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Vaccine 30 (2012) 265– 272

Contents lists available at SciVerse ScienceDirect

Vaccine

jou rn al h om epa ge: www.elsev ier .com/ locate /vacc ine

ontrol of tick infestations in cattle vaccinated with bacterial membranesontaining surface-exposed tick protective antigens

onsuelo Almazána, Orlando Moreno-Cantúa, Juan A. Moreno-Cidb, Ruth C. Galindob,ario Canalesb,c, Margarita Villarb, José de la Fuenteb,d,∗

Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Tamaulipas, Km. 5 carretera Victoria-Mante, CP 87000 Ciudad Victoria, Tamaulipas, MexicoInstituto de Investigación en Recursos Cinegéticos IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, SpainInstituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, Lisboa 1349-008, PortugalDepartmentof Veterinary Pathobiology, Center forVeterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA

r t i c l e i n f o

rticle history:eceived 9 June 2011eceived in revised form 22 October 2011ccepted 31 October 2011vailable online 12 November 2011

eywords:ickoophilusnaplasma

a b s t r a c t

Vaccines containing the Rhipicephalus (Boophilus) microplus BM86 and BM95 antigens protect cattleagainst tick infestations. Tick subolesin (SUB), elongation factor 1a (EF1a) and ubiquitin (UBQ) are newcandidate protective antigens for the control of cattle tick infestations. Previous studies showed that R.microplus BM95 immunogenic peptides fused to the Anaplasma marginale major surface protein (MSP)1a N-terminal region (BM95-MSP1a) for presentation on the Escherichia coli membrane were protec-tive against R. microplus infestations in rabbits. In this study, we extended these results by expressingSUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins on the E. coli membrane using this system anddemonstrating that bacterial membranes containing the chimeric proteins BM95-MSP1a and SUB-MSP1awere protective (>60% vaccine efficacy) against experimental R. microplus and Rhipicephalus annulatus

SP1aaccinem86m95ubolesinbiquitin

infestations in cattle. This system provides a novel, simple and cost-effective approach for the productionof tick protective antigens by surface display of antigenic protein chimera on the E. coli membrane anddemonstrates the possibility of using recombinant bacterial membrane fractions in vaccine preparationsto protect cattle against tick infestations.

© 2011 Elsevier Ltd. All rights reserved.

longation factor

. Introduction

The cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipi-ephalus annulatus, are distributed in tropical and subtropicalegions of the world [1–5]. Infestations with R. microplus and R.nnulatus economically impact cattle industry by reducing weightain and milk production, and by transmitting pathogens that causeabesiosis (Babesia bovis and Babesia bigemina) and anaplasmosisAnaplasma marginale) [2,4,6].

Although acaricides constitute a major component of integratedattle tick control strategies, their use has had limited efficacyn reducing tick infestations and is often accompanied by seri-us drawbacks, including the selection of acaricide-resistant ticks,

nvironmental contamination and contamination of milk and meatroducts with drug residues [7]. All of these considerations rein-orce the need for alternative approaches to control of cattle tick

∗ Corresponding author.E-mail addresses: jose delafuente@yahoo.com, djose@cvm.okstate.edu

J. de la Fuente).

264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2011.10.102

infestations such as the use of hosts with natural resistance to ticks,pheromone-impregnated decoys for attracting and killing ticks,biological control agents and vaccines [8–10].

In the early 1990s, vaccines containing the recombinant R.microplus BM86 and BM95 gut antigens that induced immuno-logical protection against cattle tick infestations were developedand commercialized [10–19]. These vaccines reduce the numberof engorging female ticks, their weight and reproductive capacity.Thus the greatest vaccine effect was the reduction of larval infesta-tions in subsequent generations. Vaccine field trials demonstratedthat control of cattle ticks by vaccination has the advantages ofbeing cost-effective, reducing environmental contamination andpreventing the selection of drug resistant ticks that result fromrepeated acaricide application [15,16]. In addition, these vaccinesmay also prevent or reduce transmission of pathogens by reducingtick populations and/or affecting tick vector capacity [15–17].

Despite the demonstrated effect of commercial BM86/BM95

vaccines for the control of cattle tick infestations, they showtick strain-to-strain variations in vaccine efficacy and the pro-duction of recombinant antigens is expensive and technologicallycomplex [8,10,14,15,20–24]. Adding new tick protective antigens

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66 C. Almazán et al. / V

o vaccine formulations and developing production systems thatill reduce the cost while increasing the immunogenicity of

ecombinant antigens could overcome these limitations of currentattle tick vaccines.

In a recent work, we demonstrated that a recombinant pro-ein comprising the BM95 immunogenic peptides fused to the. marginale MSP1a N-terminal region (BM95-MSP1a) is surface-xposed on the Escherichia coli membrane, resulting in a simplend cost-effective process for the production of a vaccine prepara-ion that was protective against R. microplus infestations in rabbits24–26]. These studies suggested the possibility of using recombi-ant bacterial membrane fractions containing the surface-exposedM95-MSP1a chimeric antigen as a cheaper preparation for vac-ination against cattle tick infestations [24–26]. However, theseaccination experiments were conducted as a preliminary studyn rabbits that are not the natural host for Rhipicephalus (Boophilus)pp.

In the study reported herein, we conducted a vaccination trialn cattle to demonstrate the efficacy of recombinant E. coli mem-ranes containing the BM95-MSP1a chimera for the control of R.icroplus and R. annulatus infestations and to extend to other can-idate protective antigens the possibility of using recombinantacterial membrane fractions containing surface-expose chimeras a simple and cost-effective approach for the production of tickaccines.

. Material and methods

.1. Construction and expression of tick protective antigenhimeras

The construction and expression of the BM95 chimera wasreviously reported [25]. The BM95-MSP1a chimera encoded

protein containing three BM95 (Genbank accession numberAD38381) immunogenic and protective peptides fused to A.arginale major surface protein 1a (MSP1a) N-terminal region to

urface expose the BM95 peptides when expressed in E. coli [25].or the construction of tick subolesin (SUB-MSP1a), elongation fac-or 1a (EF1a-MSP1a) and ubiquitin (UBQ-MSP1a) chimeras, protein

oding regions (amino acids 1–146, 1–462, and 45–132, for SUBDQ159964.1), EF1a (EU436163.1) and UBQ (AF506022), respec-ively) were amplified by PCR using oligonucleotide primers (SUB:MB4D8R 5′-GATGGAATTCTGTTCTGCGAGTTTGGTAGATAG-3′ and

ig. 1. Expression of recombinant UBQ-MSP1a, BM95-MSP1a, EF1a-MSP1a and SUB-MSPor the expression of chimeric proteins. 10 �g of total proteins (pellet 1 in Fig. 2) were loaSigma) were used as molecular weight (MW) markers in the electrophoresis (lanes 1 an) and transferred to a nitrocellulose membrane and probed with rabbit anti-MSP1a antecombinant UBQ-MSP1a, BM95-MSP1a, EF1a-MSP1a and SUB-MSP1a chimeric proteins

30 (2012) 265– 272

pMB4D8L 5′-CTCCTCGAGATGGCTTGCGCAACATTAAAGCGAAC-3′,EF1a: GIIR 5′-GGCTCGAGAGGCCCACGGACAAACCCCTC-3′ and GII-L5′-GGGAATTCTCAGCGGCCTTGGTGACCTTGCC-3′, UBQ: UBIQ2-L5′-GGCTCGAGATGCAAATCTTCGTTAAGACA-3′ and UBIQ-R 5′-GGGAATTCTCGAGGGTACGCCCATCTTCTAG-3′) that introducedXhoI and EcoRI restrictions sites for cloning into the pFLAG-GTCexpression plasmid [25]. In these constructs, as for the BM95-MSP1a chimera, the inserted coding region is fused to MSP1a andis under the control of the inducible tac promoter [25].

2.2. Recombinant vaccine production

For production of recombinant BM95-MSP1a, SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins, E. coli were propagatedin 10 ml Luria–Bertani (LB) broth containing 50 �g/ml ampicillinand 0.4% glucose (LBAG) for 2 h at 37 ◦C and 200 rpm and thenused to inoculate 250 ml cultures. The 250 ml cultures weregrown under the same conditions for 4 h to reach an OD600 nm = 1before inoculation of a 4 l working volume Biostat B bioreactor(B. Braun Biotech, Melsungen, Germany). Fermentation was doneas described previously [25] but continuing fermentation during5.5 h after addition of 0.5 mM final concentration of isopropyl-�-d-thiogalactopyranoside (IPTG) for induction of recombinantprotein expression. Cell growth was monitored by measuringOD600 nm throughout both propagation and fermentation steps. Thecells were harvested and disrupted using a cell sonicator (ModelMS73; Bandelin Sonopuls, Berlin, Germany) described previously[25]. After disruption, membrane-bound insoluble protein frac-tions were separated by centrifugation at 21,500 × g for 15 min at4 ◦C and stored at −20 ◦C. Protein concentrations were determinedusing the Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA).For vaccine formulation, the amount of membrane-bound insolubleprotein fraction was adjusted to contain 120 �g of the recombinantchimera that were adjuvated in Montanide ISA 50 V2 (Seppic, Paris,France) in 1 ml doses.

2.3. Recombinant protein characterization

The expression of recombinant proteins was analyzed by

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Westernblot (Fig. 1). Ten micrograms of total proteins were loaded onto a12% SDS-polyacrylamide precast gel (Expedeon Protein Solutions,Harston Cambridgeshire, UK) and electrophoresed for 3 h at 90 mA

1a fusion proteins in E. coli. Recombinant E. coli were induced for 5.5 h with IPTGded per well in a 12% SDS-polyacrylamide gel. ColorBurst electrophoresis markersd 10). Proteins were stained with Coomassie based Instant Blue (lanes 2, 4, 6 and

ibodies and developed with a goat anti-rabbit HRP conjugate (lanes 3, 5, 7 and 9). are indicated with arrows.

C. Almazán et al. / Vaccine 30 (2012) 265– 272 267

Fig. 2. Analysis of recombinant UBQ-MSP1a, BM95-MSP1a, EF1a-MSP1a and SUB-MSP1a fusion proteins expressed in E. coli. (A) The cells were grown in a biofermentor,harvested after 5.5 h induction with IPTG by centrifugation and suspended in disruption buffer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 5 mM MgCl2·6H2Oand 0.1% (v/v) Triton X-100) to lyse E. coli cells by sonication. After cell lysis, soluble (supernatant 1) and membrane-bound insoluble protein (pellet 2) fractions wereseparated by centrifugation. Pellet 2 was then washed and dissolved in 1% Triton X-100 for extraction of membrane proteins (supernatant 2). The remaining pellet 3 waswashed and dissolved in 6 M guanidine hydrochloride to extract possible inclusion bodies (supernatant 3) and produce final protein pellet 4. Protein concentrations weredetermined using bicinchoninic acid (BCA) protein assay and analyzed by SDS-PAGE and Western blot as described in Fig. 1 (Lanes: MW, molecular weight markers; 1,MSP1a control; 2, UBQ-MSP1a; 3, BM95-MSP1a; 4, EF1a-MSP1a and 5, SUB-MSP1a). Recombinant proteins were quantified by densitometric analysis of Coomassie basedI on eac d Sup

cBtBtMCmpm

nstant Blue-stained SDS-polyacrylamide gels and shown as percent of total proteinshimeras in supernatants Super 1 (0.1% Triton X-100), Super 2 (1% Triton X-100) an

onstant current. Gels were stained with Coomassie based Instantlue (No. ISB01L; Expedeon Protein Solutions) or transferredo a nitrocellulose membrane (Schleicher & Schuell, PROTRANA85, Dassel, Germany) for Western blot analysis. Proteins wereransferred to a nitrocellulose membrane during 1 h at 12 V using a

inie-Genie Electroblotter semi-dry transfer unit (Idea Scientific,

orvallis, OR, USA). The membrane was blocked with 5% skimilk for 1 h at room temperature, washed three times in TBS and

robed with rabbit antibodies against purified MSP1a [25]. Theembrane was incubated for 1 h with sera diluted 1:500 in 3%

ch fraction (column graphs). (B) Percent total proteins represented by recombinanter 3 (6 M guanidine hydrochloride).

BSA in TBS and washed three times with TBS. The membrane wasthen incubated with a goat anti-rabbit horseradish peroxidase(HRP) conjugate (Sigma, St. Louis, MO, USA) diluted 1:1000 inTBS, washed three times with TBS and finally developed withTMB stabilized substrate for HRP (Promega, Madison, WI, USA)for 20 min. An experiment was conducted to characterize the

subcellular localization of recombinant chimeric proteins bysuccessive protein extractions with 0.1% Triton X-100, 1% TritonX-100 and 6 M guanidine hydrochloride (Fig. 2A). The expres-sion of recombinant proteins was quantified by densitometric

2 accine 30 (2012) 265– 272

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Fig. 3. Antibody response in vaccinated cattle. Bovine serum antibody titers torecombinant antigens were determined by ELISA against (A) a synthetic MSP1a pep-tide, (B) recombinant BM86, or (C) recombinant subolesin in cattle vaccinated withBM95-MSP1a, SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a and adjuvant/saline con-trol. Antibody titers in immunized cattle were expressed as the OD450 nm (at differenttime points) − OD450 nm (preimmune serum) value and compared between vacci-nated and control cattle using an ANOVA test (*P < 0.05). The time of vaccinationshots and tick infestation are indicated with arrows.

68 C. Almazán et al. / V

nalysis of Coomassie based Instant Blue-stained SDS-olyacrylamide gels using molecular weight standards andhe gel analyzer ImageJ 1.44p (http://imagej.nih.gov/ij; Nationalnstitute of Health, USA).

.4. Cattle vaccination and tick infestations

A total of 20 Beefmaster × Charolais, 6 month-old heifers wereurchased from a commercial breeder with no recent historyf ectoparasite infestations, including mange and ticks. Cattleere randomly allocated into 5 groups of 4 animals each. Cat-

le were each immunized with 3 doses (days 0, 30 and 60)ontaining 120 �g/dose of E. coli membrane-bound chimeras for-ulated as described above. Negative controls were injected with

djuvant/saline alone. Cattle were injected intramuscularly with ml/dose using a 5 ml syringe and an 18G needle. Two weeks afterhe last immunization (day 75), cattle in vaccinated and controlroups were infested with 5000 B. annulatus (Mercedes, Texas, USAtrain) and B. microplus (Susceptible Media Joya, Mexico strain) lar-ae/animal applied individually to each animal in separate cottonells attached to the back of the animals [19]. Larvae were 15 daysf age at the time of infestations. Cattle were cared for in accor-ance with standards specified in the Guide for Care and Use ofaboratory Animals of the University of Tamaulipas, Mexico.

.5. Data collection and analysis

Adult female ticks dropping from cattle were daily collected,ounted and weighted. All the collected adult female ticks weressessed for oviposition and egg fertility [19]. The personnel col-ecting the ticks were ‘blinded’ as to which group animals belonged.he efficacy of vaccine formulations was evaluated by determin-ng the effect on the number of adult female ticks (DT), tick

eight (DW), oviposition (egg weight/survived tick; DO) and eggertility (larvae weight/egg weight; DF) employing the formulaeescribed previously [19]. Vaccine efficacy (E) was calculated as00 [l − (CRT × CR0 × CRF)], where CRT, CR0 and CRF represent theeduction in the number of adult female ticks, oviposition and eggertility as compared to the control group, respectively. A Student’s-test with unequal variance (P = 0.05) was used to compare theesults of adult female tick number, tick weight, oviposition andgg fertility between vaccinated and control groups.

.6. Analysis of antibody response in vaccinated cattle by ELISA

Before each immunization and tick infestation and at the endf the experiment (day 103 after the first immunization), bloodamples were collected from each cattle into sterile tubes andaintained at 4 ◦C until arrival at the laboratory. Serum was then

eparated after centrifugation and stored at −20 ◦C. Serum anti-ody titers were determined using a synthetic MSP1a peptideNH2- RSKVASVEYILAARALISVGVYAAQGEIAKSQGCAPLRV-COOH;27]) and purified recombinant BM86 [28] or subolesin [29] anti-ens in an antigen-specific indirect ELISA as described previously29]. Recombinant BM86 was used in the ELISA because BM95 andM86 differ in a single amino acid in the polypeptides expressed

n the BM95-MSP1a chimera. Antibody titers were considered pos-tive when they yielded an OD450 nm value at least twice as high ashe preimmune serum. Antibody titers in immunized cattle werexpressed as the OD450 nm (at different time points) − OD450 nmpreimmune serum) value and compared between vaccinated and

ontrol cattle using an ANOVA test (P = 0.05). A correlation analy-is was conducted in Microsoft Excel (version 12.0) to compare theumber of female ticks collected after feeding with antibody titerst time of tick infestation.

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. Results

.1. Expression and characterization of recombinant proteinhimeras

The E. coli strains expressing recombinant BM95-MSP1a, SUB-SP1a, EF1a-MSP1a and UBQ-MSP1a fusion protein chimeras grew

t a specific growth rate between 0.66 and 0.77 h−1 in the 4-itter bioreactor for 1 h to achieve an OD600 nm = 0.4 prior to thenduction with IPTG. After induction, the specific growth rateecreased to a range between 0.20 and 0.23 h−1. The recombinantM95-MSP1a, SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusionroteins were expressed at high levels after 5.5 h induction, reach-

ng 4%, 9%, 13% and 7% total cell proteins, respectively (Fig. 1).s expected, the theoretical and estimated molecular weights onDS-PAGE were similar for all recombinant proteins (67 kDa vs.5–70 kDa for BM95-MSP1a, 92 kDa vs. 85–90 kDa for SUB-MSP1a,4 kDa vs. 80–85 kDa for EF1a-MSP1a, and 71 kDa vs. 70–75 kDaor UBQ-MSP1a, respectively; Fig. 1), of which 5, 30, 22 and 9 kDaorrespond to the tick-derived protein fragment. After cell dis-uption, successive protein extractions with 0.1% Triton X-100,% Triton X-100 and 6 M guanidine hygrochloride showed thatecombinant proteins were associated with the E. coli membraneraction (Fig. 2A), as the most efficient extraction of recombinanthimeras occurred with 1% Triton X-100 (Fig. 2B). However, somef the chimeras were not extracted with 1% Triton X-100 or anyther treatment and remained associated with the insoluble pro-

ein fraction (pellet 4; Fig. 2A). A simple purification step consistingf cell disruption and centrifugation resulted in a protein frac-ion containing the membrane-bound insoluble chimeras (pellet 2;ig. 2A).

ig. 4. Antibody titers positively correlated with the reduction of tick infestations. A corumber of R. microplus female ticks collected after feeding from both vaccinated and contf tick infestation in individual cattle (A and B) or using average values per group (C). The

xis) are shown in Fig. 3C. The linear correlation coefficients (R2) are also indicated.

30 (2012) 265– 272 269

3.2. Characterization of the immune response in vaccinated cattle

The immune response varied between groups. When testedagainst a synthetic MSP1a peptide that is common to all antigens,antibody titers were low but significantly higher than controls in allgroups after the third immunization and until the end of the exper-iment (Fig. 3A). After the second immunization, antibody titersdecreased, although not significantly in the groups immunized withUBQ-MSP1a and BM95-MSP1a (Fig. 3A). In cattle immunized withSUB-MSP1a, antibody titers did not increase until after the thirdimmunization and remained lower than in the rest of the immu-nized groups (Fig. 3A). However, antibody titers had the highestlevels and were significantly different from controls at the time oftick infestation (Fig. 3A). When tested against recombinant BM86and subolesin in groups immunized with BM95-MSP1a and SUB-MSP1a, respectively, antibody titers were significantly higher thancontrols after the first immunization for BM95-MSP1a (Fig. 3B) andafter the second immunization for SUB-MSP1a (Fig. 3C). Antibodytiters decreased after the third immunization with both antigens,but were significantly higher than in the control group (Fig. 3A andB). Interestingly, antibody titers in vaccinated cattle peaked afterthe third immunization when tested against MSP1a (Fig. 3A) butpeaked after the second immunization when tested against BM86and subolesin (Fig. 3B and C).

When antibody titers at tick infestation time (day 75) werecorrelated with the number of female ticks collected after feed-ing, a positive correlation was obtained between antibody titers

and reduction of R. microplus tick infestations, particularly foranti-BM86 antibody titers (Fig. 4A–C). For R. annulatus, a pos-itive correlation was also obtained between antibody titersand reduction of tick infestations but the linear correlation

relation analysis was conducted using Microsoft Excel (version 12.0) between therol cattle and antibody titers against (A) BM86, (B) subolesin and (C) MSP1a at timeS.E. for both anti-MSP1a antibody titers (horizontal axis) and tick numbers (vertical

270 C. Almazán et al. / Vaccine 30 (2012) 265– 272

Table 1Control of R. microplus infestations in cattle vaccinated with the recombinant chimeric antigens.

Experimental groupa R. microplus (Susceptible, Mexico strain)

Percent reduction, vaccinated/controlb (average ± S.D.) Ec

DT DW DO DF

EF1a-MSP1a 38% 7% −15% 22% 38%200 223 124 0.3470 273 127 0.8370 216 119 0.9320 263 102 0.9(340 ± 112)* (244 ± 28) (118 ± 11) (0.7 ± 0.3)

UBQ-MSP1a 3% 8% 0% 0% ND620 240 100 1.0310 245 115 0.9590 262 86 1.0610 210 111 0.9(533 ± 149) (239 ± 22) (103 ± 13) (0.9 ± 0.0)

BM95-MSP1a 54% 25% −22% 22% 64%320 201 135 0.7190 215 104 0.8180 186 134 0.7320 181 130 0.6(253 ± 78)* (196 ± 15)* (126 ± 15) (0.7 ± 0.1)*

SUB-MSP1a 34% 37% 11% 67% 81%390 187 12 0.6350 151 114 0.3520 174 114 0.3180 145 128 0.2(360 ± 140)** (164 ± 20)* (92 ± 54) (0.3 ± 0.2)*

Adjuvant/saline control 620 277 123 0.9 –510 275 102 0.9580 215 99 1.0490 275 88 0.9(550 ± 61) (261 ± 30) (103 ± 15) (0.9 ± 0.0)

a Cattle were randomly assigned to experimental groups (N = 4), vaccinated and challenged with R. microplus and R. annulatus larvae.b 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. Following percent reduction are shown the data for each cattle and in parenthesis the group average ± S.D. for adult female tick number,tick weight (mg), oviposition (egg weight (mg)/tick) and egg fertility (larvae weight/egg weight) and were compared by Student’s t-test with unequal variance betweenvaccinated and control groups.

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 eggfertility as compared to the control group, respectively. E was calculated using values with statistical significance only. ND, not determined because none of the values weres

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oefficients (R2 = 0.3–0.4) were lower than for R. microplus (dataot shown).

.3. Protective efficacy of bacterial membranes containingurface-exposed protein chimeras

The results of the vaccination trial showed that EF1a-MSP1and UBQ-MSP1a chimeras had low efficacy against cattle ticknfestations, with varying effects on R. microplus and R. annula-us (Tables 1 and 2). In contrast, the SUB-MSP1a and BM95-MSP1aaccines showed high efficacy (64–81%) for the control of both R.icroplus and R. annulatus infestations (Tables 1 and 2). As expected

rom the subolesin role on tick fertility, the main effect of SUB-SP1a vaccination was on the reduction of tick fertility that was

imilar for R. microplus and R. annulatus (DF = 67%; Tables 1 and 2).s in previous experiments with BM86/BM95 vaccines, the BM95-SP1a chimera showed the highest efficacy in the reduction of tick

nfestations for both R. microplus and R. annulatus (DT = 53–54%;ables 1 and 2). The efficacy of the BM95-MSP1a vaccine was highergainst R. annulatus (74%) than R. microplus (64%) while the SUB-SP1a vaccine showed the highest efficacy for the control of R.icroplus infestations (81%) (Tables 1 and 2).

. Discussion

Commercial vaccines have been used as a complementary inter-ention for the control of cattle tick infestations, but R. microplus

and R. annulatus continue to represent a problem for the cattleindustry [7,15]. The development of improved tick vaccines willcontribute to control of cattle tick infestations but requires the dis-covery of new protective antigens and production of more effectiveand cheaper vaccine formulations [8–10,14,15]. The combination ofprotective antigens or protective epitopes derived from these anti-gens in an economically viable formulation is important towardsachieving this goal.

The tick proteins selected for this study were identified ascandidate protective antigens in RNA interference experimentsconducted in R. microplus and R. annulatus and other tick species[28]. In R. microplus and R. annulatus, EF1a, UBQ and SUB knock-down increased tick mortality and reduced oviposition [28]. Toprove the efficacy of these candidate protective antigens, vaccina-tion experiments were conducted in cattle with recombinant SUBand a synthetic UBQ peptide [28]. After vaccination, only SUB had asignificant effect on R. microplus and R. annulatus infestations in cat-tle [28]. Two limitations of this trial were that UBQ vaccination wasconducted using a synthetic peptide that may be poorly immuno-genic and EF1a was not tested because it could not be obtained insufficient quantities for vaccine formulation [28].

In previous studies, we demonstrated that the BM95-MSP1a

chimera was surface-exposed on recombinant E. coli membranesand protected against R. microplus infestations in rabbits [25,26].Therefore, this system was chosen to test the efficacy of E. colimembranes containing BM95-MSP1a, SUB-MSP1a, EF1a-MSP1a

C. Almazán et al. / Vaccine 30 (2012) 265– 272 271

Table 2Control of R. annulatus infestations in cattle vaccinated with the recombinant chimeric antigens.

Experimental groupa R. annulatus (Mission, TX strain)

Percent reduction, vaccinated/controlb (average ± S.D.) Ec

DT DW DO DF

EF1a-MSP1a 24% 21% −14% 0% ND230 241 109 0.9470 203 113 1.0210 192 107 0.8450 223 100 0.7(340 ± 139) (215 ± 22)** (107 ± 5) (0.9 ± 0.1)

UBQ-MSP1a 29% 22% −15% 22% 22%260 234 120 0.6230 210 109 0.9450 205 105 0.5320 204 98 0.7(315 ± 97) (213 ± 14)* (108 ± 9) (0.7 ± 0.2)**

BM95-MSP1a 53% 34% −26% 44% 74%290 215 120 0.6240 148 119 0.4120 181 105 0.8190 174 130 0.4(210 ± 73)* (180 ± 28)* (119 ± 10) (0.5 ± 0.2)*

SUB-MSP1a 20% 38% −15% 67% 67%400 189 107 0.5390 211 102 0.5420 142 104 0.2210 134 118 0.2(355 ± 97) (169 ± 37)* (108 ± 7) (0.3 ± 0.2)*

Adjuvant/saline control 270 313 128 0.9 –440 256 89 0.9500 238 70 0.9570 279 89 1.0(445 ± 128) (272 ± 32) (94 ± 24) (0.9 ± 0.0)

a The experimental design with 4 cattle per group is similar to that described in Table 1.b The percent reduction was calculated with respect to the control group for tick infestation (DT), tick weight (DW), oviposition (DO), and fertility (DF). Following percent

reduction are shown the data for each cattle and in parenthesis the group average ± S.D. for adult female tick number, tick weight (mg), oviposition (egg weight (mg)/tick)and egg fertility (larvae weight/egg weight) and were compared by Student’s t-test with unequal variance between vaccinated and control groups.

c Vaccine efficacy (E) was calculated as described in Table 1 using values with statistical significance only. ND, not determined because none of the values were significantlyd

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nd UBQ-MSP1a fusion proteins as candidate vaccines for the con-rol of R. microplus and R. annulatus infestations in cattle.

Although the antibody response in vaccinated cattle variedetween vaccine preparations, antibody titers were significantlyigher in vaccinated cattle when compared to controls at the timef tick infestation. The difference in the cattle antibody responsehen measured against MSP1a and tick polypeptides may reflect

he fact that tick protein epitopes were exposed on the E. coliembrane while MSP1a epitopes were mostly located in trans-embrane regions [27]. Furthermore, a positive correlation was

btained between antibody titers and reduction of tick infestationsn cattle vaccinated with BM95-MSP1a and SUB-MAP1a proteins.hese results strongly suggested, as in previous experiments withM86 [16], that vaccine efficacy on the control of cattle tick infes-ations was the result of a protective antibody response directedgainst tick protein epitopes in the recombinant chimeric antigenn vaccinated cattle.

The efficacy of the BM95-MSP1a vaccine was similar to thathown by BM86 vaccination against the same R. microplus strain butower than the 100% efficacy obtained against R. annulatus [19,28].emarkably, the efficacy of the SUB-MSP1a vaccine was higher thanhat obtained with BM86 on the control of R. microplus infestations19,28]. These results suggested that while it is difficult to improvehe efficacy of BM86 vaccines against R. annulatus, the SUB-MSP1a

accine preparation might be more effective than BM86 for theontrol of R. microplus infestations.

The results of the vaccination trial showed that the BM95-MSP1antigen had the highest effect on the reduction of tick numbers,

while the SUB-MSP1a antigen reduced more the egg fertility. Theseresults suggested that vaccination with these antigens have dif-ferent effects on tick biology, thus suggesting the possibility tocombine protective epitopes in BM86/BM95 and SUB/Akirin intoa single chimera to increase vaccine efficacy and protection againsta broader range of tick species and other hematophagous ectopar-asites [30–33].

As previously discussed, the cost and efficiency of the antigenproduction process is essential for bringing new vaccines to themarket [24]. The simplicity of the production process describedherein for MSP1a chimeras, which only involves the propagationand fermentation of the recombinant E. coli strain followed bycell harvest, disruption and debris separation, shows promise fora cost-effective process to produce a vaccine based on the bacte-rial membranes containing the surface-exposed chimeric antigenicpeptides. The high expression levels obtained for recombinantchimeras (3–12% total cell proteins), the membrane surface expo-sition of antigenic peptides that vary in molecular weight between5 and 30 kDa and the small number of steps needed for antigenpurification support the use of this E. coli expression system for ahighly simplified, efficient and cost-effective process for antigenproduction.

5. Conclusions

In summary, this is the first report showing that the recombinantE. coli membrane fractions containing the BM95-MSP1a chimerawere effective for the control of R. microplus and R. annulatus

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nfestations in cattle. The results of this study demonstratedhat the SUB-MSP1a chimera was also protective against cat-le tick infestations. Additionally, the E. coli expression systemllowed the expression of different tick polypeptides fused toSP1a, suggesting the possibility of developing tick vaccines based

n recombinant E. coli membrane fractions containing chimeraomposed of a combination of surface-exposed protective anti-ens/epitopes. These new vaccine formulations would have thedvantage of being cost-effective by using a simple and cheap pro-ess for the production of antigens for the control of cattle ticknfestations [24].

cknowledgments

This research was supported by FOMIX–TAMPS-2008-C17-07392, the Spanish Ministerio de Ciencia e Innovación (MICINN)roject BFU2008-01244/BMC, the Consejería de Educación y Cien-ia, JCCM, Spain (project PEII09-0118-8907), the Instituto Nacionale Investigación y Tecnología Agraria y Alimentaria (INIA), Spainproject FAU2008-00014-00-00), and Probiovet S.L., Spain. M. Vil-ar and M. Canales were funded by JAE-DOC program (CSIC-FSE) andunta de Comunidades de Castilla-La Mancha (Program FSE 2007-013), Spain, respectively. J.A. Moreno-Cid is a recipient of a JCCMellowship.

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