伯氏疏螺旋体外膜蛋白C基因在大肠杆菌中的表达

目的　体外表达伯氏疏螺旋体外膜蛋白Ｃ（ＯｓｐＣ） ,以制备基因工程重组抗原用于莱姆病血清学诊断研究。方法　应用聚合酶链反应技术和基因重组技术,从２ 个伯氏疏螺旋体中国分离株基因组ＤＮＡ 中扩增得到ＯｓｐＣ基因,分别克隆到表达载体ＬＫＢ２ 中,在大肠杆菌中进行表达。结果　以天然蛋白形式高效表达了ＯｓｐＣ。扫描分析显示,表达的ＯｓｐＣ 相对分子质量均在２３ ０００ 左右,表达量占菌体总蛋白的３４ ％ ～５０ ％ 。Ｗｅｓｔｅｒｎ ｂｌｏｔ 试验证明,重组ＯｓｐＣ 蛋白可与莱姆病患者血清发生特异性反应。结论　重组ＯｓｐＣ 的成功表达为制备新型莱姆病早期诊断试剂奠定了基础     

Stability of Borrelia burgdorferi outer surface protein C under immune selection pressure

Outer surface protein (Osp) C immune pressure during persistent infection with Borrelia burgdorferi was examined in relation to genetic variation of ospC. Mice were infected with clonal B. burgdorferi sensu stricto (s.s.) N40 or B. afzelii PKo and then were hyperimmunized with homologous recombinant OspC or with decorin-binding protein A (DbpA) (controls). After 6 months, B. burgdorferi isolates were subjected to restriction enzyme analysis of the amplified ospC genes and were found to have no differences among 9 B. burgdorferi s.s. N40 and 9 B. afzelii PKo isolates from OspC hyperimmune mice or among 10 B. burgdorferi s.s. N40 and 10 B. afzelii PKo isolates from DbpA hyperimmune mice, compared with input inocula. Comparison of gene sequences among 4 B. burgdorferi s.s. N40 and 9 B. afzelii PKo isolates from OspC-immunized mice revealed no ospC variation from input inocula. Variation in ospC among B. burgdorferi isolates and species during chronic infection is not likely to be an important mechanism for immune evasion.     

Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.     

Induction of an outer surface protein on Borrelia burgdorferi during tick feeding.

Lyme disease spirochetes, Borrelia burgdorferi sensu lato, are maintained in zoonotic cycles involving ticks and small mammals. In unfed ticks, the spirochetes produce one outer surface protein, OspA, but not OspC. During infection in mammals, immunological data suggest that the spirochetes have changed their surface, now expressing OspC but little or no OspA. We find by in vitro growth experiments that this change is regulated in part by temperature; OspC is produced by spirochetes at 32-37 degrees C but not at 24 degrees C. Furthermore, spirochetes in the midgut of ticks that have fully engorged on mice now have OspC on their surface. Thus two environmental cues, an increase in temperature and tick feeding, trigger a major alteration of the spirochetal outer membrane. This rapid synthesis of OspC by spirochetes during tick feeding may play an essential role in the capacity of these bacteria to successfully infect mammalian hosts, including humans, when transmitted by ticks.     

Evasion of protective immunity by Borrelia burgdorferi by truncation of outer surface protein B.

We analyzed variability in outer surface protein B (OspB) from Borrelia burgdorferi (Bb), the causative agent of Lyme disease, to determine how Bb escapes immune destruction. We have shown that vaccination with OspB from Bb strain B31 protected mice from infection with Bb B31 but not against Bb N40. The present study demonstrates that Bb N40 spirochetes which evade vaccination immunity to OspB have a truncated form of OspB, due to a TAA stop codon at nucleotide 577. In contrast, Bb N40 spirochetes that express full-length OspB are unable to infect mice immunized with OspB, analogous to our previous studies with Bb B31. Mapping of the OspB antibody response shows that epitopes in the C terminus of OspB are surface-exposed and bind protective monoclonal and polyclonal antibodies. This suggests that the C terminus of OspB is important for eliciting a protective immune response to OspB. Truncation or modification of outer surface proteins that do not bind protective antibody may be a means by which Bb evades host defenses.     

The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor.

The spirochete Borrelia burgdorferi is the causative agent of Lyme borreliosis (Lyme disease) and is transmitted to mammalian hosts by tick vectors. In humans, the bacteria induce a complex disease, which involves the skin, joints, heart, and nervous system. However, the pathogenic principles of this multisystem illness are far from being understood. To disseminate from the site of the tick bite and invade multiple organ sites, spirochetes have to penetrate normal tissue barriers, such as vascular basement membranes and other organized extracellular matrices. Substantial evidence from other invasive bacterial infections suggest that spirochetes may use endogenous or host-derived enzymes--in particular, proteinases--for this purpose. Here we show that B. burgdorferi binds human plasmin(ogen)--mainly via its outer cell surface lipoprotein A. Binding of plasminogen to spirochetal receptor leads to an accelerated formation of active plasmin in the presence of host-derived plasminogen activator. The cell-surface-associated plasmin cannot be regulated by the serum inhibitor alpha 2-antiplasmin and degrades high-molecular-weight glycoproteins, such as fibronectin. It is suggested that the acquisition of host-derived proteinase plasmin(ogen) contributes to the pathogenicity of B. burgdorferi.     

Growth-inhibiting antibody responses of humans vaccinated with recombinant outer surface protein A or infected with Borrelia burgdorferi or both

Serial serum samples from a 2-year human trial of outer surface protein (Osp) A vaccine were analyzed by Borrelia burgdorferi growth-inhibition assay (GIA) and anti-OspA ELISA to assess the antibody responses of vaccine recipients and subjects with Lyme disease. Although 74% of OspA recipients had a reciprocal GIA titer >/=64 after 3 vaccinations, none of the placebo recipients, even those with Lyme disease, had a GIA titer this high. The correlation between GIA and ELISA titers after 3 doses of vaccine was.84; however, more vaccine recipients had an elevated ELISA titer paired with low GIA titer than had a low ELISA titer with a high GIA titer. OspA-vaccine recipients who acquired Lyme disease had significantly lower serum GIA and ELISA titers after 3 immunizations than did age- and sex-matched OspA recipients without Lyme disease. Thus, vaccinated subjects had antibodies to native antigen on viable cells, and antibody assays with this specificity may predict protection of vaccinees against infection.     

Association of antibiotic treatment-resistant Lyme arthritis with T cell responses to dominant epitopes of outer surface protein A of Borrelia burgdorferi.

OBJECTIVE: To explore further the association of antibiotic treatment-resistant Lyme arthritis and T cell reactivity with outer surface protein A (OspA) of Borrelia burgdorferi, including the identification of T cell epitopes associated with this treatment-resistant course. METHODS: The responses of peripheral blood and, if available, synovial fluid lymphocytes to B burgdorferi proteins, fragments, and synthetic peptides, as determined by proliferation assay and interferon-gamma production, were compared in 16 patients with treatment-responsive and 16 with treatment-resistant Lyme arthritis. RESULTS: The maximum severity of joint swelling correlated directly with the response to OspA. Moreover, the only significant difference between patients with treatment-resistant and treatment-responsive arthritis was in reactivity with N-terminal and C-terminal fragments of OspA, OspA1 (amino acids [aa] 16-106), and OspA3 (aa 168-273). Epitope mapping showed that 14 of the 16 patients with treatment-resistant arthritis had responses to OspA peptides (usually 4 or 5 epitopes), whereas only 5 of the 16 patients with treatment-responsive arthritis had reactivity with these peptides (usually 1 or 2 epitopes) (P = 0.003). Patients with HLA-DRB1 alleles associated with treatment-resistant arthritis were more likely to react with peptide 15 (aa 154-173) and, to a lesser degree, with peptide 21 (aa 214-233) than patients with other alleles, whereas the responses to other epitopes were similar in both groups. CONCLUSION: The maximum severity of joint swelling and the duration of Lyme arthritis after antibiotic treatment are associated with T cell responses to specific epitopes of OspA.     

Recombinant outer surface protein a from Borrelia burgdorferi induces antibodies protective against spirochetal infection in mice.

The outer surface protein A (OspA) of Borrelia burgdorferi was isolated in its native form from strains ZS7 and B31 and as a recombinant protein from strain ZS7. Amino acid sequence analysis of internal peptides of native OspA (strain ZS7) revealed identity with the sequence deduced from the OspA gene. Repeated immunization of C57BL/6 and C.B-17 mice with any of the three OspA structures resulted in the generation of monospecific hyperimmune sera reactive with both native and recombinant OspA. Upon transfer of immune sera specific for either native OspA (strain B31) or recombinant OspA (strain ZS7) but not of those reactive with the recombinant 41-kDa flagellin-associated antigen, severe combined immunodeficient (SCID) mice were completely protected against infection with strain ZS7. The finding that monoclonal antibodies to OspA and to OspB but not to non-outer surface spirochetal structures such as flagellin, p20, p65, and p70 conferred protection in SCID mice makes OspA (and possibly OspB) a promising candidate vaccine against Lyme disease.     

Geographic clustering of an outer surface protein A mutant of Borrelia burgdorferi. Possible implications of multiple variants for Lyme disease persistence

DNA sequences encoding full-length outer surface protein (Osp) A were amplified from four joint fluid samples over 4.5 months from a patient with chronic Lyme arthritis, with a variant from wild type only found in sample 3. Rather than a mutation in vivo, these findings suggested a mixed infection in which BORRELIA: containing the wild-type and mutant ospA were waxing and waning in the patient's joint. If so, we reasoned that the mutant should be present in the community. We therefore took the novel epitope resulting from the mutation, expressed as a fusion protein in Escherichia coli, and performed Western blots on 80 high-titred stored sera; however, all except that of our index patient were negative. We then collected 36 stored sera from patients with Lyme disease residing within 10 miles of where the index patient had lived. An additional two sera from this circumscribed area were positive (P = 0.038). These findings show that results from single samples can be misleading, and suggest that the OspAs expressed in force late in Lyme arthritis are the same ones introduced initially into the host. Moreover, they allow a speculative mechanism for disease persistence not previously considered, in which antigenically distinct B. burgdorferi variant proteins present themselves serially to the immune system.     

HLA type and immune response to Borrelia burgdorferi outer surface protein a in people in whom arthritis developed after Lyme disease vaccination

Objective

To investigate whether persons with treatment‐resistant Lyme arthritis–associated HLA alleles might develop arthritis as a result of an autoimmune reaction triggered by Borrelia burgdorferi outer surface protein A (OspA), the Lyme disease vaccine antigen.

Methods

Persons in whom inflammatory arthritis had developed after Lyme disease vaccine (cases) were compared with 3 control groups: 1) inflammatory arthritis but not Lyme disease vaccine (arthritis controls), 2) Lyme disease vaccine but not inflammatory arthritis (vaccine controls), and 3) neither Lyme disease vaccine nor inflammatory arthritis (normal controls). HLA–DRB1 allele typing, Western blotting for Lyme antigen, and T cell reactivity testing were performed.

Results

Twenty‐seven cases were matched with 162 controls (54 in each control group). Odds ratios (ORs) for the presence of 1 or 2 treatment‐resistant Lyme arthritis alleles were 0.8 (95% confidence interval [95% CI] 0.3‐2.1), 1.6 (95% CI 0.5–4.4), and 1.75 (95% CI 0.6–5.3) in cases versus arthritis controls, vaccine controls, and normal controls, respectively. There were no significant differences in the frequency of DRB1 alleles. T cell response to OspA was similar between cases and vaccine controls, as measured using the stimulation index (OR 1.6 [95% CI 0.5–5.1]) or change in uptake of tritiated thymidine (counts per minute) (OR 0.7 [95% CI 0.2–2.3]), but cases were less likely to have IgG antibodies to OspA (OR 0.3 [95% CI 0.1–0.8]). Cases were sampled closer to the time of vaccination (median 3.59 years versus 5.48 years), and fewer cases had received 3 doses of vaccine (37% versus 93%).

Conclusion

Treatment‐resistant Lyme arthritis alleles were not found more commonly in persons who developed arthritis after Lyme disease vaccination, and immune responses to OspA were not significantly more common in arthritis cases. These results suggest that Lyme disease vaccine is not a major factor in the development of arthritis in these cases.

     

中国莱姆病螺旋体PD91外膜蛋白C表达载体的构建及序列分析

目的　构建莱姆病螺旋体PD91菌株外膜蛋白C(OspC)的表达载体 ,克隆表达OspC以便用于莱姆病诊断研究。方法　设计引物 ,用PCR从PD91扩增出ospC基因 ,定向克隆到表达载体PGEX - 5X - 1,构建重组质粒。通过PCR、酶切分析及序列测定等方法鉴定重组质粒。结果　ospC基因被正确克隆到表达载体PGEX - 5X - 1中。序列测定结果证实与国外已报道的ospC基因序列同源性在 6 5 %～ 92 %。 结论　OspC的编码基因在不同菌株间的同源性存在较大的差异。PGEX -5X - 1-ospC重组质粒的成功构建 ,为我国莱姆病特异性诊断试剂的研究奠定了基础。     

The outer surface protein A (OspA) ofBorrelia burgdorferi: A vaccine candidate and bioactive mediator

Summary In the search for a suitable vaccine candidate for Lyme borreliosis the principles of protective immunity were studied in a murine model ofBorrelia burgdorferi infection. It was found that the spirochetal outer surface protein A (lipOspA) in its native and recombinant lipidated form induces monospecific immune sera, which in passive transfer experiments protect SCID mice against experimental and tick-borne infection and disease. These and similar findings of independent groups led to the development of a vaccine formulation containing lipOspA. When tested in clinical phase I/II safety trials the recombinant lipOspA vaccine was shown to be safe, immunogenic and able to elicit borreliacidal antibodies. At present, clinical phase III efficacy trials are being conducted.B. burgdorferi infection involves the dissemination of the spirochetes from the site of the tick bite, infection of distant organs, and induction of a chronic inflammatory process. Recent studies indicate that the spirochetes may utilize host-derived enzyme systems to increase their virulence/pathogenicity. It was found that lipOspA serves as a surface receptor for the host-derived proteolytic enzyme plasmin(ogen), the central component of the so-called plasminogen activator system. Moreover, it was found that spirochetes are able to activate endothelial cells and blood-derived leukocytes, such as monocytes/macrophages, B cells and T cells, to express functions and/or secrete molecules, which are known to promote inflammatory responses. Part of these activities were exerted by the isolated lipOspA. The studies indicate an important role of lipOspA, both for the induction of a protective immune response by the host, as well as for the pathogenic processes elicited duringB. burgdorferi infection.

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Das Oberflächenprotein Outer surface protein A (OspA) vonBorrelia burgdorferi: eine mögliche Vakzine und ein biologisch aktives Mediatormolekül

Zusammenfassung Bei Untersuchungen zur Impfstoffentwicklung gegen dieBorrelia burgdorferi-Infektion haben wir grundlegende Prinzipien der protektiven Immunantwort in einem murinen Modell erarbeitet. Wir fanden, daß das Oberflächenlipoprotein outer surface protein A (lipOspA) in nativer und rekombinanter lipidierter Form monospezifische Immunseren induziert, die in passiven Transferexperimenten immuninkompetente (SCID) Mäuse gegen experimentelle und zeckenvermittelte Infektion und Erkrankung schützen. Diese Befunde zusammen mit ähnlichen Befunden anderer Arbeitsgruppen führten zur Entwicklung eines Impfstoffs auf der Basis des lipOspA. In klinischen Phase I/II Prüfungen erwies sich der rekombinante lipOspA-Impfstoff als verträglich und immunogen und fähig zur Induktion bakterizider anti-Borrelien-Antikörper. Zum gegenwärtigen Zeitpunkt werden klinische Phase III-Prüfungen zur Untersuchung der protektiven Effizienz durchgeführt. Ausgehend von der Stelle des Zeckenbisses kommt es bei derB. burgdorferi-Infektion zur Verbreitung der Spirochäten in entfernte Organe und zur Induktion einer chronischen Entzündung. Kürzlich durchgeführte Untersuchungen zeigen, daß die Borrelien möglicherweise wirtseigene humorale Enzymsysteme rekrutieren, die ihnen die Ausbreitung erleichtern. In diesem Zusammenhang fanden wir, daß das lipOspA als ein Oberflächenrezeptor für das wirtseigene proteolytische Enzym Plasmin(ogen) fungiert. Darüber hinaus konnten wir zeigen, daß Borrelien in der Lage sind, vaskuläre Endothelzellen und Leukozyten, wie Monozyten/Makrophagen, B Zellen und T Zellen, zu aktivieren. Die aktivierten Zellen exprimieren Funktionen und sezernieren Moleküle mit proinflammatorischer Wirkung. Ein Teil der stimulierenden Aktivitäten konnte auf das gereinigte lipOspA zurückgeführt werden. Unsere Studien weisen demnach darauf hin, daß lipOspA als Zielstruktur protektiver Antikörper nicht nur eine wichtige Rolle bei der Induktion einer protektiven Immunantwort, sondern auch bei den pathogenen Reaktionen im Rahmen derB. burgdorferi-Infektion spielt.

     

Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice.

We have recently shown that viable Borrelia burgdorferi organisms induce a chronic infection associated with arthritis and carditis in severe combined immunodeficiency (scid) mice but not in immunocompetent mice. The disease is similar to that found in patients suffering from Lyme disease. We now show that B. burgdorferi-specific immune mouse sera as well as a monoclonal antibody to the spirochetal outer surface antigen A (31 kDa) but not monoclonal antibodies specific for the 41-kDa antigenic component of the periplasmic flagella are able to prevent (or mitigate) the development of the disease in scid mice when passively transferred at the time of the bacterial inoculation. The identification of a B. burgdorferi-associated protective antigen suggests that the corresponding spirochetal protein should be tested as a vaccine against Lyme disease.     

Vector/host relationships of the Lyme disease spirochete, Borrelia burgdorferi

Lyme borreliosis is now occurring on several continents where its causative agent, Borrelia burgdorferi, is maintained and transmitted by ticks of the "Ixodes ricinus complex" namely I. dammini, I. pacificus, and possibly I. scapularis in North America, I. ricinus In Europe, and I. persulcatus in Asia. Because all developmental stages of these ticks feed on a large variety of hosts including humans, the vector/host relationships of this spirochete is highly complex as indicated by the voluminous literature reviewed in this article. The association of B. burgdorferi with ticks parasitizing exclusively rabbits and birds, suggests that the geographic distribution of this agent may be far greater than assumed and may include areas where the disease in humans is absent. Finally, the persistence of the Lyme disease spirochete in the midgut of its tick vectors and its invasion of other tissues during the ticks' feeding, are unique and differ from the behavior of all other arthropod-borne borreliae.