorbance readings. To correct for this, the absorbance value for each sample was subtracted from the absorbance value of fresh BSK medium. Electron microscopy. For the ultrastructural analysis of damage to B. burgdorferi exposed to MAbs, B31 cultures were centrifuged and resuspended in BSK medium to a density of 5 x 107/ml. Aliquots of 100 ,ul received MAbs or normal mouse IgG to a concentration of 2.0 ,ug/ml. After incubation at 33°C for 1 h, the spirochetes were centrifuged and the pellets were fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate (pH 7.4) at 4°C. This was followed by fixation in 1% OS04 and dehydration through a graded ethanol series. The specimens were then embedded in Epon. Staining of thin sections was done with uranyl acetate and lead citrate. Specimens were observed in a transmission electron microscope (Carl Zeiss Instruments, Thornwood, N.Y.).
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FIG. 1. Western blot reactivities of MAbs to the OspB antigen of B. burgdorferi. Lanes: 1, CB2; 2, CB2 Fab fragment; 3, CB6; 4, CB6 Fab fragment. Approximately 10 ,ug of B. bwgdorferi was loaded per gel lane. All MAbs were used at 10 pg/ml. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG, used at 1 p,g/ml. SDS-PAGE was run under reducing conditions. Molecular mass is expressed in kilodaltons.
course of adhesion studies with these MAbs, we found evidence that some of them exhibited bactericidal properties. In a series of preliminary experiments to investigate this phenomenon, B. burgdorferi B31 was cultured in the presence of a range of MAb concentrations (from 0.02 to 10.0 Fg/ml) to assess their effects on the in vitro growth of the spirochete. In these experiments, the three MAbs to OspA (two of which were of the IgM class and one of which was an IgG3, all with kappa light chains) had no effect on the growth of B. burgdorferi over a 72-h period. The growth curve of B. burgdorferi in the presence of these MAbs was similar to that obtained for B. burgdorferi grown in the presence of identical levels of normal mouse IgG (data not shown). For this reason, these MAbs were not considered further in this study. CB2 and CB6 (IgGl and IgG3, respectively, with kappa light chains), both of which are directed to OspB, showed inhibitory and bactericidal effects on the growth of B. burgdorferi cultures at all MAb levels tested. These two MAbs were therefore selected and used for all experiments described hereafter. Since the results of these experiments were dependent upon direct enumeration of spirochetes under dark-field microscopy, microagglutination of spirochetes in the presence of outer surface-specific CB2 and CB6 could result in inaccurate counts. To avoid this, we prepared Fab fragments from CB2 and CB6. Figure 1 shows the Western blots of each MAb and its corresponding Fab fragment against B.
burgdorferi. Growth of B.
bugdorferi
in the presence of MAbs. (i)
Effect(s) of MAb concentration. The effects of increasing concentrations of CB2, CB6, and their Fab fragments on the in vitro growth of B. burgdorferi B31 were investigated. One-milliliter cultures received MAbs or normal mouse IgG at concentrations ranging from 0.02 to 2.0 p,g/ml and an inoculum of 5 x 106 B. burgdorferi organisms. Growth was measured by dark-field microscopy over a period of 72 h. All
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TIME (hours) in the presence of CB2-W (W, whole immunoglobulin molecule), CB2 Fab fragment, CB6-W, and CB6 FIG. 2. Growth of B. burgdorfeC Fab fragment: effect of MAb concentration. One-milliliter cultures received a range of MAb, Fab fragment, or normal mouse IgG concentrations (0.02 to 2.0 p.g/ml) and then were inoculated with 5 x 106 B. burgdorfern organisms. Symbols: A\, control with no antibody; 0, control with normal mouse IgG at 2.0 xg/ml; 0, MAb at 0.02 ,ug/ml; *, MAb at 0.1 Fg/ml; *, MAb at 1.0 ,uLg/ml; 0, MAb at 1.5 ,ug/ml; U, MAb at 2.0 ,ug/ml. measurements were compared with the normal growth rate of this strain in medium alone. The results of a representative experiment are summarized in Fig. 2. This experiment was repeated three times as described above (and numerous times with minor variations) and in duplicate for each experiment with high reproducibility. With the exception of the culture exposed to CB6 Fab at 0.02 ,uglml, B.
burgdorferi densities in cultures exposed to all MAbs and
Fab fragnents were below those of the controls. This inhibitory effect occurred in a concentration-dependent manner over the full 72 h. In addition, we noted a bactericidal effect at the 24-h time period with CB2 and CB2 Fab at
concentrations of 1.0, 1.5, and 2.0 jig/ml. With CB6 and CB6 Fab, this bactericidal effect was apparent at concentrations of 2.0 ,ug/ml for CB6 and 1.5 and 2.0 p,g/ml for CB6 Fab. The 50% lethal dose concentrations of CB2 (0.7 jig/ml), CB2 Fab (1.0 ,ug/ml), CB6 (1.0 p.g/ml), and CB6 Fab (1.2 ,ug/ml) for B. burgdorferi for the first 24 h of incubation were calculated by probit analysis from the data in Fig. 2. To test for the possibility that the inhibitory and bactericidal activity of these MAbs could be due to nonspecific toxic substances derived from the ascitic fluid, we investigated the effect of CB2 on the in vitro growth of a related spirochete, B. hennsii, which is not recognized by this MAb.
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CB2 had no effect on the growth of B. hermsii cultures. In another experiment, CB2 also failed to inhibit the growth of B. burgdorfei BEP4, which is not recognized by this MAb. In addition, affinity-purified MAb from CB2 hybridoma supernatant had the same effect on B. burgdorferi B31 that the affinity-purified IgG from ascitic fluid did (data not
shown). To investigate the possibility that complement could be present as a contaminant of bovine serum albumin in BSK medium or in the ascitic fluid-derived MAbs and thus be responsible for the bactericidal properties noted in the assay, we heat treated the medium and the antibodies (MAbs and normal mouse IgG at 2.0 ,ug/ml) for 30 min at 56°C. After monitoring by dark-field microscopy for 72 h, we found that CB2 and CB6 retained their bactericidal properties (data not shown). (ii) Effect(s) of inoculum size. We investigated the effects of initial inoculum size (B. burgdorferi B31) on the inhibitory and bactericidal properties of CB2 and CB6. One-milliliter culture tubes containing BSK medium and CB2 and CB6 at a fixed concentration of 2.0 ,ug/ml were prepared. Control antibody tubes were also made with normal mouse IgG at 2.0 p,g/ml. To these tubes were added increasing amounts of B. burgdorferi (5 x 105, 1 x 106, 5 x 106, and 1 x 107 spirochetes per 1-ml tube). A representative experiment is summarized in Fig. 3. At all four inoculum sizes, the growth of B. burgdorferi was depressed in comparison with that of the controls. As an alternative to direct enumeration, we also assessed spirochete growth indirectly by measuring the loss of the red color of the phenol red indicator in the medium by spectrophotometry. Metabolic activity of the spirochetes exposed to CB2 and CB6 was depressed in comparison with that of the control which received normal mouse IgG. This pattern held for inocula of 5 x 105, 1 x 106, 5 x 106, and 1 x 107 organisms (Fig. 4). Ultrastructural evidence of damage to B. burgdorfeni exposed to MAbs. Because of the bactericidal activity of the MAbs noted earlier, we sought to obtain direct evidence of spirochete damage after exposure to MAbs. In this experi-
ment, 5 x 107 B. burgdorferi organisms were exposed to 2.0-,ug/ml levels of CB2 for 1 h at 330C. After being fixed, sectioned, and stained, the spirochetes were examined in a transmission electron microscope and showed evidence of damage to the spirochetal outer envelope (Fig. 5A). The control, exposed to normal mouse IgG (2.0 p,g/ml), remained unaffected (Fig. SB). Selection of OspB- escape variants. Despite the marked bactericidal and/or inhibitory effects described by the data in Fig. 2, 3, and 4, a small number of surviving organisms was often seen in the B. burgdorfen-MAb assays. To investigate the possibility that these survivors were escape variants, B. burgdorfien (B31, uncloned) was exposed to CB2 at 2.0 p,g/ml. After 72 h in culture, the surviving organisms were analyzed by SDS-PAGE and Western blotting. If survival was the result of a mutation in the gene for the epitope recognized by CB2 in OspB, the entire molecule would still be detected by SDS-PAGE, but CB2 would no longer react in Western blots. Alternatively, if the plasmid which codes for OspA and OspB was lost, this loss would be apparent in SDS-PAGE by the absence of both proteins. The survivors failed to express OspB. A Coomassie brilliant blue-stained gel of survivors which are OspA+ and OspB- is shown in Fig. 6A. Stock B. burgdorfen, but not survivors, reacted with CB2 in Western blots (Fig. 6B). Both survivors and stock organisms reacted to MAb llGl (7), confirming the presence of OspA in both populations (Fig. 6B). While the survivors failed to express OspB, they did, however, express another protein with an apparent molecular mass of 21.5 kDa not present in the parent B31 population (Fig. 6A) but which did not react with the MAbs (Fig. 6B). Survivors have grown normally in medium containing up to 10 ,ug of CB2 per ml for several passages without expression of OspB. DISCUSSION We have selected an escape variant of B. burgdorferi which does not express OspB after treatment with bacteri-
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FIG. 4. Spectrophotometric analysis of spirochete growth as determined by the change in color of the phenol red indicator in the medium. The MAb concentration was held constant at 2.0 ,ug/ml. One-milliliter cultures were inoculated with 5 x 105 (A), 1 x 106 (B), 5 x 106 (C), and 1 x 107 (D) B. burgdorferi organisms. After 72 h, the tubes were centrifuged and theA570 of the supernatants was measured. y axis (optical density) represents the absorbance of fresh BSK medium minus the absorbance of sample supernatant.
cidal MAbs directed against antigenic determinants present in this protein. The mechanism of action of these MAbs is not known at present. However, the bactericidal properties of these antibodies are not due to agglutination since Fab fragments of the affinity-purified IgG had similar effects on the organisms (Fig. 2). Furthermore, had agglutination been the reason for the decreased numbers of organisms detected after exposure to the MAbs, the indirect measurement of spirochete viability by spectrophotometric analysis of the color change of the phenol red indicator in the medium would have demonstrated metabolic activity similar to that of controls exposed to normal mouse IgG (Fig. 4). Agreement between the enumeration of the organisms and the lack of metabolic activity in the MAb-treated cultures strengthens the conclusion that the growth of the organisms was inhibited at the lower concentrations of MAbs and that the organisms were killed at the higher concentrations. The bactericidal effects of the MAbs did not appear to be due to a possible presence of nonspecific toxic compounds in the ascites. CB2 did not have an effect on the growth of B. hermsii, the agent of American relapsing fever, nor on the growth of the BEP4 strain of B. burgdorferi, which is not recognized by this MAb. Affinity-purified IgG from hybridoma supernatants of both CB2 and CB6 was found to have the same inhibitory and bactericidal action on B. burgdorferi that the affinity-purified IgG from ascitic fluid did. Lysis of B. burgdorferi by immune serum and complement
(25, 28) and immobilization of the organisms (14) have been demonstrated. The assay system used in our studies did not contain complement because the MAbs were used in the form of affinity-purified IgG from ascitic fluid and the spirochete medium was serum free. Nonetheless, medium containing MAbs was heat treated at 56°C for 30 min to inactivate any possible contaminating complement (possibly derived from the MAbs or from the bovine serum albumin in the medium). Heat treatment did not result in a decrease in inhibitory or bactericidal effects by the MAbs in the assay. Murine IgGl does not bind complement, and CB2 belongs to that subclass. From these observations, we can conclude that the inhibitory and bactericidal effects of CB2 and CB6 may be due to a unique action of these IgG antibodies on a still unknown epitope of OspB, which in turn has a disruptive effect on the outer envelope. Transmission electron microscopy of spirochetes treated with CB2 showed such a disruption of the outer envelope as compared with the intact organisms seen after treatment with normal mouse IgG (Fig. 5). Yet, the ultrastructural observations are of limited value in determining the mechanism of action of these MAbs, since some of the organisms were not bound by CB2 and others were only at one stage in the process of destruction. In this regard, recent evidence has shown that both OspA and OspB can be found in the periplasmic space, the protoplasmic cylinder, and the cytoplasmic membranes, as well as the outer enve-
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FIG. 6. (A) SDS-PAGE analysis. Lanes: 1, molecular mass standards; 2, stock B. burgdorferi B31; 3, OspB- escape variant. Arrowhead, absence of OspB; *, 21.5-kDa protein. Approximately 70 pg of antigen was loaded per gel lane. Gel contained 10% acrylamide and was run under reducing conditions. Staining was by Coomassie brilliant blue. Molecular mass is in kilodaltons. (B) Western blots confirming absence of OspB and presence of OspA in OspB- escape variant. Lanes: 1, lack of reactivity of CB2 to OspBescape variant; 2, reactivity of CB2 to stock B. burgdorferi; 3, reactivity of MAb llGl to OspB- escape variant; 4, reactivity of llGl to stock B. burgdorferi. llGl is directed against B. burgdorferi OspA and has been described previously (7). Reactivity of llGl to stock B. burgdorferi and OspB- variant confirms the presence of OspA in both strains. Approximately 10 ,ug of B. burgdorferi was loaded per gel lane. SDS-PAGE was run under reducing conditions in a gel of 10% acrylamide. MAbs were used at 10 Rg/ml. Secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG used at 1 gg/ml.
FIG. 5. (A) Thin sections of B. burgdorfeni B31 after exposure to CB2 (2.0 iLg/ml) for 1 h at 33°C; (B) control thin section after exposure of B. burgdorferi to normal mouse IgG (2.0 Fg/ml) for 1 h at 33°C. Bars, 0.2 ,um.
lope (11). This finding is relevant because if MAbs to OspB could gain entrance to the organism beyond the outer envelope, and bind internal OspB, the presence of such a complex may be sufficient to destroy the organism. It has been shown that immunogold electron microscopy of B. burgdorfen fixed in formalin after exposure to primary (MAb to OspA) and secondary antibodies showed aggregations of the gold particles. This phenomenon, not noted if the organisms were fixed before antibody treatment, was defined as patching or as a passive two-dimensional aggregation of
molecules in a fluid membrane (4). Leptospira spp. have been shown to permit longitudinal movement of antigens in the outer envelope (15), and this phenomenon may also occur in Borrelia spp. The escape variants selected by treatment with CB2 and CB6 failed to express OspB (Fig. 6). The variants also expressed a protein with a molecular mass of 21.5 kDa which was not present in the parent B31 population. Similar lower-molecular-mass proteins (21 and 18.5 kDa) in cloned strains which do not express or have altered OspB have been shown before (12). Unlike the reactivity to anti-OspB MAbs shown in this study (12), we did not find reactivity of the selecting MAbs for the 21.5-kDa polypeptide. Since both OspA and OspB are encoded by genes located in a 49-kb linear duplex plasmid with covalently closed ends (2, 23, 24), the fact that OspA was still expressed (Fig. 6) indicates that the entire plasmid was not lost and that an OspB- variant was present in the original heterogeneous B31 population. OspB- variants have been demonstrated before (3, 12, 35) as naturally occurring in various strains of B. burgdorferi. Loss of plasmids as a result of continuous in vitro cultivation has been documented and linked to a loss of infectivity of the strain for rodents (29). In fact, phenotypic expression of OspB has been lost in a heterogeneous strain of a human blood isolate after 11 to 15 in vitro passages (30, 31) and by experimentally transposable elements inserted in the OspA gene (23). If selection of OspB- variants could occur in vivo as a result of antibodies such as that which occurred in this
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experimental system, the organisms could avoid a potentially destructive antibody effect and prolong the illness. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI 27044 from the National Institutes of Health and by a grant from the G. Harold and Leila Y. Mathers Charitable Foundation to J.L.B. We appreciate the critical reviews of Jorge Galan of the Department of Microbiology and Gail S. Habicht of the Department of Pathology, State University of New York at Stony Brook. REFERENCES 1. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. 2. Barbour, A. G., and C. F. Garon. 1987. Linear plasmids of the bacterium Borrelia burgdorfeni have covalently closed ends. Science 237:409-411. 3. Barbour, A. G., S. L. Tessier, and S. F. Hayes. 1984. Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45:94-100. 4. Barbour, A. G., S. L. Tessier, and W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41:795-804. 5. Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G. Barbour, W. Burgdorfer, R. Edelman, and R. A. Kaslow. 1983. Spirochetes isolated from the blood of two patients with Lyme disease. N. Engl. J. Med. 308:740-742. 6. Benach, J. L., J. L. Coleman, J. C. Garcia-Monco, and P. C. Deponte. 1988. Biological activity of Borrelia burgdorferi antigens. Ann. N.Y. Acad. Sci. 539:115-125. 7. Benach, J. L., J. L. Coleman, and M. G. Golightly. 1988. A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete. J. Immunol. 140:265272. 8. Benach, J. L., H. B. Fleit, G. S. Habicht, J. L. Coleman, E. M. Bosler, and B. P. Lane. 1984. Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor. J. Infect. Dis. 150:497-507. 9. Bergstrom, S., V. G. Bundoc, and A. G. Barbour. 1989. Molecular analysis of linear plasmid-encoded major surface proteins, Osp A and Osp B, of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol. 3:479-486. 10. Brandt, M. E., B. S. Riley, J. D. Radolf, and M. V. Norgard. 1990. Immunogenic integral membrane proteins of Borrelia burgdorfen are lipoproteins. Infect. Immun. 58:983-991. 11. Brusca, J. S., A. W. McDowall, M. V. Norgard, and J. D. Radolf. 1991. Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorfeni. J. Bacteriol. 173:8004-8008. 12. Bundoc, V. G., and A. G. Barbour. 1989. Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi. Infect. Immun. 57:2733-2741. 13. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease-a tick-borne spirochetosis? Science 216:1317-1319. 14. Callister, S. M., R. F. Schell, and S. D. Lovrich. 1991. Lyme disease assay which detects killed Borrelia burgdorferi. J. Clin. Microbiol. 29:1773-1776. 15. Charon, N. W., C. W. Lawrence, and S. O'Brien. 1981. Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans. Proc. Natl. Acad. Sci. USA 78:71667170. 16. Coleman, J. L., and J. L. Benach. 1987. Isolation of antigenic components from the Lyme disease spirochete: their role in early diagnosis. J. Infect. Dis. 155:756-765.
INFECT. IMMUN.
17. Coleman, J. L., and J. L. Benach. 1989. Identification and characterization of an endoflagellar antigen of Borrelia burgdorferi. J. Clin. Invest. 84:322-330. 18. Coleman, J. L., and J. L. Benach. 1992. Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria. J. Infect. Dis. 165:658-666. 19. Craft, J. E., D. K. Fischer, G. T. Shimamoto, and A. C. Steere. 1986. Antigens of Borrelia burgdorfen recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J. Clin. Invest. 78:934-939. 20. Fikrig, E., S. W. Barthold, F. S. Kantor, and R. A. Flavell. 1990. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 250:553-556. 21. Garcia-Monco, J. C., B. Fernandez-Vldlar, and J. L. Benach. 1989. Adherence of the Lyme disease spirochete to glial cells and cells of glial origin. J. Infect. Dis. 160:497-506. 22. Garcia-Monco, J. C., B. Fernandez-Villar, R. C. Rogers, A. Szczepanski, C. M. Wheeler, and J. L. Benach. Borrelia burgdorfen and other related spirochetes bind to galactocerebroside. Neurology, in press. 23. Howe, T. R, F. W. LaQuier, and A. G. Barbour. 1986. Organization of genes encoding two outer membrane proteins of the Lyme disease agent Borrelia burgdorferi within a single transcriptional unit. Infect. Immun. 54:207-212. 24. Howe, T. R., L. W. Mayer, and A. G. Barbour. 1985. A single recombinant plasmid expressing two outer surface proteins of the Lyme disease spirochete. Science 227:645-646. 25. Kochi, S. K., and R. C. Johnson. 1988. Role of immunoglobulin G in killing of Borrelia burgdorferi by the classical complement pathway. Infect. Immun. 56:314-321. 26. Kurtti, T. J., U. G. Munderloh, G. G. Ahlstrand, and R C. Johnson. 1988. Borrelia burgdorfen in tick cell culture: growth and cellular adherence. J. Med. Entomol. 25:256-261. 27. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packing events. J. Mol. Biol. 80:575599. 28. Lovrich, S. D., S. M. Callister, J. L. Schmitz, J. D. Alder, and R. F. Schell. 1991. Borreliacidal activity of sera from hamsters infected with the Lyme disease spirochete. Infect. Immun. 59:2522-2528. 29. Schaible, U. E., M. D. Kramer, K. Eichmann, M. Modoleli, C. Museteanu, and M. M. Simon. 1990. Monoclonal antibodies specific for the outer surface protein A (Osp A) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci. USA 87: 3768-3772. 30. Schwan, T., and W. Burgdorfer. 1987. Antigenic changes of Borrelia burgdorferi as a result of in vitro cultivation. J. Infect. Dis. 156:852-853. 31. Schwan, T., W. Burgdorfer, and C. F. Garon. 1988. Changes in the infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect. Immun. 56:1831-1836. 32. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista. 1983. The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308:733-740. 33. Szczepanski, A., M. B. Furie, J. L. Benach, B. P. Lane, and H. B. Fleit. 1990. Interaction between Borrelia burgdorferi and endothelium in vitro. J. Clin. Invest. 85:1637-1647. 34. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 35. Wilske, B., V. Preac-Mursic, G. Schierz, and K. Busch. 1986. Immunochemical and immunological analysis of European Borrelia burgdorferi strains. Zentralbl. Bakteriol. Hyg. A 263:92102.
