Vaccination with recombinant outer surface protein A (OspA) from Borrelia burgdorferi ... zs7.a68. All three genes are located, together with ospA/B, on the linear ...
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Artificial-infection protocols allow immunodetection of novel Borrelia burgdorferi antigens suitable as vaccine candidates against Lyme disease Reinhard Wallich1, Oliver Jahraus1, Thomas Stehle2, Thi Thanh Thao Tran2, Christiane Brenner1, Heidelore Hofmann4, Lise Gern3 and Markus M. Simon2 1
Institut für Immunologie, Universitätsklinikum Heidelberg, Heidelberg, Germany Max-Planck-Institut für Immunbiologie, Freiburg, Germany Institut de Zoologie, Universite´ de Neuchatel, ´ Neuchatel, ´ Switzerland 4 Klinik für Dermatologie und Allergologie, Technische Universität München, Munich, Germany 2 3
Vaccination with recombinant outer surface protein A (OspA) from Borrelia burgdorferi provides excellent antibody-mediated protection against challenge with the pathogen in animal models and in humans. However, the bactericidal antibodies are ineffective in the reservoir host, since OspA is expressed by spirochetes only in the vector, but rarely, if at all, in mammals. Using an artificially generated immune serum (anti-108 spirochetes) with high protective potential for prophylactic and therapeutic treatment, we have now isolated from an expression library of B. burgdorferi (strain ZS7) three novel genes, zs7.a36, zs7.a66 and zs7.a68. All three genes are located, together with ospA/B, on the linear plasmid lp54, and are expressed in vitro and in ticks. At least temporarily two of them, ZS7.A36 and ZS7.A66, are also expressed during infection. The respective natural antigens are poorly immunogenic in infected normal mice but elicited antibodies in Lyme disease patients. We show that recombinant preparations of ZS7.A36, ZS7.A66 and ZS7.A68 induce functional antibodies in rabbits capable of protecting immunodeficient mice against subsequent experimental infection. These findings suggest that all three recombinant antigens represent potential candidates for a ‘second generation’ vaccine to prevent and/or cure Lyme disease. Key words: Borrelia burgdorferi / Lyme disease / Vaccine / Artificial infection
1 Introduction
10/10/02 18/12/02 14/1/03
but are able to prevent infection in naive recipients, upon passive transfer of Ab [4, 5].
Natural infection with pathogens does not always lead to resolution of infection or adequate protection against a second challenge [1]. This applies to the spirochete Borrelia burgdorferi, the causal agent of Lyme disease. Previous studies in mice and man have shown that natural infection is accompanied by the generation of specific Ab with a time-dependent increase of their diversity [2, 3]. However, in mice these pathogen-induced Ab are inefficient in resolving an established disease/infection
[I 23620] The first two authors contributed equally to this study. Supporting information for this article is available on the WWW under www.eji.de or from the author. Abbreviations: GST: Glutathione S-transferase IS: immune serum Osp: Outer surface protein PFGE: Pulse field gel electrophoresis p.i.: Post-inoculation 0014-2980/03/0303-708$17.50 + .50/0
Received Revised Accepted
Possible reasons for the insufficient eradication of spirochetes include immune escape strategies evolved by the pathogen, such as down-regulation [6] or variation [7] of outer surface proteins (Osp). As a consequence, bactericidal Ab may be generated too late or only at suboptimal concentrations. In fact, it was found that the pattern of osp gene expression in B. burgdorferi dramatically changes depending on the microenvironment and that some osp genes even cease expression in mammals. The latter is the case for ospA, which is dominantly expressed in spirochetes harbored in the gut of unfed ticks, but not in those seeding the vertebrate host [8–11]. In contrast, expression of other osp genes, e.g. ospC, bbk32 and bbk50, is first detectable on spirochetes in ticks after engorgement, but are also on spirochetes present in several mouse tissues [8, 11, 12] Other spirochetal gene products, such as eppA, pG, p21, bbK2.10, 35-kDa, and lp6.6, seem to be selectively expressed in © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the mammalian environment, but not the tick environment, at least temporarily [13, 14]. A recent report indicating that homogeneous populations of B. burgdorferi within the tick gut give rise to transmittable progenies with extensive antigenic heterogeneity upon feeding adds a further dimension to the problem of immune surveillance [15].
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2 Results 2.1 Immune serum raised following artificial infection with high numbers of B. burgdorferi contains Ab with prophylactic and therapeutic potential
Thus it is surprising that not only Ab (including OspC-, pG-, DpbA- and p35/37-specific Ab) that accumulate during natural infection but also Ab (such as OspAspecific Ab) that are hardly, if at all, detectable have the potential to protect mice against subsequent challenge [6, 10, 13, 16–18]. The fact that OspA-specific Ab, which block transmission of spirochetes by targeting and killing B. burgdorferi in the midgut of feeding ticks [10], are generated exclusively under artificial conditions emphasizes the notion that “efficient vaccines may need additional antigenic stimuli not available during natural infection” [1]. On the basis of such studies, a monovalent vaccine, consisting of the OspA lipoprotein, was developed [16] and shown to be efficacious in humans [19].
Immune serum (IS) from mice infected via ticks (i.e. “ § tick” IS) or infected experimentally with either low ( § 103) or high doses ( § 108) of B. burgdorferi (ZS7) contain Ab that are able to protect, upon passive transfer, naive recipients against subsequent challenge [4, 5]. Besides Ab to OspA and OspB [16], which are only observed in § 108 but not in § tick and § 103 IS (Fig. 1A; and [4]), all three IS contain Ab with protective potential that are specific for other known spirochetal structures, including OspC and DbpA, though at different levels (Fig. 1A). Whereas § 108 IS contains high amounts of DbpA-specific Ab and lower levels of OspC-specific Ab, the reverse is true for § 103 IS. § tick IS, on the other hand expresses, if at all, only marginal amounts of Ab to OspC and DbpA.
Ab induced by the OspA vaccine are unable to resolve B. burgdorferi infection within the mammalian host for reasons given above [9, 20]. However, our recent observation of a close correlation between high titers of serum Ab to OspC and resolution of infection in susceptible AKR/N mice suggested that spirochetes were susceptible to Ab in vivo as long as sufficient amounts of relevant Ab are available [20]. This is supported by the finding that passive transfer of OspC-specific Ab prevents and resolves experimental and tick-borne B. burgdorferi infections in immunodeficient mice and in immunocompetent mice [11, 20]. Thus, eradication of spirochetes during chronic infection may be feasible by artificially enhancing Ab responses to in-vivo-expressed B. burgdorferi antigens.
Among protective Ab species described so far, including pG [13], Ab to OspA, OspB, OspC and DbpA seem to be the most potent ones in preventing infection in mice [16–18, 20, 21]. It was thus surprising to find in a number of initial experiments that following depletion of Ab to OspA, OspB, OspC, pG, P39, P83/100 and/or DbpA, the two IS ( § 103 and § 108) derived from experimentally infected mice retained protective activity(ies). Experiments with serial dilutions of IS showed that from the two pre-absorbed IS, § 108 IS had the strongest potential to prevent infection in mice (data not shown) and, most importantly, was the only one to also readily clear spirochetes from SCID mice as demonstrated by negative ear cultures.
In the present study we used an artificially generated immune serum with high prophylactic and therapeutic potential to identify three novel B. burgdorferi antigens of strain ZS7 (B. burgdorferi s.s.). We report on their expression patterns in ticks, in mice and in vitro, their immunogenicity during infection and their ability to induce protective Ab.
As shown in Table 1 (two independent experiments), passive transfer of § 108 IS to infected SCID mice on four occasions, starting at day 26 post-inoculation (p.i.), resulted in resolution of joint swelling and clearance of spirochetes. Previous experiments [11, 20, 22] and our initial experiments (data not shown) had already demonstrated that § 103 IS or normal mouse sera had no or only minimal effects on established B. burgdorferi infection in SCID mice.
2.2 Identification and characterization of three novel genes from B. burgdorferi strain ZS7 To identify the target structures recognized by the residual protective Ab contained within the § 108 IS (see above), a B. burgdorferi ZS7 expression library was
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Eur. J. Immunol. 2003. 33: 708–719 Fig. 1. Western blot analysis of immune sera from infected or immunized mice. (A) Pooled IS from BALB/c mice experimentally infected with either 108 (IS § 108, diluted 1:400) or 103 (IS § 103, diluted 1:50) ZS7 spirochetes or challenged via pre-infected ticks (IS § tick, diluted 1:25) were tested on whole cell lysate (ZS7, 5×106 cell equivalents/lane, lane a), rOspA (500 ng/lane, lane b), rOspC (500 ng/lane, lane c), and rDbpA (500 ng/lane, lane d). (B) IS § 108 (1:150), IS § 103 (1:150), and IS § tick (1:50) were tested on preparations of rZS7.A36 (lane 1), rZS7.A66 (lane 2), and rZS7.A68 (lane 3). (C) Pooled IS (diluted 1:200) from BALB/c mice immunized with preparations of rZS7.A36, rZS7.A66 and rZS7.A68 were tested on rZS7.A36, (lane 1), rZS7.A66 (lane 2), and rZS7.A68 (lane 3). B. burgdorferi antigens are indicated by arrows. (D) Pooled rabbit IS § ZS7.A36 (diluted 1:25, lane 1), § ZS7.A66 (diluted 1:25, lane 2), § ZS7.A68 (diluted 1:25, lane 3), § OspC (diluted 1:6000, lane 4), and normal rabbit serum (diluted 1:1000, lane 5), were tested on whole spirochetal cell lysates (strain ZS7, 2.5×106 cell equivalents/lane). A mix of mAb directed against OspA, OspB, OspC, P39, flagellin and HSP60 is shown in lane S.
zs7.a36, zs7.a66 and zs7.a68 have open reading frames of 636, 1227, and 753 nucleotides. zs7.a36 encodes a unique protein with a molecular mass of 22 kDa, as detected with specific IS (Fig. 1C). ZS7.A36 represents a
screened with either pre-adsorbed or non-adsorbed § 108 IS. Clones encoding ospA–C, pG, and dbpA were identified by Southern blot hybridization and excluded from further analysis (data not shown). Among a number of newly isolated clones, five (pUEX 15, 52.1, 72, 83 and 89) were characterized in more detail and shown to represent three distinct genes (Table 2). Comparative sequence analyses of the five clones revealed that the corresponding genes are homologues of the putative open reading frames bba36, bba66 and bba68 in the TIGR database of B. burgdorferi, strain B31 (www.TIGR.org) and are hereinafter termed zs7.a36, zs7.a66 and zs7.a68. Pulse field gel electrophoresis (PFGE) demonstrated that all three ZS7 genes, as their B31 counterparts, are located together with ospA/B, dbpA/B and p35 on the 54-kb linear plasmid (lp54) of B. burgdorferi ZS7 (Fig. 2A, and www.TIGR.org).
Fig. 2. PFGE separating the chromosomal and plasmid DNA (A) and Southern blot analysis of HindIII-digested (B) DNA from B. burgdorferi ZS7 (lane 1), B. afzelii MMS (lane 2), and B. garinii ZQ1 (lane 3). DNA was electrophoresed through agarose gels, transferred to a nylon membrane and hybridized with the indicated gene probes. Marker sizes are in kilobases.
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Table 1. Therapeutic effect of mouse immune serum ( § 108 B.b. ZS7) on established experimental B. burgdorferi infection of C.B17.SCID mice Exp.
1
2
a)
Clinical arthritisa), days p.i.
Mouse
Ear culture, days p.i.
number
26
49
96
153
221
26
74
221
1
++/+
+/±
±/(±)
–/–
–/–
+
–
–
2
+/+
+/±
(±)/(±)
(±)/–
–/–
+
–
–
3
+/++
±/+
–/(±)
–/–
–/–
+
–
–
4
+/±
+/(±)
+/–
±/(±)
(±)/–
+
–
–
1
++/++
+/±
(±)/–
–/–
–/–
+
–
–
2
+/+
–/(±)
–/–
–/–
–/–
+
–
–
3
+/+
+/+
±/±
(±)/(±)
–/–
+
–
–
4
++/++
(±)/±
(±)/–
–/–
–/–
+
–
–
On days 26, 30, 34 and 38 p.i. (10 B.b.), each mouse was given 100 ? l of § 10 -IS (i.p.). Concentration of B.b.-specific Ab (ELISA): G 500 ? g/ml (308.0 ? g/ml specific for OspA, 5.5 ? g/ml specific for OspC and 28.0 ? g/ml specific for LA-2 equivalents). Arthritis scoring: – none; (±) reddening and/or mild swelling; ± moderate swelling; + severe swelling; ++ extreme swelling of tibiotarsal joint. 3
8
typical prokaryotic lipoprotein localized to the cell envelope of the spirochete and exhibits no significant similarity to any other known B. burgdorferi protein (data not shown). zs7.a66 encodes a 45-kDa protein (Fig. 1C), which belongs to a large paralogous gene family (gbb54) (www.TIGR.org). zs7.a68, another member of the gbb54 (TIGR database) paralogous gene family, encodes a putative lipoprotein likely to be surface localized with a predicted molecular mass of 28 kDa (Fig. 1C). zs7.a36 and zs7.a66 as well as ospA (control) but not zs7.a68 are also detectable in the genome of the other two human pathogenic B. burgdorferi species, i.e. B. garinii (ZQ1) and B. afzelii (MMS; Fig. 2A). The different signal intensities for zs7.a36 and zs7.a66 in strains ZQ1 and MMS as compared with strain ZS7 suggest varying
degrees of similarity between the homologous genes, as reported before for ospA [23]. The latter results were confirmed by differential patterns obtained employing RFLP analysis (Fig. 2B) and DNA sequencing (data not shown).
2.3 Expression patterns of zs7.a36, zs7.a66 and zs7.a68 in cultured spirochetes, tick associated spirochetes or mouse tissue associated spirochetes, and immunogenicity of the respective proteins In contrast to zs7.ospA-specific mRNA, which is readily expressed in cultured spirochetes [11], transcripts for zs7.a36, zs7.a66 and zs7.a68 were undetectable by
Table 2. Identification of three novel B. burgdorferi clones by screening a ZS7 expression library employing a pre-adsorbed protective mouse immune serum Insert size (kbp)
Protein size (MW)
Name
Gene localizationa)
TIGR designationb)
15
1.8
28 kDa
ZS7.A68
lp 54
BBA68
52.1
2.8
45 kDa
ZS7.A66
lp 54
BBA66
72
2.3
28 kDa
ZS7.A68
lp 54
BBA68
83
3.0
22 kDa
ZS7.A36
lp 54
BBA36
89
1.5
45 kDa
ZS7.A66
lp 54
BBA66
pUEX clone
a) b)
Indicates the plasmid localization as revealed by Southern blot analysis (lp, linear plasmid). See www.TIGR.org for details.
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Northern blot analysis in all three spirochetal strains (data not shown). By applying RT-PCR to the RNA preparation of ZS7, marginal or low amounts of mRNA specific for zs7.a36 and zs7.a68, respectively, but none for
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zs7.a66 were detectable whereas ospA was highly expressed. (Fig. 3A, lane 1). As expected, all four genes were contained in the spirochetal genome (Fig. 3A, lane 3).
Fig. 3. PCR analysis of gene transcripts expressed in B. burgdorferi ZS7 organisms propagated in vitro (A), derived ex vivo from infected ticks (B), or from mouse tissue (C). Total genomic DNA and cellular RNA samples isolated from in vitro-propagated spirochetes (A), or from infected ticks (B), were tested for zs7.a36, zs7.a66, zs7.a68, and ospA expression. Specific mRNA transcripts were amplified by RT-PCR (RNA + RT, lane 1; RNA + RT + RNase, lane 2). Control samples containing total genomic DNA (lane 3) or no template (lane 4) were amplified by PCR. Marker sizes are in bp indicated on the left. (C) Total cellular RNA from heart and joint tissues of three individual C.B-17.SCID mice previously infected with B. burgdorferi ZS7 (days 7, 30, 58 and/or 90 p.i.) was tested for ospA, ospC, zs7.a36, zs7.a66 and zs7.a68 expression (control: flagellin, fla) by RT-PCR in the presence (+RT) or absence (–RT) of reverse transcriptase as described in Sect. 4. Accordingly, total genomic DNA isolated from identical tissue samples at different time points was analyzed by PCR. For positive controls, titration of plasmid DNA harboring the indicated genes was performed and followed by PCR analysis as described: 4×104 to 4×102 plasmid copies (from left to right) for zs7.a36 and zs7.a66; 5×105 to 5×103 plasmid copies for zs7.a68 and ospC.
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When expression of zs7.a36, zs7.a66 and zs7.a68 was analyzed in ticks previously fed on B. burgdorferi (strain ZS7)-infected mice, specific transcripts for all three genes were found (Fig. 3B). Next we analyzed for the presence of spirochetes and the expression pattern for zs7.a36, zs7.a66, zs7.a68 and/ or ospC and ospA in heart and joint tissue of experimentally infected (103 ZS7/mouse) C.B-17.SCID mice. As shown in Fig. 3C (left panel), spirochetal DNA was hardly detectable at day 7 p.i., using PCR primers for ospA, ospC, zs7.a36, zs7.a68 and zs7.a66, with somewhat increasing intensities at later time points (days 30, 58 and 90). RT-PCR analysis of the same preparations at day 7 p.i. revealed specific mRNA for ospC and zs7.a36, in particular in the joint, but no DNA or mRNA for zs7.a66, zs7.a68 or ospA. When assayed at later timepoints (days 30, 58 and 90 p.i.) significant transcription was also seen, in both tissues, for zs7.a36 and zs7.a66 but, with one exception, not for zs7.a68. Using ospCspecific primers and spirochetal DNA as template, two PCR fragments were detected (days 7, 30 and 90 p.i.). Similarly, two RNA transcripts could be detected at day 7 but not at day 58 and day 90 p.i. in all subsequent experiments and may be due to the existence of more than one ospC gene ([24, 25] and Wallich et al., in preparation).
2.4 Immunogenicity of ZS7.A36, ZS7.A66 and ZS7.A68 during infection and protective potential of IS generated against the respective recombinant proteins To test whether the proteins encoded by zs7.a36, zs7.a66 or zs7.a68 are immunogenic during experimental or tick-borne infection, IS from mice infected experimentally with either 108 or 103 spirochetes or via infected ticks were tested for reactivity with the respective recombinant proteins on Western blots. As shown in Fig. 1B, significant amounts of Ab with specificity for rZS7.A36, rZS7.A66 or rZS7.A68 were only seen in § 108 IS, with a preponderance for § ZS7.A36 Ab. In contrast, § 103 IS and § tick IS contained, if at all, only marginal amounts of the respective Ab. The polyclonal monospecific mouse and rabbit IS to ZS7.A36, ZS7.A66 or ZS7.A68 were shown to be specific for the respective recombinant proteins (Fig. 1C; only shown for mouse IS). As expected from the in vitro expression of B. burgdorferi antigens (Fig. 3A), rabbit IS § ZS7.A.66 did not react with a ZS7 cell lysate. From the two other rabbit IS, only § ZS7.A68 IS, but not § ZS7.A36 IS, showed a specific band (Fig. 1D). Obviously, the amount of ZS7.A36 in the ZS7 cell lysate was too low to be detectable. A similar reactivity pattern was seen with the respective mouse IS (data not shown).
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We next examined the potential of polyclonal and monospecific mouse and rabbit IS generated against ZS7.A36, ZS7.A66 or ZS7.A68 to protect C.B-17.SCID mice against experimental challenge with B. burgdorferi. Since initial experiments indicated a higher protective potential in rabbit IS as compared with mouse IS (data not shown), only the former preparations were used in all subsequent experiments. As shown in Table 3, passive transfer of rabbit IS specific for ZS7.A68 and, with one exception each, also those with specificity for ZS7.A36 or ZS7.A66 into recipients before experimental challenge led to complete protection against disease and infection. Rabbit and mouse IS to OspC served as positive controls and normal rabbit serum as a negative control [11, 20]. In essence similar results were obtained with mouse IS (data not shown). Finally, the respective rabbit IS were also tested for their potential to clear spirochetes as demonstrated by negative ear culture (Table 4). As shown before [11], repeated transfer to C.B-17.SCID mice of OspC-specific rabbit IS, starting at day 11 p.i., resulted in prevention of development of arthritis in four out of five and of infection in three out of five recipients. In contrast, from the rabbit IS to ZS7.A36, ZS7.A66 or ZS7.A68, only that with specificity for ZS7.A36 resulted in a delayed onset of arthritis, whereas the other two had no effect at all. With one exception each, none of the three latter IS was able to clear disseminated spirochetes.
2.5 Antibodies with specificity for ZS7.A36, ZS7.A66 and ZS7.A68 in sera of patients with Lyme disease A panel of individual sera from patients representing various clinical symptoms of B. burgdorferi infection, including EM, Lyme arthritis, Neuroborreliosis and ACA, were tested for the presence of Ab with specificities for ZS7.A36, ZS7.A66 and ZS7.A68. As seen in Fig. 4A (only shown for ZS7.A36), sera from selected patients with different clinical manifestations but not sera from healthy individuals reacted with ZS7.A36, though at different levels. Specific ZS7.A36 Ab were found in 30% of patients with arthritis (3 out of 10), in 13% of those with neurological symptoms (3 out of 23) and in 58% (7 out of 12) of those with ACA. For comparison, sera were also reacted against whole spirochetal cell lysates (Fig. 4B). Ab specific for ZS7.A66 and ZS7.A68 could be observed in 20% (5 out of 20) and 4% (1 out of 25), respectively, of Lyme disease patients but were also detectable in sera from healthy individuals ( e 10%; data not shown).
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Table 3. Prevention of experimental B. burgdorferi infection in C.B-17.SCID mice by passive transfer of immune sera specific for ZS7.A36, ZS7.A66, ZS7.A68 or OspC Immune serum transferreda)
Dose
Rabbit § -ZS7.A36
1 ml
Rabbit § -ZS7.A66
Clinical arthritisb), days p.i.
Mouse number
Ear culturec)
7
15
24
55
86
126
1 2 3
–/– –/– –/–
–/– (±)/– –/–
–/– –/– –/–
(±)/± –/– –/–
++/++ –/– –/–
++/++ –/– –/–
+ – –
1 ml
1 2 3 4
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– (+)/± –/– –/–
(±)/(±) ++/++ –/– –/–
–/– ++/++ –/– –/–
–/– ++/+ –/– –/–
– + – –
Rabbit § -ZS7.A68
1 ml
1 2 3 4
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/– –/–
(±)/– –/– –/– –/–
– – – –
Rabbit § -OspC/GST
38 ? g
1 2 3 4
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/– –/–
–/– –/– –/–
– – – –
–/– –/– –/–
Not done Not done Not done
Mouse § -OspC/GST
12 ? g
1 2 3
–/– –/– –/–
–/– –/– –/–
–/– –/– –/–
–/– –/– –/–
–/– –/– –/–
Normal rabbit serum
1 ml
1 2 3
–/– –/– –/–
(±)/(±) ±/(±) –/–
++/+ ++/++ ++/+
++/++ ++/++ ++/++
e)
a)
b)
c) d) e)
e) e)
d)
+ + +
Rabbit immune sera were given i.p. 2 h before infection with B.b. ZS7 (1×103/mouse, s.c., base of tail). Titers of ZS7.A36/66/ 68-specific antibodies (ELISA) were 1:464,000/ 1:420,000/ 1:200,000, respectively. Arthritis scoring: – none; (±) reddening and/or mild swelling; ± moderate swelling; + severe swelling; ++ extreme swelling of tibiotarsal joint. Ear biopsy for recultivation of B. burgdorferi ZS7 was taken at day 53 p.i. This mouse died. These mice were killed.
3 Discussion The most intriguing finding of the present study is the fact that promising vaccine candidates against the B. burgdorferi pathogen can be discovered when artificial immunization regimens with intact spirochetes were used that do not reflect conditions of natural infection. We showed here that IS from mice experimentally infected with abnormally high doses of spirochetes (108) contain protective Ab with specificities for B. burgdorferi antigens that are expressed, but non-immunogenic, during experimental infection. Ab generated to B. burgdorferi during natural infection are able to prevent pathogenic processes in outbred and laboratory mice, but in most cases do not eradicate the spirochetes [26]. Thus, spirochetes have evolved strategies to escape natural immunity and to persist in their
mammalian hosts. However, the previous finding that a fraction of experimentally challenged AKR/N mice with high levels of OspC-specific Ab was able to resolve infection [20] and that IS from tick- or needle-infected mice prevent subsequent infection in naive recipients [4, 5] suggested that inherent spirochetal defense mechanisms can be breached by more-potent and/or morewell-timed Ab responses. This has been most convincingly documented for Ab to OspA and OspC [10, 16, 20]. These two lipoproteins are known to be differentially expressed on spirochetes in the vector and mammalian hosts [9, 14]. Because of down-regulation of OspA in the mammalian host, Ab to OspA are rarely produced during natural infection of mice and man [9, 27]. However, IS that are induced by immunization with the OspA protein convey protection against subsequent infection by targeting spirochetes within vector ticks, thereby blocking transmission to the mammalian host [10]. On the other
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Table 4. Therapeutic effect of immune sera specific for ZS7.A36, ZS7.A66, ZS7.A68 or OspC on established experimental B. burgdorferi infection of C.B-17.SCID mice Immune serum transferreda)
Dose
Rabbit § -ZS7.A36
1 ml
Rabbit § -ZS7.A66
Rabbit § -ZS7.A68
Clinical arthritisb), days p.i.
Mouse number
Ear culturec)
11
24
35
55
86
151
1 2 3 4 5
–/– –/– –/– –/– –/–
–/– –/– –/– –/– ±/±
–/– ±/(±) –/– –/– ±/(±)
(±)/– –/– –/– –/– ++/++
+/+ ++/± –/– ++/– ±/+
+/+ +/± –/– ++/++ +/(±)
+ + – + +
1 ml
1 2 3 4 5
–/– –/– –/(±) –/– –/–
–/± +/+ +/++ +/± –/–
–/(±) +/+ ++/+ ++/+ –/–
–/+ ++/+ +/+ ++/++ –/–
–/– ++/++ +/+ ++/++ –/–
+/– +/+ ++/± ++/++ –/–
+ + + + –
1 ml
1 2 3 4 5
–/(±) (±)/(±) –/– –/– –/–
++/+ +/++ –/– +/± +/±
++/+ ++/++ –/– ++/+ ++/+
++/++ ++/++ –/– ++/++ ++/++
++/++ ++/++ –/– ++/++ ++/++
++/++ ++/++ –/– ++/++ ++/+
+ + – + +
Rabbit § -OspC/GST
1800 ? g
1 2 3 4 5
–/– –/– –/– –/– –/–
–/– –/– –/– –/– –/–
–/– –/– –/– –/– –/+
–/– –/– –/– –/– (±)/++
–/– –/– –/– –/– ±/++
–/– –/– –/– –/– ±/++
– – – + +
Normal rabbit serum
500 ? l
1 2 3 4
–/– –/– (±)/(±) –/–
+/+ +/+ +/+ +/(±)
++/++ ++/++ ++/+ ++/+
d)
a)
b)
c) d)
d) d) d)
+ + + +
Immune sera were given i.p. on days 11, 15, 19 and 24 post infection with B. burgdorferi ZS7 (1x103/mouse, s.c., base of tail). Amount of B.b.-specific Ab as in legend to Table 3. Arthritis scoring: – none; (±) reddening and/or mild swelling; ± moderate swelling; + severe swelling; ++ extreme swelling of tibiotarsal joint. Ear biopsy for recultivation of B.b. ZS7 was taken at day 53 p.i. These mice were killed.
Fig. 4. Detection of ZS7.A36-specific antibodies in sera derived from Lyme disease patients. (A) Antigen strips containing 1 ? g of rZS7.A36 were reacted either against patient’s sera (lanes 1–4, ACA; lane 5, arthritis; lanes 6 and 7, neuroborreliosis) or sera (lanes 8–11) from healthy individuals. The position of ZS7.A36 is indicated by an arrow. (B) Antigen strips containing whole ZS7 cell lysates (5×106 cell equivalents) were processed accordingly.
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hand, Ab generated to OspC, a lipoprotein expressed by spirochetes in ticks and vertebrates, at least temporarily [9, 11], were found not only to prevent but also to resolve spirochetal infections, when passively transferred to recipients in sufficient amounts [11, 20]. The three novel B.burgdorferi ZS7 genes — zs7.a36, zs7.a66 and zs7.a68 — identified with the artificial IS ( § 108) and characterized in the present study are differentially expressed in vitro and in vivo. All three genes are activated in tick-associated spirochetes but only two of them, i.e. zs7.a36 and zs7.a68, are expressed in cultured spirochetes. Upon their experimental transmission to mice, spirochetes cease expression of zs7.a68, a phenomenon reminiscent of ospA [9, 11, 14]. On the other hand, spirochetes express ZS7.A36- and also ZS7.A66specific mRNA, though at low levels, in heart and/or joint tissue during the entire observation period (up to day 90 p.i.). However, in contrast to ospC [11], no or only marginal Ab responses to either ZS7.A36 or ZS7.A66 antigens were observed following natural challenge or experimental infection with low numbers of spirochetes (Fig. 1A). It is tempting to speculate that in the natural reservoir host, spirochetes either block translation of zs7.a36-, zs7.a66- and zs7.a68-specific mRNA or express the respective antigens, ZS7.A36 and ZS7.A66, in only suboptimal and/or non-immunogenic amounts to escape immunity. Nonetheless, the finding that both antigens are immunogenic in Lyme disease patients, at least to some extent, again emphasizes the potential of B. burgdorferi to readily adapt, via phenotypic changes, to different environmental conditions. Anyway, the fact that IS to rZS7.A36 and rZS7.A66 are able to transfer partial or total protection to naive recipients against subsequent experimental challenge clearly demonstrates that both antigens are expressed on spirochetes in mice at levels sufficient to serve as targets for protective Ab. Questions regarding the extent to which spirochetes express ZS7.A36 and ZS7.A66 in other organs, such as skin, joints and spleen, or at later stages of infection, and whether they are always accessible and susceptible to specific Ab are subject to further experimentation. The finding that the IS to ZS7.A68 was also able to prevent experimental infection, in spite of the absence of the respective antigen on spirochetes in the mammalian host, was not surprising in light of previous experiences with OspA. Like zs7.a68, it was shown before that ospA is also expressed in cultured spirochetes and in tickassociated spirochetes but not in host-associated spirochetes [8, 9]; it was also shown that OspA Ab are able to prevent, upon adoptive transfer, experimental and natural infection [10, 16, 28]. Thus, it is possible that ZS7.A68
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represents another vector-specific transmissionblocking agent. However, as indicated before with another vaccine candidate, i.e. decorin binding protein (DbpA; [29]), it is not clear a priori whether the results obtained with needle-inoculation vaccination can be extended to tick-challenged mice. Finally, since comprehensive data regarding the heterogeneity of the three novel antigens among members of the three human pathogenic species are lacking it is not known whether the effective prophylactic treatment with the respective IS seen here is of general applicability for all species of the B. burgdorferi sensu lato complex. In contrast to OspC IS, ZS7.A36- and ZS7.A66-specific IS had no or only a limited potential to clear spirochetes from chronically infected SCID mice; the reason for this is not known. In light of the finding that resolution of joint-swelling and clearance of spirochetes were only achieved with IS containing high levels of OspC Ab, it is possible that the ZS7.A36- and ZS7.A66-specific IS contained only suboptimal amounts of protective antibodies. Alternatively, a mixture of Ab with specificities to various B. burgdorferi antigens, distinct from OspC, may be required for optimal therapeutic protection. The latter assumption is likely in the context of a recent study indicating that homogeneous populations of B. burgdorferi within the tick gut give rise to transmittable progenies with extensive antigenic heterogeneity [15]. Most notably, zs7.a36, zs7.a66 and zs7.a68 are expressed together with ospA/B, DbpA/B and p35 on the linear plasmid lp54 of B. burgdorferi ZS7, and are also differentially regulated in vivo and in vitro. The plasticity displayed by the lp54-encoded Osp molecules may be used by the spirochetes to adjust to the particular environments and to escape the immune defense. Although the mechanisms underlying the differential expression of Osp genes are unknown, it is clear that environmental factors must be involved and that these factors distinctly regulate the various genes associated with lp54 of ZS7. In fact it was shown before that synthesis of OspC, which is encoded by a gene located on a circular plasmid of B. burgdorferi (www.TIGR.org), is influenced by pathogen density, growth phase and temperature in vitro as well as by adaptation to the microenvironment of the host tissue, including pH and immune selection processes in vivo [8, 14, 30, 31]. Moreover it was shown that expression of other Osp genes could limit Ab access to particular antigens, such as P66 [32]. The present findings add another component to the notion that potent vaccines have to do more than just mimicking natural infection [1]. The fact that the application of an artificial-infection protocols results in the elucidation of pathogen structures, including those that are
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expressed but non-immunogenic under natural conditions, encourages further attempts to identify suitable vaccine candidates for prevention and/or cure of Lyme disease.
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(Pharmacia, Freiburg, Germany) in accordance with the manufacturer’s recommendations.
4.4 Expression and purification of recombinant B. burgdorferi proteins
4 Materials and methods 4.1 Mice, spirochetes, ticks and infection Adult female mice of strains C.B-17 SCID (H-2d) were bred under specific pathogen-free conditions at the Max-PlanckInstitut für Immunbiologie, Freiburg, Germany. Animals between 6 and 8 weeks of age were used throughout the experiments. Mice were challenged either by needle inoculation with 103–108 viable B. burgdorferi organisms (s.c.) or by previously infected Ixodes ricinus nymphs [33]. The virulent low-passage (4–5 in vitro passages) B. burgdorferi s.l. tick isolates, ZS7 (B. burgdorferi s.s.), ZQ1 (B. garinii) and MMS (B. afzelii) were grown in Barbour-Stoenner-Kelly medium at 33°C for 48–72 h and harvested as previously described [34]. Uninfected larval I. ricinus were placed on the head of infected (strain ZS7) BALB/c mice. Until molting had become completed, engorged larvae were maintained in glass vials at room temperature and 100% humidity. A direct immunofluorescence assay was used to detect spirochetes in nymphal ticks derived from challenged larvae as described [33].
Recombinant ZS7.A36, ZS7.A66 and ZS7.A68 were expressed as fusion proteins with glutathione S-transferase (GST). The zs7.a36, zs7.a66, and zs7.a68 genes lacking their leader sequences were subcloned into the pGEX-2T vector (Pharmacia). The ligation mixtures were used to transform Escherichia coli DH5 § as described [13]. Expression of the respective recombinant B. burgdorferi GST fusion proteins, affinity purification on Glutathione-Sepharose columns and endoproteinase thrombin cleavage of the GST fusion proteins were performed as recommended by the manufacturer (Pharmacia, Freiburg, Germany).
4.5 PCR analysis Details on PCR and RT-PCR analysis of culture-, tick-, and mouse-tissue-derived spirochetes, including the respective 5’ and 3’ primer pairs of individual Osp genes are available on request or as supporting information (www.eji.de).
4.6 Generation of immune sera and analysis of antibodies by ELISA and Western blot
4.2 Screening of a B. burgdorferi expression library DNA was isolated from infectious B. burgdorferi ZS7 passage 3 and cloned into the bacterial expression vector pUEX1 as previously described [35]. Immunoreactive clones were identified using either pre-adsorbed or non-adsorbed protective IS ( § 103 and § 108) at a dilution of 1:2000 as described previously [13]. To enrich for antibodies specific for novel infection-associated antigens, IS were adsorbed with different recombinant antigens (OspA, OspB, OspC, pG, P39, P83/100 and DbpA) conjugated to Sepharose 4B (Pharmacia). The adsorption was repeated three times until the adsorbed IS lost reactivity with the indicated antigens derived from B. burgdorferi ZS7 as determined by immunoblotting (data not shown).
4.3 Southern blotting, PFGE analysis and DNA sequencing Southern blotting of total genomic DNA was done as described previously [13]. Random primed gene probes were prepared by using the Random Primer Labeling system (GIBCO-BRL) and hybridization was conducted as previously described [23]. PFGE was performed using the CHEF Mapper System (Bio-Rad, Munich, Germany) as described [13]. B. burgdorferi DNA fragments cloned in pUEX1 plasmid derivatives were sequenced by using a T7 sequencing kit
Details on the generation of mouse and rabbit IS and analysis of quantity and quality of B. burgdorferi-specific Ab are available on request or as supporting information (www.eji.de).
4.7 Disease model and protection experiments For passive immunization, C.B-17 SCID mice were injected i.p. with IS specific for individual recombinant proteins or with the OspA-specific Ab LA-2 [34] 2 h before infection. Control mice received 50 ? l of undiluted anti-GST IS or were left untreated. For treatment of established infection by passive immunization, mice were first infected s.c. and subsequently given repeatedly (four times at 3–4-day intervals) various amounts of polyclonal IS specific for individual recombinant proteins (i.p.) at indicated time-points relative to infection. C.B-17 SCID mice were monitored for the development of clinical arthritis in the tibiotarsal joints under double-blind conditions. The severity of arthritis was scored in the right and left tibiotarsal joint [20]. The status of infection of disease-resistant BALB/c mice was determined by testing for the outgrowth of spirochetes from cultured biopsies, as described [11, 36].
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4.8 Human sera Sera from culture-confirmed Lyme disease patients were obtained from the Dermatologischen Klinik an der Technischen Universität Munich (Germany). All patients were diagnosed in the Bavarian area of Germany. The sera were collected at different times (1–5 months) after disease onset and all were treated with antibiotics within 2 months of onset. To serve as negative controls, the Heidelberg University blood bank provided sera from healthy individuals who live in areas where Lyme disease is not known to be endemic. 4.9 Nucleotide sequence accession numbers The nucleotide sequences of ZS7.A36, ZQ.A36, MMS.A36, ZS7.A66, ZQ.A66, MMS.A66, and ZS7.A68 have been deposited in the EMBL/Genbank databases under accession numbers AJ430846, AJ430847, AJ430848, AJ430850, AJ430851, AJ430849 and AJ430845, respectively. Acknowledgements: This study was supported in part by the Deutsche Forschungsgemeinschaft (Si 214/9–1) and Glaxo SmithKline.
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Eur. J. Immunol. 2003. 33: 708–719 24 Ryan, J. R., Levine, J. F., Apperson, C. S., Lubke, L., Wirtz, R. A., Spears, P. A. and Orndorff, P. E., An experimental chain of infection reveals that distinct Borrelia burgdorferi populations are selected in arthropod and mammalian hosts. Mol. Microbiol. 1998. 30: 365–379. 25 Tilly, K., Lubke, L. and Rosa, P., Characterization of circular plasmid dimers in Borrelia burgdorferi. J. Bacteriol. 1998. 180: 5676–5681. 26 Magnarelli, L. A., Anderson, J. F., Hyland, K. E., Fish, D. and McAninch, J. B., Serologic analyses of Peromyscus leucopus, a rodent reservoir for Borrelia burgdorferi, in northeastern United States. J. Clin. Microbiol. 1988. 26: 1138–1141. 27 Craft, J. E., Fischer, D. K., Shimamoto, G. T. and Steere, A. C., Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J. Clin. Invest. 1986. 78: 934–939. 28 Kurtenbach, K., Dizij, A., Voet, P., Hauser, P. and Simon, M. M., Vaccination of natural reservoir hosts with recombinant lipidated OspA induces a transmission-blocking immunity against Lyme disease spirochaetes associated with high levels of LA-2 equivalent antibodies. Vaccine 1997. 15: 1670–1674. 29 Hagman, K. E., Yang, X., Wikel, S. K., Schoeler, G. B., Caimano, M. J., Radolf, J. D. and Norgard, M. V., Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks. Infect. Immun. 2000. 68: 4759–4764. 30 Carroll, J. A., Garon, C. F. and Schwan, T. G., Effects of environmental pH on membrane proteins in Borrelia burgdorferi. Infect. Immun. 1999. 67: 3181–3187.
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31 Liang, F. T., Jacobs, M. B., Bowers, L. C. and Philipp, M. T., An immune evasion mechanism for spirochetal persistence in Lyme borreliosis. J. Exp. Med. 2002. 195: 415–422. 32 Bunikis, J. and Barbour, A. G., Access of antibody or trypsin to an integral outer membrane protein (P66) of Borrelia burgdorferi is hindered by Osp lipoproteins. Infect. Immun. 1999. 67: 2874–2883. 33 Gern, L., Toutoungi, L. N., Hu, C. M. and Aeschlimann, A., Ixodes (Pholeoixodes) hexagonus, an efficient vector of Borrelia burgdorferi in the laboratory. Med. Vet. Entomol. 1991. 5: 431–435. 34 Schaible, U. E., Kramer, M. D., Eichmann, K., Modolell, M., Museteanu, C. and Simon, M. M., Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci. U S A 1990. 87: 3768–3772. 35 Wallich, R., Moter, S. E., Simon, M. M., Ebnet, K., Heiberger, A. and Kramer, M. D., The Borrelia burgdorferi flagellumassociated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect. Immun. 1990. 58: 1711–1719. 36 Sinsky, R. J. and Piesman, J., Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J. Clin. Microbiol. 1989. 27: 1723–1727.
Correspondence: Markus M. Simon, Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany Fax: +49–761–5108–529 e-mail: simon — immunbio.mpg.de
