Selection of an Escape Variant of Borrelia burgdorferi by Use

Transcription

1 INFECTION ND IMMUNITY, ug. 1992, p /92/8398-7$2./ Copyright X 1992, merican Society for Microbiology Vol. 6, No. 8 Selection of an Escape Variant of Borrelia burgdorferi by Use of Bactericidal Monoclonal ntibodies to OspB JMES L. COLEMN,' RENE C. ROGERS,' ND JORGE L. BENCHl 2* State ofnew York Department of Health1 and Department ofpathology,2 State University ofnew York at New York Stony Brook, Stony Brook; Received 28 January 1992/ccepted 7 May 1992 Two immunoglobulin G (IgG) monoclonal antibodies (Mbs) 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 Mbs showed the same effects, indicating that they were not caused by agglutination of the organisms by the intact Mbs. The inhibition of spirochete growth in cultures containing Mbs was also detected by spectrophotometric analysis of the media. CB2 did not inhibit the growth ofborrelia hermsii or the BEP4 strain of B. burgdorferi, neither of which is recognized by the Mb. ffinity-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 Mbs 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 Osp 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, Osp and OspB (3, 4, 7). lthough these two lipoproteins (9, 1) 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). Osp has been proposed as a suitable vaccine candidate since these outer surface proteins are recognized readily by the immune system in rodent models (2, 29). Protection conferred by Osp 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 Osp 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 (Mbs) to Osp and OspB to attempt selective inhibition of the binding process. Through the course of these investigations, we noted that some of these Mbs 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 Mbs to OspB and the * Corresponding author. 398 selection of an escape variant which does not express this antigen. MTERILS ND 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). Mb production and purification. Whole, washed B. burgdorferi B31 spirochetes were used to immunize BLB/c mice. The immunization regimen, fusion, screening, subcloning, and isotyping of clones have been described previously (7, 17, 18). Mbs 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 affinity chromatography (ImmunoPure Fab Preparation Kit; Pierce, Rockford, Ill.). Mbs of the immunoglobulin M (IgM) class were partially purified by precipitation in 4% ammonium sulfate. The Mbs were characterized for their antigenic specificities by Western blots (immunoblots) against wholecell lysates of B. burgdorferi. SDS-PGE and Western blotting. B. burgdorferi organisms were harvested from the medium by centrifugation at 7, x g for 2 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-PGE) was done by using a Laemmli buffer system (27) in gels of 1% acrylamide. ll other conditions have been described previously (7, 17, 18). West-

2 VOL. 6, 1992 ESCPE VRINTS SELECTED BY Mbs TO B. BURGDORFER 399 em blotting was also done as described previously (7, 17, 18, 34) Ġrowth of B. burgdorferi in the presence of Mbs. B. burgdorferi organisms (5 x 16) were added to sterile tubes (Sarstedt, Newton, N.C.) containing 1 ml of serum-free BSK medium with a range of affinity-purified Mb concentrations (.2,.1, 1., 1.5, and 2.,ug/ml) and incubated at 33 C. Protein determinations of Mbs were made by using a kit (BC 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 Mb and normal mouse IgG concentrations were held at 2.,ug/ml and the size of the B. burgdorferi inoculum was varied (5 x 15, 1 x 16, 5 x 16, and 1 x 17 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 Mbs or normal mouse IgG was heated at 56 C for 3 min to inactivate any possible complement contaminant from the bovine serum albumin present in the medium or from the ascitic fluid from which the Mbs 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). 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. wavelength of 57 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, x g for 1 min and the 57 for each sample was determined. t 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 Mbs, B31 cultures were centrifuged and resuspended in BSK medium to a density of 5 x 17/ml. liquots of 1,ul received Mbs or normal mouse IgG to a concentration of 2.,ug/ml. fter incubation at 33 C for 1 h, the spirochetes were centrifuged and the pellets were fixed in 3% glutaraldehyde in.2 M sodium cacodylate (ph 7.4) at 4 C. This was followed by fixation in 1% OS4 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.). RESULTS Mbs. The Mbs used in this study were all directed against either Osp or OspB of B. burgdorfen. During the kda l l l l U U * - FIG. 1. Western blot reactivities of Mbs to the OspB antigen of B. burgdorferi. Lanes: 1, CB2; 2, CB2 Fab fragment; 3, CB6; 4, CB6 Fab fragment. pproximately 1,ug of B. bwgdorferi was loaded per gel lane. ll Mbs were used at 1 pg/ml. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG, used at 1 p,g/ml. SDS-PGE was run under reducing conditions. Molecular mass is expressed in kilodaltons. course of adhesion studies with these Mbs, 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 Mb concentrations (from.2 to 1. Fg/ml) to assess their effects on the in vitro growth of the spirochete. In these experiments, the three Mbs to Osp (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 Mbs 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 Mbs 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 Mb levels tested. These two Mbs 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 Mb and its corresponding Fab fragment against B. burgdorferi. Growth of B. bugdorferi in the presence of Mbs. (i) Effect(s) of Mb 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 Mbs or normal mouse IgG at concentrations ranging from.2 to 2. p,g/ml and an inoculum of 5 x 16 B. burgdorferi organisms. Growth was measured by dark-field microscopy over a period of 72 h. ll

3 C/) 31 COLEMN ET L. c 5 o) I3Iol 1 1 E INFECr. IMMUN. TIME (hours) Growth of B. burgdorfecin the presence of CB2-W (W, whole immunoglobulin molecule), CB2 Fab fragment, CB6-W, and CB6 FIG. 2. Fab fragment: effect of Mb concentration. One-milliliter cultures received a range of Mb, Fab fragment, or normal mouse IgG concentrations (.2 to 2. p.g/ml) and then were inoculated with 5 x 16 B. burgdorfern organisms. Symbols: \, control with no antibody;, control with normal mouse IgG at 2. xg/ml;, Mb at.2,ug/ml; *, Mb at.1 Fg/ml; *, Mb at 1.,uLg/ml;, Mb at 1.5,ug/ml; U, Mb at 2.,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.2,uglml, B. burgdorferi densities in cultures exposed to all Mbs 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., 1.5, and 2. jig/ml. With CB6 and CB6 Fab, this bactericidal effect was apparent at concentrations of 2.,ug/ml for CB6 and 1.5 and 2. p,g/ml for CB6 Fab. The 5% lethal dose concentrations of CB2 (.7 jig/ml), CB2 Fab (1.,ug/ml), CB6 (1. 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 Mbs 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 Mb.

4 VOL. 6, 1992 ESCPE VRINTS SELECTED BY Mbs TO B. BURGDORFERI 311 E e r 175 = - 15 NMG C CB2 co 1 O CB 4).n M, 75- ) co 25- Or O-d _ inoculum size in millions FIG. 3. Effect of inoculum size on growth of B. burgdorferi in the presence of CB2 and CB6. One-milliliter cultures received a fixed concentration of Mbs (2.,ug/ml) and then were inoculated with increasing numbers of B. burgdorferi (5 x 15, 1 x 16, 5 x 16, and 1 x 17). Counts were made at 24 (), 48 (B), and 72 (C) h. 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 Mb. In addition, affinity-purified Mb 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 Mbs and thus be responsible for the bactericidal properties noted in the assay, we heat treated the medium and the antibodies (Mbs and normal mouse IgG at 2.,ug/ml) for 3 min at 56 C. fter 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.,ug/ml were prepared. Control antibody tubes were also made with normal mouse IgG at 2. p,g/ml. To these tubes were added increasing amounts of B. burgdorferi (5 x 15, 1 x 16, 5 x 16, and 1 x 17 spirochetes per 1-ml tube). representative experiment is summarized in Fig. 3. t all four inoculum sizes, the growth of B. burgdorferi was depressed in comparison with that of the controls. s 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 15, 1 x 16, 5 x 16, and 1 x 17 organisms (Fig. 4). Ultrastructural evidence of damage to B. burgdorfeni exposed to Mbs. Because of the bactericidal activity of the Mbs noted earlier, we sought to obtain direct evidence of spirochete damage after exposure to Mbs. In this experiment, 5 x 17 B. burgdorferi organisms were exposed to 2.-,ug/ml levels of CB2 for 1 h at 33C. fter 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. 5). The control, exposed to normal mouse IgG (2. 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-mb assays. To investigate the possibility that these survivors were escape variants, B. burgdorfien (B31, uncloned) was exposed to CB2 at 2. p,g/ml. fter 72 h in culture, the surviving organisms were analyzed by SDS-PGE 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-PGE, but CB2 would no longer react in Western blots. lternatively, if the plasmid which codes for Osp and OspB was lost, this loss would be apparent in SDS-PGE by the absence of both proteins. The survivors failed to express OspB. Coomassie brilliant blue-stained gel of survivors which are Osp+ and OspB- is shown in Fig. 6. Stock B. burgdorfen, but not survivors, reacted with CB2 in Western blots (Fig. 6B). Both survivors and stock organisms reacted to Mb llgl (7), confirming the presence of Osp 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. 6) but which did not react with the Mbs (Fig. 6B). Survivors have grown normally in medium containing up to 1,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-

5 312 COLEMN ET L. INFECT. IMMUN. * NMIgG._ n.3 - E El CB2 CB C.) =X--._ U) a(._ C.) I- C FIG. 4. Spectrophotometric analysis of spirochete growth as determined by the change in color of the phenol red indicator in the medium. The Mb concentration was held constant at 2.,ug/ml. One-milliliter cultures were inoculated with 5 x 15 (), 1 x 16 (B), 5 x 16 (C), and 1 x 17 (D) B. burgdorferi organisms. fter 72 h, the tubes were centrifuged and the57 of the supernatants was measured. y axis (optical density) represents the absorbance of fresh BSK medium minus the absorbance of sample supernatant. cidal Mbs directed against antigenic determinants present in this protein. The mechanism of action of these Mbs 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 Mbs, 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). greement between the enumeration of the organisms and the lack of metabolic activity in the Mb-treated cultures strengthens the conclusion that the growth of the organisms was inhibited at the lower concentrations of Mbs and that the organisms were killed at the higher concentrations. The bactericidal effects of the Mbs 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 merican relapsing fever, nor on the growth of the BEP4 strain of B. burgdorferi, which is not recognized by this Mb. ffinity-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 ofb. burgdorferi by immune serum and complement. I I B D (25, 28) and immobilization of the organisms (14) have been demonstrated. The assay system used in our studies did not contain complement because the Mbs were used in the form of affinity-purified IgG from ascitic fluid and the spirochete medium was serum free. Nonetheless, medium containing Mbs was heat treated at 56 C for 3 min to inactivate any possible contaminating complement (possibly derived from the Mbs or from the bovine serum albumin in the medium). Heat treatment did not result in a decrease in inhibitory or bactericidal effects by the Mbs 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 Mbs, 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 Osp and OspB can be found in the periplasmic space, the protoplasmic cylinder, and the cytoplasmic membranes, as well as the outer enve-

6 VOL. 6, 1992 ESCPE VRINTS SELECTED BY Mbs TO B. BURGDORFERI 313 B I1-84- Yt.. 4;F t:*4.w-, FIG. 5. () Thin sections ofb. burgdorfeni B31 after exposure to CB2 (2. ilg/ml) for 1 h at 33 C; (B) control thin section after exposure of B. burgdorferi to normal mouse IgG (2. Fg/ml) for 1 h at 33 C. Bars,.2,um. lope (11). This finding is relevant because if Mbs 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 (Mb to Osp) 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 FIG. 6. () SDS-PGE analysis. Lanes: 1, molecular mass standards; 2, stock B. burgdorferi B31; 3, OspB- escape variant. rrowhead, absence of OspB; *, 21.5-kDa protein. pproximately 7 pg of antigen was loaded per gel lane. Gel contained 1% 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 Osp 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 Mb llgl to OspB- escape variant; 4, reactivity of llgl to stock B. burgdorferi. llgl is directed against B. burgdorferi Osp and has been described previously (7). Reactivity of llgl to stock B. burgdorferi and OspB- variant confirms the presence of Osp in both strains. pproximately 1,ug of B. burgdorferi was loaded per gel lane. SDS-PGE was run under reducing conditions in a gel of 1% acrylamide. Mbs were used at 1 Rg/ml. Secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG used at 1 gg/ml. 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 Mbs shown in this study (12), we did not find reactivity of the selecting Mbs for the 21.5-kDa polypeptide. Since both Osp and OspB are encoded by genes located in a 49-kb linear duplex plasmid with covalently closed ends (2, 23, 24), the fact that Osp 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 (3, 31) and by experimentally transposable elements inserted in the Osp gene (23). If selection of OspB- variants could occur in vivo as a result of antibodies such as that which occurred in this

7 314 COLEMN ET L. experimental system, the organisms could avoid a potentially destructive antibody effect and prolong the illness. CKNOWLEDGMENTS This work was supported by Public Health Service grant I 2744 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,. G Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57: Barbour,. G., and C. F. Garon Linear plasmids of the bacterium Borrelia burgdorfeni have covalently closed ends. Science 237: Barbour,. G., S. L. Tessier, and S. F. Hayes Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45: Barbour,. G., S. L. Tessier, and W. J. 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