Identification of a synthetic peptide inducing cross-reactive antibodies binding to Rhipicephalus ( Boophilus) decoloratus, Rhipicephalus ( Boophilus) microplus, Hyalomma anatolicum

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Vaccine 28 (2010) 261–269

Contents lists available at ScienceDirect

Vaccine

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dentification of a synthetic peptide inducing cross-reactive antibodies binding tohipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus,yalomma anatolicum anatolicum and Rhipicephalus appendiculatus BM86omologues

adja Koppa, Diana Diaza, Mario Amackerd, David O. Odongoc,e, Konstantin Beierb,ordula Nitschb, Richard P. Bishopc, Claudia A. Daubenbergera,∗

Swiss Tropical Institute, Department of Medical Parasitology and Infection Biology, Socinstrasse 57, 4002 Basel, SwitzerlandDepartment of Neuroanatomy, Institute of Anatomy, University of Basel, SwitzerlandInternational Livestock Research Institute, P.O. Box 30709, Nairobi 00100, KenyaPevion Biotech Ltd., Worblentalstrasse 32, 3063 Ittigen, SwitzerlandSchool of Biological Sciences, University of Nairobi, Kenya

r t i c l e i n f o

rticle history:eceived 12 May 2009eceived in revised form 5 August 2009

a b s t r a c t

The BM86 antigen, originally identified in Rhipicephalus (Boophilus) microplus, is the basis of the onlycommercialized anti-tick vaccine. The long-term goal of our study is to improve BM86 based vaccinesby induction of high levels of tick gut binding antibodies that are also cross-reactive with a range of

ccepted 21 September 2009vailable online 4 October 2009

eywords:ick subunit vaccine developmentynthetic peptide

BM86 homologues expressed in other important tick species. Here we have used a BD86 derived syn-thetic peptide, BD86-3, to raise a series of mouse monoclonal antibodies. One of these mAbs, named12.1, recognized BM86 homologues in immuno-histochemical analyses in four out of five tick speciesincluding R. (B.) microplus, Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum anatolicum andRhipicephalus appendiculatus. Our results indicate that broadly cross-reactive tick gut binding antibodies

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mmuno-histochemistryross-reactivity

can be induced after imm

. Introduction

Tick borne diseases (TBD) affect about 80% of the world’s cat-le population and are widely distributed throughout the world,articularly in the tropics and sub-tropics. It has been estimatedhat the annual global costs associated with ticks and TBD in cat-le amounted up to 18.7 billion US$ [1]. The costs associated withBD include both direct losses (from mortality and reduced produc-ion) and expenses associated with control and treatment. Heavyick infestations can result in anemia, particularly in small animals,kin ulceration with secondary bacterial infections and the stressaused by the presence of large numbers of ticks can lead to a sig-ificant loss of weight and condition [2]. Ixodid ticks with seriousirect effects on infested animals include species of Rhipicephalus

Boophilus), Amblyomma, Hyalomma and Rhipicephalus. Hyalommanatolicum anatolicum and Rhipicephalus appendiculatus are three-ost ticks transmitting the important cattle pathogens Theileriannulata and Theileria parva, respectively. The one-host tick Rhipi-

∗ Corresponding author. Tel.: +41 61 284 82 17; fax: +41 61 271 86 54.E-mail address: [email protected] (C.A. Daubenberger).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.09.085

tion with a synthetic peptide derived from the protein BD86.© 2009 Elsevier Ltd. All rights reserved.

cephalus (Boophilus) decoloratus can efficiently transmit Babesiabigemina and R. (B.) microplus is a competent vector of B. bigem-ina, B. bovis and Anaplasma marginale. Amblyomma variegatum isthe main vector for Ehrlichia ruminantium, the causative agent ofheart water disease [3].

Currently, the means of controlling ticks and TBD rely mainlyon tick control. Chemical acaricides are widely used since theyare readily available, efficacious if used properly and farmers arefamiliar with them since they are actively promoted by commer-cial companies [4]. However, development of chemical resistancein tick populations is a serious and increasing global problem andthe presence of chemical residues in the environment and food isincreasingly an issue of concern [5–7]. The only currently marketedcommercial vaccine against an ecto-parasitic arthropod is based onthe glycoprotein BM86 present on tick gut cells of R. (B.) microplus,expressed as a recombinant protein in the methylotrophic yeastPichia pastoris (TickGard PlusTM (Intervet) and GavacTM (Heber

Biotech)) [8]. The BM86 molecule is encoded by 650 amino acidswith a predicted molecular weight of 71.7 kDa as an unprocessedprotein; it has a N-terminal leader peptide and a single C-terminaltrans-membrane segment [9]. In the mature protein, this trans-membrane sequence is replaced by a glycosyl-phosphatidyl inositol

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nchor [10]. The amino acid sequence contains 66 cysteine residuesith eight EGF-like domains predicted, likely rendering the three-imensional structure of the protein highly complex [10,11]. Itas been shown that the antigen localizes to the surface of tickut digest cells [12,13]. In contrast to naturally acquired immunitygainst ticks, the protection induced by BM86 is mediated by hostntibodies which bind to and damage gut cells, interfering withick feeding [14]. Major effects of the vaccine were on reproductiveutput of female ticks, such that the vaccine acts by controllingick populations. The BM86 gut protein is a so-called ‘concealed’ntigen, since it is not normally exposed to the host’s immune sys-em and this is presumed to be an important factor contributingo the success of this vaccination strategy [15]. Direct correlationetween vaccine induced anti-BM86 antibody levels and effects onick moulting weight and egg laying capacity is described [14,16].M86 based vaccines currently require multiple boosting (as manys four inoculations of the antigen) in order to achieve sufficientlyigh antibody tires to control the tick populations. We have previ-usly described the detailed analysis of cross-reactive antibodies inera of TickGard PlusTM immunized Bos indicus animals binding toeptides within the BM86 homologue present in R. (B.) decoloratus17]. Several cross-reactive, linear and immuno-dominant aminocid sequences were identified [17]. In the current study, two ofhese linear peptides were used to immunize mice. Several mono-lonal antibodies were established and employed to characterize inetail the expression and localization BM86 homologues in a rangef tick species.

. Materials and methods

.1. Peptide synthesis

Immuno-dominant B-cell epitopes within BD86 (the BM86omologue in R. (B.) decoloratus) have been identified and twof these linear epitopes were located between BD86 amino acidesidues 36–50 and 404–418 [17]. These synthetic peptides,amely BD86-1 (CTAESSICSDFGNEFCRDAECEVVPG-amide) andD86-3 (IATKPLSKHVVKKLQACEHPIGEWC-amide) were producedommercially by Biosynthan, Berlin, Germany. Peptides wereurified using HPLC and purity determined using MALDI-TOFnalysis with a purity of >85%. The predicted calculated molecularasses of BD86-1 and BD86-3 are 2768.1 and 2815.4 Da with

he measured masses [M+H] found as 2768.9 and 2816.2 Da,espectively. The C-terminal ends of the peptides were modi-ed by incorporation of an amide-group. The cysteine residuest the C- or N-terminal end were used for coupling of theeptides to a maleimide-modified phosphatidylethanolamine1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-

aleimidomethyl)cyclohexane-carboxamide]; Avanti Polaripids, Alabaster AL, USA).

.2. Preparation of peptide-loaded virosomes

Immuno-potentiating reconstituted influenza virosomesIRIVs) were prepared as described previously [18]. Briefly,2 mg of egg phosphatidylcholine (PC) (Lipoid, Ludwigshafen,ermany) and 8 mg of 1-oleoyl-3-palmitoyl-rac-glycero-2-hosphoethanolamine (PE) (Bachem, Bubendorf, Switzerland)ere dissolved in 3 ml of PBS, 100 mM octaethyleneglycol-mono-

n-dodecyl) ether (OEG-PBS) (Sigma, Buchs, Switzerland). Twoilligrams influenza haemagglutinin of inactivated influenza

train A/H1N1 virus was centrifuged at 100,000 × g for 1 h at 4 ◦C,nd the pellet was dissolved in 1 ml of OEG-PBS. The detergent-olubilized phospholipids and viruses were mixed with theeptide-PE conjugates and sonicated for 1 min. This mixture wasentrifuged at 100,000 × g for 1 h at 18 ◦C. The supernatant was

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used and virosomes were formed by detergent removal using1.25 g of wet SM2 Bio-Beads (BioRad, Reinach, Switzerland) for 1 hat room temperature with shaking and three times for 30 min with625 mg of SM2 Bio-Beads each. Finally, the virosomes were sterilefiltered (0.2 �m).

2.3. Mouse immunogenicity studies

Balb/c mice were immunized intramuscularly with 1 �ghaemagglutinin of inactivated influenza A/H1N1 virus. Three weekslater the mice were immunized with peptide-loaded virosomesthrice with a 3-weeks interval. Blood was collected 2 weeks afterthe final inoculation and tested in ELISA.

2.4. Generation of hybridoma cell lines and production of mAbs

Hybridomas were generated from spleen cells of mouse 04/157-3 after a booster immunization with BD86-3 loaded IRIVs. PAImouse myeloma cells served as fusion partner and hybrido-mas were selected in hypoxanthin–aminopterin–thymidine (HAT)medium. Wells were analyzed after 10 days and cells that secretedantibodies binding to BD86 recombinant protein identified inELISA were expanded. Individual clones were established by lim-iting dilution three consecutive times and cultivated in Isocove’smodified Dulbeco’s medium, completed with 10% (v/v) heat-inactivated fetal calve serum (Sigma), l-glutamine (Sigma) andpenicillin/streptomycin (Sigma) and 0.34% (v/v) mercaptoethanol(Sigma) at 37 ◦C, 5% CO2 and 95% humidity. For large scale mAbproduction, hybridoma cell lines were cultured in tissue cultureflasks and mAbs were purified by protein A affinity chromatogra-phy. Purified mAbs were sampled in PBS and concentrated usingAmicon Ultra-15 (Millipore).

2.5. ELISA

Enzyme-linked immunosorbent assay (ELISA) analyses wereessentially performed as described [17]. Briefly, 96-well plates(Maxisorp, Nunc) were coated with 0.5 �g recombinant BD86 pro-tein per well and incubated overnight at 4 ◦C. After washing platesthrice with PBS containing 0.5% (v/v) Tween 20 using Easy Wash2000 (Bioreba), wells were blocked with 5% (w/v) skimmed milk inphosphate-buffered saline (MPBS) for 30 min, washed again andincubated with graded dilutions of 100 �l of mouse hybridomacell culture supernatant or purified mouse monoclonal antibodies(adjusted for all mAbs to a stock concentration of 1 mg/ml, startingdilution of 1/100) for 2 h at 37 ◦C. Following another washing step,50 �l alkaline phosphatase-conjugated goat anti-mouse gammaheavy chain secondary antibody (Milian analytica) was added perwell for 1 h at 37 ◦C. Preceding the addition of 50 �l substrate bufferper well (1 mg of p-nitrophenyl phosphate per ml in a pH 9.8 buffersolution with 10% (v/v) diethanolamine and 0.02% (w/v) MgCl2), thewells were washed once more. Plates were incubated for 80 minin the dark and optical density assessed using the ELISA readerSunrise (Tecan) at 405 nm. As negative controls, wells incubatedin the absence of primary antibodies were always included intothe experiment. Isotypes of the anti-BD86 mAb were determinedby using alkaline phosphatase labeled goat antibodies specific formouse immunoglobulin �1, �2a, �2b, �3, � or � chains (South-ern Biotechnology). MAb DD1.1 is directed against a Plasmodiumfalciparum derived asexual blood stage protein and has been estab-lished in our laboratory. DD1.1 served as isotype matched controlantibody (IgG1/�) for ELISA and Western blot analyses [19].

2.6. Western blot analysis

LDS-PAGE (lithium dodecyl sulphate-polyacrylamide gel elec-trophoresis) was performed with either recombinant BD86 protein

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Fig. 1. (A) ELISA of purified mAbs 14.1, 12.1, 2.3 and DD1.1 using plates coated withrecombinant BD86. Purified mAbs were concentrated to 1 mg/ml and starting witha concentration of 10 �g/ml tested in graded dilutions in ELISA plates coated withrecombinant BD86. (B) Western blot analysis of the binding specificities of anti-BD86 mAbs 12.1 (lanes 1 and 2), 14.1 (lanes 3 and 4), 2.3 (lanes 5 and 6). MAb DD1.1has been included as isotype matched negative control antibody (lanes 7 and 8).Antibodies were tested as cell culture supernatants (lanes 1, 3, 5 and 7) and purifiedmAb after protein A affinity chromatography purification (lanes 2, 4, 6 and 8). In lane9, secondary antibody only was applied. A single band of the predicted molecularweight of 75 kDa is recognized. Recombinant BD86 was separated by gradient SDS-PAGE, transferred to a nitrocellulose membrane and cut into individual strips thatwere incubated with different antibody preparations. Blots were developed usingthe ECL development system. The molecular weight marker is given on the left. (C)Western blot analysis of the reactivity of anti-BD86 mAbs 12.1 (lanes 1 and 2), 14.1(lanes 3 and 4) and 2.3 (lanes 5 and 6) tested as cell culture supernatants (lanes 1, 3and 5) and purified mAb after protein A affinity chromatography purification (lanes

N. Kopp et al. / Vac

r total lysate of R. (B.) decoloratus tick gut material. Briefly, anPG well pre-cast gradient gel (NuPAGE 4–12% Bis–Tris ZOOM Gel,nvitrogen) was loaded with 2.5 mg recombinant BD86 and subse-uently blotted with the iBlotTM (Invitrogen). Before loading, BD86rotein was mixed with LDS sample buffer and the reducing agentInvitrogen), heated for 5 min at 95 ◦C and quickly chilled on ice.fter transfer, membranes were blocked with 5% skimmed milk inBS (MPBS) containing 0.1% (v/v) Tween (MPBST) for 1 h at 37 ◦C.embranes were cut in strips and each strip was incubated with an

ndividual primary antibody preparation diluted with PBST for 1 hn the shaker at RT. Strips were washed four times with PBST formin before adding the horseradish peroxidase conjugated goatnti-mouse secondary antibody (Southern Biotech) for 1 h. Blotsere developed using the ECL system following the instructions of

he manufacturer (Pierce).Dissected R. (B.) decoloratus whole gut preparations (day 20)

ere snap frozen in liquid nitrogen immediately after isolationnd transported on dry ice to Basel. Two whole guts were usedor the total lysate production. 300 �l of tissue lysis buffer (5% (v/v)riton-X 100 in 10 mM Tris/HCl buffer pH 8.0 including proteinasenhibitor (Roche)) was added and the tissues were homogenized byonication using a Branson Sonifier 250 on ice. Centrifugation for0 min at 10,000 × g at 4 ◦C removed cellular debris and the super-atant was transferred to fresh tubes and kept at −80 ◦C. 10 �l ofotal lysate was mixed with LDS sample buffer and the reducinggent (Invitrogen), heated for 5 min at 95 ◦C and quickly chilled once. Seeblue plus (Invitrogen) was employed as a molecular weight

arker in all experiments.

.7. Maintenance of tick colony at ILRI

The R. (B.) decoloratus ticks used originated from Rusinga Islandn Western Kenya in the 1980s, while the R. (B.) microplus ticks origi-ated from Zanzibar and have subsequently been maintained in the

LRI tick-unit in an incubator at 28 ◦C with 80% relative humidity.he R. appendiculatus ticks used were the Muguga stock originallyollected from the central highlands of Kenya in the 1950s andere maintained initially at the East African Veterinary Researchrganization-Kenya Agricultural Research Institute (EAVRO-KARI)nd subsequently at the ILRI tick-laboratories [20] in an incubatort 24 ± 1 ◦C with 80% relative humidity. H. a. anatolicum adult ticksere originally collected from sheep in Sudan, north of Khartoum

n the 1990s and a tick colony established and maintained at ILRIs described [21].

.8. Immuno-histochemistry of tick gut tissue with anti-BD86Ab

R. (B.) decoloratus and R. (B.) microplus tick guts were dissectedrom semi-engorged adult female ticks that were fed on naive cattleetween days 18 and 20 post larval infestation. Both R. appendic-latus and H. a. anatolicum tick gut samples were dissected fromemi-engorged adult female ticks that had been fed on naive rab-its for 4 days post infestation. Dissected gut samples were, fixed

n Bouin’s Solution and embedded in paraffin using standard pro-edures. Blocks with were cut in sections of 5 �m with a watericrotome (Microm) and left to dry over night. The sectioned gutsere placed onto glass slides and fixed by placing the slides on aeating block at 59 ◦C for 5 min. For removal of paraffin, slides were

ncubated thrice 10 min in Ultraclear (Medite) and the sectionse-hydrated in descending alcohol concentrations for 2 min each

tarting from twice in 100%, and once in 95%, 90%, 70% and finallyn ddH2O. Antigen retrieval and immuno-histochemical staining

ere carried out according to the Vectastain elite ABC kit pro-ocol (Vector). The slides were washed with PBS before applyingew agents and incubation times longer than 5 min were per-

2, 4 and 6) with total lysates of whole tick guts of R. (B.) decoloratus ticks (20 daysfed on cattle). In lanes 7 and 8, recombinant BD86 is loaded with lane 7 incubatedwith mAb 12.1 as positive control and in lane 8, the primary antibody was omitted.The molecular weight markers are given on the left.

formed on a shaker. Antigen retrieval involved heating the slides in10 mM citrate buffer (Sigma) pH 6.0 in the microwave for 4.5 min.A cycle of 5 min cooling and heating for 30 s was repeated twicebefore letting the slides cool down in the 10 mM citrate buffer for20 min. Subsequently, endogenous peroxidase activity was blockedby applying 3% (v/v) H2O2 directly onto the slides and left for5 min, followed by blocking using 1.5% normal horse serum (Vector)for 20 min. Serum was removed, antibodies applied at a concen-tration of 10 �g/ml and incubated for 2 h. The biotinylated horse

anti-mouse secondary antibody (Sigma) was diluted 1:200 in 15%Normal Horse Serum and incubated for 30 min. The avidin–biotinhorseradish-peroxidase complex (Vector) was added for an addi-tional 30 min followed by the reaction with DAB or NovaRedTM

substrate kit for peroxidase (Vector) as chromogen and H2O2 as

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Fig. 2. (A) Analyses of tick gut binding properties of anti-BD86 mAb. Immuno-histochemical analysis of whole gut section of R. (B.) decoloratus (20 days fed) stained withmAbs 12.1 (I), 14.1 (II) and 2.3 (III). The scale bar is 100 �m, magnification 200×. (B) BD86 expression detected by mAb 12.1 is restricted to the surface of cells lining the tickgut. Immuno-histochemical analysis of sequential sections of whole gut tissue of R. (B.) decoloratus (20 days fed) stained with mAb 12.1. The scale bar is 25 �m, magnification400×. (C) BD86 protein is detectable by mAb 12.1 from days 8 to 24 after tick attachment. Immuno-histochemical analysis of whole gut sections of R. (B.) decoloratus ticksfed for 8 (I), 20 (II), 21 (III), 22 (IV), 23 (V) and 24 (VI) days on cattle. The picture shown in VII is an enlarged fragment of section shown in VI. As isotype control, mAb 2.3was used throughout the analyses and as a representative result R. (B.) decoloratus tick fed for 20 days on cattle is shown in VIII. The scale bar for pictures I–V and VII is50 �m, magnification 400×. The scale bar in picture VI is 200 �m, magnification 100×. Scale bar in picture VI is 200 �m, magnification 100×. The scale bar for picture VIII is100 �m, magnification 200×. (D) MAb 12.1 recognizes BD86 homologues in a range of ixodid tick species. Foregut and midgut sections of R. (B.) decoloratus, R. (B.) microplus,H. a. anatolicum, R. appendiculatus and A. variegatum ticks were stained with mAb 12.1 (columns 2 and 3). The scale bar for foregut and midgut stainings using mAb 12.1 is100 �m, magnification 200×. In the immuno-histochemistry stainings using mAb 2.3 with foregut sections (column 1) the scale bar is 50 �m for R. (B.) decoloratus foregutand 100 �m for the remaining pictures. Magnification: 400× for R. (B.) decoloratus foregut and 200× for the other pictures.

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Fig. 2. (Continued ).

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ig. 3. Amino acid sequence alignments using ClustalW program of peptide BD86-3eposited at NCBI Genbank (search conducted May 2009). The Genbank accession nure given in brackets.

ubstrate. After a counter-staining with haematoxylin, the slidesere analyzed under a Zeiss Axiophot microscope equipped withdigital camera.

. Results

.1. Establishment of R. (B.) decoloratus tick gut bindingntibodies

Two synthetic peptides delineated from BD86 that haveeen named BD86-1 (CTAESSICSDFGNEFCRDAECEVVPG-amide)nd BD86-3 (IATKPLSKHVVKKLQACEHPIGEWC-amide) were syn-hesized, coupled to the surface of IRIVs and used for immunizationf mice [22]. In contrast to mice immunized with synthetic peptideD86-1 or the IRIV carrier alone, BD86-3 induced strong humoral

mmune responses specific for the recombinant BD86 (data nothown). Spleen cells of one of these animals were used to establishnumber of hybridoma cell lines secreting mAb specific for BD86.hree hybridoma clones, namely 14.1, 12.1 and 2.3 were chosen forurther analysis and mAbs were purified from cell culture mediumsing protein A sepharose affinity chromatography.

Using ELISA, the mAbs 14.1, 12.1 and 2.3 were isotyped asgG1/� antibodies (data not shown). Each antibody preparation

as adjusted to 1 mg/ml and mAbs 14.1, 12.1, 2.3 and DD1.1ere analyzed by ELISA using BD86 recombinant protein as the

arget antigen. The three anti-BD86 mAbs recognized the plas-ic bound recombinant BD86 protein yielding similar OD valuesFig. 1A) while the isotype matched control mAb DD1.1 did notind BD86. Identical mAb preparations were tested in Westernlot analysis using recombinant BD86 and total protein lysate ofhole tick gut tissue of female R. (B.) decoloratus ticks fed 20 days

n cattle (Fig. 1B and C). Recombinant BD86 was recognized byhe three anti-BD86 mAbs as demonstrated by the detection of

distinct band at the predicted molecular weight of 68 kDa. In

ontrast, total lysates of whole tick gut tissue of B. decoloratusere recognized strongly by mAb 12.1 but incubation with mAb

4.1 and mAb 2.3 resulted in weak or no signals, respectively.he apparent molecular weight of the single band of approxi-ately 90 kDa recognized by mAbs 12.1 and 14.1 is consistent

or monoclonal production and fragments of its homologues of different tick speciess and the number of identical amino acid positions compared to the BD86-3 peptide

with reports describing the BM86 homologue of R. (B.) microplus[13].

Next we wanted to analyze by immuno-histochemistry the tickgut binding properties of these mAbs using tick gut tissue sec-tions of R. (B.) decoloratus. A novel method to embed and stain tickgut preparations was developed as described in Section 2. Stain-ing with mAb 12.1 resulted in specific signals of distinct surfacerestricted regions in cells lining the tick gut while mAb 14.1 andmAb 2.3 showed weak and no tick gut tissue binding, respectively(Fig. 2A). Since mAbs 12.1, 14.1 and 2.3 share the antibody isotype,results obtained with mAb 2.3 were used as negative controls inour immuno-histochemistry analyses.

According to Agyei and Runham, distinct cell types can be recog-nized in tick guts, stem cells, digest cells and secretory cells [23]. Tofollow in greater detail the surface restricted expression of BD86,we performed immuno-histochemistry of serial sections of twosessile digest cells using mAb 12.1 (Fig. 2B). Clearly, the proteinrecognized is confined to the surface area of the cells and coversmainly the luminal surface area. In summary, we have identifiedmAb 12.1 that was raised against a synthetic peptide derived fromthe BD86 amino acid sequence. MAb 12.1 binds to a single pro-tein in total tick gut protein lysates in Western blot analysis andin immuno-histochemical analyses; this protein is restricted to thesurface area of gut tissue sections.

3.2. BD86 expression during tick feeding of R. (B.) decoloratus

R. (B.) decoloratus larvae were applied on naive cattle and moni-tored for attachment. Beginning 8 days post infestation and every 3days thereafter, samples of ticks were manually detached from thehost animal and individual gut samples were dissected and fixed inBouin’s fixative. Feeding stages of female R. (B.) decoloratus wholetick gut sections were analyzed to define the level and cellular dis-tribution of BD86 expression. Whole tick gut sections of days 8, 11,

20, 21, 23 and 24 post application of larvae are shown in Fig. 2C. Asexpected, the tick gut lumen expanded considerably with feedingtime when days 8 and 24 were compared. In all stages analyzed,BD86 expression could be observed on nearly all visible cells liningthe tick gut displaying a strong surface confined staining pattern. In

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ur sections, BD86 expression was observed on the surface of ses-ile and detached digest cells mainly on the luminal side of theseells.

.3. Detection of BD86 homologues in R. (B.) microplus, R.ppendiculatus and H. a. anatolicum tick gut sections

Several investigators have reported cross-protection conferredy immunization with TickGardTM against additional tick species

ncluding R. (B.) decoloratus, R. (B.) microplus and H. a. anatolicum24,25]. This cross-protection is most likely explained by existencef homologues of the BM86 protein constituting the TickGardTM

accine that are recognized by vaccine induced cross-reactive anti-odies. Therefore, we analyzed mAb 12.1 for staining of foregutnd midgut preparations of female R. (B.) decoloratus (20 days fed),emale R. (B.) microplus (20 days fed), female R. appendiculatus adult4 days fed), female H. a. anatolicum adult (4 days fed) and female. variegatum (4 days fed) for potential cross-reactivity, expres-ion level and cellular distribution of BM86 homologues. Fig. 2Dummarizes results of these immuno-histochemical analyses withtrong indications that mAb 12.1 recognized homologues in R. (B.)icroplus, R. appendiculatus and H. a. anatolicum in addition toD86 (Fig. 2D, columns 2 and 3). Similar surface confined stain-

ng signals were obtained with all tick species analyzed except. variegatum, which remained negative in all sections analyzed.omparison of foregut and midgut staining in H. a. anatolicum and. appendiculatus showed a tendency for stronger signals to origi-ate in the foreguts (Fig. 2D, columns 2 and 3). In R. (B.) decoloratusnd R. (B.) microplus these differences in BD86 homologue expres-ion between foreguts and midguts could not be detected by thisechnique. A general tendency of more cells staining positive in. (B.) decoloratus when compared with R. (B.) microplus tick gutissue was observed (Fig. 2D). In column 1 of Fig. 2D, stainingesults with isotype matched, non-tick gut binding mAb 2.3 arehown.

Amino acid sequences of BM86 homologues from six tick speciesncompassing the amino acid sequence stretch used to raise mAb2.1 were aligned to gain insight into potential conserved primarymino acid sequence binding sites (Fig. 3). A stretch of eight aminocids (KLQACEHP) was 100% conserved between the sequencesligned suggesting that it constitutes at least part of the bindingite of mAb 12.1.

. Discussion

Several tick species co-exist and possibly co-feed on cattle infrican countries. A detailed study carried out in Southern Sudanhowed that 4 tick genera and 11 species were identified in totalncluding A. variegatum, H. a. anatolicum, R. (B.) decoloratus and R.ppendiculatus [26,27]. Our studies aim to develop novel tick con-rol measures that provide broad cross-protection against a rangef Ixodid tick species co-infesting cattle [28].

A number of groups have reported on technical challenges toxpress recombinant the full length BM86 and its homologues inifferent tick species in bacterial, yeast or viral protein expressionystems [29,30] (Olds et al., unpublished observation). Seven EGF-ike domains are highly conserved in BM86 homologues across

range of several tick species suggesting that these domainsulfill crucial functions [8]. The reconstitution of such complexhree-dimensional structures in subunit vaccine development ishallenging but very likely to be essential for optimal induction of

ntibody responses targeting the naive protein after vaccination.ynthetic peptide-based vaccines do not consist of recombinantroteins but of peptides comprising a selected number of epi-opes presented in a conformation mimicking closely the nativerotein structure of the pathogen [31–33]. We hypothesize that

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immunization regimes using synthetic peptides would allow us tofocus humoral immune responses on functionally important pro-tein domains resulting in higher protection levels. Even thoughthe identification and use of synthetic peptides as vaccines tofocus the humoral immune response on neutralizing domains arestill in its infancy, several examples in recent years have demon-strated the successful induction of potentially protective antibodyresponses against HIV [34,35], malaria [36], cancer [37] and inallergy [38].

Two linear, synthetic peptide sequences of BD86 that were iden-tified as immuno-dominant in TickGardTM immunized cattle [17]were coupled onto the surface of IRIV and used to immunize mice.The BD86-3 synthetic peptide induced strong antibody responsesand a range of hybridoma cell lines were established from one ofthese animals. Two hybridoma cell lines, named 12.1 and 14.1,secreted antibodies recognizing recombinant BD86 (68 kDa) [17]and a single band of about 90 kDa in total tick gut lysates of R.(B.) decoloratus in Western blot analysis. The calculated molecu-lar weight of the sequenced fragment of BD86 is 68 kDa [17]. Thisapparent higher molecular weight of the protein detected in totaltick gut lysates could be explained by heavy N-linked glycosylationof BD86 as described for BM86 [9,13]. MAb 12.1 was used to followBD86 expression by immuno-histochemistry throughout tick feed-ing starting from day 8 to day 24 after tick application. In all tissuesections strong staining on the surface of cells lining the tick gutlumen could be found. The sub-cellular distribution of the stainingobserved is consistent with the reports of Willadsen, and Goughand Kemp, describing the expression of BM86 as being mainly con-fined to the surface of digest cells in the tick gut of R. (B.) microplus[8,12]. These analyses emphasized that antibodies binding to BD86can detect their target at least from at least day 8 post applicationup to final engorgement of the ticks.

Next, we compared staining of foregut and midgut sections ofA. variegatum, R. (B.) decoloratus, R. (B.) microplus, R. appendiculatusand H. a. anatolicum. All sections except tissue from A. variegatumticks displayed the characteristic surface confined staining pat-tern with mAb 12.1 described in R. (B.) microplus (Fig. 2D). Thelack of reactivity in A. variegatum is not surprising consideringthat the Amblyominae are phylogenetically very distinct from theother three genera [39]. Hence, mAb 12.1 detected BM86 homo-logues in four out of five different tick species analyzed. de Vos etal. tested TickGardTM for cross-protection against H. a. anatolicumdemonstrating a 50% reduction in total weight of nymphs engorg-ing on vaccinated calves [24] presumably indicating that vaccineinduced antibodies bind to HA86 and mediate cross-protectionagainst H. a. anatolicum. No significant cross-protection againstchallenge with R. appendiculatus nymphs has been described aftervaccination with TickGardTM [17,24]. The fact that we obtained across-reactive antibody binding to R. (B.) decoloratus and R. appen-diculatus gut tissue strongly suggests that by focusing the humoralimmune response to the epitope recognized by mAb 12.1 cross-protection could be expanded to R. appendiculatus. Final proof ofthis hypothesis will be addressed by immunization of cattle withthe BD86-3 synthetic peptide and challenge with R. appendiculatusticks.

Several factors in addition to amino acid sequence variationbetween BM86 homologues have been implicated in different out-comes of TickGardTM based tick challenge experiments. Theseincluded localization and expression level of BM86, quantity andquality of the blood meals determining the amount of anti-BM86antibody uptake per tick and environmental factors [8,40,41].

Interestingly, when midgut sections of R. (B.) decoloratus andR. (B.) microplus were compared, we found a tendency towardsstronger signals and higher numbers of BD86 expressing cellsin R. (B.) decoloratus. This observation could be explained by (i)higher affinity of mAb 12.1 to the BD86 or (ii) differences in the

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xpression level of BD86 homologues between these tick species.f differences in BD86 homologues expression levels exist theyould directly translate into vaccine efficacy against different tickpecies.

Well defined synthetic peptides have the potential to mimiche structure of native proteins and in many cases this has beenhown to constitute an important feature for a synthetic peptide toehave as a suitable immunogen [33,42]. Conformational restric-ion of a peptide may induce higher binding affinities of antibodiesnd a greater degree of specificity in peptide–receptor interaction31,33,43].

Only antibodies binding native BD86 protein confer immuneffector mechanisms against ticks. The BD86-3 synthetic peptideepresents a structure that needs further development to be anptimal synthetic peptide since mAbs 2.3 and 14.1 recognizedecombinant BD86 in ELISA and Western blot analysis but did notind native BD86. As only antibodies binding to the native BD86rotein confer immune effector mechanisms against ticks we areurrently comparing the binding of mAbs 12.1 and 2.3 to randomeptide phage display libraries to define better the sequences tohich they bind and the concomitant conformational requirements

35]. Taken together, the availability of the broadly reactive mAb2.1 generated in this study should enhance biological investiga-ions into the biological function of BD86 and its homologues int least four tick species. Functional assays including in vitro tickeeding experiments to assess the threshold concentration of anti-odies required to affect a substantial number of ticks feeding are

n progress [44].

cknowledgments

We would like to thank Marco Tamborrini for help in perform-ng ELISA on polyclonal mouse sera. This project received financialupport from the Research Fellow Partnership Programme for Agri-ulture, Forestry and Natural Resources (RFPP) of the North-Southentre, Zürich, Switzerland. ILRI publication number is IL-200906.

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