Vaccine 23 (2005) 5294–5298

Vaccination with recombinant tick antigens for the control ofIxodes scapularis adult infestations

Consuelo Almazana, Katherine M. Kocana, Edmour F. Blouina, Jose de la Fuentea,b,∗a Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, 250 McElroy Hall,

Oklahoma State University, Stillwater, OK 74078, USAb Instituto de Investigacion en Recursos Cinegeticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain

Received 15 June 2005; accepted 1 August 2005Available online 15 August 2005

Abstract

Antigens protective againstIxodes scapularis infestations were identified by cDNA expression library immunization (ELI) and analysisof expressed sequenced tags (EST). Three cDNAs protective against larval tick infestations, 4F8, with homology to a nucleotidase, and4D8 and 4E6 of unknown function, were characterized and obtained as recombinant proteins for immunization studies. Vaccination trials

thend the

ntrol groupnant tick33–71%.stations.

gnedtar-

ogens

-hostca-trol

n theens

aryncedsed

gytion,

with recombinant proteins demonstrated an effect of these antigens againstI. scapularis larvae in a mouse model. Herein, we evaluatedeffect of recombinant antigens onI. scapularis adult infestations on immunized sheep. Vaccination with recombinant 4D8, 4F8, 4E6 acombination of all three antigens reduced adult tick infestations by 58, 12, 20, and 16%, respectively, when compared to the cobut was statistically significant for 4D8 and 4F8 only. Oviposition was reduced by 22–49% in all groups immunized with recombiantigens (P < 0.05). The overall efficacy of vaccine formulations considering the effect on tick infestations and oviposition averagedThese antigens, and especially 4D8, appear to be good candidates for continued development of a vaccine for control of tick infe© 2005 Elsevier Ltd. All rights reserved.

Keywords: Tick vaccine; Expression library immunization; Recombinant protein; Sheep; Development

1. Introduction

Ixodes scapularis (Acari: Ixodidae) is one of the mostimportant vector of pathogens affecting humans and wild anddomestic animals in North America[1,2]. Members of theIxodes ricinus complex, which includesI. scapularis, I. paci-ficus andI. ricinus, are the primary tick vectors ofBorreliaburgdorferi andAnaplasma phagocytophilum, the causativeagents of Lyme disease and human granulocytic anaplasmo-sis, respectively[1,2].

Tick vaccines offer an alternative approach for the controlof tick infestations (reviewed by[3,4]). Control of ticks byvaccination would avoid environmental contamination andthe selection of drug resistant ticks that result from repeated

∗ Corresponding author. Tel.: +1 405 744 0372; fax: +1 405 744 5275.E-mail addresses: josedelafuente@yahoo.com,

djose@cvm.okstate.edu (J. de la Fuente).

acaricide applications. Tick vaccines could also be desito include multiple tick and pathogen antigens that mayget a broad range of tick species and associated path(reviewed by[3]).

The recent development of vaccines against oneBoophilus spp. has provided new possibilities for identifition of protective antigens for use in vaccines for the conof tick infestations (reviewed by[3,4]). However, the limitingstep for the development of new tick vaccines has beeidentification and characterization of tick protective antig(reviewed by[3,4]).

Recently, the combination of cDNA expression librimmunization (ELI) and analysis of expressed sequetags (EST) in a mouse model of tick infestations was ufor the identification of cDNAs protective againstI. scapu-laris larvae[5,6]. Three of these cDNAs, 4F8 with homoloto a nucleotidase, and 4D8 and 4E6 of unknown funcwere characterized and expressed inEscherichia coli for

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2005.08.004

C. Almazan et al. / Vaccine 23 (2005) 5294–5298 5295

immunization studies[7]. 4D8 and 4F8 antigens wereselected because immunization of mice with these cDNAsresulted in 50 and 40% inhibition ofI. scapularis larval infes-tations, respectively[5,6]. 4E6 cDNA, although inhibited tickinfestations at a lower level (20%) compared to 4D8 and 4F8[6], was selected for further evaluation because it encodesa small protein of 38 amino acids which could be poten-tially used in chimeric polypeptides or in combination withother antigens for vaccination against ticks[7]. Vaccinationtrials with recombinant proteins demonstrated an effect ofthese antigens againstI. scapularis larvae in a mouse modeland preliminary experiments in rabbits suggested a protectiveeffect against nymphs[7]. However, the effect of vaccina-tion with recombinant 4D8, 4F8 and 4E6 antigens on adultI. scapularis has not been demonstrated. For a three-hosttick such asI. scapularis, vaccine efficacy against immaturestages as well as adult tick stages is important for optimalcontrol of tick infestations. Herein, we describe the results ofa vaccination trial using recombinant 4D8, 4F8 and 4E6 pro-tective antigens in which we demonstrated control of adultI.scapularis infestations in sheep.

2. Materials and methods

2.1. Ticks

theO andn eep.O ho-t

2v

eree asd

2s

per-i ls toe tionw ins,t rd eepw n theG erei 0, 6,1 ,U sep-a withF

(week 16) after the last immunization, sheep were infestedwith 100I. scapularis females and 50 males per sheep, usingcotton stockinette cells glued to the side of the animal. Theunattached ticks were counted and removed 48 h after infes-tation, and engorged ticks were collected daily for 7 daysfrom each sheep, counted and weighted. The engorged tickswere held in a humidity chamber for 40 days, after whichoviposition was evaluated. The inhibition of tick infestation(I) and the inhibition of oviposition (O) for each test groupwith respect to the BSA-immunized controls and the overallefficacy of the vaccine (E) were calculated[5,7].

2.4. Characterization of the immune response inimmunized sheep by ELISA

The antibody response against recombinant 4F8, 4D8 and4E6 tick antigens in test and control sheep was evaluatedby indirect ELISA. Sheep sera for ELISA were obtainedat weeks 0, 6, 12 and 16, before each immunization shotand before tick infestation, respectively. Purified recombi-nant proteins or totalE. coli proteins (0.1�g/well) were usedto coat ELISA plates overnight at 4◦C. Sera were seriallydiluted to 1:100–1:1600 in PBST (PBS/0.5% Tween 20, pH7.2) and 10% fetal bovine serum (Sigma). The plates wereincubated with the diluted sera for 1 h at 37◦C and thenincubated with 1:10,000 rabbit anti-sheep IgG–HRP con-j sd ndt tesw u-n ter-m y fore hent ast zeds mean((

2

df[ -bi heepi pers ers (f ls as[ sp ofe acyow(

I. scapularis females and males were obtained fromklahoma State University Tick Rearing Facility. Larvaeymphs were fed on rabbits and adult ticks were fed on shff-host ticks were maintained in a 12 h light:12 h dark p

operiod at 22–25◦C and 95% relative humidity.

.2. Production of recombinant tick antigens andaccine formulations

I. scapularis 4D8, 4F8 and 4E6 protective antigens wxpressed inE. coli and purified for vaccine formulationsescribed previously[7].

.3. Immunization of sheep and infestation with I.capularis adults

Five groups of four sheep each were used in this exment. A randomized list was created to assign animaach group. Experimental groups included immunizaith 100�g/dose of 4F8, 4D8 or 4E6 recombinant prote

he combination of all three antigens (50�g each antigen peose) and 100�g/dose BSA to serve as control. The shere cared for in accordance with standards specified iuide for Care and Use of Laboratory Animals. Sheep w

njected subcutaneously with each antigen at weeks2 and 14 with 500�l/dose in FIA (Sigma, St. Louis, MOSA) using a 6 ml syringe and an 18.5 G needle. Inrate experiments we have shown that immunizationIA did not have an effect on tick feeding[7]. Two weeks

ugates (Sigma) for 1 h at 37◦C. The color reaction waeveloped with 3,3′,5,5′-tetramethylbenzidine (Sigma) a

he OD450 nm was determined. After incubation the plaere washed with PBST. Antibody titers in the group immized with the combination of all three antigens were deined by averaging the titers determined independentlach antigen. Antibody titers were considered positive w

hey yielded an OD450 nm value at least twice as highhe negative control serum. Antibody titers in immuniheep were expressed as the inverse of the geometricmean± S.D.) serum dilution at which a positive OD450 nmODtest sheep− ODcontrol) was obtained.

.5. Data analysis

The inhibition of tick infestation (I) was calculateor female I. scapularis as described previously[6,8] as1 − ((RLn/RLi )/(RLnc/RLic))] × 100, whereRLn is the numer of replete ticks recovered per sheep in test groups,RLnc

s the average number of replete ticks recovered per sn controls,RLic is the average number of ticks attachedheep in controls, andRLi is the number of attached ticks pheep for each test group. The inhibition of ovipositionO)or each test group was calculated with respect to contro1 − (Otest/Ocontrol)] × 100, whereOtest is the weight of egger tick in test groups andOcontrol is the average weightggs per tick in the control group. The protection efficf the vaccine was calculated asE = 100× [1 − (RI × RO)]hereRI (reduction in tick infestation) = 1− I/100 andRO

reduction in oviposition) = 1− O/100.

5296 C. Almazan et al. / Vaccine 23 (2005) 5294–5298

The number of ticks attached per animal (RLi ), the ratioRLn/RLi of the number of replete ticks and the ticks attachedper sheep, tick weights and the weight of eggs/tick were com-pared by Student’st-test between tick antigen-immunizedand control BSA-immunized animals. The linear regressionanalysis of the cumulative number of replete ticks collectedper day in each experimental group was done with MicrosoftOffice Excel 2003.

3. Results

An experiment was conducted to evaluate the effect ofvaccination with recombinant tick protective antigens 4D8,4F8 and 4E6 on adultI. scapularis infestations in sheep.

Antibody levels in sheep serum samples were monitoredby ELISA until tick infestation at week 16 of the experiment(Fig. 1). The sheep responded to immunizations with recom-binant tick protective antigens with increasing antibody titers,which varied among sheep immunized with the same antigen(Fig. 1). Antibody titers against totalE. coli proteins weredetermined on each animal to evaluate the specific responseto tick protective antigens and were lower than 1:100 at week16 (data not shown).

After infestation with adultI. scapularis, tick attach-m9c -i callys cci-n nsw chedar i-n lativen -p

F heep.A verseo sitiveO

Fig. 2. Repletion period for femaleI. scapularis. The cumulative numberof replete ticks collected per day was plotted for each experimental groupvaccinated with 4D8, 4F8, 4E6, the combination of all three antigens (All) orBSA for controls. The equation of the linear regression (y = ax + b; a, slope;b, intercept) is shown for each treatment.

The weight of the replete ticks did not vary(P > 0.05) between femaleI. scapularis recovered from test(198± 23 mg/tick) and control sheep (199± 24 mg/tick).However, the weight of the egg mass oviposited per tickwas statistically lower (P < 0.05) for groups immunized with4E6, 4F8, 4D8 and the combination of all three tick antigens(Table 1).

The inhibition of tick infestations caused by the vacci-nation with tick protective antigens varied between 12 and58% (Table 1). The effect of vaccination on tick reproductiveperformance resulted in the reduction of oviposition rangingfrom 22 to 49% (Table 1). The overall efficacy of the vaccineon femaleI. scapularis was then calculated considering thereduction in tick infestations and oviposition and resulted in33–71% efficacy (Table 1).

4. Discussion

The identification and characterization of tick protectiveantigens has been a growing area of research since the vac-cines for the control ofB. microplus infestations reached themarket in the early 1990s (reviewed by[3,4]). The feasibilityof controlling tick infestations through immunization of hostswith tick antigens has been demonstrated forBoophilus spp.u wedb t ofn har-aF nti-g evalu-a ns(

olo-g ities

ent was homogeneous in all infested sheep (average± S.D.,4± 7) and did not differ (P > 0.05) between test (93± 7) andontrol (96± 2) animals (Table 1). However, tick antigenmmunized sheep had lower infestation rates with statistiignificant reduction in tick infestations in the groups vaated with 4F8 and 4D8 (Table 1). Sheep-to-sheep variatioere observed within test groups in the number of attand replete ticks recovered after infestation (Table 1). Theepletion of femaleI. scapularis was delayed in 4D8 vaccated sheep (slope of the linear regression of the cumuumber of replete ticks collected per day,a = 28) when comared to the other test and control groups (a = 51–58) (Fig. 2).

ig. 1. Antibody response to recombinant tick antigens in immunized sntibody titers were determined by ELISA and expressed as the inf the geometric mean (mean + S.D.) serum dilution at which a poD450 nm(ODtest sheep− ODcontrol) was obtained.

sing the recombinant Bm86 and Bm95 proteins (reviey [3,4]). However, the limiting step for the developmenew tick vaccines continues to be the identification and ccterization of tick protective antigens (reviewed by[3,4]).ew of the tick proteins identified as putative protective aens have been obtained as recombinant proteins andted in vaccination trials for the control of tick infestatioreviewed by[3,4]).

The development of high throughput screening technies and bioinformatic tools has provided new opportun

C. Almazan et al. / Vaccine 23 (2005) 5294–5298 5297

Table 1Effect of vaccination with recombinant tick antigens on the control ofI. scapularis adult infestations

Groupa Sheep no. RLib RLn

c RLn/RLi I (%)d Eggs (g) Eggs/tick (g)e O (%)f E (%)g

Control 28 97 59 0.61 – 2.343 0.040 – –38 94 54 0.57 – 2.375 0.044 – –42h – – – – – – – –50 98 54 0.55 – 2.483 0.046 – –Average± S.D. 96± 2 56± 3 0.58± 0.03 – 2.400± 0.073 0.043± 0.003 – –

4E6 27 95 75 0.79 −37 2.272 0.030 30 633 76 16 0.21 64 0.558 0.035 19 7137 81 22 0.27 53 0.504 0.023 47 7640 94 54 0.57 1 2.186 0.040 6 9Average± S.D. 87± 9 42± 28 0.46± 0.27 20± 41 1.380± 0.981 0.032± 0.007* 26± 15 40± 38

4F8 29 97 44 0.45 22 1.750 0.040 8 2930 87 46 0.53 9 1.715 0.037 14 2244 91 46 0.51 13 1.342 0.029 33 4245 99 53 0.54 7 1.554 0.029 32 38Average± S.D. 94± 6 47± 4 0.51± 0.04* 12± 6 1.590± 0.186 0.034± 0.005* 22± 11 33± 9

4D8 26 99 6 0.06 90 0.033 0.006 87 9931 97 54 0.56 4 1.990 0.037 15 1939 98 19 0.19 66 0.269 0.014 67 8949 99 16 0.16 72 0.586 0.037 15 77Average± S.D. 98± 1 24± 21 0.24± 0.22* 58± 32 0.720± 0.877 0.023± 0.016* 46± 32 71± 36

All 36 88 49 0.56 4 1.088 0.022 49 5141 99 30 0.30 48 0.621 0.021 52 7543 95 58 0.61 −6 1.099 0.019 56 5446 99 46 0.46 20 1.204 0.026 39 52Average± S.D. 95± 5 46± 12 0.48± 0.13 16± 20 1.003± 0.260 0.022± 0.003* 49± 6 58± 11

a Groups of 4 sheep each were vaccinated with recombinant 4E6, 4F8, 4D8 and the combination of all three antigens (All) or BSA for controls.b RLi , number of ticks attached per sheep.c RLn, number of replete ticks recovered per sheep.d I, reduction of tick infestations with respect to the control group.e Calculated by dividing the weight of eggs laid by the number of replete ticks collected from each sheep.f O, reduction of tick oviposition with respect to the control group.g E, vaccine efficacy considering the effect on tick infestations and oviposition.h Sheep no. 42 died before completion of the vaccination scheme.* P < 0.05 (Student’st-Test).

for the identification of tick protective antigens.I. scapularisprotective antigens were identified using a combination ofELI and EST characterization in the mouse model of tickinfestations[5,6] and these results were confirmed recentlyby RNA interference in female ticks[8]. Three of theseantigens, 4D8, 4F8 and 4E6 were expressed inE. coli andcharacterized[7]. Vaccination trials with recombinant anti-gens demonstrated control ofI. scapularis larvae in miceand preliminary experiments in rabbits suggested an effecton I. scapularis nymphs[7]. These results encouraged theevaluation of 4D8, 4F8 and 4E6 for the control ofI. scapu-laris adults in vaccinated sheep. The possibility of controllinginfestations by all tick developmental stages in a three-hosttick such asI. scapularis would be an important contributionto tick vaccine development.

The results reported herein demonstrated the effect ofrecombinant 4D8, 4F8 and 4E6 antigens on the control of tickinfestations. The vaccination with tick protective antigensreducedI. scapularis adult infestations and oviposition. Thereduction of tick infestations was shown for all tick protectiveantigens but was statistically significant in the groups immu-

nized with 4F8 and 4D8 proteins only. The lack of statisticalsignificance in the groups immunized with 4E6 and the com-bination of all three antigens could be due to sheep-to-sheepvariations in the response to vaccination and tick infestation.These differences, which resulted in one animal on each of the4E6 and all three antigens combined groups with infestationlevels higher than controls (negative “I” on Table 1), are com-mon in vaccination experiments with tick protective antigens[7,9,10]. Alternatively, vaccination with 4E6 could be inef-fective to control adultI. scapularis infestations. However,experiments involving larger number of animals are requiredto fully evaluate the influence of animal-to-animal variationsin the efficacy of tick vaccine formulations in the field[9].

The effect of vaccination withI. scapularis 4D8, 4F8 and4E6 protective antigens on tick infestations and/or oviposi-tion were similar to those obtained with Bm86 and Bm95 anti-gens for the control ofBoophilus spp. infestations (reviewedby [3,4]). However, the reduction of tick oviposition, whichaffects tick progeny and thus reduces the fertility of ticks, wasnot the result of the reduction in tick weight as it has been sug-gested forB. microplus gut antigens[9]. The host response to

5298 C. Almazan et al. / Vaccine 23 (2005) 5294–5298

vaccination with Bm86/Bm95 antigens results in the lysis oftick gut epithelial cells through anti-Bm86/95 antibodies withthe possible involvement of the complement and other effec-tor mechanisms, which result in reduced tick survival, weightand fertility[9–13]. In the experiments described herein withI. scapularis, the typical red coloration observed in adultB.microplus that feed on Bm86/Bm95 vaccinated cattle[10–12]was not observed. These results suggest that the mechanismsby whichI. scapularis 4D8, 4F8 and 4E6 antigens conferredprotection against adultI. scapularis infestations may be dif-ferent from that proposed for Bm86 and Bm95 antigens.

The effect of vaccination on tick infestations and oviposi-tion were more pronounced in 4D8-vaccinated sheep with anaverage overall vaccine efficacy of 71%. Furthermore, 4D8delayed the repletion of those ticks that completed feedingon sheep. These results justify a role for 4D8 on tick growthand development ([7,8], unpublished results). The combinedefficacy of the 4D8 vaccine, considering the effect on larvae(71% efficacy, ref.[7]) and on adults reported here (71%),was 92%. If the suggested reduction onI. scapularis nymphalinfestations[7] proves to be true, then the overall efficacy ofthe 4D8 vaccine may be even higher.

In summary, the results reported herein demonstrated theeffect of recombinant 4D8, 4F8 and 4E6 protective antigensin the control ofI. scapularis adult infestations on vacci-nated sheep. These results and particularly those obtainedw effi-c 4D8a

A

alth,K M.K tionP erA ndP rs.J hoolo ndJ tateU ofr spec-t ann

(Oklahoma State University, OK, USA) are acknowledgedfor providing ticks and/or for technical assistance during thevaccination trial.

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cknowledgments

This research was supported by Pfizer Animal Healamazoo, MI, USA, the Sitlington Endowed Chair (K.ocan) and the Oklahoma Agricultural Experiment Staroject 1669. Consuelo Almazan was supported by Pfiznimal Health, a grant-in-aid from the CONACYT aromep (University of Tamaulipas), Mexico. We thank D.C. Garcia-Garcia (The Johns Hopkins University Scf Medicine, Baltimore, MD, USA), Shalu Ayalew aeremiah T. Saliki (Veterinary Pathobiology, Oklahoma Sniversity, OK, USA) for assistance with the purification

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RNA interference screening in ticks for identification of protecantigens. Parasitol Res 2005;96:137–41.

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13] Garcıa-Garcıa JC, Soto A, Nigro F, Mazza M, Joglar M, HechevaıaM, et al. Adjuvant and immunostimulating properties ofrecombinant Bm86 protein expressed inPichia pastoris. Vaccine1998;16:1053–5.