Serum reactivity against Borrelia burgdorferi OspA in patients with rheumatoid arthritis

Lyme arthritis and rheumatoid arthritis share common clinical features and synovial histology. It is unclear whether they also share similar pathogenesis. Previous studies have shown that the severity and duration of Lyme arthritis correlate directly with serum concentrations of antibody against outer surface protein A (OspA) of the causative pathogen Borrelia burgdorferi. We tested the sera of 68 subjects with rheumatoid arthritis, 147 subjects with other autoimmune diseases, and 44 healthy subjects who had never had Lyme disease, as well as sera of 16 patients who had Lyme disease, for reactivity against the B. burgdorferi OspA protein. The sera of about a quarter of the rheumatoid arthritis patients and a 10th of the autoimmune disease and Lyme disease patients reacted against OspA antigen. Of 50 rheumatoid arthritis patients who could be evaluated for disease severity, a 28-joint count disease activity score of >2.6 was noted for 11 of 15 (73%) patients whose sera reacted against OspA antigen and 13 of 35 (37%; P < 0.05) whose sera were nonreactive. Serum reactivity against OspA antigen is associated with the pathogenesis of rheumatoid arthritis.     

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.     

Tripalmitoyl-S-glyceryl-cysteine-dependent OspA vaccination of toll-like receptor 2-deficient mice results in effective protection from Borrelia burgdorferi challenge

Toll-like receptor 2 (TLR2) is a transmembrane signal transducer for tripalmitoyl-S-glyceryl-cysteine (Pam(3)Cys)-modified lipoproteins, including OspA from the Lyme disease spirochete Borrelia burgdorferi. The Pam(3)Cys modification provides adjuvant activity for inducing humoral responses, suggesting that TLR2 could function as the adjuvant receptor for the OspA vaccine. The importance of TLR2 in the humoral response to OspA was confirmed, because overall levels of immunoglobulin G (IgG) were reduced in TLR2-deficient mice, when compared with those in wild-type mice. However, the levels of production of IgG1 were similar in both mouse strains, and the levels of induction of protective immunity were comparable. Unlipidated OspA was not immunogenic in wild-type or TLR2-deficient mice, indicating the lipid modification was active in the absence of TLR2. These findings indicate that the Pam(3)Cys modification of bacterial lipoprotein has adjuvant properties independent of TLR2 signaling.     

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.     

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.     

An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.     

Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi.

Twenty-two specific-pathogen-free beagles were vaccinated with recombinant OspA (ospA gene derived from Borrelia burgdorferi B31) alone or with adjuvant (QuilA, Montanide ISA25, or aluminum hydroxide) at 6 weeks of age. Thirteen dogs were used as nonvaccinated controls with or without adjuvant. Three dogs were kept as contact controls and received neither vaccine nor challenge. Six weeks or 6 months after the first vaccination, the vaccinated (20 of 22) and nonvaccinated dogs (13) were challenged by exposure to adult ticks (Ixodes scapularis) naturally that were infected with B. burgdorferi (tick infection rate, > or = 60%) and that were collected from Westchester County, N.Y. Protection from infection was evaluated by culture for B. burgdorferi from skin biopsies taken near the sites of tick bites. Skin biopsies were taken at monthly intervals for 3 months. B. burgdorferi was not isolated from any of the vaccinated dogs. In contrast, 12 of 13 control dogs challenged by exposure to the ticks were culture positive. The histopathology of the joint capsules 3 months after the challenge was used to evaluate protection from arthritis. Eight of 13 control dogs showed synovitis in single or multiple joints, while only 1 of the 22 vaccinated dogs had a single focus of mild inflammation in a single joint. At the time of the challenge, the vaccinated dogs had antibody to B. burgdorferi, which was demonstrable by kinetic enzyme-linked immunosorbent assay, Western blotting (immunoblotting), and a serum growth inhibition assay. The vaccinal antibody declined gradually after the challenge, especially in dogs vaccinated with OspA without adjuvants. Antibodies in the challenge control dogs were only detectable by 4 to 6 weeks after the challenge and remained at high levels until the termination of the study. Contact control dogs showed no antibody responses or histopathologic lesions and were culture negative. By Western blot analysis, antibodies to OspA first appeared in the sera of vaccinated dogs 3 weeks after the first vaccination. The absence of additional bands after the challenge suggests that infection in vaccinated dogs was blocked. Results from this study show that vaccination with recombinant OspA protected dogs against infection and disease after an experimental challenge with B. burgdorferi by exposure to ticks.     

Borrelia burgdorferi enzyme-linked immunosorbent assay for discrimination of OspA vaccination from spirochete infection.

Recombinant Lyme disease vaccines based on purified preparations of outer surface protein A (OspA) have been shown to be effective in preventing transmission of Borrelia burgdorferi in experimental animal models and are now being tested in humans. Since the most widely used screening tests for Lyme disease are based on a whole-cell sonicate of B. burgdorferi, serologic false positivity in vaccinated persons could result from reactivity to OspA within the antigen preparation. In order to avoid serologic false positivity in vaccinated subjects, we developed an immunoassay based on a low-passage-number, naturally occurring variant of B. burgdorferi which lacks the plasmid encoding OspA and OspB. The use of an antigen preparation derived from this organism provided sensitive and specific detection of B. burgdorferi seropositivity in experimental animals and in human Lyme disease cases. The OspA-B-negative enzyme-linked immunosorbent assay (ELISA) also appeared to be capable of discriminating the vaccinated state from vaccine failure and natural infection in experimental animals. Sera from human subjects participating in a vaccine trial gave false-positive results with an ELISA based on an OspA-containing strain, but no such reactivity was observed when the OspA-negative ELISA was used. We conclude that low-passage-number OspA-B-negative isolates in immunoassays may become useful for the immunologic discrimination of the vaccinated state, natural infection, and vaccine failure.     

Myositis in mice inoculated with Borrelia burgdorferi.

The authors describe the appearance of myositis in immunocompetent and immunodeficient mice after subcutaneous inoculation with Borrelia burgdorferi by histology and immunohistology. Experimental infection of mice 1) causes inflammation of striated but not smooth muscles, 2) affects the entire musculoskeletal system, and 3) is characterized by perivascular and interfacicular infiltration of mononuclear leukocytes in the striated muscle leading to necrosis as well as disruption of muscle fibers. The lesions found in striated muscle specimens were most pronounced in immunodeficient (SCID), less severe in T-cell-deficient nu/nu (BALB/c, C57BL/6) and marginal to moderate or almost not present in immunocompetent AKR/N and C.B-17 mice, respectively.     

A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.     

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.     

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.     

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.     

Killing of Borrelia burgdorferi by Antibody Elicited by OspA Vaccine Is Inefficient in the Absence of Complement

A Lyme disease vaccine, based on the Borrelia burgdorferi lipoprotein OspA, has recently undergone phase III trials in humans. The results of one of these trials indicate that vaccine efficacy positively correlates with anti-OspA antibody titer. Spirochete killing within the tick vector midgut, upon which vaccine efficacy appears to depend, may occur chiefly via a mechanism that involves antibody alone, as it has been reported that complement is degraded by tick saliva decomplementing factors. We compared the in vitro killing efficiencies of anti-OspA antibody elicited in rhesus monkeys by the OspA vaccine, in the presence and in the absence of monkey complement. Killing in the absence of complement was between 14 and 3,800 times less efficient than with complement present, depending on the spirochete strain. The relative inefficiency of the complement-independent killing mechanism by anti-OspA antibody may explain why OspA vaccine efficacy is critically dependent on antibody titer.     

DbpA, but Not OspA, Is Expressed by Borrelia burgdorferi during Spirochetemia and Is a Target for Protective Antibodies

DbpA is a target for antibodies that protect mice against infection by cultured Borrelia burgdorferi. Infected mice exhibit early and sustained humoral responses to DbpA and DbpB, suggesting that these proteins are expressed in vivo. Many antigens expressed in mammals by B. burgdorferi are repressed in vitro at lower growth temperatures, and we have now extended these observations to include DbpA and DbpB. To confirm that the protective antigen DbpA is expressed in vivo and to address the question of its accessibility to antibodies during infection, we examined B. burgdorferi in blood samples from mice following cutaneous inoculation. B. burgdorferi was visualized by dark-field microscopy in plasma samples from spirochetemic mice, and an indirect immunofluorescence assay showed that these spirochetes were DbpA positive and OspA negative. We developed an ex vivo borreliacidal assay to show that hyperimmune antiserum against DbpA, but not OspA, killed these plasma-derived spirochetes, demonstrating that DbpA is accessible to antibodies during this phase of infection. Blood transferred from spirochetemic donor mice readily established B. burgdorferi infection in naive recipient mice or mice hyperimmunized with OspA, while mice hyperimmunized with DbpA showed significant protection against challenge with host-adapted spirochetes. Antiserum from persistently infected mice had borreliacidal activity against both cultured and plasma-derived spirochetes, and adsorption of this serum with DbpA substantially depleted this killing activity. Our observations show that immunization with DbpA blocks B. burgdorferi dissemination from the site of cutaneous inoculation and suggest that DbpA antibodies may contribute to control of persistent infection.     

Borrelia burgdorferi strain-specific Osp C-mediated immunity in mice.

Antibodies to the outer surface proteins (Osps) A, B, and C of the spirochete Borrelia burgdorferi can prevent infection in animal models of Lyme borreliosis. We have previously demonstrated that immune serum from mice infected with B. burgdorferi N40 can also prevent challenge infection and induce disease regression in infected mice. The antigens targeted by protective and disease-modulating antibodies are presently unknown, but they do not include Osp A or Osp B. Because Osp C antibodies are present in immune mouse serum, we investigated the ability of hyperimmune serum to recombinant Osp C (N40) to protect mice against challenge infection with N40 spirochetes. In both active and passive immunization studies, Osp C (N40) antiserum failed to protect mice from challenge infection with cultured organisms. Mice actively immunized with recombinant Osp C (N40) were susceptible to tick-borne challenge infection, and nymphal ticks remained infected after feeding on Osp C-hyperimmunized mice. In contrast, similar immunization studies performed with Osp C (PKo) antiserum prevented challenge infection of mice with a clone of PKo spirochetes pathogenic for mice. Both Osp C (N40) and Osp C (PKo) antisera showed minimal in vitro borreliacidal activity, and immunofluorescence studies localized Osp C beneath the outer membrane of both N40 and PKo spirochetes. We conclude that Osp C antibody-mediated immunity is strain specific and propose that differences in Osp C surface expression by spirochetes in vivo may account for strain-specific immunity.     

Experimental Borrelia burgdorferi infection of outbred mice.

Infectivity of Borrelia burgdorferi strain 297 in normal outbred ddY mice was examined. Strain 297 was inoculated intraperitoneally in 3-week-old outbred ddY mice. B. burgdorferi was routinely cultured from the heart and urinary bladder 5 to 84 days postinoculation. The combined isolation rate from both heart and urinary bladder was significantly higher than the rate from spleen, kidney, liver, urine, and blood samples. Three- and 10-week-old mice were infected with inocula of 10(4) and 10(5) or more, respectively. Passive transfer of undiluted and 10-fold-diluted anti-297 rabbit serum and active immunization of 50 to 100 micrograms of lyophilized whole cells completely protected mice from infection with B. burgdorferi. These results suggest that the outbred mouse is a convenient model for experimental infection with B. burgdorferi.