Identification and characterization of Borrelia antigens as potential vaccine candidates against Lyme borreliosis

Vaccine 30 (2012) 4398–4406

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Identification and characterization of Borrelia antigens as potential vaccine candidates against Lyme borreliosis Albina Poljak, Pär Comstedt, Markus Hanner, Wolfgang Schüler, Andreas Meinke, Benjamin Wizel ∗ , Urban Lundberg Intercell AG, Campus Vienna Biocenter 3, A-1030 Vienna, Austria

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Article history: Received 30 June 2011 Received in revised form 19 October 2011 Accepted 26 October 2011 Available online 18 November 2011 Keywords: Borrelia Lyme borreliosis Antigen Genomic screen

a b s t r a c t The three Borrelia species, Borrelia afzelii, Borrelia burgdorferi and Borrelia garinii are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. By applying a genomic approach relying on human antibodies we have identified 122 antigenic Borrelia proteins associated with Lyme borreliosis, including already known and published protective antigens. The heterogeneity of the Borrelia species causing Lyme borreliosis makes the search for conserved antigens providing broad protection challenging. Using several in vitro assays we narrowed down the selection to 15 vaccine candidates. These antigens were further analyzed for antigenicity and cross-reactivity using sera from mice infected with the three pathogenic Borrelia species. All antigens analyzed showed a high degree of cross-reactivity between the three Borrelia species, essential for providing cross-protection. We also investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses. For a selection of these antigens, we observed preferentially a pro-inflammatory response in C3H/HeN mice, while in contrast also a type 2 T cell response was seen in the Borrelia-resistant mouse strain BALB/c. Thus, antigens mounting a type 2 or mixed type 2/type 1 T cell response might be preferred vaccine candidates for evaluation in animal models of Lyme borreliosis. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Lyme borreliosis is the most prevalent tick-borne zoonosis in the northern hemisphere and an important emerging infectious disease in Europe, North America and Far Eastern countries. The causative agents of Lyme borreliosis are pathogenic Borrelia species, from which B. afzelii, B. garinii and B. burgdorferi are responsible for the vast majority of cases and can cause different disease manifestations. The primary characteristics of Lyme borreliosis are similar worldwide, and the infection usually begins with erythema migrans, the red skin rash caused by localized infection of the skin. In general, Lyme borreliosis occurs in stages, with different clinical manifestations at each stage [1]. Early infection (stage 1) consists of localized infection of the skin, followed within days or weeks by disseminated infection (stage 2), and months to years later by persistent and chronic infection (stage 3). Persistent infection of the central nervous system with B. garinii causes more severe neurological symptoms (neuroborreliosis), and a persistent infection of the skin with B. afzelii results in acrodermatitis chronica atrophicans [2]. The most common symptom in

∗ Corresponding author. Tel.: +43 1 20620 1174; fax: +43 1 20620 81174. E-mail addresses: [email protected], [email protected] (B. Wizel). 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.10.073

North America is Lyme arthritis, caused by persistent infection with B. burgdorferi. Borrelia belongs to the family Spirochaetaceae and is a spiral shaped vigorously motile bacterium that grows under microaerophilic conditions. Its cell wall consists of a cytoplasmic membrane surrounded by peptidoglycan and a loosely associated outer membrane, which is fluid, lacks lipopolysaccharides [3] and consists of phospholipids and outer membrane proteins [4]. The outer membrane proteins include outer surface lipoproteins and integral membrane spanning proteins. Outer surface lipoproteins are the most abundant outer membrane proteins [5] and play a major role in host inflammatory response activation and tissue inflammation. They also play an important role in adaptive bacterial responses and Borrelia pathogenicity, which include antigenic variation, complement evasion, and adherence mechanisms. The Borrelia outer membrane proteins do also facilitate Borrelia–host interactions which are important for the dissemination of the spirochetes in the body [6]. Borrelia is an extracellular pathogen and thus attacked by the innate immune system and the humoral arm of the adaptive immunity, resulting in macrophage- and antibody-mediated killing of the spirochete [7]. As B cell mitogens, spirochetal lipoproteins, which are major PAMPs (pathogen associated molecular patterns) and pro-inflammatory agonists, cross-link B cell receptors directly

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leading to a T cell independent B cell response [8,9]. Humoral immune responses to non-lipidated spirochetal proteins are more likely to be T cell dependent. Macrophages and dendritic cells present spirochetal peptides to CD4+ and CD8+ T cells [7]. The primary role of CD4+ T cells is to prime T cell dependent B cell responses, and CD8+ T cells appear to be an important source of IFN-␥ [10]. In vivo T cell depletion studies in susceptible and resistant mouse strains showed that CD4+ T cells contribute to the control of spirochete growth and amelioration of arthritis, while CD8+ T cells exacerbate infection with increased arthritic severity and spirochete loads [11]. That cytokines mediate many of these T cell effects is supported by cytokine inhibition studies showing that IL-4 has protective effects, whereas IFN-␥ and IL-17 have disease-promoting effects [12,13]. However, a strong type 1 T helper response at a very early stage of the infection is also reported as crucial for eradicating spirochetes [14]. In order to identify vaccine candidates naturally recognized by the human immune system, we applied the ANTIGENome technology [15,16] for the identification of novel Borrelia vaccine candidates. While the primary selection step of this technology is based on the recognition by human antibodies, T cells have also been shown to play critical roles for Borrelia infections in mice. As IL-4, IFN-␥ and IL-17 are soluble mediators which define different cytokine-producing T cell subsets, and these cytokines modulate spirochete growth and disease development, we investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses to a selected panel of Borrelia antigens identified by the ANTIGENome technology.

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individuals with occupational exposure to ticks (e.g. forest workers). Serum samples were kindly provided by Dr. Dagmar Hulinska (National Institute of Public Health, Czech Republic), Prof. Gerold Stanek (Medical University of Vienna, Austria) and Prof. Rudolf Stauber (Medical University of Graz, Austria). The disease symptoms recorded and medical diagnosis of these patients included acute Lyme borreliosis (erythema migrans), neuroborreliosis, Lyme arthritis, and cardiological and dermatological symptoms (acrodermatitis chronica atrophicans). All human serum samples were analyzed by ELISA using whole cell lysates from B. burgdorferi strain B31, disease specific sera with high titer were pooled and IgG isolated as described elsewhere [24]. 2.4. Gene distribution analysis Oligonucleotide pairs for all identified ORFs, were designed using the online tool Primer3 (http://frodo.wi.mit.edu). Genomic DNA from Borrelia isolates including the most prevalent OspC types associated with human invasive and non-invasive disease was used as template for PCR and DNA products were subsequently visualized by electrophoresis. All Borrelia strains used in this study are listed in Supplementary Table 1. 2.5. Peptide ELISA

2. Materials and methods

ELISA tests were conducted using 25-mer peptides with a five amino acid overlap and spanning the sequence of epitope-bearing fragments. Assays were conducted using a Gemini 160 ELISA robot (TECAN, Austria) as previously described [24]. Results are expressed as ELISA units (A405 (sample-blank) × 1000).

2.1. Borrelia strains and culture conditions

2.6. Surface staining of B. afzelii K78 cells

Borrelia afzelii strain K78 used for generation of bacterial surface display libraries and as source for cloning of antigens, was isolated from a skin biopsy of a Lyme borreliosis patient by Prof. Gerold Stanek (Medical University of Vienna, Austria). For in vivo studies, low passage isolates of Borrelia burgdorferi strain N40 [17,18] and Borrelia garinii strain LU185, outer surface protein A (OspA) serotype 6 [19], were kindly provided by Prof. Sven Bergström (Umeå University, Sweden). B. afzelii isolate IS1 used to infect Ixodes ricinus ticks, and subsequently for challenge of mice, is a field isolate and has never been passaged in vitro. The ticks were derived from a colony bred on B. afzelii IS1-infected gerbils (Insect Services, Germany). The Borrelia strains used for the gene distribution studies were mainly isolated from patients with different symptoms of Lyme borreliosis. All Borrelia strains used in this study are listed in Supplementary Table 1. The Borrelia species of the isolates have been determined by sequencing of the 16S–23S intergenic spacer region [20] and ospA [21]. In addition the ospC gene was sequenced from strains used for gene distribution studies [22]. Bacteria were grown at 34 ◦ C in Barbour–Stoenner–Kelly II (BSK-II) medium, supplemented with 6% (vol/vol) heat inactivated rabbit serum (Sigma–Aldrich, Austria).

Immune sera were generated in NMRI mice (Harlan, Italy) by immunization with pools of up to five total bacterial lysates (corresponding to 5 × 107 cells) prepared from MACS (magnetic cell sorting) screen-selected clones expressing adjacent epitopebearing fragments. Approximately 105 spirochetes in 100 ␮l BSK-II medium, were incubated with an equal volume of heat inactivated mouse sera (diluted 1:25 with BSK-II) at room temperature (RT) for 1 h before detection with PE-conjugated goat anti-mouse IgG (H + L; Beckman Coulter, Austria) antibodies. After fixation with 1% paraformaldehyde, surface staining was detected by flow cytometry (Cytomics FC500; Beckman Coulter), and data were analyzed using the analysis software CXP (Beckman Coulter).

2.2. Generation of bacterial surface display libraries in E. coli and magnetic cell sorting Generation of bacterial surface display libraries in Escherichia coli and subsequent identification of the Borrelia ANTIGENome was performed as described previously [15,23]. 2.3. Human serum samples A collection of 1411 human sera was obtained from patients with medical diagnosis of Lyme borreliosis as well as from healthy

2.7. Cloning and expression of recombinant proteins The gene fragments of interest were amplified from B. afzelii strain K78 (prefix “BA”) or B. burgdorferi strain N40 (prefix “BB”) genomic DNA by PCR and cloned into the pET28b(+) vector (Novagen, USA). Proteins were expressed in BL21-CodonPlus (DE3)RIPL cells (Invitrogen, USA). His-tagged proteins were purified over a Ni-sepharose column (Ni Sepharose 6 Fast Flow; GE Healthcare, UK). Insoluble proteins were solubilized in 8 M urea in 50 mM Tris–HCl, pH 8 prior to affinity purification. Bound proteins were eluted with Imidazole. 2.8. Animal challenge studies All animal experiments were conducted in accordance with Austrian law (BGB1 No. 501/1989) and approved by “Magistratsabteilung 58”. Two species of inbred mice; C3H/HeN (H-2k ) and BALB/c (H-2d ), were used for the immunological studies (Harlan). Prior to each challenge, groups of five 8 week-old female mice were bled via the tail vein and pre-immune sera were prepared and

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pooled. In order to infect mice with B. afzelii, the hair of the back of each mouse was removed by Veet® Cream (Reckitt Benckiser, UK) and a small ventilated container was glued to the skin with super glue (Pattex, Germany). Thereafter, two I. ricinus nymphs infected with B. afzelii (strain IS1) were applied per mouse, let to attach and feed to depletion. To infect mice with B. burgdorferi, a low passage glycerol stock of strain N40 was thawed and inoculated in BSK-II medium. The culture was grown to mid logarithmic phase before the titer was determined using a phase contrast microscope and a Petroff–Hausser counting chamber (Hausser Scientific, USA). The culture was diluted in fresh BSK-II medium to a final concentration of 5 × 105 spirochetes/ml. For challenge, 100 ␮l (equivalent to 5 × 104 spirochetes) of the subsequently prepared dilution was injected subcutaneously (s.c.) in each mouse. The same procedure was used when infecting mice with B. garinii strain LU185, however, a challenge dose of 1 × 104 spirochetes was used. Borrelia infected mice, as determined by VlsE (IR6) ELISA, were sacrificed by cervical dislocation and the blood was collected by orbital bleeding. 2.9. VlsE (IR6) ELISA Four weeks post-challenge, the infection status of each mouse was determined by ELISA with the Invariable Region 6 (IR6) of the Variable major protein-Like Sequence E protein (VlsE). A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK) derived from the sequence of B. garinii strain IP90 was used for the analysis [25]. Streptavidin pre-coated 96-well ELISA plates (Nunc, Denmark), were coated with 100 ␮l/well (1 ␮g/ml) peptide in PBS supplemented with 0.1% Tween 20 (PBS/0.1 T). The plates were incubated overnight at 4 ◦ C. After coating with the peptide, the plates were washed once with PBS/0.1 T. The plates were then blocked for one hour at RT with 100 ␮l/well of PBS + 2% BSA, before being washed again with PBS/0.1 T. Reactivity of post challenge sera to the peptide was tested at 1:200, 1:400 and 1:800 dilutions in PBS + 1% BSA. Plates were incubated for 90 min at RT before being washed three times with PBS/0.1 T. Each well then received 50 ␮l of 1.3 ␮g/ml polyclonal rabbit anti-mouse IgG conjugated to HRP (Dako, Denmark) in PBS + 1% BSA. The plates were then incubated for 1 h at RT. After three washes with PBS/0.1 T, ABTS (50 ␮l/well) was added as substrate (Sigma–Aldrich) and color was allowed to develop for 30 min. Absorbance was measured at 405 nm. All sera were tested in duplicates; negative controls included PBS instead of sera as well as plates not coated with the peptide. Sera from mice shown to be culture positive for Borrelia infection were used as positive controls. 2.10. Immunoblotting Binding of post challenge sera to recombinant proteins was analyzed by immunoblotting. Briefly, 1 ␮g of each recombinant protein was separated by SDS-PAGE under reducing conditions using 4–12% Tris–Glycine ZOOM gels (Invitrogen). Separated proteins were transferred onto nitrocellulose membrane using the iBlot® Dry blotting system (Invitrogen). After overnight blocking in 5% milk, post-challenge sera (pooled from five mice) were added at 1:1000 dilution and polyclonal rabbit anti-mouse IgG conjugated to HRP (Dako) was used for detection. The immunoblots were visualized with Amersham ECL PlusTM Western blotting detection reagents (GE Healthcare) and Kodak BioMax films (Kodak, USA). 2.11. Cytokine ELISpot assays Enumeration of Borrelia antigen-specific, IFN-␥-, IL-4-, and IL-17-producing cells was performed by ELISpot. Capture and detection antibody pairs were AN-18/R4-6A2 for IFN-␥,

11B11/BVD6-24G2 for IL-4, and TC11-18H10/TC11-8H4.1 for IL-17 (BD Biosciences, Austria). Single spleen cell (SC) suspensions from B. afzelii-infected C3H/HeN and BALB/c mice (Harlan) were prepared by homogenizing spleens in cell strainers (BD Biosciences). After lysing red blood cells, SC were washed and resuspended in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated FCS, 20 mM HEPES, 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 20 ␮g/ml gentamicin and 50 ␮M 2-mercaptoethanol (all from PAA Laboratories GmbH, Austria). PVDF-backed 96-well plates (MultiScreenHTS -IP MSIPS4510; Millipore, Germany) were coated overnight at 4 ◦ C with 75 ␮l/well of capture antibody (2–5 ␮g/ml) in 0.1 M NaHCO3 (pH 9.2–9.5). After washing with PBS, the wells were blocked for 1 h at 37 ◦ C with 100 ␮l of complete medium. SC were then plated in triplicate wells at 5 × 105 and 1 × 106 (100 ␮l/well) and different Borrelia antigens diluted in complete media were added to the appropriate wells at 5 ␮g/ml (100 ␮l/well). Ovalbumin (5 ␮g/ml) was used as an irrelevant stimulus and complete medium alone was used as a control for antigen-independent cytokine secretion. As positive controls, SC were stimulated with Concanavalin A (1 ␮g/ml) and PMA (50 ng/ml)/ionomycin (500 ng/ml) (Sigma–Aldrich). After incubation at 37 ◦ C in 5% CO2 for 24 h (IFN-␥) or 48 h (IL-4 and IL-17), plates were washed three times with PBS, followed by three times with PBS/0.05% Tween 20 (PBS/0.05 T). Wells then received 75 ␮l of a solution of biotinylated detection antibody (1–3 ␮g/ml) in PBS/0.05 T/0.1% BSA. After incubation at 37 ◦ C for 2 h, plates were washed six times with PBS/0.05 T, and 100 ␮l HRP-conjugated Streptavidin (Roche Diagnostics, Austria) was added (1:3000 in PBS/0.05 T) to each well. Following 1 h incubation at 26 ◦ C, wells were washed four times with PBS/0.05 T and twice with PBS. Spots were developed with 75 ␮l/well of a 3,3 -diaminobenzidine (Sigma–Aldrich) substrate solution and the reaction was stopped after 20 min by washing the plates in tap water. Dried plates were analyzed using an ELISpot plate reader (BIOREADER 5000; BioSys, Germany). Net spot numbers of negative control (spots of OVA-stimulated SC − spots counted in the presence of complete medium only) were subtracted from the net spot numbers of each Borrelia antigen-stimulated sample (spots of test antigenstimulated SC − spots in the presence of complete medium). If the latter was lower than the former, the signal was considered as 0 or below background, and the results were expressed as the net number of cytokine-producing cells per 1 × 106 total SC (mean of triplicates). 2.12. Cytometric bead array-based cytokine measurements Antigen-stimulated production of IFN-␥, IL-4 and IL-17 by SC from B. afzelii-infected mice was assessed by flow cytometry using a cytometric bead array kit (FlowCytomix, eBioscience, Austria). SC were plated at 1 × 106 /well and stimulated for 48 h at 37 ◦ C in 5% CO2 in the presence of individual borrelial antigens (5 ␮g/ml) and appropriate negative and positive controls. After stimulation, supernatants were removed and analyzed for the presence of the indicated cytokines according to the manufacturer’s protocol. Samples were measured on a FACSCalibur (BD Biosciences). The data was analyzed and interpreted using the FlowCytomix Pro Software. 3. Results 3.1. Characterization and selection of human serum samples for genomic antigen screens A large collection of human sera from 1411 patients with a confirmed medical diagnosis of Lyme borreliosis was collected in

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two different countries (Austria and Czech Republic). The recorded disease symptoms and medical diagnosis of the patients included acute borreliosis, Lyme arthritis, as well as cardiological, neurological and dermatological symptoms of Lyme borreliosis. In order to select sera with a high titer of Borrelia-specific antibodies, all serum samples were analyzed by ELISA using whole cell extracts from B. burgdorferi. A broad range of responses were detected (data not shown). The sera with the highest titer were selected for subsequent antigen identification and validation studies. Six different serum pools were prepared, encompassing 25 high titer sera specific for distinct patient groups, neuroborreliosis (NB [P3077, P3084, P3286, P3288, P3337]), Lyme arthritis (LA [P3120, P3239, P3251]), acrodermatitis chronica atrophicans (ACA [P3183, P3209, P3336]), erythema migrans (EM [P3055, P3144, P3186, P3219]), and Lyme borreliosis (LB-I [P1755, P1766, P1772, P1806, P1811] and LB-II [P3016, P3150, P3301, P3344, P3394]). Prior to library screening, sera were absorbed with E. coli DH5␣ whole cells in order to prevent nonspecific selection of clones by E. coli-specific antibodies and thus increase selection of clones by antibodies recognizing Borrelia epitopes. Subsequently, IgGs were purified from pooled sera and tested for immune reactivity towards Borrelia proteins using whole bacterial extracts in Western blot. This confirmed that the selected IgG pools recognized large arrays of proteins in extracts of two different Borrelia species, B. burgdorferi strain B31 and B. afzelii strain K78 (data not shown). 3.2. Selection of Borrelia antigens by human sera: the ANTIGENome To express Borrelia-derived epitope-bearing peptide fragments on the surface of E. coli, two different genomic libraries were generated from the B. afzelii strain K78 using two platform proteins for surface expression, LamB and FhuA. Our choice of Borrelia species was motivated by the fact that B. afzelii is the most prevalent pathogen causing human borreliosis in Europe. In order to generate the LamB library, genomic DNA of the strain K78 was digested with DNase I into approximately 50 bp-long fragments. By contrast, the FhuA library was generated by sonication of DNA resulting in 150–300 bp-long fragments. The fragments were first cloned into a frame selection vector as described previously [15]. To assess the quality of the libraries, approximately 500 randomly chosen clones were sequenced from each of the frame selection libraries. Bioinformatic analysis showed that the actual insert length was close to the intended size (average length, FhuA 170 bp and LamB 51 bp) and that the majority of clones fell within a narrow size range (data not shown). After quality control of the frame selection libraries, inserts were transferred into the display vectors, creating bacterial surface display libraries that present epitope-bearing peptide fragments from Borrelia on the surface of E. coli [15]. In order to identify immunoreactive Borrelia proteins, both LamB and FhuA bacterial surface display libraries were incubated with human IgG pools as described previously [15]. Using streptavidin-coated microbeads, clones carrying inserts recognized by antibodies were separated by MACS (magnetic cell sorting) sorting. Selected E. coli clones were analyzed by Western blotting to confirm their antigenicity (data not shown) and subsequently, approximately 8000 clones were picked from a total of 12 screens and their inserts were sequenced. The sequences were then mapped to the published genomes of B. burgdorferi strain B31 [26,27] and B. garinii strain PBi [28]. This resulted in the identification of 122 open reading frames which were identified at least twice (Supplementary Table 2). The most antigenic among the 122 selected candidates, identified 313 times, was the hypothetical protein BAP001 (homolog to the B. burgdorferi gene BB0844). The second most frequently recognized candidate, with 147 hits, was protein P35 (BAP024, which is the homolog to the B. burgdorferi

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gene BBK32). Another highly recognized candidate was the flagellar filament core protein (BA0149; homolog to the B. burgdorferi gene BB0147). In general, hypothetical proteins were most frequently selected among the highly immunoreactive candidates, and with 51 out of 122 (42%), they formed the largest group of the Borrelia ANTIGENome. The percentage of hypothetical proteins identified is similar to the percentage found in the Borrelia genome (41%). When analyzing selected antigens with known function, lipoproteins and outer surface proteins formed a considerable group, among which many proteins already known to be involved in dissemination or immune evasion of Borrelia were included (e.g. OspC, P35, DbpA, and P66). It is worth mentioning that we did not identify OspA in our selection since the ANTIGENome technology will only detect antigens that are expressed in the infected patient and the expression of OspA is down regulated before the Borrelia spirochetes are transmitted from the tick [29]. 3.3. Selection of 12 vaccine candidates based on in vitro validation assays Several in vitro validation steps were applied in order to select the most promising antigens as vaccine candidates. As a first step we investigated the presence of the antigen encoding genes in a collection of human pathogenic Borrelia strains. A panel of 30 different Borrelia isolates, comprising 24 different ospC types associated with invasive and non-invasive human disease [30], was tested by PCR for the presence of the respective genes. Of the 121 genes analyzed, 64 were present in more than 85% of the strains tested, of which 44 genes were identified in all tested strains (Supplementary Table 2). The identification of epitope-bearing peptide fragments in the bacterial surface display libraries is dependent on the level and affinity of antibodies in the applied serum pools. In order to assess the extent of antigenicity, the presence of antibodies in individual human sera specific for the identified epitope-bearing fragments was determined by peptide ELISA. The human sera used for this analysis were mainly those included in the six serum pools applied for the identification of antigens by bacterial surface display. In total, 537 overlapping synthetic biotin-labeled peptides covering the epitope-bearing fragments (clone regions) identified in 119 ORFs, were used to coat streptavidin ELISA plates. Many of the peptides were confirmed to be antigenic (data not shown). For some of the antigens, it was observed that peptides representing different regions showed various degrees of reactivity, further delineating the immunoreactive region of the respective antigen (Fig. 1). As an example the results for BAP001 (homolog to the B. burgdorferi gene BB0844) are shown in Fig. 1. On the other hand, peptides from some antigens showed no reactivity with the human sera, such as BA0210 (homolog to the B. burgdorferi gene BB0210) (data not shown). The selection of such antigens by our screens despite this lack of ELISA reactivity might be explained by the possibility that the antibodies against these antigens recognized conformational rather than linear epitopes. Although these antigens could also have vaccine potential, their selection and testing in efficacy studies will only proceed if those candidates that were chosen through a strategy that includes additional criteria fail to associate with protection. A further important criterion for the selection of a vaccine candidate is its surface exposure and thus accessibility for binding of human antibodies. In order to address this issue, immune sera against epitope-bearing fragments were generated in mice by immunization with total bacterial lysates prepared from MACS screen-selected clones. Sera against 107 antigens were successfully generated and of those 95 (89%) showed reactivity with one or more of the corresponding peptides in ELISA (data not shown). Testing the sera by flow cytometry with live in vitro grown B. afzelii K78 cells indicated surface exposure of 32 antigens (Supplementary Table 2), from which 11 showed

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Fig. 1. Immunoreactive regions of BAP001 (A). Arrows above the gene BAP001 show the location of the epitope-bearing fragments identified by AIP (Black; FhuA clones, Gray; LamB clones). Biotinylated peptides used for the peptide ELISA are depicted above the epitope-bearing fragments. (B) Peptide ELISA with human sera. Disease indications are shown above the serum identification numbers: LB, Lyme borreliosis; EM, erythema migrans; NB, neuroborreliosis; ACA, acrodermatitis chronica atrophicans. Immune reactivities of human sera for BAP001 are shown. Peptides are named as follows: BAP001 followed by the peptide number, 01–07. Coding of reactivity: <50 ELISA units (white), 50–100 ELISA units (light gray), 100–200 ELISA units (dark gray), >200 ELISA units (black).

prominent shifts of over 70% of cells (representative example shown in Fig. 2). Among these surface exposed antigens were some hypothetical proteins (e.g. BA0056, BAP019 and BAP037), flagellar proteins (e.g. BA0221), known immunoreactive proteins (e.g. BA0024 and BA0737) and membrane associated proteins (e.g. BA0210 and BAP033). However, it should be kept in mind that some surface exposed antigens, very likely were missed by our analysis, because they are not expressed in in vitro grown cells, or because the selected peptides may not be readily accessible on the surface. Based on the results of the different in vitro validation steps (prevalence of the antigen in the tested Borrelia strains, and/or their high reactivity with human sera and/or confirmation to be surface located), 12 of the 122 identified candidates were selected for further studies, in addition we also included three (BB0062, BB0193 and BBA73) antigens with lower ranking (Table 1).

Fig. 2. Surface staining of B. afzelii strain K78 cells with an immune sera generated by immunizing mice with a bacterial lysate consisting of clones expressing adjacent epitope-bearing fragments from the surface-located membrane protein 1, BA0210.

3.4. Antigen-specific antibody and T cell cytokine responses induced in mice infected with B. afzelii through tick challenge To determine whether the 15 selected Borrelia antigens elicit protective immune responses, and thus qualify as candidates with vaccine potential, immunization-challenge studies will need to be conducted in mice. In addition to a needle-challenge model, we have established a mouse model where animals are exposed to an infectious challenge using B. afzelii-infected I. ricinus ticks. The tick-challenge model has been validated microbiologically through the isolation of B. afzelii from the ears and urinary bladder of infected mice. As an effort to further validate the model from an immunologic standpoint, we evaluated the ability of infected mice to generate antibody and T cell responses to Borrelia antigens. The panel of 15 recombinant Borrelia antigens were tested for antibody recognition by Western blot using sera from B. afzelii (tick)-, B. burgdorferi (needle)-, and B. garinii (needle)-infected C3H/HeN mice. Eleven of these antigens were also evaluated for their ability to recall T cell responses by cytokine ELISpots using splenocytes from C3H/HeN and BALB/c mice with confirmed B. afzelii infection after challenge with infected ticks. Results from the Western blot showed that 14 out of 15 candidates reacted to all three species-specific post-challenge sera tested, confirming their expression during murine infection

Table 1 List of proteins selected for further analysis. Hits; total number of hits in all 12 screens, GD; gene distribution, FC; percentage of cells shifted in flow cytometry, PE; median ELISA titer of the most reactive peptide per antigen analyzed, nt; not tested. Antigens (amino acid sequence)

B. burgdorferi B31 homolog

HITS

GD

FC

PE

BA0210 (27–709) BA0221 (1–408) BA0327 (22–527) BA0507 (399–870) BA0644 (189–374) BA0675 (391–753) BA0737 (23–663) BA0759 (2–348) BA0792 (23–181) BAP025 (1–319) BAP034 (21–111) BB0077 (19–342) BB0062 (2–238) BB0193 (20–246) BBA73 (21–296)

BB0210 BB221 BB0329 BB0512 BB0649 BB0680 BB0744 BB0765 BB0796 BBQ13 BBO25 – – – –

39 11 40 8 7 12 54 12 6 14 8 4 9 9 6

93% 100% 77% 100% 95% 100% 87% 100% 100% 100% 50% 93% 100% 80% 70%

80% 87% 77% 21% <20% <20% 83% <20% <20% 22% 39% <20% <20% <20% <20%

0 98 94 130 76 143 18 16 117 128 0 135 30 15 nt

A. Poljak et al. / Vaccine 30 (2012) 4398–4406 Table 2 Reactivity of immune sera collected from mice infected with three different Borrelia species to recombinant proteins. Coding of post challenge sera reactivity to recombinantly expressed proteins by immunoblotting: strong reactivity (+++), medium reactivity (++), low reactivity (+), no reactivity (−). Antigens

B. afzelii (IS1)

B. burgdorferi (N40)

B. garinii (LU185)

BA0210 BA0221 BA0327 BA0507 BA0644 BA0675 BA0737 BA0759 BA0792 BAP025 BAP034 BB0062 BB0077 BB0193 BBA73

+++ ++ +++ ++ + + +++ +++ + +++ + ++ ++ + +++

++ + +++ +++ + + ++ + + +++ + + + + +++

++ + +++ +++ + ++ +++ +++ + +++ − + ++ + +

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of this cytokine in this mouse strain. Interestingly, IL-4 production was recalled from the splenocytes of BALB/c mice, and this response was positive upon stimulation with only 5 antigens (BA0759, BA792, BAP025, BB0062 and BBA73). One (BBA73) of these 5 antigens, stimulated the secretion of only IL-4, and the remaining four induced the production of all three cytokines. As for IL-4, the IL-17 response was also mostly observed in BALB/c mice, with measurable precursor frequencies observed for 7 antigens, of which two (BB0077 and BB0193) also elicited the production of IFN-␥, and 4 (BA0759, BA0792, BAP025, and BB0062) were associated with the simultaneous induction of IFN-␥ and IL-4 production. The aforementioned cytokine secretion patterns were confirmed by flow cytometry through a cytometric bead assay using antigenstimulated splenocytes from infected C3H/HeN and BALB/c mice (data not shown). Altogether, these results show that for the tested antigens, a type 1 T cell pro-inflammatory response is prevalent in C3H/HeN mice, and although such a pro-inflammatory cytokine profile is also evident in BALB/c mice, the response in the latter appears to be also accompanied by the production of a type 2 T cell cytokine. Thus, antigens inducing a type 2 or mixed type 2/type 1 T cell cytokine response could probably be the most suitable candidates for a Lyme borreliosis vaccine.

and strongly suggesting cross-reactivity (Table 2). However, the immunoreactive intensity varied significantly for a given serum to the 15 different antigens. In contrast, the intensity of reactivity to a given protein was similar for the three different post-challenge sera, providing evidence for a high degree of epitope conservation across species for the tested antigens. The most pronounced specific- as well as cross-reactivity was seen for the candidates BA0210, BA0327, BA0507, BA0737 and BAP025. By contrast, a weak yet detectable reactivity was noted for the candidates BA0644, BA0792, BAP034 and BB0193. This reactivity suggested either low antigenicity or limited expression during infection. The intensity of the reactivity noted for two of the candidates, namely BBA73 and BA0759 was highly variable when probed against post-challenge sera to each of the three Borrelia species. While BBA73 was only weakly detected by sera from B. garinii-infected mice, BA0759 was weakly immunoreactive using sera from B. burgdorferi-infected mice. Eleven of the 15 selected Borrelia antigens were further tested for their ability to stimulate the secretion of IFN-␥, IL-4 and IL17 (Table 3), of which ten antigens elicited a recall response of at least one cytokine from the splenocytes of one or both B. afzelii tick-infected mouse strains. In infected C3H/HeN mice, 9 antigens elicited a recall IFN-␥ response. The single antigen (BA0792) that stimulated production of IL-17 also elicited an IFN-␥ response. None of the tested antigens were capable of recalling an IL-4 response. In BALB/c mice, all but one of the antigens that elicited a IFN-␥ response in C3H/HeN, were also associated with a response

4. Discussion Considering the diagnostic difficulties, severe health problems caused by chronic infection, progression of Lyme borreliosis in endemic regions, the development of a prophylactic vaccine would be of great benefit. Because Borrelia has a very complex biology and alters expression of outer surface proteins according to temperature, pH, and other environmental stimuli, identification of novel vaccine candidates bears a great challenge. Besides OspA, which was used as vaccine antigen in the LYMErix vaccine [31], alternative Borrelia outer membrane proteins have been evaluated as vaccine antigens in various studies [32,33]. We have used the ANTIGENome technology [24] as a tool for identification of novel vaccine candidates as well as diagnostic markers and to aid in the development of a novel vaccine with broad coverage against Lyme borreliosis caused by the three pathogenic species; B. burgdorferi, B. afzelii and B. garinii. The technology relies on the human humoral immune response by screening bacterial surface display libraries with serum IgGs isolated from patients suffering from different manifestation of Lyme borreliosis. Theoretically the bacterial surface display libraries allow expression of all potential antigenic determinants as epitope-bearing fragments and enables detection of in vivo expressed antigens under in vitro culture conditions [34]. This is important since Borrelia spirochetes are known

Table 3 Enumeration of cytokine-producing cells in spleens from B. afzelii tick-infected mice. Values represent net numbers of spot-forming cells for the cytokines IFN-␥, IL-4, and IL-17 as measured by ELISpot using 1 × 106 SC from B. afzelii tick-infected mice. SC were stimulated with a panel of recombinant Borrelia antigens (5 ␮g/ml) as described in Materials and Methods. A signal was considered positive when the specific net number of spot-forming cells was ≥10. Mean number of spots for OVA-stimulated SC from infected C3H/HeN and BALB/c mice were respectively 5 and 6 for IFN-␥, 9 and 16 for IL-4, and 16 and 15 for IL-17. Mean number of spots for medium only-stimulated SC were for both mouse strains and all 3 cytokines below the mean number of spots obtained for OVA-stimulated SC. Antigens

IFN-␥ BA0221 BA0644 BA0675 BA0759 BA0792 BAP025 BAP034 BB0062 BB0077 BB0193 BBA73

BALB/c

C3H/HeN

2 34 39 62 95 52 51 42 126 34 2

IL-4 0 0 0 4 0 0 0 0 0 0 1

IL-17

IFN-␥

IL-4

IL-17

6 1 2 7 20 7 5 2 4 0 7

0 13 8 46 85 37 32 59 94 37 0

0 6 0 15 24 10 0 22 7 0 14

0 0 16 25 25 70 2 18 30 11 0

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to adapt their surface protein expression in response to different environmental stimuli. The ANTIGENome technology is thus not influenced by the in vitro expression profile of potential antigens, but also not by predictive algorithms of bioinformatic approaches. It was therefore interesting to note that hypothetical proteins constituted 42% of the Borrelia ANTIGENome. The selected antigens with known function could be divided mainly into 4 different groups: outer membrane lipoproteins (e.g. OspC, OspG, and OspF); flagellar proteins (e.g. flagellar hook and hook-associated proteins); heat shock proteins (e.g. heat shock proteins 70 and 90); and various oligopeptide ABC transporters. Many of the selected antigenic Borrelia proteins are involved in pathogenesis, such as decorin binding protein A [35], the Borrelia direct repeat (Bdr) and OspEF-related Erp proteins, important for complement inactivation [36]. However, these proteins are very variable among different Borrelia strains and are therefore not suitable as vaccine candidates possibly conferring broad protection. Our ANTIGENome screens were though able to select most of the proteins previously identified as antigens, especially among the highly immunoreactive candidates. For example, the immunoreactive proteins P35 (BAP024; [37]) and p83/100 (BA0737; [38]) were selected with 147 and 54 hits, respectively. Earlier studies have shown the diagnostic value of both P35 [39,40] and p83/100 [41–43] which is in agreement with our antigen selection. The selection of the chromosome encoded protein BA0737 did confirm our technology, as it is widely used in serological tests as a marker for stage 3 infection, since BA0737 cross-reacting antibodies are generated upon infection by the Borrelia species causing Lyme borreliosis. Outer surface proteins A and B, previously identified as protective antigens [44], were not selected by screening the bacterial surface display libraries with human IgGs. This was not surprising though, as OspA and OspB are mainly expressed during infection of the tick and expression of these antigens is downregulated before the spirochetes enter the mammalian host. Thus, they do not elicit an immune response upon infection of humans [29]. Accordingly, the presence of OspA specific antibodies has only been reported in few patients with late stage Lyme arthritis [45]. After defining the Borrelia ANTIGENome, several in vitro validation assays were applied in order to select the most promising vaccine candidates for further analysis. The first step, the determination of gene prevalence in clinical isolates, was extremely important for a genetically heterogeneous bacterium such as Borrelia [46,47]. The gene prevalence analysis resulted in a significant reduction of the number of possible vaccine candidates, as from 121 only 64 antigens showed a prevalence of ≥85% in the 30 Borrelia strains analyzed. However, it should be emphasized that a negative result for one strain does not necessary mean that the gene is absent, but could be due to a sequence variation at the binding site of at least one of the primers used for the analysis. The second validation step was to determine immunoreactivity of human sera to selected antigens by peptide ELISA. This positive selection criterion identified highly immunoreactive peptide-bearing epitopes encoded by the corresponding ORFs. The presence of high levels of antibodies strongly suggests in vivo expression of the vaccine candidate by the pathogen during infection and allowed the selection of 54 promising candidates (data not shown). Another important criterion for a vaccine candidate is its accessibility for antibody binding, e.g. exposure on the surface. To address this issue, immune sera were generated by immunizing mice with lysates of E. coli clones selected by Borrelia-specific antibodies. The sera were characterized for immunoreactivity by ELISA and flow cytometry with the library strain B. afzelii K78, which indicated surface exposure of 32 candidates. By assessing the reactivity of immune sera collected from mice infected with the three most important Borrelia species with regards to human infection (B. afzelii, B. garinii and B. burgdorferi), to our candidate proteins, we aimed to not only verify our selection

of target antigens but also to study cross reactivity. Since almost all (14/15) selected antigens could be detected by the three different post challenge sera pools tested (Table 2), we confirmed that the ANTIGENome technology selected valid antigenic proteins also generating a clear antibody response upon natural infection in mice. Furthermore, the high degree of cross-reactivity observed, suggested that epitopes expressed during infection are conserved between the three different species of Borrelia. However, some immune sera reacted with a decreased intensity to certain proteins, suggesting a lower degree of conservation or decreased immunogenicity. Other studies assessing the reactivity of immune sera from Lyme borreliosis patients in US (i.e. B. burgdorferi infected subjects) to purified outer membrane proteins or recombinant polypeptides, have also identified BB0329 (BA0327 homolog), BB0649 (BA0644 homolog) and BB0744 (BA0737 homolog) among others [48,49]. In addition, the same two studies also detected BB0329 and BB0649 by using sera collected from tick infected white-footed mice (P. leucopus). As further support for antigens being expressed during infection of mice or when bacteria were grown under in vivo likeconditions, previous studies have reported the following proteins: BB0077, BB0680 (BA0675 homolog) and BBA73 [50–54]. It should be noted here that the reactivity of sera from Borrelia-infected mice to recombinant proteins does not necessarily indicate that the antigens are surface-accessible, but only that they are expressed during murine infection. The high degree of cross-reactivity observed for most of the candidates indicates that the conservation of the antigens is sufficient to promote the induction of antibodies recognizing proteins from diverse Borrelia strains. While numerous studies have confirmed a role for specific antibodies in resistance to Borrelia infection [33,55,56], there is evidence showing that T cells and their associated cytokines can also influence the outcome of such infection [11–13,57]. As reported for B. burgdorferi infections [18,58], we observed that C3H/HeN and BALB/c mice were respectively also more susceptible and resistant to B. afzelii infection following tick challenge. Our results showed that in B. afzelii-infected C3H/HeN and BALB/c mice, in vitro cytokine recall responses to a panel of 11 Borrelia antigens were clearly pro-inflammatory in C3H/HeN mice, with IFN-␥ production detected upon stimulation with 9 antigens, and both pro- and anti-inflammatory in BALB/c mice, with IFN-␥, IL-4 and IFN-␥/IL4 production shown after stimulation with 4, 1, and 5 antigens, respectively. For the tested antigens, the cytokine profiles in both strains of mice may represent a snapshot of the overall type 1 and type 2 T cell cytokine responses and infection susceptibility to B. afzelii, and as such, our findings would be in agreement with studies that showed a deleterious outcome of infection in mice depleted in vivo of the type 2 cytokine IL-4 and an increased resistance to infection in mice depleted of the type 1 cytokine IFN-␥ [12]. Other studies have also supported the protective effects of IL-4 and disease-promoting effects of IFN-␥ [59,60]. IL-4 is likely to play a beneficial role by launching a type 2 T cell response that directs immunoglobulin class switching to IgG1 and IgE [61,62] and by stimulating the proliferation and differentiation of mast cells [63]. Borrelia antigen-specific IgE have been detected in infected individuals and these antibodies are reported to potentially contribute to protective immunity against Borrelia infection [64]. It is important to note, however, that because experimental Lyme arthritis can also take place in IFN-␥-deficient mice [65], IFN-␥ production alone is not solely responsible for the pathologic changes that occur during Borrelia infection. Thus, based on a number of studies that correlate the production of IL-17 with the genesis of Lyme arthritis and other pathological manifestations of Lyme borreliosis [13,57], we have also assessed IL-17 production after stimulation with Borrelia vaccine candidate antigens. Interestingly, for the tested panel of antigens, IL-17 production was noted almost exclusively in B. afzelii-infected BALB/c mice, with 7 of 11 antigens recalling an IL-17

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response in vitro. By contrast, only one antigen stimulated IL-17 production by the splenocytes of infected C3H/HeN mice. With a few exceptions, the same antigens that stimulated production of IL-17 also stimulated production of IFN-␥. From the limited set of available data, it is tempting to speculate that an antigen could have Borrelia vaccine potential if it is capable of inducing the production of a predominant type 2 T cell cytokine profile or a balanced type 2/type 1 T cell cytokine profile, with minimal or absent induction of IL-17 production. We will further evaluate the T cell cytokine response of mice infected with B. afzelii or B. burgdorferi through tick or needle challenge to support the validation of the antigens identified by our ANTIGENome technology aimed for testing in immunization-challenge studies. Conflict of interest statement The authors declare potential conflict of financial interest being employees of Intercell AG, a biotechnology company. Acknowledgements The authors wish to express their gratefulness to Birgit Noiges, Christine Triska, Ulrike Stierschneider, Christina Satke, Dagmar Zierer, Ivan Gomez, Sandra Jost, Gregory Bouchard and Dieter Gelbmann for their technical support. This study was funded by the European Union (512598-BOVAC; grant FA794A0101). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2011.10.073. References [1] Steere AC. Lyme disease. N Engl J Med 1989;321:586–96. [2] Wilske B. Diagnosis of Lyme borreliosis in Europe. Vector Borne Zoonotic Dis 2003;3:215–27. [3] Takayama K, Rothenberg RJ, Barbour AG. Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi. Infect Immun 1987;55:2311–3. [4] Barbour AG, Hayes SF. Biology of Borrelia species. Microbiol Rev 1986;50:381–400. [5] Haake DA. Spirochaetal lipoproteins and pathogenesis. Microbiology 2000;146:1491–504. [6] Coburn J, Fischer JR, Leong JM. Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete. Mol Microbiol 2005;57:1182–95. [7] Steere AC, Glickstein L. Elucidation of Lyme arthritis. Nat Rev Immunol 2004;4:143–52. [8] Ma Y, Weis JJ. Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess B-cell mitogenic and cytokine-stimulatory properties. Infect Immun 1993;61:3843–53. [9] Whitmire WM, Garon CF. Specific and nonspecific responses of murine B cells to membrane blebs of Borrelia burgdorferi. Infect Immun 1993;61:1460–7. [10] Dong Z, Edelstein MD, Glickstein LJ. CD8+ T cells are activated during the early Th1 and Th2 immune responses in a murine Lyme disease model. Infect Immun 1997;65:5334–7. [11] Keane-Myers A, Nickell SP. T cell subset-dependent modulation of immunity to Borrelia burgdorferi in mice. J Immunol 1995;154:1770–6. [12] Keane-Myers A, Nickell SP. Role of IL-4 and IFN-gamma in modulation of immunity to Borrelia burgdorferi in mice. J Immunol 1995;155:2020–8. [13] Burchill MA, Nardelli DT, England DM, DeCoster DJ, Christopherson JA, Callister SM, et al. Inhibition of interleukin-17 prevents the development of arthritis in vaccinated mice challenged with Borrelia burgdorferi. Infect Immun 2003;71:3437–42. [14] Zeidner NS, Schneider BS, Rutherford JS, Dolan MC. Suppression of Th2 cytokines reduces tick-transmitted Borrelia burgdorferi load in mice. J Parasitol 2008;94:767–9. [15] Etz H, Minh DB, Henics T, Dryla A, Winkler B, Triska C, et al. Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus. Proc Natl Acad Sci U S A 2002;99:6573–8. [16] Giefing C, Meinke AL, Hanner M, Henics T, Bui MD, Gelbmann D, et al. Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies. J Exp Med 2008;205:117–31.

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