Effects of OspA vaccination on Lyme disease serologic testing.
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This study presents the effects of OspA vaccination on two-step testing for Borrelia burgdorferi antibodies. Although vaccinees developed enzyme-linked immunosorbent assay reactivity, immunoblots did not fulfill Centers for Disease Control and Prevention criteria for positivity. Furthermore, OspA reactivity did not interfere with interpretation of immunoblots with sera from patients who developed early Lyme disease despite vaccination. (+info)
Population dynamics of a naturally occurring heterogeneous mixture of Borrelia burgdorferi clones.
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Two unique isolates of Borrelia burgdorferi, differing in plasmid content and outer surface protein C expression, were cultured on sequential captures of a single free-living Peromyscus leucopus mouse and were examined for differences in transmissibility. Both isolates were transmissible from inoculated C.B-17 mice to larval Ixodes scapularis ticks and, subsequently, from infected nymphal ticks to C3H/HeJ mice. Plasmid and protein analyses suggested that the original isolates were a mixed population of B. burgdorferi, and cloning by limiting dilution resulted in the identification of two clonal groups. In addition to being heterogeneous in plasmid and genomic macrorestriction analyses, the clones varied with respect to the electrophoretic mobilities and antigenicity of their OspC proteins, as shown by their reactivity to a panel of monoclonal antibodies. Plasmid analysis of sequential isolates from C3H mice experimentally infected with the primary isolate or various mixtures of its subclones showed an apparently random fluctuation in clonal dominance in the majority of mice. Surprisingly, mice infected with each subclone were permissive to superinfection with the heterologous subclone, despite the presence of anti-B. burgdorferi antibodies at the time of the secondary challenge. These results show conclusively that mice captured at Lyme disease enzootic sites may be infected by mixed populations of genetically and antigenically distinct B. burgdorferi clones and that these infections can be acquired by coinfection or by sequential infection. The lack of cross-immunization between clones existing within a naturally occurring population may play a role in the maintenance of the genetic heterogeneity of B. burgdorferi in nature. (+info)
Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family.
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We previously reported on the existence of a family of lipoprotein genes, designated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of Borrelia burgdorferi 297. A distinguishing feature of the 2.9 lipoproteins were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed herein mlp, for multicopy lipoprotein genes. Computer analyses and immunoblotting revealed that Mlp-9 segregated with the antigenic class I lipoproteins, whereas Mlp-8 and Mlp-10 were members of class II. Northern blotting showed that all three of the mlp genes were expressed when B. burgdorferi was cultivated in vitro at 34 degrees C, although mlp-9 and mlp-10 transcripts were expressed at very low levels. Additional combined immunoblotting and comparative reverse transcription-PCR analyses performed on borreliae cultivated in vitro at 23, 34, or 37 degrees C indicated that although Mlp-8 was substantially more abundant than Mlp-9 or Mlp-10, all three of the mlp genes were upregulated during B. burgdorferi replication at 37 degrees C. Expression of the same three lipoproteins was further enhanced upon growth of the spirochetes within dialysis membrane chambers (DMCs) implanted intraperitoneally in rats (i.e., spirochetes in a mammalian host-adapted state), suggesting that temperature alone did not account for maximal upregulation of the mlp genes. That certain mlp genes are likely expressed during the growth of B. burgdorferi in mammalian tissues was supported by findings of antibodies against all three Mlp lipoproteins in mice after challenge with Ixodes scapularis nymphs harboring B. burgdorferi 297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashion (e.g., OspA/OspC), at least three mlp genes are simultaneously upregulated by temperature (37 degrees C) and some other mammalian host factor(s). The findings have importance not only for understanding alternative modes of differential antigen expression by B. burgdorferi but also for assessing whether one or more of the Mlp lipoproteins represent new candidate vaccinogens for Lyme disease. (+info)
An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi.
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Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable Ags such as the variable major protein of Borrelia hermsii, the variant surface glycoprotein of African trypanosomes, and the pilin of Neisseria gonorrhoeae include an immunodominant variable domain and one or more invariable domains that are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain. VlsE (variable major protein-like sequence, expressed), the variable surface Ag of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR1-6) that together amount to half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Surprisingly, unlike the invariable regions of variable major protein, variant surface glycoprotein, and pilin, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. IR6 is exposed on the surface of VlsE, as assessed by immunoprecipitation experiments, but is inaccessible to Ab on the spirochete's outer membrane, as demonstrated by immunofluorescence and in vitro killing assays. VlsE thus significantly departs from the antigenic variation paradigm, whereby immunodominance is only manifest in variable portions. We submit that IR6 may act as a decoy epitope(s) and contribute to divert the Ab response from other, perhaps protective regions of VlsE. (+info)
Clinical evaluation of guidelines and two-test approach for lyme disease.
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OBJECTIVE: The diagnosis of Lyme disease should be based on objective clinical signs and symptoms. In a clinical study, we have evaluated whether the recommended two-step approach for serodiagnosis of Lyme disease is useful in daily clinical practice and can influence clinical decision making. METHODS: The signs and symptoms of patients with ongoing musculoskeletal complaints, assumed by their referring physician or themselves to be attributable to active or chronic Lyme disease, and of patients diagnosed as having Lyme disease, were evaluated. On the basis of clinical evaluation only, patients were classified into three groups: previous Lyme disease, active Lyme disease and no Lyme disease. Antibodies to Borrelia burgdorferi were determined by means of an enzyme-linked immunosorbent assay (ELISA), followed, when positive, by immunoblotting. RESULTS: One hundred and three patients (41 males and 62 females, mean age 48.7 yr) participated in the study. Of the 49 patients classified as previous Lyme disease, 25 (51%) had antibodies to B. burgdorferi. All 10 patients with active Lyme disease had positive antibodies and 12 of the 44 patients (27%) classified as no Lyme disease had positive antibodies. No statistically significant differences were found between the percentage of positive immunoblots from patients with previous Lyme disease (72%) and patients with active Lyme disease (100%). In the group of no Lyme disease, five out of 12 patients had a negative immunoblot. Concerning serological testing, immunoblotting could have added additional information. However, immunoblotting did not influence clinical decision making in this group of patients. CONCLUSION: Immunoblotting did not influence clinical decision making for the 47 patients with antibodies to B. burgdorferi in this study. (+info)
Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.
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Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion. (+info)
Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE.
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VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects. (+info)
Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi.
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VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen. (+info)