Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum

Vaccine 28 (2010) 1786–1795

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Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum José de la Fuente a,b,∗ , Raúl Manzano-Roman a , Victoria Naranjo a,b , Katherine M. Kocan a , Zorica Zivkovic c , Edmour F. Blouin a , Mario Canales b , Consuelo Almazán d , Ruth C. Galindo b , Douglas L. Step e , Margarita Villar b a

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain c Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584CL Utrecht, The Netherlands d Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Tamaulipas, Km. 5 carretera Victoria-Mante, CP 87000 Cd. Victoria, Tamaulipas, Mexico e Veterinary Clinical Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA b

a r t i c l e

i n f o

Article history: Received 30 September 2009 Received in revised form 1 December 2009 Accepted 2 December 2009 Available online 16 December 2009 Keywords: Amblyomma americanum Lone star tick Tick Vaccine RNA interference

a b s t r a c t The lone star tick, Amblyomma americanum, vectors pathogens of emerging diseases of humans and animals in the United States. Currently, measures are not available for effective control of A. americanum infestations. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, the limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, we report the application of RNA interference (RNAi) for screening an A. americanum cDNA library for discovery of tick protective antigens that reduce tick survival and weights after feeding. Four cDNA clones, encoding for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7), were selected for vaccine studies in cattle, along with subolesin, a tick protective protein identified previously. In vaccinated cattle, an overall efficacy (E) > 30% was obtained when considering the vaccine effect on both nymphs and adults, but only 2D10, 2G7 and subolesin affected both tick stages. The highest efficacy of control for adult ticks (E > 55%) was obtained in cattle vaccinated with recombinant 2G7 or subolesin. These collective results demonstrated the feasibility of developing vaccines for the control of lone star tick infestations. The use of RNAi for identification of tick protective antigens proved to be a rapid and cost-effective tool for discovery of candidate vaccine antigens, and this approach could likely be applied to other parasites of veterinary and medical importance. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important arthropod vector of pathogens in North America [1]. Populations of the lone star tick, Amblyomma americanum (Linnaeus), previously considered to be localized in the southeastern and Gulf Coast regions of the USA, have rapidly increased in numbers and range, and now extend into the northeastern USA [2–9]. A. americanum is rapidly becoming one of the most common ticks within its host range found

∗ Corresponding author at: Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, 250 McElroy Hall, Oklahoma State University, Stillwater, OK 74078, USA. Tel.: +1 405 744 0372. E-mail addresses: jose [email protected], jose.de la [email protected], [email protected] (J. de la Fuente). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.12.007

on dogs, white-tailed deer and cattle [8,10,11]. Parasitized animals often have heavy tick burdens and the clustering of ticks at feeding sites causes large skin lesions that are susceptible to secondary bacterial infection. Historically, A. americanum was considered to be a relatively insignificant vector of human disease. In the past two decades, public health interest in A. americanum has increased and this tick has become recognized as an important component of the natural history and zoonotic transmission of several recently emerging pathogens, including those causing human monocytic ehrlichiosis, granulocytic ehrlichiosis, southern tick associated rash illness (STARI) and spotted fevers associated with Rickettsia spp. [8,12]. Currently, control measures are not available for effective control of A. americanum infestations. Acaricides application constitutes a major component of integrated tick control strategies [13,14]. However, use of acaricides has had limited efficacy in reducing tick infestations and is often accompanied by serious

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Fig. 1. Procedure for the screening of the A. americanum cDNA library by RNAi and results of positive clones selected for further analysis.

drawbacks such as the selection of acaricide-resistant ticks, contamination of the environment and presence of drug residues in milk and meat product [13]. All of these issues reinforce the need for alternative approaches for control of tick infestations, such as the use of hosts with natural resistance, pheromone-impregnated decoys that both attract and kill ticks, biological control agents and vaccines [15–17]. In the early 1990s, vaccines inducing immunological protection against cattle tick Rhipicephalus (Boophilus) spp. infestations were developed [15,17]. These vaccines reduce tick numbers, weight and reproductive capacity that in turn, results in control of tick infestations in subsequent generations. Vaccine trials using Bm86-based vaccines demonstrated that tick control by vaccination has the advantages of being cost-effective, reducing environmental contamination, preventing the selection of drug resistant ticks that result from repeated acaricide application and reducing the transmission of some tick-borne pathogens [18]. Recently, new tick protective antigens have been discovered and characterized [15,17,19]. However, the only tick vaccines currently marketed are those containing Bm86 and Bm95 antigens [18]. Despite recent efforts to develop methods for rapid screening of tick cDNA libraries for identification of new antigens [20], the main obstacle for the development of improved tick vaccines remains the discovery of protective antigens. RNA interference (RNAi) was proposed as a method for screening for tick protective antigens [21] and has been used for the characterization of tick genes with potential applications in vaccine development [22]. This study is the first report of RNAi for screening tick cDNAs for the identification of protective antigens. The results demonstrated the possibility of using RNAi for discovery of tick protective antigens for the development of vaccines against A. americanum infestations. This approach will likely be applicable for identification of candidate antigens for vaccine development for other parasites of veterinary and medical importance. 2. Materials and methods 2.1. Ticks For cDNA library construction, gene expression studies, RNAi and vaccination experiments, A. americanum ticks were obtained

from the laboratory colony maintained at the Oklahoma State University Tick Rearing Facility. Larvae and nymphs were fed on rabbits and adult ticks were fed on sheep. Animals were housed at the Tick Rearing Laboratory with the approval and supervision of the OSU Institutional Animal Care and Use Committee. Off-host ticks were maintained in a 12 h light:12 h dark photoperiod at 22–25 ◦ C and 95% relative humidity. The A. americanum strains Athens, GA (field collected by S. Little); Barnsfall, OK (field collected by H. Gann) and Springfield, MO (field collected by J. Kocan) and other Amblyomma species, A. cajenense, Mexico (provided by C. Almazán from a laboratory colony maintained at the University of Tamaulipas, Mexico); A. maculatum, OK (obtained from a laboratory colony maintained at OSU) and A. hebraeum (kindly provided by R. Kaufmann from a laboratory colony maintained at the Department of Biological Sciences, University of Alberta, Canada), were used for gene polymorphism analysis. 2.2. Construction and screening by RNAi of an A. americanum cDNA library Total RNA was extracted from 375 whole unfed adult ticks (150 males and 225 females) using the RNAeasy kit (Qiagen, Valencia, CA, USA). The cDNA library was synthesized using the CloneMiner cDNA Library Construction Kit in the vector pDONR222 (Invitrogen, Carlsbad, CA, USA). The library had >90% recombinants with inserts >250 bp. Oligonucleotide primers (pDONT75: 5 -TAATACGACTCACTATAGGGTACTCAACAAATTGATGAGCAATGC- 3 and pDONT73: 5 -TAATACGACTCACTATAGGGTACTGATAAGCAATGCTTTCTTATA-3 ) were synthesized specific for vector DNA sequences flanking the tick cDNA insert and containing T7 promoter sequences (in italics) for in vitro transcription and synthesis of dsRNA. PCR reactions were performed from individual cDNA clones in 96-well plates using the Access RT-PCR system (Promega, Madison, WI, USA) in a 50-␮l reaction mixture. The resultant amplicons were purified using the Wizard 96-well PCR purification system (Promega). Screening of the A. americanum cDNA library was conducted by RNAi according to the methods of de la Fuente et al. [21], and the experimental design is described in Fig. 1. For the primary screen, 384 cDNA clones were screened in 8 pools of 48 cDNA clones each.

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Pools contained 1 ␮l from each PCR were precipitated with ethanol and used as templates for dsRNA synthesis. Two pools were then selected for the secondary screen. From these two pools containing 96 cDNA clones, 10 sub-pools of 8 clones each were made and analyzed. From the secondary screen, six sub-pools were selected for the final (tertiary) screen of 48 individual clones. In vitro transcription and purification of dsRNA was done using the Megascript RNAi kit (Ambion, Austin, TX, USA). The dsRNA was quantified by spectrometry. Ticks were injected with approximately 0.5 ␮l of dsRNA (5 × 109 –5 × 1011 molecules per ␮l) in the lower right quadrant of the ventral surface of the exoskeleton of female ticks. The injections were done with a Hamilton syringe with a 1-in., 33 gauge needle. Control ticks were injected with vector DNA, injection buffer (10 mM Tris–HCl, pH 7, 1 mM EDTA) or subolesin dsRNA (positive control; [23]). Twenty female ticks were used in each group. After injection with dsRNA, the female ticks were held in a humidity chamber for 1 day after which they were allowed to feed on cattle with 10 male ticks. Unattached ticks were removed 2 days after infestation. Females that fed to repletion or those that were removed from the cattle after 10 days of feeding were collected, counted to evaluate mortality and weighed to evaluate repletion. The engorgement weights (average ± S.D.) were compared by Analysis of Variance (ANOVA) followed by a series of Tukey’s post-hoc tests for pair comparisons between vector-injected and subolesin dsRNA-injected controls and test dsRNA-injected ticks. Protective pools (primary screen), sub-pools (secondary screen) and individual clones (tertiary screen) were selected based on lower tick weights and/or higher mortality as compared with the subolesin dsRNA-injected and vector-injected control ticks. 2.3. Sequence analysis The DNA from the 48 individual cDNA clones analyzed in the tertiary screen was purified using Wizard SV 96 plasmid DNA purification system (Promega) and partially sequenced with a 5 vector-specific primer (5 -CAACAAATTGATGAGCAATGC-3 ) at the Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University, using ABI Prism dye terminator cycle sequencing protocols developed by Applied Biosystems (PerkinElmer Corp., Foster City, CA, USA). In most cases a sequence larger than 500 nucleotides was obtained. The cDNA Annotation System software (CAS; Bioinformatics and Scientific IT Program (BSIP), Office of Technology Information Systems (OTIS), National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA) (http://exon.niaid.nih.gov) was used for automated sequence clean up, blasting against multiple sequence databases (ncbi non-redundant sequence database and databases of tickspecific sequences http://www.ncbi.nlm.nih.gov/; http://www. vectorbase.org/index.php) and gene ontology (GO) assignments. Nucleotide sequences were aligned using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD, USA). Gene sequences were deposited in the GenBank with accession numbers GT091165–GT091208. 2.4. Gene expression and polymorphism analysis by reverse transcriptase (RT)-PCR Total RNA was isolated from A. americanum (OSU colony) eggs (three batches of approximately 3000 eggs each), larvae (three pools of 100 larvae each), nymphs (three pools of 100 nymphs each) and unfed and fed adult male and female salivary glands and guts (10 individual ticks each) using TriReagent (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions. Two primers were designed based on the sequences determined for 2C9, 2D10, 2B7 and 2G7 genes using

the optimized primer sequences designed with Primer3 (http://fokker.wi.mit.edu/primer3/input.htm) for real-time RTPCR analysis of mRNA levels in A. americanum (OSU colony) tissues and developmental stages. Real-time RT-PCR was done using the QuantiTec SYBR Green RT-PCR kit (Qiagen, Valencia, CA, USA) and a Bio-Rad iQ5 thermal cycler (Hercules, CA, USA) following manufacturer’s recommendations. mRNA levels were normalized against tick beta-actin (Genbank accession number DT044040) using the comparative Ct method [24]. mRNA levels were compared between different samples using the Student’s t-test (P = 0.05). For gene polymorphism analysis in other A. americanum strains and in other Amblyomma species, total RNA was isolated from whole unfed adults of A. americanum strains (Athens, Barnsfall and Springfield) and from A. cajenense, A. maculatum and A. hebraeum as described previously. Oligonucleotide primers were synthesized homologous to the A. americanum (OSU colony) sequences and used for RT-PCR using the Access RT-PCR system (Promega). Amplified fragments were resin purified (Wizard, Promega) and cloned into the pGEM-T vector (Promega) for sequencing both strands. At least two independent clones were sequenced for each PCR. The sequences of 2C9, 2D10, 2B7 and 2G7 genes obtained from the other A. americanum strains and Amblyomma species were deposited in the GenBank with accession numbers GU014517–GU014525. 2.5. Expression, purification and formulation of recombinant proteins The A. americanum 2C9, 2D10, 2B7 and 2G7 cDNA fragments and subolesin (DQ159962) were amplified by PCR using oligonucleotide primers (5 –3 ) 2C9QEG5: GGCC ATG GTG GCA GCC AAG ACC and 2C9QEG3: GGAGATCT TTA TAG GCT GAC GAA TTC TTG, 2D10QEG5: GGCC ATG GGT GGG TTT TCA CGG and 2D10QEG3: GGAGATCT TTA GTA GCC GTA GCT CTT GAT, 2B7QEG5: GGCC ATG GCG TGT CTG CCG CCG and 2B7QEG3: GGAGATCT TTA TGC TCG CTT TCC CCA AAG, 2G7QEG5: GGCC ATG GTC CGT GTT ATG TCA and 2G7QEG3: GGAGATCT TTA CAG GAC AAN TCT GCC GTC, 4D8QEG5: GGCC ATG GCT TGC GCA ACA TTA and 4D8QEG3: GGAGATCT TTA AAT AAT TTG GTC GTA CGT, respectively, and the Access RT-PCR system (Promega). The PCR primers introduced NcoI and BglII restriction sites for cloning into the expression vector pQE-60 fused to a 6×Histag (Qiagen). Recombinant proteins were expressed in Escherichia coli JM109 as described previously [25]. For affinity purification of recombinant proteins, Ni-NTA spin columns (Qiagen) were used following manufacturer’s protocol. The eluted proteins were subjected to 10–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using the HisDetector Western Blot Kit HRP (KPL, Gaithersburg, MD, USA). Vaccine doses were prepared by mixing the recombinant protein and adjuvant, anhydromannitoletheroctodecenoate (Montanide ISA 50V; Seppic, Paris, France) in a batch-by-batch process using a high-speed mixer Heidolph DIAX 900 (Heidolph Elektro, Kelheim, Germany) at 8000 rpm. Twenty ml of vaccine was filled manually into glass bottles (Wheaton, Millville, NJ, USA) under sterile conditions at a concentration of 100 ␮g/2 ml dose. Quality control was done by testing the mechanical and thermal stability of vaccine emulsions as described by Canales et al. [26]. 2.6. Immunization of cattle and infestation with A. americanum nymphs and adults Cattle were randomly assigned to experimental groups. Three crossbred calves per group (18, total) were each immunized with 3 doses (weeks 0, 4 and 6) containing 100 ␮g/dose of purified recombinant proteins formulated as described previously. Negative controls were injected with adjuvant/saline alone. Cattle were

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injected subcutaneously with 2 ml/dose using a 5-ml syringe and an 18G needle. Two weeks after the last immunization, cattle in vaccinated and control groups were each infested with 200 adult male/female pairs and 500 nymphs applied in separate cotton cells glued to the side of the animals. Cattle were maintained in accordance with regulations of the Guide for Care and Use of Laboratory Animals and with the approval and supervision of the OSU Institutional Animal Care and Use Committee. Unattached adult female ticks were removed 2 days after infestation. Engorged nymphs were collected from days 4 to 7 after infestation. Adult female ticks dropping from cattle were daily collected during 12 days, counted and weighted. All the collected nymphs were assessed for molting and adult female ticks were assessed for oviposition and egg fertility [27–31]. The personnel collecting the ticks were ‘blinded’ to the identity of the experimental groups. The effect of the vaccine on adult and nymphal ticks was determined as follows [27,28]: the effect on the number of adult female ticks (DT) = 100[l − (NTV/NTC)], where NTV is the number of adult female ticks in the vaccinated group and NTC is the number of adult female ticks in the control group; the effect on tick weight (DW) = 100[1 − (WTV/WTC)], where WTV is the average adult female tick weight in the vaccinated group and WTC is the average adult female tick weight in the control group; the effect on oviposition (DO) = 100[1 − (PATV/PATC)], where PATV is the average weight of the eggs per survived tick in the vaccinated group and PATC is the average weight of the eggs per survived tick in the control group; the effect on egg fertility (DF) = 100[1 − (PPLOV/PPLOC)], where PPLOV is the average weight of the larvae per mg of eggs in the vaccinated group and PPLOC is the average weight of the larvae per mg of eggs in the control group; and the effect on nymph molting (DM) = 100[1 − (MV/MC)], where MV is the average number of adult ticks obtained in the vaccinated group and NTC is the average number of adult ticks obtained in the control group. For adult ticks, vaccine efficacy (E) was calculated as 100[l − (CRT × CR0 × CRF)], where CRT = NTV/NTC, CR0 = PATV/PATC and CRF = PPLOV/PPLOC that represent the reduction in the number of adult female ticks, oviposition and egg fertility as compared with the control group, respectively. For nymphs, E was calculated as 100[l − (CRT × CRM)], where CRT and CRM are the reduction in the number of nymphs and molting as compared to the control group, respectively. Only values with statistical significance were use to calculate E. The overall vaccine E was calculated considering E values for both tick stages. A Student’s t-test with unequal variance (P = 0.05) was used to compare the results of tick weight, oviposition, molting and egg fertility between vaccinated and control groups. Tick numbers were compared between vaccinated and control groups by Chi2 -test as implemented in Mstat 4.01 (Chi < 0.01). 2.7. Characterization of the antibody response in immunized cattle by ELISA Serum samples were collected from each animal before each immunization, at 2 weeks after the last immunization (just before tick infestation) and after completion of tick feeding. Blood samples were collected from each calf using 10 ml syringes with an 18-gauge needle, dispensed into sterile tubes and stored at 4 ◦ C. The serum was then separated by centrifugation and stored at −20 ◦ C. Serum antibody titers were determined using an antigen-specific indirect ELISA. Purified recombinant antigens (0.1 ␮g/well) were used to coat ELISA plates overnight at 4 ◦ C. Sera were serially diluted to 1:10, 1:100 and 1:1000 in PBST (PBS/0.5% Tween 20, pH 7.2) and 10% fetal bovine serum (Sigma). The plates were incubated with the diluted sera for 1 h at 37 ◦ C and then incubated with 1:10,000 rabbit anti-bovine IgG–HRP conjugates (Sigma) for 1 h at 37 ◦ C. The color reaction was developed with 3,3 ,5,5 -tetramethylbenzidine

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Table 1 Results of the primary and secondary screen by RNAi of A. americanum cDNA library. Group Primary screen 1 2 3 4 5 6 7 8 Saline Vector Subolesin Secondary screen 1 2 3 4 5 6 7 8 9 10 Saline Vector Subolesin

Tick mortality (%)

Tick weight (mg)

71 94 64 100 94 0 100 50 0 0 41

27 ± 1* Only one tick survived 13 ± 1* All ticks died Only one tick survived 9 ± 0* All ticks died 226 ± 10 559 ± 79 617 ± 91 9 ± 0*

58 45 78 78 41 21 0 47 94 78 0 0 50

7 ± 1*, ** 30 ± 25* 8 ± 2*, ** 7 ± 2*, ** 8 ± 2*, ** 22 ± 15* 173 ± 150* 17 ± 5* Only one tick survived 17 ± 5*, ** 475 ± 193 496 ± 186 29 ± 5*

The screening of the A. americanum cDNA library was conducted by RNAi in 20 female ticks per group using the procedure described in Fig. 1. The engorgement weights (average ± S.D.) were compared by ANOVA followed by a series of Tukey’s post-hoc tests for pair comparisons between test dsRNA-injected ticks and vector-injected (*P < 0.001) and subolesin dsRNA-injected controls (**P < 0.05 for values lower than that in subolesin dsRNA-injected ticks). Protective pools (primary screen) and subpools (secondary screen) were selected based on a lower tick weight and/or higher mortality when compared to subolesin dsRNA-injected and vector-injected control ticks. The two pools from the primary screen and the 6 sub-pools from the secondary screen selected for further screening are shown in bold-italic letters.

(Sigma) and the OD450nm was determined. After incubation the plates were washed with PBST. Antibody titers were considered positive when they yielded an OD450nm value at least twice as high as the pre-immune serum. Antibody titers in immunized cattle were expressed as the OD450nm value for the highest serum dilution (1:1000) and compared between vaccinated and control cattle using an ANOVA test (P = 0.05). 3. Results 3.1. Screening of the A. americanum cDNA library by RNAi The screening of the A. americanum cDNA library by RNAi is described in Fig. 1. A total of 384 cDNA clones were analyzed in the first screen in 8 pools of 48 clones each. Two pools (96 clones) were selected based on 100% tick mortality (Table 1). Of these 96 clones, 80 were analyzed in the secondary screen in sub-pools of 8 clones each. Six sub-pools (48 clones) were selected with >40% tick mortality and significantly lower tick weights as compared with vector and subolesin dsRNA-injected ticks (Table 1). These 48 clones were selected for sequence analysis and for the tertiary screen of individual clones (Table 2). After the tertiary screen, 7 cDNA clones were selected for re-screening (Table 2). The rescreening resulted in 4 clones with an average tick weight <9 mg and/or higher mortality when compared with the subolesin dsRNAinjected and vector-injected control ticks that were selected for further studies (Table 3). These cDNA clones encoded for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7).

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Table 2 Results of the tertiary screen by RNAi of A. americanum cDNA library. Clone (sub-pool)

Sequence identity (Genbank accession No.)

E-value

2A7 (1) 2B7 (1) 2C7 (1) 2D7 (1) 2E7 (1) 2F7 (1) 2G7 (1) 2H7 (1) 2A9 (3) 2B9 (3) 2C9 (3) 2D9 (3) 2E9 (3) 2F9 (3) 2G9 (3) 2H9 (3) 2A10 (4) 2B10 (4) 2C10 (4) 2D10 (4) 2E10 (4) 2F10 (4) 2G10 (4) 2H10 (4) 2A11 (5) 2B11 (5) 2C11 (5) 2D11 (5) 2E11 (5) 2F11 (5) 2G11 (5) 2H11 (5) 4A5 (9) 4B5 (9) 4C5 (9) 4D5 (9) 4E5 (9) 4F5 (9) 4G5 (9) 4H5 (9) 4A6 (10) 4B6 (10) 4C6 (10) 4D6 (10) 4E6 (10) 4F6 (10) 4G6 (10) 4H6 (10) Saline Vector Subolesin

A. americanum 5.8S, ITS2 and 28S rRNA (AF548532) Sphaerius sp. 60S ribosomal protein L13e (CAJ17270) A. americanum mt cytochrome oxidase subunit 1 (cox1) (DQ168131) Similar to 4C6 Vector sequences Unknown G. gallus interfase cytoplasmic foci protein 45 (NP 001025787) Similar to 2B7 I. scapularis 60S ribosomal protein L9 (DQ066193) A. variegatum myosin alkali light chain (TC310) H. sapiens threonyl-tRNA synthetase (BAD96524) S. purpuratus gp330 precursor (LDL receptor) (XP 794505) G. gallus translation initiation factor 4 gamma, 2 (eIF4G) (NP 001036814) I. scapularis ISUFL14 ADP/ATP translocase (DQ066215) E. ocellatus C-type lectin (FM177949) Vector sequences Similar to 4C6 G. gallus similar to CDNA sequence BC021608 (XP 417895) Unknown A. melifera 60S ribosomal protein L13a (XP 623813) Vector sequences Similar to 2F9 R. (Boophilus) microplus Oligopeptide transport ATP-binding protein (TC1891) A. americanum NADH dehydrogenase subunit 1 (DQ168139) Unknown Vector sequences Similar to 2D10 A. americanum mt 12S rRNA (AF150050) Unknown Similar to 4C6 Unknown Unknown I. scapularis DNAJA5 (ribosome-associated chaperone) (XM 002414981) A. americanum mitochondrial mRNA (DQ168139) Unknown Unknown D. variabilis 40S ribosomal protein S5 Unknown Unknown D. persimilis isocitrate dehydrogenase (XM 002026272) Similar to 4C6 A. mellifera thyroid hormone receptor-associated protein 5 (XP 623610) A. americanum mt 16S rRNA (DQ168139) Unknown Unknown Similar to 4D6 Unknown R. (Boophilus) microplus (TC187) – – A. americanum (DQ159962)

1E+00 3E−68 1E+00 – – – 4E-39 – 6E−115 8E−70 3E−93 5E−05 1E−31 1E+00 3E−13 – – 5E−32 – 2E−30 – – 9E−36 6E−100 – – – 5E−140 – – – – 1E−74 1E+00 – – 5E−110 – – 4E−80 – 1E−18 8E−146 – – – – 5E−22 – – 1E+00

Tick mortality (%) 89 83 72 13 0 0 45 60 31 39 61 0 0 0 0 0 0 0 0 100 0 62 0 10 0 0 75 100 0 0 0 5 15 79 80 15 10 95 0 25 0 0 0 0 37 0 5 10 0 0 62

Tick weight (mg) 1 ± 0*, ** 3 ± 2*, ** 17 ± 10* 370 ± 233 302 ± 277 202 ± 171 6 ± 4* 67 ± 55* 7 ± 1* 7 ± 1* 6 ± 2* 90 ± 61* 52 ± 22* 61 ± 17* 8 ± 2* 403 ± 242 193 ± 58 378 ± 135 298 ± 141 All ticks died 502 ± 201 23 ± 7* 274 ± 201 516 ± 197 532 ± 114 548 ± 167 35 ± 32* All ticks died 314 ± 221 528 ± 122 590 ± 177 575 ± 111 125 ± 112* 65 ± 19* 156 ± 150* 468 ± 172 557 ± 172 Only one tick survived 559 ± 157 571 ± 275 512 ± 147 414 ± 114 411 ± 125 44 ± 35* 16 ± 9* 81 ± 11* 514 ± 105 521 ± 87 393 ± 173 498 ± 137 17 ± 10*

The screening of the A. americanum cDNA library was conducted by RNAi in 20 female ticks per group using the procedure described in Fig. 1. The engorgement weights (average ± S.D.) were compared by ANOVA followed by a series of Tukey’s post-hoc tests for pair comparisons between test dsRNA-injected ticks and vector-injected (*P < 0.001) and subolesin dsRNA-injected controls (**P < 0.05 for values lower than that in subolesin dsRNA-injected ticks). Protective clones were selected based on an average tick weight <7 and/or higher mortality when compared to subolesin dsRNA-injected and vector-injected control ticks. The 7 clones selected for further characterization are shown in bold-italic letters.

3.2. Expression profile and sequence polymorphism of selected 2G7, 2B7, 2C9 and 2D10 genes The four selected genes (2G7, 2B7, 2C9 and 2D10) were found to be expressed in all tick developmental stages (Figs. 2 and 3). In tick immature stages, gene expression levels increased as tick development proceeded from eggs to nymphs (P < 0.01; Fig. 2). Expression levels for all analyzed genes in male ticks were higher in guts than in salivary glands (P < 0.01; Figs. 3A–D). However, in female ticks only 2B7 levels were higher in the guts than in salivary glands (P < 0.01; Fig. 3B). For the rest of the genes, expression levels in female ticks were similar in guts and salivary glands (Fig. 3A,C,D). Changes in gene expression levels were observed after tick feeding in salivary glands and guts of both male and female ticks (Fig. 4). For 2D10, 2G7 and 2B7 genes, tick feeding downregulated the expression in male

salivary glands while expression in other samples was upregulated or remained unchanged after feeding (Fig. 4). For 2C9, tick feeding resulted in gene downregulation in salivary glands and guts of both male and female ticks (Fig. 4). For the analysis of gene polymorphism, PCR was used to amplify ortholog sequences from A. americanum strains and other Amblyomma species (Supplementary Fig. 1). 2B7 sequences were amplified from A. americanum strains only and had a 99% identity. 2C9 sequences were amplified from A. americanum strains and from A. cajennense and A. hebraeum with 93–98% identity. The 2D10 PCR produced amplicons for A. hebraeum and A. maculatum but not for other A. americanum strains and these sequences had 93–94% identity. Finally, 2G7 sequences could not be amplified from other A. americanum strains or Amblyomma species. However, a sequence with a fragment of 326 bp showing a 72% identity to A. americanum

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(OSU strain) 2G7 was found in the newly released Ixodes scapularis genome (Genbank accession number DS649308). 3.3. Control of A. americanum tick infestations after immunization with recombinant proteins

Fig. 2. Gene expression analysis in A. americanum immature tick stages using real-time RT-PCR. Total RNA was purified and analyzed from eggs (three batches of approximately 3000 eggs each), larvae (three pools of 100 larvae each) and nymphs (three pools of 100 nymphs each). Amplification efficiencies were normalized against tick beta-actin (Genbank accession number DT044040) using the comparative Ct method. Bars show normalized average ± S.D. mRNA levels. mRNA levels were compared for each gene between different samples by Student’s t-test and were significantly different (P < 0.01).

The cDNA clones selected after the re-screening (2C9, 2D10, 2B7, 2G7) and A. americanum subolesin were expressed and purified from recombinant E. coli (Fig. 5). To evaluate the protective capacity of recombinant antigens against A. americanum nymphs and adult infestations, antibody responses of cattle vaccinated with the recombinant proteins were compared to cattle that received the adjuvant/saline control. The vaccinated animals, but not the controls, developed antibodies against recombinant proteins (Fig. 6). Differences were observed between recombinant antigens in the antibody response, suggesting that in cattle 2D10 and 2B7 were more immunogenic than the other antigens. Nonetheless, antibody titers were significantly higher for all antigens after each immunization in vaccinated cattle as compared with the controls (Fig. 6). An average of 87% of the nymphs was recovered after engorgement in control cattle (Table 4). However, a significant reduction in nymph infestations was obtained in groups immunized with 2G7 (33% reduction) and subolesin (12% reduction) when compared with control animals (Table 4). Although nymphs ingest smaller amounts of blood, a significant reduction of 10% in engorged nymph weight was obtained in ticks collected from groups immunized

Fig. 3. Gene expression analysis in A. americanum female and male ticks using real-time RT-PCR. Total RNA was purified and analyzed from unfed adult male and female salivary glands and guts (10 individual ticks each). Amplification efficiencies were normalized against tick beta-actin (Genbank accession number DT044040) using the comparative Ct method. Bars show normalized average ± S.D. mRNA levels. mRNA levels were compared for each gene between guts and salivary glands by Student’s t-test (*P < 0.01).

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Table 3 Re-screening by RNAi of selected A. americanum cDNA clones. Clone

Putative function

Expression silencing (%)

Tick mortality (%)

Tick weight (mg)

2A7 2B7 2G7 2C9 2D10 2D11 4F5 Subolesin Vector

5.8S, ITS2 and 28S rRNA 2B7 60S ribosomal protein L13e Interphase cytoplasm foci protein 45 Threonyl-tRNA synthetase 60S ribosomal protein L13a Mitochondrial 12S rRNA Unknown Regulatory factor –

80 ± 21 84 ± 14* 90 ± 14* 82 ± 11* 81 ± 19* 87 ± 51 ND 95 ± 3* –

45 85 50 65 100 45 80 85 0

10 ± 3** 3 ± 2**, *** 8 ± 3** 4 ± 1**, *** All ticks died 9 ± 5** 11 ± 2** 18. ± 9** 395 ± 148

Twenty female ticks were injected per group. The mRNA levels were determined by real-time RT-PCR in 5 ticks collected 4 days after dsRNA injection. Gene expression silencing was calculated by comparing the normalized Ct values between dsRNA- and vector-injected ticks (*P < 0.05; Student’s t-test). The engorgement weights (average ± S.D.) were determined after 10 days of feeding and compared by Student’s t-test between test dsRNA-injected ticks and vector-injected (**P < 0.001) and subolesin dsRNA-injected controls (***P < 0.05 for values lower than that in subolesin dsRNA-injected ticks). Clones for vaccination experiments were selected based on an average tick weight <9 and/or higher mortality when compared to subolesin dsRNA-injected and vector-injected control ticks. The 4 clones selected for further characterization are shown in bold-italic letters.

Fig. 4. Gene expression analysis in unfed and fed A. americanum female and male ticks using real-time RT-PCR. Total RNA was purified and analyzed from unfed and fed adult male and female salivary glands and guts (10 individual ticks each). Results were recorded as the ratio of the average of fed to unfed ticks after normalization for tick beta-actin (Genbank accession number DT044040) using the comparative Ct method.

with 2D10 and 2G7 antigens when compared to controls (Table 4). The molting of nymphs to adult ticks was reduced by 2–9% as compared with controls, with significant reduction in groups immunized with 2D10 (5% reduction) and 2G7 (9% reduction) (Table 4). Considering the significant effect of vaccination on nymph infestations and molting, the vaccine efficacy on nymphs was calculated for 2D10 (5% efficacy), 2G7 (93%) and subolesin (12%) only (Table 4). The engorgement of adult female ticks produced three different patterns in vaccinated cattle (Supplementary Fig. 2). The groups immunized with 2B7 and 2D10 showed a pattern similar to con-

trols with most replete ticks dropping on day 4. In contrast, the highest percentage of replete ticks was obtained on day 3 in groups immunized with 2C9 and 2G7. Finally, a delay was observed in the subolesin-immunized cattle in tick engorgement with the high-

Fig. 5. Expression and purification of recombinant proteins in E. coli. His-tag recombinant subolesin (Sub), threonyl-tRNA synthetase (2C9), ribosomal protein L13a (2D10), interphase cytoplasm foci protein 45 (2G7) and ribosomal protein L13e (2B7) were expressed in E. coli and purified by affinity to Ni. E. coli cells transformed with the expression vector alone were used as control. The eluted proteins (10 ␮g) were subjected to 10–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using the HisDetector Western Blot Kit HRP (KPL, Gaithersburg, MD, USA). The position of recombinant proteins is indicated with arrows. Abbreviation: MW, molecular weight marker.

Fig. 6. Antibody response in vaccinated cattle. Bovine serum antibody titers to recombinant proteins were determined by ELISA in cattle vaccinated with subolesin, threonyl-tRNA synthetase (2C9), ribosomal protein L13a (2D10), interphase cytoplasm foci protein 45 (2G7) and ribosomal protein L13e (2B7) and adjuvant/saline controls. Antibody titers in immunized cattle were expressed as the OD450nm value for the highest serum dilution (1:1000) and compared between vaccinated and control cattle using an ANOVA test. For all antigens antibody titers were significantly higher after each immunization in vaccinated cattle when compared to controls (P < 0.05). The time of Immunizations and tick infestation are indicated with arrows.

J. de la Fuente et al. / Vaccine 28 (2010) 1786–1795

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Table 4 Control of A. americanum nymphs in cattle vaccinated with the recombinant antigen preparations. Experimental groupa

Amblyomma americanum nymphs Percent reduction (vaccinated/control)b

Ec

DT

DW

DM

No. 1 Threonyl-tRNA synthetase (2C9) No. 2 60S ribosomal protein L13a (2D10) No. 3 60S ribosomal protein L13e (2B7) No. 4Interfase cytoplasmic foci protein 45 (2G7) No. 5 Subolesin

7% (406 ± 89) 10% (394 ± 42) 23% (338 ± 154) 33%(293 ± 233)* 12% (384 ± 89)*

0% (10 ± 2) 10%(9 ± 0)** 10% (9 ± 1) 10%(9 ± 0)** 10% (9 ± 1)

4% (93 ± 4) 5%(92 ± 2)** 2% (95 ± 3) 9%(88 ± 6)** 5% (92 ± 6)

ND 5% ND 39% 12%

Adjuvant/saline control

(437 ± 23)

(10 ± 0)

(97 ± 1)

–

a

Cattle were randomly assigned to experimental groups (N = 3), vaccinated and challenged with 500 nymphs of A. americanum. b The percent reduction was calculated with respect to the control group: DT, % reduction in tick infestation; DW, % reduction in tick weight; DM, % reduction in molting. In parenthesis are shown the average ± S.D. for nymph tick number, nymph weight (mg) and percent molting. Tick number was compared between vaccinated and control groups by Chi2 -test (*Chi < 0.01). Tick weight and molting were compared between vaccinated and control groups by Student’s t-test with unequal variance (**P ≤ 0.05). c Vaccine efficacy (E) was calculated as 100[l − (CRT × CRM)], where CRT and CRM are the reduction in the number of nymphs and molting as compared to the control group, respectively. E was calculated using values with statistical significance only. ND, not determined because none of the values were significantly different from controls.

est percentage of ticks completing feeding on day 5. A significant reduction in adult tick infestations, varying from 7% to 32%, was obtained in cattle immunized with 2C9, 2D10, 2G7 and subolesin when compared with control cattle (Table 5). The weight of replete female ticks was significantly reduced by 33–42% in ticks collected from cattle immunized with 2C9, 2G7 and subolesin (Table 5). Oviposition was inhibited by 36% and 41% in ticks collected from cattle immunized with 2G7 and subolesin, respectively (Table 5). The fertility of laid eggs was significantly reduced (33–44%) in all groups except for ticks collected from 2G7-immunized cattle (Table 5). When considering the significant effect of vaccination on adult female tick infestations, oviposition and fertility, vaccine efficacy varied between 33% and 66% with the highest efficacy for 2G7 (56%) and subolesin (66%) (Table 5). To summarize the effect of vaccination on both nymph and adult ticks, an overall vaccine efficacy was calculated considering the effect on both tick stages and resulted in 49% (2C9), 43% (2D10), 33% (2B7), 73% (2G7) and 70% (subolesin).

4. Discussion Tick vaccines have shown to be an effective alternative for tick control, which reduces the use of acaricides and thus the selection

of acaricide-resistant ticks [18]. However, the only commercially available vaccines with recombinant Bm86 antigens are designed for the control of cattle tick, Rhipicephalus (Boophilus) spp. infestations [18]. Development of vaccines for the control of other tick species of medical importance, such as A. americanum, requires the discovery and characterization of new antigens, a process that constitutes the limiting step for advancing this area of research [15–17,19]. The utility of using molecular biology and genomics approaches for the screening of tick protective antigens was demonstrated by Almazán et al. [20]. These experiments used expression library immunization and resulted in the identification of the tickprotective antigen, subolesin. Subolesin was shown by both RNAi gene knockdown and immunization trials using the recombinant protein to protect hosts against tick infestations, reduce tick survival, feeding and reproduction, cause degeneration of tick gut, salivary gland, reproductive tissues and embryos, affect tick vector capacity and function in the control of gene expression in ticks [20,23,25,28,29,32–35]. The results reported herein further demonstrated that subolesin plays a role in the control of tick feeding and fertility and its potential effect as a tick protective antigen for control of A. americanum infestations. As proposed in previous experiments [21,22], RNAi offers a new method for the screening and characterization of tick protective

Table 5 Control of A. americanum adult infestations in cattle vaccinated with the recombinant antigen preparations. Experimental groupa

Amblyomma americanum adult females Percent reduction (vaccinated/control)b

No. 1 Threonyl-tRNA synthetase (2C9) No. 2 60S ribosomal protein L13a (2D10) No. 3 60S ribosomal protein L13e (2B7) No. 4 Interfase cytoplasmic foci protein 45 (2G7) No. 5 Subolesin Adjuvant/saline control a

Ec

DT

DW

DO

DF

9% (147 ± 16)*

36%(340 ± 90)**

30% (0.22 ± 0.05)

44%(259 ± 41)**

49%

11%(144 ± 20)*

0% (530 ± 70)

8% (0.29 ± 0.06)

33%(311 ± 34)**

40%

0% (177 ± 6)

0% (534 ± 133)

7% (0.30 ± 0.09)

33%(312 ± 46)**

33%

32%(109 ± 68)*

42%(308 ± 36)**

36%(0.20 ± 0.01)*

25% (349 ± 94)

56%

7% (150 ± 41)*

33%(356 ± 92)**

41%(0.19 ± 0.08)*

38%(285 ± 35)**

66%

(161 ± 17)

(530 ± 111)

(0.32 ± 0.07)

(463 ± 73)

–

Cattle were randomly assigned to experimental groups (N = 3), vaccinated and challenged with 200 pairs of adult A. americanum. The percent reduction was calculated with respect to the control group: DT, % reduction in tick infestation; DW, % reduction in tick weight; DO, % reduction in oviposition; DF, % reduction in egg fertility. In parenthesis are shown the average ± S.D. for adult female tick number, tick weight (mg), oviposition (egg weight (mg)/tick) and egg fertility (larvae weight/egg weight). Tick number was compared between vaccinated and control groups by Chi2 -test (*Chi < 0.01). Tick weight, oviposition and egg fertility were compared between vaccinated and control groups by Student’s t-test with unequal variance (**P ≤ 0.05). c Vaccine efficacy (E) was calculated as 100[l − (CRT × CR0 × CRF)], where CRT, CRO and CRF are the reduction in the number of adult female ticks, oviposition and egg fertility as compared to the control group, respectively. E was calculated using values with statistical significance only. b

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antigens. In this research, we used RNAi to screen an A. americanum cDNA library and select genes encoding for tick protective antigens. These results demonstrated that RNAi is a powerful tool for rapid identification of tick protective antigens and for analysis of molecules in an inexpensive manner when compared with previous methods. However, one drawback of this approach is the necessity of beginning the screening process with large pools of dsRNAs in order to increase the number of clones analyzed while reducing the number of treatments. The results demonstrated that dsRNA pools provided better results than individual clones. This effect could be due to synergistic and/or additive effect between different clones in the pool, a result that has been reported previously in RNAi studies [36] and in other tick vaccine and screening experiments [20,37,38]. However, the results of the screening reported herein demonstrated that individual clones could be identified that were protective against tick infestations by both gene silencing by RNAi and in immunization trials using recombinant proteins. Several genes, in addition to subolesin, proved to have an effect on tick survival and/or female engorgement weights after RNAi. These genes encoded for proteins with putative functions in protein translation (38%), unknown function (29%), enzymatic activity (13%), protein binding (8%), DNA/RNA metabolism (4%), cell proliferation (4%), and structural protein (4%). These functions have been shown previously to be affected by RNAi in ticks [22,25,39]. Together with subolesin, four genes were selected for expression of recombinant proteins and vaccination studies in cattle based on the effect of RNAi in A. americanum ticks. These genes encoded for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7). In vaccinated cattle, all antigens had an overall efficacy >30% when considering the effect on both nymph and adult ticks. However, only 2D10, 2G7 and subolesin affected both tick stages in vaccinated cattle. The highest efficacy was obtained for vaccine preparations containing recombinant 2G7 and subolesin. The protection capacity of ribosomal proteins and components has been documented previously in ticks and other organisms [20,38,40–48]. However, vaccination of sheep with recombinant ribosomal protein L23a was not protective against Haemaphysalis qinghaiensis infestations [49]. Recently, Fumagalli et al. [50] demonstrated that cell proliferation and ribosome formation are closely linked by showing that impaired assembly of ribosomes can cause cell-cycle arrest and disease. In this study, a special role for 60S ribosomal protein L11 was demonstrated in this process in which the binding of L11 to MDM2 prevented the molecule from degrading p53, which in turn resulted in cell-cycle arrest in mouse cells with impaired ribosome assembly. These results suggest that some ribosomal proteins may be essential for ribosome formation and cell proliferation and therefore high tick mortality would result after RNAi and vaccination with the recombinant protein, as shown here for the A. americanum ribosomal protein L13a. The interphase cytoplasm foci protein 45 (ICF45) is a cytoplasmic 45 kDa protein discovered in human HeLa cells [51]. ICF45 gene knockdown by RNAi inhibited cell growth and proliferation and induced apoptosis and up-regulation of p53 expression [51]. These experiments suggested that ICF45 is expressed in a cell-cycledependent manner and may be involved in cell-cycle progression and cell proliferation. Although the function of the putative interphase cytoplasm foci protein 45 (2G7) in ticks is unknown, it is possible that this protein has a function similar to that described for ICF45 in human cells which would explain its effect on reducing A. americanum infestations and feeding after both RNAi and vaccination with the recombinant protein. Threonyl-tRNA synthetase (2C9) is essential for tRNAThr synthesis and protein translation. Interestingly, sequence identity to the Ixodes scapularis conserved hypothetical protein EEC02417 was also found with the cDNA clone 2G7 (E-value = 3e−56), with a

region encoding for a putative tRNAHis guanylyltransferase (Thg1). Thg1 is an essential protein required for aminoacylation of tRNAHis by histidyl-tRNA synthetase that may have an additional uncharacterized role in RNA or DNA repair or metabolism [52]. Thus, inhibition of tRNA synthetases by RNAi or vaccination with the recombinant proteins would affect tRNA synthesis and protein translation resulting in impaired tick development and fertility. All proteins identified to be protective against A. americanum infestations appear to be ultimately involved in the control of gene expression, protein synthesis and regulation of cell cycle with potential impact on important biological processes such as cell growth and maintenance. However, these proteins are intracellular and may be evolutionarily highly conserved which may pose a problem for their use in vaccine formulations for the control of tick infestations. As discussed previously, safety issues may arise when protein ortholog sequences are used for vaccination as they may have the potential of inducing autoimmune responses damaging to the host [31]. However, the antibody response is expected to be primarily directed against non-selfepitopes, thus reducing the possibility of detrimental effects to the host when protein ortholog sequences are highly conserved among arthropod vectors and vertebrate hosts. Furthermore, immunization with intracellular proteins has been effective in ticks and other invertebrate organisms and suggests a low risk of inducing autoimmune responses in vertebrate hosts [23,28,29,31,43]. Additionally, although not addressed in these studies, the immune response in vaccinated cattle may be directed against both vaccine antigens and/or cross-reactive epitopes in other tick antigens [53]. These experiments demonstrated the potential effect of tick proteins identified herein and most notably the interphase cytoplasm foci protein 45 (2G7) and subolesin as antigens for the control of A. americanum infestations. Although not evaluated in the present experiments, these proteins may also reduce larval infestations as shown previously for subolesin [20,28], thus increasing the overall efficacy of vaccine preparations containing these antigens. In addition, development of vaccine formulations containing several antigens may increase the vaccine efficacy. The results of these studies support the development of vaccines for the control of A. americanum infestations, which will likely improve animal and human health by reducing tick populations through vaccination of cattle and dogs in tick infested areas. Finally, the approach described herein for the identification of A. americanum protective antigens could be used for discovery of protective antigens in other tick species and/or parasites that affect human and animal health. Acknowledgements We thank S. Little, H. Gann, J. Kocan and R. Kaufmann for providing A. americanum tick strains. We also thank P. Ruybal, D. Clawson, Ryan Grant and Jason Thorne for technical assistance. This research was supported by grants from NRI-USDA (project No. 2007-04613); INIA, Spain (project FAU2008-00014-00-00); the Consejería de Educación y Ciencia, JCCM, Spain (project PEII09-0118-8907); the Oklahoma Agricultural Experiment Station (project 1669) and the Walter R. Sitlington Endowed Chair for Food Animal Research (K. M. Kocan, Oklahoma State University). Raúl Manzano-Roman was funded by Ministerio de Educación y Ciencia, Spain. V. Naranjo was founded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007-2013), Spain. M. Canales was funded by the Wellcome Trust under the “Animal Health in the Developing World” initiative (project 0757990). R.C. Galindo was funded by Ministerio de Educación y Ciencia, Spain. M. Villar was funded by the JAE-DOC program (CSIC-FSE), Spain.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.12.007. References ˜ A, Venzal JM, Kocan KM, Sonenshine DE. Overview: [1] de la Fuente J, Estrada-Pena ticks as vectors of pathogens that cause disease in humans and animals. Front Biosci 2008;13:6938–46. [2] Mather TN, Mather ME. Intrinsic competence of three ixodid ticks (Acari) as vectors of the Lyme disease spirochete. J Med Entomol 1990;27:646– 50. [3] Ginsberg HS, Ewing CP, O’Connell Jr AF, Bosler EM, Daly JG, Sayre MW. Increased population densities of Amblyomma americanum (Acari: Ixodidae) on Long Island, New York. J Parasitol 1991;77:493–5. [4] Mount GA, Haile DG, Barnard DR, Daniels E. New version of LSTSIM for computer simulation of Amblyomma americanum (Acari: Ixodidae) population dynamics. J Med Entomol 1993;30:843–57. [5] Means RG, White DJ. New distribution records of Amblyomma americanum (L.) (Acari: Ixodidae) in New York State. J Vector Ecol 1997;22:133–45. [6] Kierans JE, Lancombe EH. First records of Amblyomma americanum, Ixodes (Ixodes) dentatus, and Ixodes (Ceratixodes) uriae (Acari: Ixodidae) from Maine. J Parasitol 1998;84:629–31. [7] Felz JW, Chandler Jr FW, Oliver JH, Rahn DW, Schriefer ME. Solitary erythema migrans in Georgia and South Carolina. Arch Dermatol 1999;135:1317–26. [8] Childs JE, Paddock CD. The ascendancy of Amblyomma americanum as a vector of pathogens affecting humans in the United States. Ann Rev Entomol 2003;48:307–37. [9] Yabsley MJ, Wimberly MC, Stallknecht DE, Little SE, Davidson WR. Spatial analysis of the distribution of Ehrlichia chaffeensis, causative agent of human monocytotropic ehrlichiosis, across a multi-state region. Am J Trop Med Hyg 2005;72:840–50. [10] Patrick CD, Hair JA. Seasonal abundance of lone star ticks on white-tailed deer. Environ Entomol 1977;6:263–9. [11] Koch HG. Suitability of white-tailed deer, cattle and goats as hosts for the lone star ticks. Amblyomma americanum (Acari: Ixodidae). J Can Entomol Soc 1988;61:251–7. [12] Goddard J, Varela-Stokes AS. Role of the lone star tick, Amblyomma americanum (L.), in human and animal diseases. Vet Parasitol 2009;160:1–12. [13] Graf JF, Gogolewski R, Leach-Bing N, Sabatini GA, Molento MB, Bordin EL, et al. Tick control: an industry point of view. Parasitology 2004;129:S427–42. [14] Carroll JF, Benante JP, Klun JA, White CE, Debboun M, Pound JM, et al. Twelve-hour duration testing of cream formulations of three repellents against Amblyomma americanum. Med Vet Entomol 2008;22:144–51. [15] de la Fuente J, Kocan KM. Strategies for development of vaccines for control of ixodid tick species. Parasite Immunol 2006;28:275–83. [16] Sonenshine DE, Kocan KM, de la Fuente J. Tick control: further thoughts on a research agenda. Trends Parasitol 2006;22:550–1. [17] Willadsen P. Tick control: thoughts on a research agenda. Vet Parasitol 2006;138:161–8. [18] de la Fuente J, Almazán C, Canales M, Pérez de la Lastra JM, Kocan KM, Willadsen P. A ten-year review of commercial vaccine performance for control of tick infestations on cattle. Anim Health Res Rev 2007;8:23–8. [19] de la Fuente J, Kocan KM. Advances in the identification and characterization of protective antigens for development of recombinant vaccines against tick infestations. Expert Rev Vac 2003;2:583–93. [20] Almazán C, Kocan KM, Bergman DK, Garcia-Garcia JC, Blouin EF, de la Fuente J. Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization. Vaccine 2003;21:1492–501. [21] de la Fuente J, Almazán C, Blouin EF, Naranjo V, Kocan KM. RNA interference screening in ticks for identification of protective antigens. Parasitol Res 2005;96:137–41. [22] de la Fuente J, Kocan KM, Almazán C, Blouin EF. RNA interference for the study and genetic manipulation of ticks. Trends Parasitol 2007;23:427–33. [23] de la Fuente J, Almazán C, Blas-Machado U, Naranjo V, Mangold AJ, Blouin EF, et al. The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction. Vaccine 2006;24:4082–95. [24] Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H. Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula. J Mol Med 2006;84:901–10. [25] de la Fuente J, Maritz-Olivier C, Naranjo V, Ayoubi P, Nijhof AM, Almazán C, et al. Evidence of the role of tick subolesin in gene expression. BMC Genomics 2008;9:372. [26] Canales M, Enriquez A, Ramos E, Cabrera D, Dandie H, Soto A, et al. Large-scale production in Pichia pastoris of the recombinant vaccine GavacTM against cattle ticks. Vaccine 1997;15:414–22. [27] de la Fuente J, Rodríguez M, Montero C, Redondo M, García-García JC, Méndez L, et al. Vaccination against ticks (Boophilus spp.): the experience with the Bm86based vaccine GavacTM . Genet Anal Biomol Eng 1999;15:143–8.

1795

[28] Almazán C, Blas-Machado U, Kocan KM, Yoshioka JH, Blouin EF, Mangold AJ, et al. Characterization of three Ixodes scapularis cDNAs protective against tick infestations. Vaccine 2005;23:4403–16. [29] Almazán C, Kocan KM, Blouin EF, de la Fuente J. Vaccination with recombinant tick antigens for the control of Ixodes scapularis adult infestations. Vaccine 2005;23:5294–8. [30] Canales M, Almazán C, Naranjo V, Jongejan F, de la Fuente J. Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations. BMC Biotechnol 2009;9:29. [31] Canales M, Naranjo V, Almazán C, Molina R, Tsuruta SA, Szabó MPJ, et al. Conservation and immunogenicity of the mosquito ortholog of the tick protective antigen, subolesin. Parasitol Res 2009;105:97–111. [32] de la Fuente J, Blouin EF, Manzano-Roman R, Naranjo V, Almazán C, Perez de la Lastra JM, et al. Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale. Genomics 2007;90:712–22. [33] Nijhof AM, Taoufik A, de la Fuente J, Kocan KM, de Vries E, Jongejan F. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference. Int J Parasitol 2007;37:653–62. [34] Kocan KM, Manzano-Roman R, de la Fuente J. Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA. Parasitol Res 2007;100:1411–5. [35] Galindo RC, Doncel-Pérez E, Zivkovic Z, Naranjo V, Gortazar C, Mangold AJ, et al. Tick subolesin is an ortholog of the akirins described in insects and vertebrates. Dev Comp Immunol 2009;33:612–7. [36] de la Fuente J, Almazán C, Naranjo V, Blouin EF, Kocan KM. Synergistic effect of silencing the expression of tick protective antigens 4D8 and Rs86 in Rhipicephalus sanguineus by RNA interference. Parasitol Res 2006;99:108–13. [37] Willadsen P, Smith D, Cobon G, McKenna RV. Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91. Parasite Immunol 1996;18:241–6. [38] Almazán C, Kocan KM, Bergman DK, Garcia-Garcia JC, Blouin EF, de la Fuente J. Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags. Gene Ther Mol Biol 2003;7:43–59. [39] Gong H, Liao M, Zhou J, Hatta T, Huang P, Zhang G, et al. Gene silencing of ribosomal protein P0 is lethal to the tick Haemaphysalis longicornis. Vet Parasitol 2008;151:268–78. [40] Schalla WO, Johnson W. Immunogenicity of ribosomal vaccines isolated from group A, type 14 Streptococcus pyogenes. Infect Immun 1975;11:1195–202. [41] Swendsen CL, Johnson W. Humoral immunity to Streptococcus pneumoniae induced by a pneumococcal ribosomal protein fraction. Infect Immun 1976;14:345–54. [42] Tewari RP, Lynn M, Birnbaum AJ, Solotorovsky M. Characterization of the immunoprotective antigen of ribosomal preparations from Haemophilus influenzae. Infect Immun 1978;19:58–65. [43] Elad D, Segal E. Immunogenicity in calves of a crude ribosomal fraction of Trichophyton verrucosum: a field trial. Vaccine 1995;13:83–7. [44] Melby PC, Ogden GB, Flores HA, Zhao W, Geldmacher C, Biediger NM, et al. Identification of vaccine candidates for experimental visceral leishmaniasis by immunization with sequential fractions of a cDNA expression library. Infect Immun 2000;68:5595–602. [45] Cassataro J, Velikovsky CA, Giambartolomei GH, Estein S, Bruno L, Cloeckaert A, et al. Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA. Vaccine 2002;20:1660–9. [46] Rajeshwari K, Patel K, Nambeesan S, Mehta M, Sehgal A, Chakraborty T, et al. The P domain of the P0 protein of Plasmodium falciparum protects against challenge with malaria parasites. Infect Immun 2004;72:5515–21. [47] Iborra S, Carrion J, Anderson C, Alonso C, Sacks D, Soto M. Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice. Infect Immun 2005;73:5842–52. [48] Luo D, Ni B, Li P, Shi W, Zhang S, Han Y, et al. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice. Infect Immun 2006;74:2734–41. [49] Gao J, Luo J, Li Y, Fan R, Zhao H, Guan G, et al. Cloning and characterization of a ribosomal protein L23a from Haemaphysalis qinghaiensis eggs by immuno screening of a cDNA expression library. Exp Appl Acarol 2007;41:289– 303. [50] Fumagalli S, Di Cara A, Neb-Gulati A, Natt F, Schwemberger S, Hall J, et al. Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction. Nat Cell Biol 2009;11:501–8. [51] Guo D, Hu K, Lei Y, Wang Y, Ma T, He D. Identification and characterization of a novel cytoplasm protein ICF45 that is involved in cell cycle regulation. J Biol Chem 2004;279:53498–505. [52] Jackman JE, Phizicky EM. tRNAHis guanylyltransferase catalyzes a 3 –5 polymerization reaction that is distinct from G-1 addition. Proc Natl Acad Sci U S A 2006;103:8640–5. [53] Trimnell AR, Davies GM, Lissina O, Hails RS, Nuttall PA. A cross-reactive tick cement antigen is a candidate broad-spectrum tick vaccine. Vaccine 2005;23:4329–41.