Immunogenicity of Borrelia burgdorferi arthritis-related protein

Immunization against arthritis-related protein (Arp) elicits antibody in mice that resolves arthritis but is not protective against challenge with Borrelia burgdorferi. In mice immunized against Arp, an unrelated 37-kDa protein (P37-42), outer surface protein A (OspA), or glutathione S-transferase (GT) and then challenged by syringe or tick, only OspA conferred protection. Passive transfer of Arp antiserum into infected SCID mice induced arthritis resolution, but antisera to P37-42, OspA, GT, or six overlapping Arp peptide fragments did not. Results suggest that the arthritis-resolving immunogenicity is specific to Arp, but the relevant epitopes may be conformational.     

Antigenic stability of Borrelia burgdorferi during chronic infections of immunocompetent mice.

Mice were actively immunized by intradermal inoculation with 10(4) cloned Borrelia burgdorferi bacteria and then cured of the B. burgdorferi infection with an antibiotic after 90 days. They were resistant to intradermal 10(2)- or 10(4)-bacterium challenge infection with either the original cloned B. burgdorferi or B. burgdorferi isolated from each punch biopsies at 90 days of infection (prior to antibiotic treatment), including autologous B. burgdorferi isolates. In contrast, sham-infected (nonimmune) mice were susceptible to challenge infection with both early and late B. burgdorferi isolates. Since there was a potential for in vitro modification of the spirochetes during the 2-week culture period which would obscure results, an alternate means of challenge infection, using tissue transplants, was implemented. By using the same approach, mice were immunized by infection, treated with antibiotics, but challenged by subcutaneous transplantation of ear skin pieces biopsied and frozen prior to antibiotic treatment. Mice were infected for 15, 90, or 180 days before biopsy and antibiotic treatment and then transplant challenged with autologous infected tissue. Sham-immunized mice received infected tissue, and immune mice received uninfected tissue as controls. Mice infected for only 15 days, but not mice infected for 90 or 180 days, could be reinfected by autografts, whereas nonimmune mice became infected with tissues collected at each of these intervals and immune mice transplanted with normal skin were uninfected. These results indicate that immunity to B. burgdorferi is effective against the original inoculum, late isolates of the spirochete, or infected tissues collected at intervals of up to 180 days, suggesting that there is no significant antigenic change in B. burgdorferi during chronic 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.     

Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals.

Borrelia burgdorferi expresses a conserved, species-specific 39-kDa protein (P39) that can stimulate antibodies during human infection. To confirm that anti-P39 antibodies are produced consistently in animals exposed to infectious spirochetes, white-footed mice, Peromyscus leucopus, and laboratory white mice, Mus musculus (strain BALB/c), were experimentally inoculated with either infectious or noninfectious B. burgdorferi and the antibody response to P39 was determined by immunoblot at 21 days postinoculation. All mice inoculated with approximately 10(7) infectious B. burgdorferi produced anti-P39 antibodies and were cultured positive for this spirochete. Mice inoculated with similar numbers of inactivated or viable noninfectious B. burgdorferi still producing P39 did not induce anti-P39 antibodies. By contrast, putative antiflagellin antibodies were detected in less than 18% of the infected animals, which supports the notion that antibody reactive with flagellin may not be reliable as a marker for B. burgdorferi exposure as was originally thought. Mice infected with B. burgdorferi following exposure to ticks (Ixodes dammini) produced anti-P39 antibodies no later than 7 days postinfection, indicating that P39 is an effective immunogen in natural infections. Notably, anti-P39 antibodies were the predominant B. burgdorferi reactive antibodies detected early in the infection. Our results indicate that anti-P39 antibodies are produced in response to an active infection and are therefore reliable markers for infection in experimentally and naturally inoculated animals.     

Antibody response in white-footed mice (Peromyscus leucopus) experimentally infected with the Lyme disease spirochete (Borrelia burgdorferi).

White-footed mice (Peromyscus leucopus), the primary reservoir for Borrelia burgdorferi in the northern midwest and northeastern United States, were experimentally inoculated with an infectious strain or a noninfectious strain of the Lyme disease spirochete and examined for their specific antibody response with the enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Immunoglobulin M (IgM) anti-B. burgdorferi antibodies were detected in mice 1 to 2 days after inoculation with either the infectious or noninfectious strain of spirochetes and peaked on days 4 and 5. Mice inoculated with the infectious strain of spirochete had a secondary increase in IgM 21 days after inoculation. Mice also produced both IgG1 and IgG2 antibodies beginning 5 to 7 days after inoculation and they increased in titer until 84 days after inoculation when the experiment was terminated. Western blot analysis of sequential plasma samples from mice inoculated with the infectious strain of spirochete demonstrated the development of IgM, IgG1, and IgG2 antibodies to numerous spirochetal antigens, whereas mice inoculated with the noninfectious strain had reduced blot patterns with antibodies reactive primarily to the 31,000-kilodalton outer surface protein A. Persistent spirochetal infection in some mice, in spite of a strong and diverse antibody response, deserves further investigation.     

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.     

Specificity of Infection-Induced Immunity among Borrelia burgdorferi Sensu Lato Species

The specificity of infection-induced immunity in mice infected with cultured or host-adapted Borrelia burgdorferi sensu lato, the agent of Lyme disease, was examined. Sera obtained from mice following infection with high and low doses of cultured B. burgdorferi sensu stricto, transplantation of infected tissue (host-adapted spirochetes), or tick-borne inoculation all showed protective activity in passive immunization assays. Infection and disease were similar in mice infected with cultured spirochetes or by transplantation. Thus, the adaptive form of inoculated spirochetes did not influence the immune response during active infection. Mice infected with B. burgdorferi sensu stricto and then cured of infection with an antibiotic during early or late stages of infection were resistant to challenge with high doses of homologous cultured spirochetes for up to 1 year. In contrast, actively immune mice infected with different Borrelia species (B. burgdorferi sensu lato, B. burgdorferi sensu stricto cN40, Borrelia afzelii PKo, and Borrelia garinii PBi) and then treated with an antibiotic were resistant to challenge with cultured homologous but not heterologous spirochetes. Similar results were achieved for actively immune mice challenged by transplantation and by passive immunization with sera from mice infected with each of the Borrelia species and then challenged with cultured spirochetes. Arthritis and carditis in mice that had immunizing infections with B. afzelii and B. garinii and then challenged by transplantation with B. burgdorferi sensu stricto were equivalent in prevalence and severity to those in nonimmune recipient mice. These results indicate that protective immunity and disease-modulating immunity that develop during active infection are universal among species related to B. burgdorferi sensu lato but are species specific.     

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.     

OspA antibodies inhibit the acquisition of Borrelia burgdorferi by Ixodes ticks.

Ixodes ticks are infected by Borrelia burgdorferi when larvae feed on spirochete-infected mice. We studied the acquisition of B. burgdorferi by larval ticks, characterized the production of outer surface protein A (OspA) by spirochetes entering larvae, and examined the effects of OspA antibodies on the establishment of B. burgdorferi infections in ticks. Most larvae were infected by spirochetes 24 to 48 h after placement on mice. OspA antibodies stained the first spirochetes observed in larvae, suggesting that OspA is synthesized early during the colonization of the vector. When OspA antibodies were administered to B. burgdorferi-infected mice and larvae were then placed on the animals, the severity of larval infection and the number of infected ticks (7 of 16) were decreased compared with that of controls (15 of 16). The inhibitory effects of OspA antibodies were observed with passive antibody transfer as well as active host-generated immunity. The lower larval infection rate observed in the presence of OspA antibodies was exacerbated after the larval molt since only 1 of 12 nymphs was infected, and none of the mice that were fed upon by these nymphs became infected with B. burgdorferi. Therefore, an OspA antibody response in mice altered the reservoir competence of the vertebrate host by inhibiting the movement of B. burgdorferi from the host to the vector.     

A guinea pig model for Lyme disease.

We report that outbred Hartley guinea pigs are susceptible to Borrelia burgdorferi. We recovered spirochetes from 57 of 60 (95%) guinea pigs inoculated when < or = 3 months of age. In contrast, animals inoculated when > or = 6 months of age were resistant to infection as defined by recovery of organisms at > or = 4 weeks postinoculation. Infection was widely disseminated: B. burgdorferi was recovered from 83% of bladders, 64% of knee joints, 57% of hearts, 48% of spleens, and 38% of spinal cords examined within 4 weeks of inoculation. Histopathologic changes were common in the heart (88%) (preferential involvement of perineural tissues near the annulus fibrosus) and bladder (76%) and were also noted in a minority of spinal cords (13%) and knee joints (9%). Western immunoblots demonstrated an immunoglobulin G response to B. burgdorferi, particularly to the 24-, 31- (OspA), 39-, and 41-kDa (flagellin) antigens. Infection was cleared from most tissues with the passage of time; spirochetes were recovered from 63% of tissues removed from guinea pigs at < or = 4 weeks after inoculation but from only 32% at > or = 8 weeks postinoculation (P < 0.001). An exception was the failure to clear spirochetes from infected knees, 90% of which were culture positive even when evaluated at > or = 8 weeks postinoculation. The guinea pig provides a new model useful for studying host-spirochete interactions in Lyme disease.     

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.     

OspA vaccination of mice with established Borrelia burgdorferi infection alters disease but not infection.

C3H mice were actively immunized with outer surface protein A (OspA) at different intervals after infection with Borrelia burgdorferi to determine the effect of postexposure vaccination on the course of murine Lyme borreliosis. Mice were vaccinated with an OspA-glutathione transferase fusion protein or glutathione transferase (control) in complete Freund's adjuvant; vaccination was followed by two weekly booster injections in incomplete adjuvant. Two weeks after the final booster injection, organs were cultured for B. burgdorferi (blood, spleen, skin, and bladder) and examined for histopathology (joints and hearts). When vaccination was commenced in the early stages (5 to 14 days) of infection, active immunization with OspA partially cleared spirochetes from the bloodstream but did not eliminate them from other tissues or alter the course of joint or heart disease. Commencement of vaccination at 60 days after infection (at which time joint or heart disease is resolving), however, reduced both the number of mice and individual joints with arthritis, a result suggesting an acceleration of the resolution phase of the disease. Postexposure immunization with OspA may partially alter the course of murine Lyme arthritis but does not eliminate infection.     

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.     

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.     

Infectivity of the highly transformable BBE02- lp56- mutant of Borrelia burgdorferi, the Lyme disease spirochete, via ticks

Infectious Borrelia burgdorferi strains that have increased transformability with the shuttle vector pBSV2 were recently constructed by inactivating the gene encoding BBE02, a putative restriction-modification gene product expressed by the linear plasmid lp25 (Kawabata et al., Infect. Immun. 72:7147-7154, 2004). The absence of the linear plasmid lp56, which carries another putative restriction-modification gene, further enhanced transformation rates. The infectivity of these mutants was assessed previously in mice that were inoculated with needle and syringe and was found to be equivalent to that of wild-type spirochetes. Here we examined the infectivity of spirochetes to ticks after capillary inoculation of Ixodes scapularis nymphs and the subsequent spirochetal infectivity to mice via ticks by using B. burgdorferi B31 clonal isolates lacking lp56 and/or BBE02. The absence of lp56 (but not BBE02) correlated with a lower number of spirochetes in ticks after feeding on mice; this plasmid thus may play a role, albeit not an essential one, in supporting spirochetal survival in the feeding tick. Importantly, however, the absence of lp56 and BBE02 did not detectably influence infectivity to mice via ticks.     

Passive immunizing activity of sera from mice infected with Borrelia burgdorferi.

A single injection of serum from C3H mice at 90 days after intradermal inoculation with 10(4) Borrelia burgdorferi spirochetes protected naive mice when administered subcutaneously at -18 h relative to intradermal challenge inoculation with 10(4) B. burgdorferi spirochetes. When immune serum was given at intervals (-18, 0, +24, +48, and +96 h) relative to intradermal challenge with 10(4) B. burgdorferi spirochetes, it was protective if given before or at the time of challenge but not at later times. Protection with 90-day serum given at -18 h was effective at dilutions up to 1:32, but not 1:64, on the basis of culture or disease at either 5 or 15 days after challenge. Passive immunizing activity was also present in sera from mice at 21 days after intradermal challenge with 10(4), 10(2), or 10(1) spirochetes, indicating that the immunizing component was not dose dependent and probably not related to antibody to outer surface protein A. Passive immunizing titers of sera from mice at days 1, 15, 30, 90, 180, and 360 after intradermal B. burgdorferi inoculation appeared as early as day 15, were highest on day 30, and then declined progressively on days 90, 180, and 360. Immunizing titers of sera from mice at 360 days after intradermal B. burgdorferi inoculation were identical in passively immunized mice challenged with the original inoculum or with B. burgdorferi isolated at 360 days after inoculation, suggesting that there was no antigenic discrimination between the original inoculum and late isolates. These results suggest that protective antibody is produced early in the course of B. burgdorferi infection and is unrelated to antibody to outer surface protein A. In addition, the decline of protective serum titers over time despite persistent infection suggests that the antigens eliciting the protective response are either modified or suppressed, but antigenic modification could not be demonstrated.     

Infection of mice with lyme disease spirochetes constitutively producing outer surface proteins a and B

Outer surface protein A (OspA) of the Lyme disease spirochete is primarily produced in the tick vector. OspA, which is a receptor for attaching spirochetes to the tick gut, is down regulated as the spirochetes leave the tick and enter the mammalian host. Although OspA is not a major antigen produced in the mammal, the protein appears to be produced under some conditions and production has been linked to more severe disease. A Lyme disease vaccine based on recombinant OspA has been approved for human use. However, the vaccine is no longer available, in part because of fears that OspA causes arthritis in people. To further understand the consequences of OspA production in the host, we created a Borrelia burgdorferi mutant that was unable to down regulate OspA. C3H/HeN mice infected with this mutant developed a specific anti-OspA immune response, and the spirochetes were unable to persist in these mice. In contrast, immunodeficient SCID mice were persistently infected with the mutant. We conclude that spirochetes producing OspA and B from the flaB promoter in immunocompetent mice stimulate an immune response that clear the bacteria without any signs of disease development in the mice.     

Infection of Ixodes scapularis (Acari: Ixodidae) with Borrelia burgdorferi using a new artificial feeding technique

To study interactions between Ixodes scapularis (Say) and Borrelia burgdorferi, an artificial feeding system was refined to allow controlled manipulation of single variables. The feeding system uses a mouse skin mounted on a water-jacketed glass membrane feeder. I. scapularis were infected using either BSK-H-cultured B. burgdorferi spirochetes or a B. burgdorferi-infected mouse skin as the source of spirochetes. Sixty-six percent of nymphs successfully fed to repletion using the artificial feeding systems with at least 75% of nymphs becoming infected with B. burgdorferi. Strain B31 B. burgdorferi spirochetes from passages 2-17 were equally infectious to nymphal ticks. At concentrations of one spirochete per microliter, 12% of nymphs acquired infection and 14 and 100 spirochetes per microliter resulted in 50 and 100% infection rates, respectively. Eighty-nine percent of nymphs fed by artificial feeding molted to the adult stage. When subsequently fed as adults, these I. scapularis successfully transmitted infectious B. burgdorferi spirochetes to mice.     

Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunized mice.

We determined whether Borrelia burgdorferi outer surface proteins (Osps) E and F could elicit immune responses useful for a Lyme disease vaccine. Thirty days after challenge with B. burgdorferi, mice produced antibodies to OspE but not OspF, whereas antibodies to OspF were present in sera of mice obtained 90 days after infection. Examination of sera from patients with Lyme disease revealed antibodies to OspF in a small number (14%) of early-stage disease patients but in a majority (58%) of patients with late-stage disease, while antibodies to OspE were rarely detected in patients. Mice immunized with recombinant OspE or OspF produced high titers of antibodies to OspE or OspF, respectively. OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity. B. burgdorferi organisms were, however, substantially destroyed within ticks that engorged on either OspE- (75% reduction in the number of spirochetes within the ticks, compared with controls) or OspF (90% reduction in the number of spirochetes within the ticks)-immunized mice.