Protection of C3H/HeN mice from challenge with Borrelia burgdorferi through active immunization with OspA, OspB, or OspC, but not with OspD or the 83-kilodalton antigen.

Recent advances in the development of animal models for Lyme borreliosis have provided means of identifying potential targets for the design of a subunit vaccine to prevent this disease. The C3H/HeN mouse model was used to study several Borrelia burgdorferi antigens from a single isolate for their ability to elicit borreliacidal and protective antibodies. The ospA, ospB, ospC, ospD, and 83-kDa genes from a California isolate, SON 188, were cloned and expressed in Escherichia coli as proteins fused to the C-terminal end of maltose-binding protein. Active immunization of mice with these fusion proteins elicited high titers of antibodies that recognized the homologous SON 188 antigens upon immunoblotting. Antibodies generated to the OspA and OspB fusion proteins, but not to the OspC, OspD, and the 83-kDa fusion proteins, demonstrated in vitro borreliacidal activity. Challenge of all actively immunized mice with 10(7) SON 188 spirochetes resulted in infection in all mice receiving the OspD or 83-kDa immunogens but not in any mice receiving the OspA, OspB, or OspC fusion proteins. These results demonstrate the potential of OspA, OspB, and OspC as components of a subunit vaccine for the prevention of Lyme borreliosis.     

Protective immunity is induced by a Borrelia burgdorferi mutant that lacks OspA and OspB.

A mutant of virulent Borrelia burgdorferi 297 was apparently selected for by long-term storage at 5 degrees C. This mutant was found to lack the plasmid which encodes outer surface protein A (OspA) and OspB. In addition to the loss of the OspA and OspB proteins, the mutant lacked two lipoproteins, of 20 and 7.5 kDa, that were observed in the wild type. Since the mutant was not recovered from the tissues or blood of hamsters injected with the mutant, the mutant was determined to be noninfectious. Hamsters vaccinated with noninfectious mutant 297 were protected completely from challenge with virulent wild-type 297 spirochetes. Prechallenge sera from hamsters vaccinated with mutant 297 lacked antibodies to OspA and OspB, while those from hamsters vaccinated with virulent wild-type 297 or avirulent 297 exhibited antibodies to these proteins. Hamsters vaccinated with virulent wild-type 297 or mutant 297 elicited antibodies to OspC and a 39-kDa protein (P39), whereas hamsters vaccinated with avirulent 297 lacked these antibodies. These results suggest that OspC and/or P39 are important for the development of a protective immune response. Study of this mutant may elucidate factors important to the development of a Lyme disease vaccine.     

Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi.     

Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess B-cell mitogenic and cytokine-stimulatory properties.

Sonicated Borrelia burgdorferi was previously reported to possess both B-cell mitogenic and interleukin-6 (IL-6) stimulatory activities. In this report, two outer surface lipoproteins, OspA and OspB, were purified from B. burgdorferi and assessed for the presence of these functions. OspA was purified from two strains, an OspB-deficient variant of HB19 and N40, while OspB was purified from the N40 strain. All lipoprotein preparations were free of endotoxin contamination, and polymyxin B failed to inhibit responses, indicating that media contamination was not contributing to biological assays. All three preparations were able to stimulate proliferation of mononuclear cells from naive C3H/HeJ and BALB/c mice. Depletion experiments indicated that the responding cells were B lymphocytes and not T lymphocytes. Purified OspA and OspB stimulated immunoglobulin M production by splenocyte cultures from naive mice, a property also previously attributed to sonicated B. burgdorferi. OspA and OspB also stimulated the production of IL-6 and tumor necrosis factor alpha by bone marrow-derived macrophages from BALB/c and C3H/HeJ mice. Cytokine production was enhanced by the presence of gamma interferon in the cultures, indicating that the magnitude of responses to these lipoproteins may be modulated by cytokines in the microenvironment of infected tissues. Human endothelial cells produced IL-6 when incubated with OspA and OspB, indicating that non-hematopoietic lineage cells can respond to the lipoproteins. Purified OspA and OspB had approximately equal activity, with responses detected in the range of 10 ng of lipoprotein per ml to 1 microgram of lipoprotein per ml. Comparison with published dose responses for lipoproteins purified from Escherichia coli indicates that OspA and OspB purified from B. burgdorferi are much more potent. The high potency of the B. burgdorferi lipoproteins and the ability of the spirochete to invade tissues and persist argue that they could be important in the localized events contributing to the pathology of Lyme disease.     

Selection of variant Borrelia burgdorferi isolates from mice immunized with outer surface protein A or B.

A nonclonal population of Borrelia burgdorferi N40 (passage 3) that survived protective immunity following challenge inoculation of outer surface protein (Osp) A- or B-hyperimmunized mice were characterized for the molecular basis of evasion of immunity. Two of six B. burgdorferi isolates, cultured from OspA-immunized mice, had antigenic diversity in the carboxyl terminus of OspA and did not bind to the protective OspA monoclonal antibody designated IXDII. However, OspA-immunized mice challenged with these variants were fully protected. Moreover, B. burgdorferi isolates with a point mutation in ospB, which results in a truncated OspB that does not bind to protective OspB monoclonal antibody 7E6C, were frequently enriched after infection of OspB-immunized mice. These studies suggest that the incomplete efficacy of an OspA- or OspB-based vaccine may be partly due to immunomediated in vivo selective pressure, resulting in the persistence of some spirochetes that do not bind to protective antibodies.     

Specific and nonspecific responses of murine B cells to membrane blebs of Borrelia burgdorferi.

Lymphocyte blastogenesis assays and immunoblotting were used to investigate and compare murine B-cell responses to preparations of extracellular membrane blebs (BAg) and spirochetes (Ag) of Borrelia burgdorferi. Immunoblotting BAg, Ag, and medium control preparations with serum from naive and infected C57BL/10 mice revealed that BAg and Ag had similar specific reactivity profiles except that major antigens of 83, 60, and 41 kDa were detected in Ag but not in BAg. It was determined that 1 microgram (dry weight) of Ag contained 0.0051 and 0.0063 microgram of outer surface proteins A (OspA) and OspB, respectively, whereas 1 microgram (dry weight) of BAg contained 0.0024 microgram of OspA and 0.0015 microgram of OspB. Both BAg and Ag caused blastogenesis in cultures of spleen cells from both groups of mice, but BAg-stimulated lymphocytes exhibited significantly greater (P < or = 0.05) blastogenesis after 2 or 6 days of culture than did lymphocytes stimulated by Ag or medium control. Flow cytometry and antibody capture enzyme-linked immunosorbent assays identified responding lymphocytes as B cells which secreted polyclonal immunoglobulin M (IgM) but not IgG or IgA. Treatment of BAg and lipopolysaccharide controls with polymyxin B resulted in as much as 20.7 and 54.3% mean decreases in blastogenesis, respectively. Fractionation of BAg or Ag by ultracentrifugation before culture with spleen cells from naive mice indicated that B-cell blastogenesis was probably associated with spirochetal membranes. The results of this study demonstrate that specific humoral responses are directed towards extracellular membrane blebs which lack the 83-, 60-, and 41-kDa antigens of intact spirochetes and that blebs also possess significant nonspecific mitogenic activity for murine B cells. This activity was not due entirely to typical lipopolysaccharide or OspA and OspB lipoproteins.     

Borrelia burgdorferi in Tick Cell Culture Modulates Expression of Outer Surface Proteins A and C in Response to Temperature

The Lyme disease spirochete Borrelia burgdorferi sensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding. The switching of these proteins correlates with increased spirochetal infectivity for the mammal. We examined the effect of temperature on differential expression of OspA and OspC by B. burgdorferi cocultivated with a cell line isolated from the vector tick Ixodes scapularis. The effect of incubation at 31, 34, or 37 degrees C on expression of OspA and OspC by B. burgdorferi JMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting. The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31 degrees C and declined with increasing temperature in both strains. OspC production was enhanced in spirochetes cocultivated with tick cells at 37 degrees C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37 degrees C, but OspA content was not appreciably affected by temperature. Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.     

Association of treatment-resistant chronic Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi.

Chronic Lyme arthritis that is unresponsive to antibiotic therapy is associated with an increased frequency of the HLA-DR4 specificity. To determine whether the immune response to a particular polypeptide of Borrelia burgdorferi may be associated with treatment-resistant chronic Lyme arthritis, we correlated the clinical courses and HLA-DR specificities of 128 patients with Lyme disease with their antibody responses to spirochetal polypeptides. Antibody reactivity was determined by Western blotting (immunoblotting) with sonicated whole B. burgdorferi and recombinant forms of its outer surface proteins, OspA and OspB, as the antigen preparations. Of 15 patients monitored for 4 to 12 years, 11 (73%) developed strong immunoglobulin G responses to both OspA and OspB near the beginning of prolonged episodes of arthritis, from 5 months to 7 years after disease onset. When single serum samples from 80 patients with Lyme arthritis, were tested, 57 (71%) showed antibody reactivity to recombinant Osp proteins; in contrast, none of 43 patients who had erythema migrans or Lyme meningitis (P < 0.00001) and 1 of 5 patients who had chronic neuroborreliosis but who never had arthritis (P = 0.03) showed antibody reactivity to these proteins. Among the 60 antibiotic-treated patients with Lyme arthritis, those with the HLA-DR4 specificity and Osp reactivity had arthritis for a significantly longer time after treatment than those who lacked Osp reactivity (median duration, 9.5 versus 4 months; P = 0.009); a similar trend was found for the HLA-DR2 specificity. For other HLA-DR specificities, arthritis resolved within a median duration of 2 months in both Osp-reactive and nonreactive patients. We conclude that the combination of the HLA-DR4 specificity and OspA or OspB reactivity is associated with chronic arthritis and the lack of a response to antibiotic therapy.     

Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice.

Vaccination with recombinant outer surface protein A (OspA) has been shown to protect mice from infection with Borrelia burgdorferi, the Lyme disease agent. To determine whether antibodies to B. burgdorferi proteins other than OspA are involved in protective immunity, antibodies to OspA were removed from protective anti-B. burgdorferi serum; the residual serum was still protective. Absorption of OspA and OspB antibodies from anti-B. burgdorferi serum eliminated the protective effect. Therefore, active immunization experiments were performed to determine the roles of OspB and flagellin in protective immunity and to determine whether protective immunity induced by OspA is dose dependent. Active immunization with recombinant OspA protected mice from infection with an inoculum of 10(4) spirochetes, but this protection could be overcome with a challenge of 10(7) spirochetes; OspB protected mice from infection with an inoculum of 10(3) spirochetes but was insufficient to fully protect against 10(4) organisms; and immunization with flagellin had no protective effect. These studies suggest that OspA and OspB, but not flagellin, play roles in protective immunity to spirochete infection.     

Resistance to tick-borne spirochete challenge induced by Borrelia burgdorferi strains that differ in expression of outer surface proteins.

Hamsters were immunized with thimerosal-killed Borrelia burgdorferi 297 or a mutant of 297 (M297) that lacks the 49-kb linear plasmid and expression of outer surface proteins A and B (OspA and OspB). Ixodes scapularis nymphs infected with either the B. burgdorferi sensu stricto strain 297 or JMNT, similar in OspA and OspB but differing in OspC expression, were used to evaluate protection. In a homologous challenge, 24 hamsters were vaccinated, 8 each with 297 or M297 and 8 sham (adjuvant)-vaccinated controls. Hamsters vaccinated with either bacterin were completely protected against a natural tick bite or subcutaneous (s.c.) inoculation of 297. Borreliae were effectively eliminated from 80 to 90% of the 297-infected ticks that fed on four hamsters immunized with the 297 bacterin. Cultures of spirochetes isolated from the ticks that remained infected were infectious and induced joint inflammation in naive hamsters. There was no reduction of strain 297 spirochetes in ticks that fed on four hamsters immunized with M297, but the hamsters were protected. Results with the M297 bacterin indicate that proteins other than OspA or OspB can protect hamsters against a tick challenge without eliminating B. burgdorferi in the tick. In a heterologous challenge, 36 hamsters were vaccinated, 12 with each bacterin and 12 controls. None of the hamsters immunized with either bacterin were protected from a challenge involving JMNT-infected ticks, while two of four were protected against an s.c. challenge. Hamsters challenged s.c. with strain 297 spirochetes were protected. There was partial elimination of JMNT spirochetes in ticks that fed on the group of four hamsters immunized with the 297 bacterin, and infection rates were reduced by 50 to 60%. JMNT spirochetes reisolated from the ticks that fed on 297-vaccinated hamsters also remained infectious for hamsters. In the JMNT-infected ticks that fed on four M297-immunized hamsters, there was no decline in the proportion of infected ticks. Destruction of spirochetes in ticks that fed on the hamsters vaccinated with the 297 bacterin suggests that antibodies to OspA and OspB may have been responsible, since the mutant did not induce this activity.     

Specific adherence of Borrelia burgdorferi extracellular vesicles to human endothelial cells in culture.

Borrelia burgdorferi produces extracellular vesicles which contain some of the outer surface proteins of the bacterium (e.g., OspA and OspB). Borrelial vesicles, isolated by differential centrifugation and filtration, were tested for the ability to bind to cultured human umbilical vein endothelial (HUVE) cells in culture. The recently described lipoprotein OspD was expressed on vesicles. Vesicles exhibited differential expression of OspB and OspD in a relationship with passage number and medium serum supplement type, respectively. Qualitative immunoblotting analyses demonstrated dose-dependent, passage number-dependent adsorption of vesicles by HUVE cells. This adsorption was demonstrated to be dependent upon a borrelial component of the vesicle and not due to the presence of minor contamination with intact spirochetes. Quantitative experiments examining inhibition of B. burgdorferi-HUVE association as a function of prior vesicle-HUVE association demonstrated dependence upon (i) a borrelial component(s) in the vesicle, (ii) low passage number, and (iii) vesicle protein concentration. However, vesicle pretreatment of the HUVE cell monolayer was not requisite for this inhibition. Vesicles from highly passaged borrelias were noninhibitory for B. burgdorferi-HUVE cell association, regardless of the serum used to supplement the medium. The use of vesicles as a tool for studying B. burgdorferi pathogenesis and/or physiology is proposed.     

Comparison of protection in rabbits against host-adapted and cultivated Borrelia burgdorferi following infection-derived immunity or immunization with outer membrane vesicles or outer surface protein A

In this study, infection-derived immunity in the rabbit model of Lyme disease was compared to immunity following immunization with purified outer membrane vesicles (OMV) isolated from Borrelia burgdorferi and recombinant outer surface protein A (OspA). Immunization of rabbits with OMV isolated from virulent strain B31 and its avirulent derivative B313 (lacking OspA and DbpA) conferred highly significant protection against intradermal injection with 6 x 10(4) in vitro-cultivated virulent B. burgdorferi. This is the first demonstration of protective immunogenicity induced by OMV. While immunization with OspA and avirulent B31 OMV provided far less protection against this challenge, rabbits with infection-derived immunity were completely protected. Protection against host-adapted B. burgdorferi was assessed by implantation of skin biopsies taken from rabbit erythema migrans (a uniquely rich source of B. burgdorferi in vertebrate tissue) containing up to 10(8) spirochetes. While all of the OMV- and OspA-immunized rabbits were fully susceptible to skin and disseminated infection, rabbits with infection-derived immunity were completely protected. Analysis of the antibody responses to outer membrane proteins, including DbpA, OspA, and OspC, suggests that the remarkable protection exhibited by the infection-immune rabbits is due to antibodies directed at antigens unique to or markedly up-regulated in host-adapted B. burgdorferi.     

Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete.

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in hypertonic sucrose followed by isopycnic ultracentrifugation of the plasmolyzed cells. The unilamellar vesicles thus obtained were shown to be OMs by the following criteria: (i) they contained OspA and OspB; (ii) they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture electron microscopy was identical to that of OMs of intact organisms. Consistent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small proportions of the total cellular content of OspA or OspB were OM associated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [3H]palmitate or 35S-amino acids demonstrated that the OMs contained both nonlipidated and lipidated proteins. This fractionation procedure was also used to isolate OMs from virulent and avirulent isolates of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluorography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of antibody reactivity was directed against intracellular proteins. The availability of isolated OMs should facilitate efforts to elucidate the complex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.     

Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice

The Lyme disease-associated spirochete, Borrelia burgdorferi, is maintained in enzootic cycles involving Ixodes ticks and small mammals. Previous studies demonstrated that B. burgdorferi expresses outer surface protein A (OspA) but not OspC when residing in the midgut of unfed ticks. However, after ticks feed on blood, some spirochetes stop making OspA and express OspC. Our current work examined the timing and frequency of OspA and OspC expression by B. burgdorferi in infected Ixodes scapularis nymphs as they fed on uninfected mice and in uninfected I. scapularis larvae and nymphs as they first acquired spirochetes from infected mice. Smears of midguts from previously infected ticks were prepared at 12- or 24-h intervals following attachment through repletion at 96 h, and spirochetes were stained for immunofluorescence for detection of antibodies to OspA and OspC. As shown previously, prior to feeding spirochetes in nymphs expressed OspA but not OspC. During nymphal feeding, however, the proportion of spirochetes expressing OspA decreased, while spirochetes expressing OspC became detectable. In fact, spirochetes rapidly began to express OspC, with the greatest proportion of spirochetes having this protein at 48 h of attachment and then with the proportion decreasing significantly by the time that the ticks had completed feeding. In vitro cultivation of the spirochete at different temperatures showed OspC to be most abundant when the spirochetes were grown at 37 degrees C. Yet, the synthesis of this protein waned with continuous passage at this temperature. Immunofluorescence staining of spirochetes in smears of midguts from larvae and nymphs still attached or having completed feeding on infected mice demonstrated that OspA but not OspC was produced by these spirochetes recently acquired from mice. Therefore, the temporal synthesis of OspC by spirochetes only in feeding ticks that were infected prior to the blood meal suggests that this surface protein is involved in transmission from tick to mammal but not from mammal to tick.     

Characterization of the physiological requirements for the bactericidal effects of a monoclonal antibody to OspB of Borrelia burgdorferi by confocal microscopy.

A confocal microscopy study was undertaken to characterize the bactericidal effects of the Fab fragments of CB2, an immunoglobulin G1kappa murine monoclonal antibody, to an epitope in the carboxy region of the outer surface protein B (OspB) of Borrelia burgdorferi. Simultaneous direct labeling of both fixed and live spirochetes with fluorochrome-labeled Fab-CB2 and 11G1, and an immunoglobulin Mkappa monoclonal antibody to OspA, showed that OspA and OspB seem to colocalize in dead spirochetes but do not appear to be physically associated when the organisms are alive. A polar bleb composed of a Fab-CB2-OspB complex, followed by incorporation of 11G1-OspA, precedes the formation of a spheroplast. The spheroplasts contain both OspA and OspB and are a terminal stage in the bactericidal process induced by Fab-CB2. Outer membrane destabilization by Fab-CB2, but not cell wall or cytoplasmic membrane alterations, was demonstrated experimentally by the sequential treatment of spirochetes with Fab-CB2 and monoclonal antibodies to flagellin and DnaK. The action of Fab-CB2 is epitope specific, as another monoclonal antibody to an epitope in the amino terminus of OspB was not bactericidal. The bactericidal effect of Fab-CB2 is not dependent on the induction of spirochetal proteases but is dependent on the presence of Ca2+ and Mg2+. Supplementation of Ca2(+)- and Mg2(+)-free medium with these cations restored the bactericidal effects of Fab-CB2. The mechanism by which a Fab fragment of an antibody destroys a bacterium directly may represent a novel form of antibody-organism interaction.     

Evaluation of whole-cell and OspC enzyme-linked immunosorbent assays for discrimination of early lyme borreliosis from OspA vaccination

A recombinant Lyme borreliosis vaccine consisting of outer surface protein A (OspA) is commercially available for vaccination of humans against infection with Borrelia burgdorferi. Vaccination with OspA induces an antibody response that makes serologic interpretation of infection with B. burgdorferi difficult, especially by screening tests based on whole-cell preparations of B. burgdorferi. We show that an enzyme-linked immunosorbent assay with B. burgdorferi sensu stricto 50772, which lacks the plasmid encoding OspA and OspB, or a full-length recombinant OspC protein can identify patients infected with B. burgdorferi. We found that 69 and 65% of serum samples from patients with case-defined early Lyme borreliosis had anti-B. burgdorferi sensu stricto 50772 and anti-OspC reactivities, respectively. In addition, little or no reactivity was detected with sera obtained from individuals vaccinated with OspA. Unfortunately, 51 and 33% of sera from healthy patients and sera from patients with other illnesses were also reactive against B. burgdorferi sensu stricto 50772 and OspC, respectively. Although these assays can discriminate B. burgdorferi infection from vaccination with OspA, their lack of specificity highlights the necessity for confirming equivocal or positive reactivities with more specific serodiagnostic tests.     

Characterization of the cellular and humoral immune response to outer surface protein C and outer surface protein 17 in children with early disseminated Lyme borreliosis

OspC and Osp17 are immunodominant proteins of Borrelia burgdorferi eliciting a clear humoral immune response in adult patients with systemic Lyme disease. In this study, the cellular immune response to B. burgdorferi and the major outer surface proteins OspC and Osp17 was investigated in children during the course of early disseminated B. burgdorferi infection. Lymphoproliferative responses to recombinant proteins were compared to the protein-specific humoral immune reaction. OspC induced a clear antibody response but elicited an even stronger cellular immune response. In contrast, a cellular as well as humoral immune reaction to Osp17 was only rarely detected. Follow-up examinations demonstrated that the lymphoproliferative response to B. burgdorferi and OspC persisted for several months after antibiotic therapy. Here, we show that in early disseminated Lyme disease of childhood, OspC is a potent antigen influencing both the humoral and cellular immunity, while Osp17 plays only a minor role in immune activation.     

Complement receptor 3 binds the Borrelia burgdorferi outer surface proteins OspA and OspB in an iC3b-independent manner

Persistence of borreliae within the vertebrate host depends on the fate of interactions between the spirochetes and target cells. The present work demonstrates the direct binding of the Borrelia burgdorferi outer surface proteins OspA and OspB to CR3 and that this binding is independent of iC3b.     

Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi.

We describe the cloning, expression, and molecular characterization of a novel polymorphic Borrelia burgdorferi lipoprotein recognized by monoclonal antibody LA7. Sequence analysis revealed an open reading frame encoding a 21,866-Da polypeptide (IpLA7). Comparison with other known proteins indicated sequence similarity between IpLA7 signal peptides and those of other prokaryotic lipoproteins, including the immunodominant B. burgdorferi outer surface proteins OspA, OspB, pC, and OspD. Both natural IpLA-7 and recombinant IpLA-7 could be biosynthetically labeled with [3H]palmitate. Upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114, IpLA7 was extracted together with other lipoproteins into the detergent phase. Indirect immunolabeling studies indicated that the epitope recognized by monoclonal antibody LA7 is mainly located in the periplasmic space. Two-dimensional gel electrophoresis and immunoblotting confirmed the calculated acidic pI of 5.7 for IpLA-7. The LA7 gene was shown to be species specific and to be located on the linear chromosome of B. burgdorferi. The analysis of 40 individual spirochetal isolates on the basis of restriction fragment length polymorphisms revealed considerable genotypic heterogeneity of LA7 corresponding to that previously found for ospA. Native IpLA-7 and recombinant IpLA-7 were recognized by immune sera from infected mice as well as some human sera derived from infected but healthy donors and may thus prove useful as an additional marker for the serodiagnosis of Lyme disease.