Does host complement kill Borrelia burgdorferi within ticks?

The Lyme disease spirochete, Borrelia burgdorferi, inhabits the gut lumen of the tick vector. At this location the spirochete is exposed to host blood when a tick feeds. We report here on studies that were done with normal and complement-deficient (C3-knockout) mice to determine if the host complement system killed spirochetes within the vector. We found that spirochete numbers within feeding nymphs were not influenced by complement, most likely because host complement was inactivated within the vector. The Lyme disease outer surface protein A (OspA) vaccine is a transmission-blocking vaccine that targets spirochetes in the vector. In experiments with mice hyperimmunized with OspA, complement was not required to kill spirochetes within nymphs and to block transmission from nymphs to the vaccinated host. However, host complement did enhance the ability of OspA antibody to block larvae from acquiring spirochetes. Thus, the effects of OspA antibody on nymphal transmission and larval acquisition appear to be based on different mechanisms.     

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.     

Reservoir targeted vaccine for lyme borreliosis induces a yearlong, neutralizing antibody response to OspA in white-footed mice

Lyme disease is caused by the spirochete Borrelia burgdorferi. The enzootic cycle of this pathogen requires that Ixodes spp. acquire B. burgdorferi from infected wildlife reservoirs and transmit it to other uninfected wildlife. At present, there are no effective measures to control B. burgdorferi; there is no human vaccine available, and existing vector control measures are generally not acceptable to the public. However, if B. burgdorferi could be eliminated from its reservoir hosts or from the ticks that feed on them, the enzootic cycle would be broken, and the incidence of Lyme disease would decrease. We developed OspA-RTV, a reservoir targeted bait vaccine (RTV) based on the immunogenic outer surface protein A (OspA) of B. burgdorferi aimed at breaking the natural cycle of this spirochete. White-footed mice, the major reservoir species for this spirochete in nature developed a systemic OspA-specific IgG response as a result of ingestion of the bait formulation. This immune response protected white-footed mice against B. burgdorferi infection upon tick challenge and cleared B. burgdorferi from the tick vector. In performing extensive studies to optimize the OspA-RTV for field deployment, we determined that mice that consumed the vaccine over periods of 1 or 4 months developed a yearlong, neutralizing anti-OspA systemic IgG response. Furthermore, we defined the minimum number of OspA-RTV units needed to induce a protective immune response.     

Outer surface protein A protects Lyme disease spirochetes from acquired host immunity in the tick vector

The Lyme disease spirochete Borrelia burgdorferi alters the expression of outer surface protein (osp) genes as the bacterium cycles between ticks and mammals. OspA is produced as borreliae enter the tick vector and remains a major surface antigen during midgut colonization. To elucidate the role of OspA in the vector, we created an insertional deletion of ospA in strain B31-A3. The ospA mutant infects mice when it is injected intradermally and is acquired by larval ticks fed on these mice, where it persists through the molt to the nymph stage. Bacterial survival rates in artificially infected tick larvae fed on naïve mice were compared with those in the vector fed on immune mice. The ospA mutant proliferates in larvae if it is exposed to blood from naïve mice, but it declines in density after larval feeding if the blood is from immune mice. When uninfected larvae are fed on B-cell-deficient mice infected with the ospA mutant, larvae show borrelial densities and persistence that are significantly greater than those fed on infected, immunocompetent mice. We conclude that OspA serves a critical antibody-shielding role during vector blood meal uptake from immune hosts and is not required for persistence in the tick vector.     

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.     

Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.     

Intrinsic competence of three ixodid ticks (Acari) as vectors of the Lyme disease spirochete

We compared the intrinsic vector competence of Ixodes dammini Spielman et al., Dermacentor variabilis (Say), and Amblyomma americanum (L.) for the Lyme disease spirochete (Borrelia burgdorferi Johnson et al.) on Prudence Island, Rhode Island, a Lyme disease-endemic site where all three ticks occur together. Natural and experimental spirochete infection rates were determined in those ticks and their degree of contact with white-footed mice (Peromyscus leucopus), the principal reservoir host, was compared. Host-seeking nymphal and adult I. dammini and A. americanum, collected by flagging, were nearly equally abundant, as were adult D. variabilis, but only I. dammini were infected with B. burgdorferi. Larvae and nymphs of both I. dammini and D. variabilis were found infesting mice, but A. americanum never were found on this host. Furthermore, although larvae of all three tick species became infected by ingesting spirochetes while feeding on experimentally infected mice, only I. dammini remained infected following the transstadial molt. These findings suggest that of these three tick species, only I. dammini is competent as a vector of the Lyme disease spirochete.     

Role of attached lipid in immunogenicity of Borrelia burgdorferi OspA.

OspA is a protective antigen of the Lyme disease spirochete Borrelia burgdorferi. Expression of the full-length B. burgdorferi B31 OspA gene in Escherichia coli produces a protein that is processed posttranslationally by signal peptidase II and contains an attached lipid moiety. The recombinant OspA lipoprotein has been purified by detergent extraction and ion-exchange chromatography. Priming and boosting with OspA lipoprotein, either with no adjuvant or adsorbed to alum, elicited a strong, dose-dependent immunoglobulin G response. Serum from vaccinated mice inhibited spirochetal growth in vitro. Mice immunized twice with as little as 0.4 micrograms of OspA lipoprotein were protected against an intradermal challenge with 10(4) infectious spirochetes. The ability of the purified recombinant lipoprotein to induce a strong protective response in the absence of toxic adjuvants makes it an excellent candidate antigen for a human vaccine against Lyme disease. By contrast, no serum immunoglobulin G or growth inhibitory response to OspA nonlipoprotein was seen at any dose. The difference in immunogenicities of the lipoprotein and nonlipoprotein forms of OspA is not due to any difference in the antigenicities of the two proteins. These results suggest that posttranslational lipid attachment is a critical determinant of the immunogenicity of the OspA protein.     

Abilities of OspA proteins from different seroprotective groups of Borrelia burgdorferi to protect hamsters from infection.

The ability of vaccination with recombinant OspA from six seroprotective groups of Borrelia burgdorferi sensu lato to induce protection against infection with homologous and other Lyme spirochetes was examined in hamsters. Antisera generated against the OspA proteins of B. burgdorferi sensu stricto S-1-10 and C-1-11 (seroprotective groups 1 and 2, respectively), Borrelia afzelii BV1 (seroprotective group 4), and Borrelia garinii LV4 (seroprotective group 5) were able to kill the homologous spirochete in vitro but not other isolates. Surprisingly, antisera against B. afzelii PKo (seroprotective group 6) and B. burgdorferi sensu lato LV5 (seroprotective group 3) OspA proteins were unable to kill the homologous organism, although LV5 OspA antisera killed the heterologous isolates S-1-10 and LV4. In vivo vaccination studies supported the in vitro findings, confirming that vaccination with a single OspA protein does not provide complete protection against challenge with all Lyme disease spirochetes. In addition, OspA antibodies from some isolates may not protect against the homologous isolate. The induction of protective antibodies against other B. burgdorferi proteins may be necessary to insure a comprehensive Lyme disease vaccine.     

Natural Antibody Affects Survival of the Spirochete Borrelia burgdorferi within Feeding Ticks

Natural antibodies are those immunoglobulin molecules found in mammalian serum that arise in the absence of exposure to environmental pathogens and may comprise an early host defense against invading pathogens. The spirochete Borrelia burgdorferi first encounters natural antibodies when its arthropod vector, Ixodes scapularis, begins feeding on a mammalian host. Natural antibodies may therefore have an impact on pathogens within blood-sucking vectors, prior to pathogen transmission to the mammal. In this study, we investigated whether natural antibodies influenced the number and/or phenotype of B. burgdorferi organisms within feeding I. scapularis nymphs. Using a competitive PCR, we found that ticks ingesting a blood meal from B-cell-deficient mice, which lack all immunoglobulins, contained fivefold more spirochete DNA than ticks feeding on control mice. Spirochete DNA levels could be reduced to that of controls with passive transfer of normal mouse serum or polyclonal immunoglobulin M (IgM), but not IgG, into B-cell-deficient mice prior to placement of infected ticks. At 48 h of tick feeding, 90% of spirochetes within salivary glands of ticks removed from B-cell-deficient mice were found by confocal immunofluorescence microscopy to express outer surface protein A (OspA), compared to only 5% of salivary gland spirochetes from ticks detached from control mice. Taken together, these results show that ingestion of natural antibodies limits the spirochete burden within feeding ticks. Because OspA is normally downregulated when spirochetes moved from the tick midgut to the salivary gland, our findings suggest that OspA-expressing midgut spirochetes may be particularly susceptible to the borrelicidal effects of these molecules.     

Interactions of OspA monoclonal antibody C3.78 with Borrelia burgdorferi within ticks

The Borrelia burgdorferi outer surface protein A (OspA) vaccine induces antibodies that prevent transmission from the tick to the host. Here we describe studies with an OspA monoclonal antibody (C3.78) to understand the mechanism by which antibodies entering the tick block Borrelia transmission. Host complement in the tick's blood meal did not contribute to protection because the antibody was equally effective whether infected ticks fed on normal or complement-deficient mice. Antibody-mediated cross-linking of bacteria or cross-linking of OspA molecules was not required for protection because C3.78 Fab' fragments were as effective as whole antibody molecules. At low C3.78 concentrations, transmission was blocked despite the presence of many live spirochetes within the tick, confirming that clearance of Borrelia organisms was not required to block transmission. We propose that OspA antibody binding to the surface of spirochetes blocks transmission by a mechanism that does not require bacterial killing.     

Competitive Advantage of Borrelia burgdorferi with Outer Surface Protein BBA03 during Tick-Mediated Infection of the Mammalian Host

Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.     

Influence of Outer Surface Protein A Antibody on Borrelia burgdorferi within Feeding Ticks

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick’s salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 μg/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 μg/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induce ospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.     

Long-term protection of mice from Lyme disease by vaccination with OspA.

Mice vaccinated with recombinant outer surface protein A (OspA) have been shown to be protected from infection with Borrelia burgdorferi, the agent of Lyme disease, when sacrificed 14 days after challenge with an intradermal inoculum of the spirochete. To determine whether infection was not merely delayed and that protection was long-lasting, we sacrificed vaccinated mice 60, 120, and 180 days after challenge; and to determine whether vaccinated mice retained their immune state over long periods, we challenged mice with B. burgdorferi 60, 90, 120, and 150 days after vaccination. The results of both groups of experiments show that the mice remained free from infection and disease and extend the usefulness of OspA as a vaccine candidate for Lyme borreliosis.     

Antibody-mediated disease remission in the mouse model of lyme borreliosis

In the mouse model of Lyme borreliosis, the host immune response during infection with Borrelia burgdorferi results in the remission of carditis and arthritis, as well as global reduction of spirochete numbers in tissues, without elimination of infection. These events were recapitulated by passive transfer of immune serum from infected immunocompetent mice or T-cell-deficient mice to severe combined immunodeficient (SCID) mice. Previous studies have shown that immune serum is reactive against arthritis-related protein (Arp) and that Arp antiserum induces arthritis remission. However, although immune serum from T-cell-deficient mice induced disease remission, it was not reactive against Arp, suggesting that antibody to another antigen may be responsible. T-cell-deficient mouse immune serum was reactive to decorin binding protein A (DbpA). Therefore, DbpA antiserum was tested to determine its ability to induce disease remission in SCID mice. Antisera to Arp or DbpA induced both carditis and arthritis remission but did not significantly reduce spirochete numbers in tissues, based upon quantitative flaB DNA analysis, nor did treatment affect RNA levels of several genes, including arp and dbpA. Immunohistochemical labeling of spirochetes in hearts and joints during disease remission induced by adoptive transfer of lymphocytes, passive transfer of immune serum, or passive transfer of DbpA antiserum revealed that such treatment resulted in elimination of spirochetes from heart base and synovium but not vascular walls, tendons, or ligaments. These results suggest that Arp and DbpA antibodies may be active as disease-resolving components in immune serum but antibody against other antigens may be involved in reductions of spirochetes in tissues.     

Motility Is Crucial for the Infectious Life Cycle of Borrelia burgdorferi

The Lyme disease spirochete, Borrelia burgdorferi, exists in a zoonotic cycle involving an arthropod tick and mammalian host. Dissemination of the organism within and between these hosts depends upon the spirochete''s ability to traverse through complex tissues. Additionally, the spirochete outruns the host immune cells while migrating through the dermis, suggesting the importance of B. burgdorferi motility in evading host clearance. B. burgdorferi''s periplasmic flagellar filaments are composed primarily of a major protein, FlaB, and minor protein, FlaA. By constructing a flaB mutant that is nonmotile, we investigated for the first time the absolute requirement for motility in the mouse-tick life cycle of B. burgdorferi. We found that whereas wild-type cells are motile and have a flat-wave morphology, mutant cells were nonmotile and rod shaped. These mutants were unable to establish infection in C3H/HeN mice via either needle injection or tick bite. In addition, these mutants had decreased viability in fed ticks. Our studies provide substantial evidence that the periplasmic flagella, and consequently motility, are critical not only for optimal survival in ticks but also for infection of the mammalian host by the arthropod tick vector.     

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.     

Lack of detectable variation at Borrelia burgdorferi vlsE locus in ticks

VlsE is a surface exposed lipoprotein of the Lyme disease spirochete, Borrelia burgdorferi. Spirochetes are able to generate many antigenic variants of VlsE by DNA recombination at the vlsE locus. Novel VlsE antigenic variants are readily observed in mice infected with B. burgdorferi. We followed a clonal population of spirochetes through a tick transmission cycle and report that unlike in vertebrates, the vlsE locus is stable in ticks.