CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice

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Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

pG-ODN combined with Neospora caninum lysate, but not withxcreted-secreted antigen, enhances protection against infection in mice

âmaso P. Ribeiroa, Marina M.P. Freitasa, Mariana R.D. Cardosoa, Ana C.A.M. Pajuabaa,eide M. Silvab, Tiago W.P. Mineoa,c, João S. Silvac, José R. Mineoa, Deise A.O. Silvaa,∗

Laboratory of Immunoparasitology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará 1720, Uberlândia 38400-902, MG, BrazilLaboratory of Immunopathology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará 1720, Uberlândia 38400-902, MG, BrazilLaboratory of Immunoparasitology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto 14049-900, SP, Brazil

r t i c l e i n f o

rticle history:eceived 29 November 2008eceived in revised form 5 February 2009ccepted 6 February 2009vailable online xxx

eywords:eospora caninumpG-ODNdjuvantaccination

a b s t r a c t

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens,but there is limited information concerning their use in immunization protocols against neosporosis.This study aimed to evaluate the potential of CpG-ODN combined with Neospora lysate antigen (NLA)or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninuminfection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals withNLA, NLA + CpG, NcESA, NcESA + CpG, CpG (adjuvant control) or PBS (infection control). Serological assaysshowed an increased specific IgG2a response in animals immunized with either antigen plus adjuvantand elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferativeresponses upon antigen stimulation were higher in groups immunized with NLA or NcESA combined withCpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA + CpG demonstratedhigher IFN-� levels and IFN-�/IL-10 ratio. After lethal challenge, mice immunized with NLA + CpG orNLA had lower morbidity score and body weight changes in comparison to other groups, and animals

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did not succumb during acute infection. In contrast, NcESA + CpG or NcESA groups exhibited the highestmorbidity scores, body weight impairment and mortality rates, associated with greatest brain parasiteburden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immuneresponse with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-drivencellular immune response and total protection only when combined with NLA. Vaccination with NcESAalone or combined with CpG resulted in a strong cellular immune response associated with high levels

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. Introduction

Neospora caninum is an obligate intracellular protozoan parasite,hich was first identified as a cause of neuromuscular diseases inogs [1]. Nowadays, N. caninum is recognized to infect a wide rangef animal species and to be an important cause of abortion in cattleorldwide, leading to significant economic losses [2]. The para-

ite can perpetrate the infection by two major manners. First, cattle

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Please cite this article in press as: Ribeiro DP, et al. CpG-ODN combinantigen, enhances protection against infection in mice. Vaccine (2009

cquire infection horizontally by exposure and ingestion of oocystsxcreted by canine definitive hosts [3]. Second, during pregnancy,ossibly as a result of imbalance of the immune system by fetal-erived regulatory cytokines, such as IL-10 and IL-4, bradyzoites

∗ Corresponding author at: Laboratório de Imunologia, Instituto de Ciênciasiomédicas, Universidade Federal de Uberlândia, Av. Pará, 1720, Uberlândia, 38400-02, MG, Brazil. Tel.: +55 34 3218 2195 fax: +55 34 3218 2333.

E-mail address: [email protected] (D.A.O. Silva).

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264-410X/$ – see front matter © 2009 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2009.02.028

dering mice more susceptible to parasite challenge.© 2009 Published by Elsevier Ltd.

contained within tissue cysts recrudesce and differentiate, resultingin tachyzoite parasitemia [4]. Parasites may cross the placenta andinfect the foetus, causing abortion or congenital infection, depend-ing on the time during gestation when the fetus becomes infected[5]. This mode of transmission accounts for the vast majority ofinfection, and vertical transmission occurs during repeated preg-nancies with up to 95% of calves born from infected dams testingpositive [6].

Protective immune responses against experimentally inducedneosporosis in mice have shown to be mainly associated with thedevelopment of a Th1-type immune response, dominated by IgG2aantibody production and increased IFN-� synthesis [7,8]. The roleof B cells and antibodies in a Neospora protective immune response

ed with Neospora caninum lysate, but not with excreted-secreted), doi:10.1016/j.vaccine.2009.02.028

has been recognized to control the spread of extracellular stages of 46

parasites [9]. Also, innate immunity plays a crucial role in protection 47

of protozoan infections, once antigen-presenting cells distinguish 48

infectious agents through conserved pathogen-associated molecu- 49

lar patterns by Toll-like receptors (TLRs). Stimulation of TLRs causes 50

dx.doi.org/10.1016/j.vaccine.2009.02.028

http://www.sciencedirect.com/science/journal/0264410X

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n immediate protective response, including production of diversentimicrobial peptides and cytokines [10].

Considering the economic impact of neosporosis, biologicalontrol measures have been widely investigated to prevent itsransmission and infection or to reduce the severity of the disease11]. At the present time, there is no vaccine capable of inducingatisfactory protective immunity in cattle. Therefore, studies usingurine models have contributed to characterize novel antigens

nd strategies for successful vaccination procedures. In this con-ext, several immunization protocols against N. caninum have usedive, �-irradiated, or live attenuated tachyzoites [12–14], whole. caninum tachyzoite lysates [15], recombinant proteins, includ-

ng surface antigens (NcSRS2 and NcSAG1) [16,17], micronemeNcMIC3, NcMIC1, NcMIC4) [18–20], rhoptry (NcROP2) [11] andense granule (NcGRA7, NcGRA6 and NcDG1) proteins [21–23] orven mixture of recombinant proteins (GRA1, GRA2, MIC10 and24B) [24]. All these formulations have shown that protection isometimes partial and depends on the type of antigen and adju-ant used, as well the vaccination strategies, including expressionn plasmid DNA or recombinant virus or bacterial vectors.

Oligodeoxynucleotides (ODN) containing unmethylated CpGotifs are recognized by Toll-like receptor 9 (TLR9) and act as a

anger signal by the immune system of mammals, since they areften present in pathogen or immune complexes containing selfNA [25]. The interaction of TLR9 with CpG motifs triggers a cas-ade of events in antigen presenting cells resulting in the activationf B and T helper (Th1) cells with subsequent secretion of Th1-ype cytokines, such as IL-12, IFN-� and TNF-� [26]. Thus, syntheticpG ODNs possess immunostimulatory properties and have beenvaluated as immune adjuvant in vaccines against several intracel-ular pathogens, including Leishmania, Toxoplasma and Plasmodium27–29].

As in other apicomplexan parasites, N. caninum excreted-ecreted antigen (NcESA) represents a rich source of mosticronemal and rhoptry proteins that have been shown to playcrucial role in the host cell invasion [30] and could constitutepotential candidate to multi-component vaccines. As vaccina-

ion with a single protein has been considered less effective than aombination of proteins [17], vaccines containing a complete com-lement of protective parasite antigens, such as Neospora lysatentigen (NLA) and NcESA, could be a more rational choice for effec-ive vaccines against neosporosis [31]. In the present study, wevaluated the potential of CpG-ODN combined with NLA or NcESAn inducing enhanced immune response and protection against N.aninum infection in mice.

. Material and methods

.1. Parasites and antigens

N. caninum tachyzoites (NC-1 isolate) [1] were maintainedy serial passages in bovine monocytes cultured in RPMI 1640edium supplemented with 2% heat-inactivated calf fetal serum

CFS), 2 mM glutamine, 100 U penicillin/ml and 100 �g strepto-ycin/ml in a 5% CO2 atmosphere, at 37 ◦C. Parasite suspensionsere obtained as previously described [32]. Briefly, tachyzoitesere harvested by scraping off the cell monolayer after 2–3 days

f infection, passed through a 26-gauge needle to lyse any remain-ng intact host cells, and centrifuged at low speed (45 × g) for 1 mint 4 ◦C, in order to remove host cell debris. The supernatant con-aining parasite suspension was collected and then washed twice
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720 × g, 10 min, 4 ◦C) in phosphate buffered saline (PBS, pH 7.2) andhe resulting pellet was resuspended in PBS for antigen preparation.

N. caninum lysate antigen (NLA) was prepared as previouslyescribed [32]. Parasite suspension was treated with protease

nhibitors and lysed by freeze-thaw cycles followed by ultrasound

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on ice. The presence of probable live parasites was monitoredby light microscopy under Trypan blue staining. After centrifuga-tion (10,000 × g, 30 min, 4 ◦C), supernatant was collected, filteredon 0.22 �m membrane and its protein concentration determined[33]. Different batches were done for NLA preparation and pooledtogether to obtain the required protein concentration. NLA aliquotswere stored at −20 ◦C until using in immunization, serological testsand proliferation assays.

Preparation of N. caninum excreted-secreted antigen (NcESA)was carried out as described elsewhere [34]. Freshly liberatedNC-1 tachyzoites (108 parasites/ml) were washed twice in PBS,resuspended in Hanks saline solution and incubated for 30 min at37 ◦C, at mild agitation. Parasites were then centrifuged (720 × g,10 min, 4 ◦C), the supernatant was collected and again centrifuged(10,000 × g, 4 ◦C, 30 min). The final supernatant was filtered on0.22 �m membranes and its protein concentration determined [33].Different batches were done for NcESA preparation and pooledtogether to obtain the required protein concentration. Parasitebreakdown during NcESA preparations was monitored through theexpression of p97, a cytosolic protein of T. gondii, which is con-served in apicomplexan parasites, as previously described [35,36].NcESA aliquots were stored at −20 ◦C until using in immunizationprocedures.

N. caninum whole antigens were prepared as previouslydescribed [37]. Parasite suspension was treated with 1% formalde-hyde, for 30 min at room temperature. After washing in PBS,parasites were dry-fixed in microscopic slides and stored at −20 ◦Cuntil using in indirect fluorescent antibody test (IFAT).

2.2. Oligodeoxynucleotide (ODN)

The ODN used in this study was CpG ODN 1826 (5′-TCCAT-GACGTTCCTGACGTT-3′), synthesized with a nuclease-resistantphosphorothioate backbone (Coley Pharmaceuticals, Wellesley,MA), containing two CpG motifs with known immunostimulatoryeffects in murine species [38].

2.3. Animals, immunization and challenge

All experiments were carried out with 8–12 week old maleC57BL/6 mice obtained from University of Campinas, Brazil. Ani-mals were maintained under standard conditions in the BioterismCenter and Animal Experimentation, Federal University of Uberlan-dia, Brazil. All procedures were conducted according to institutionalguidelines for animal ethics.

Six groups of 11 mice were immunized subcutaneously threetimes at 2-week intervals, with 200 �l/animal of the following for-mulations: 25 �g of NLA combined with 50 �g CpG in sterile PBS(NLA + CpG group); 25 �g of NcESA added to 50 �g CpG in sterilePBS (NcESA + CpG group); 25 �g of NLA alone (NLA group); 25 �gof NcESA alone (NcESA group); 50 �g CpG alone (CpG group); anddiluent only (PBS group). Blood samples were collected at 0, 14, 28,42 and 56 days after immunization, and the sera analyzed for thepresence of specific antibodies. Two weeks after the last immuniza-tion, three mice in each group were euthanized and their spleenswere removed aseptically for proliferation assays.

The remaining animals were challenged intraperitoneally 4weeks after the last immunization with 2 × 107 NC-1 tachyzoites.Negative controls included non-immunized and unchallenged mice(n = 3). Animals were observed daily for mortality, morbidityand body weight changes. Morbidity scores were calculated as


described elsewhere [39], as follows: sleek/glossy coat, bright and 171

active (score 0); ruffled coat, hunched, tottering gait, reluctance to 172

move (score 1); stary stiff coat (score 2). All surviving animals were 173

euthanized at 32 days after challenge, when brain tissues were col- 174

lected, sliced longitudinally and half of them was stored at −70 ◦C 175


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or analysis by polymerase chain reaction (PCR). The remainingissue was fixed in 10% buffered formalin, embedded in paraffinnd routinely processed for histological and immunohistochemicalssays.

.4. Antibody levels and IgG isotype determination

N. caninum-specific total IgG, IG1and IgG2a antibodies wereeasured by ELISA as described elsewhere [32], with modifications.igh affinity microtiter plates were coated with NLA (0.5 �g/well),ashed with PBS plus 0.05% Tween 20 (PBS-T) and blocked with 5%

kim milk in PBS-T (for IgG detection) or 1% BSA in PBS-T (for IgG1nd IgG2a detection). Serum samples were diluted 1:25 in blockinguffer and incubated for 1 h (IgG) or 2 h (IgG1 and IgG2a) at 37 ◦C.fter washing, peroxidase-labeled goat anti-mouse IgG (1:1000;igma Chemical Co., St Louis, MO) or biotinylated anti-mouse IgG11:4000) or anti-mouse IgG2a (1: 2000) antibodies (Caltag Lab. Inc.,outh San Francisco, CA) were added and incubated for 1 h at 37 ◦C.ext, streptavidin-peroxidase (1:1000; Sigma) was added whenppropriate. The assays were developed with 0.01 M 2,2′-azino-bis--ethyl-benzthiazoline sulfonic acid (ABTS; Sigma) and 0.03% H2O2,nd absorbance was determined in a plate reader at 405 nm.

IFAT was also carried out to verify N. caninum immunostain-ng with mouse sera at 42 days after immunization as previouslyescribed [32]. Slides containing formolized tachyzoites were

ncubated with serum samples and then with FITC-labeled goatnti-mouse IgG (diluted 1:50; Sigma). Slides were overlaid withuffered glycerol and examined in epifluorescence microscope.

.5. SDS-PAGE and immunoblot

NLA and NcESA were submitted to electrophoresis in a 12% poly-crylamide gel (SDS-PAGE) under non-reducing conditions [40].els were stained with silver nitrate or proteins were electrophoret-

cally transferred to nitrocellulose membranes [41]. Immunoblotssays were carried out to verify the N. caninum reactivity pro-le exhibited by mouse sera at 42 days after immunization [32].itrocellulose strips were blocked with 5% skim milk in PBS-T,

ncubated with mouse sera diluted 1:50 and then with peroxidase-oat anti-mouse IgG (diluted 1:1000; Sigma). The reaction waseveloped with by adding 0.03% H2O2 and 3,3′-diaminobenzidineetrahydrochloride (DAB; Sigma). The apparent molecular massesf antigenic bands were determined in relation to a standard molec-lar weight curve.

.6. Proliferation assay and cytokine quantification

Spleen cell suspensions were washed in RPMI medium, treatedn lysis buffer (0.16 M NH4Cl and 0.17 M Tris–HCl, pH 7.5), washedgain and resuspended in complete RPMI medium containing 10%FS. For proliferation assays, 2 × 105 cells were cultured in triplicate

n 96-well culture plates (200 �l/well) in the presence of 10 �g/mlLA, 5 �g/ml Concanavalin A (Con A) or medium alone and incu-ated at 37 ◦C in 5% CO2 for 72 h. Cells were pulsed with 0.5 �Ci/well[H] thymidine (New England Nuclear, Bucknghamshire, UK) forhe last 12 h, harvested on glass fiber filters, and counted in a liq-id scintillation �-counter (Packard, Downers Grove, IL) in countser minute (cpm). Results were expressed as stimulation index (SI),sing the following formula: SI = cpm of stimulated wells/cpm ofon-stimulated wells. Values of SI > 2 were considered positive [42].

For cytokine analyses, spleen cells (4 × 106) were cultured in
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riplicate in 24-well plates (500 �l/well) and stimulated as aboveescribed. Cell free supernatants were collected after 48 h (IL-2) or 72 h (IFN-�, IL-4 and IL-10) and stored at −70 ◦C. Cytokineeasurements were performed by sandwich ELISAs according toanufacturer’s instructions (IL-12 and IFN-�, PeProtech Mexico,

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Veracruz, México; IL-4 and IL-10, R&D Systems, Minneapolis, MN).The limit of detection for each assay was 31 pg/ml (IL-12), 39 pg/ml(IFN-�), 15 pg/ml (IL-4), and 31 pg/ml (IL-10). Intra-assay and inter-assay coefficients of variation were below from 20% and 10%,respectively.

2.7. Quantitative real-time PCR

Brain parasite burden in mice was determined by quantita-tive real-time PCR as previously described [20], using primerpairs (sense 3′-GCTGAACACCGTATGTCGTAAA-5′; antisense 3′-AGAGGAATGCCACATAGAAGC -5′) designed to detect the N. caninumNc-5 sequence through SYBR green detection system (Invitrogen,San Francisco, CA). DNA extraction was performed from 20 mgof murine brain tissues using the Wizard SV Genomic DNA kit(Promega Co., Madison, WI) following manufacturer’s instructions.DNA concentrations were determined by UV spectrophotometry(260 nm) and adjusted to100 ng/�l with sterile DNAse free water.Assays to determine N. caninum tachyzoite loads were performedthrough real-time PCR (ABI 7000, Applied Biosystems, Foster City,CA) and parasite counts were calculated by interpolation from astandard curve (101 to 10−8 ng) with DNA equivalents extractedfrom 107 NC-1 tachyzoites included in each run. As negative con-trol, brain tissue from non-immunized and unchallenged mice wasanalyzed in parallel.

2.8. Immunohistochemical and histopathological assays

Brain tissue parasitism was also determined by immunohis-tochemistry as previously described [43]. Deparaffinized sectionswere incubated with 3% hydrogen peroxide and then with 0.2 Mcitrate buffer (pH 6.0) for a 7-min cycle in microwaves to rescueantigenic sites. Next, sections were blocked with 2% non-immunegoat serum and then with primary antibody (pooled sera from miceexperimentally infected with N. caninum) diluted 1:100 in PBS plus1% bovine serum albumin. As negative controls, paired tissue sec-tions were incubated in the absence of primary antibodies (diluentonly) or with non-immune mouse serum. Subsequently, secondarybiotinylated goat anti-mouse IgG antibody (1:300; Sigma) and thestreptavidin-biotinylated peroxidase complex (1:250; DAKO Corpo-ration, Carpinteria, CA) were added and the reaction was developedwith 0.03% H2O2 and 3,3′-diaminobenzidine tetrahydrochloride(DAB; Sigma). Slides were counterstained with Harris haematoxylinand examined under light microscopy.

Histological changes were analyzed in two cerebral noncon-tiguous sections (40 �m distance between them) stained withhaematoxylin and eosin obtained from each mouse and from atleast three mice per group as previously described [44]. For theinflammatory score in the brain, the total numbers of focal or dif-fuse inflammatory foci were counted in a sagittal section and thecuffing of blood vessels and inflammatory cells infiltration in themeninges were analyzed. All analyses were done in a magnificationof 1 × 40 in a blind manner by two researchers. The inflammatoryscore was represented as arbitrary units: 0–2, mild; 2–4, moder-ate; 4–6, severe. Negative controls included cerebral tissue fromnon-immunized and unchallenged mice.

2.9. Statistical analysis

The Kaplan–Meier method was applied to estimate the per-centage of mice surviving at each time point after challenge and


survival curves were compared using the log rank test. Differences 291

between groups were analyzed using the ANOVA or Kruskal–Wallis 292

test when appropriate, and the Tukey or Dunn multiple comparison 293

tests, respectively, were applied to examine all possible pairwise 294

comparisons. Student t test was used for comparison of IgG isotypes, 295


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Fig. 1. Levels of total IgG (A), IgG1 (B) and IgG2a (C) to N. caninum determined byELISA in sera of C57BL/6 mice immunized subcutaneously three times (gray arrows)with Neospora lysate antigen (NLA) alone or NLA plus CpG, N. caninum excreted-secreted antigen (NcESA) alone or NcESA plus CpG, or CpG (adjuvant control) orPBS (infection control). Mice were challenged with 2 × 107 tachyzoites of NC-1 iso-

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ytokine levels and stimulation index in different groups. Statisti-al analysis was carried out using GraphPad Prism 4.0 (GraphPadoftware Inc., San Diego, CA). A value of P < 0.05 was consideredtatistically significant.

. Results

.1. Humoral immune responses after immunization and parasitehallenge

Mice immunized with NLA plus CpG presented total IgG levelso N. caninum significantly higher in comparison to all other groupsrom 14th to 56th days after immunization (d.a.i.) (Fig. 1A). Theame pattern was observed for mice immunized with NLA alone inelation to the remaining groups (P < 0.05). Mice immunized withcESA + CpG had significantly increased total IgG levels at 42 and6 d.a.i. as compared to NcESA or controls. For IgG1 isotype assayFig. 1B), NLA alone induced increased IgG1 levels from 28th to6th d.a.i. in comparison with NcESA alone or combined with CpGP < 0.05). Mice immunized with NLA + CpG also presented highergG1 levels at 28 and 56 d.a.i. in relation to other groups (P < 0.05),ut lower IgG1 levels at 42 d.a.i. as compared to NLA alone (P < 0.05).

gG2a isotype responses (Fig. 1C) were similar to those found forotal IgG, with increased IgG2a levels in mice immunized withLA + CpG or NLA alone at 28 and 42 d.a.i. (P < 0.05). Before chal-

enge (56 d.a.i.), similar IgG2a levels were found in mice immunizedith NcESA + CpG and NLA, but significantly higher than NcESA and

ontrols (P < 0.05).Seroconversion and antibody isotype responses were compared

efore and after parasite challenge in all experimental groupsFig. 2). Levels of IgG2a were significantly higher than IgG1 in micemmunized with either antigen (NLA or NcESA) combined withpG, while the use of the antigens alone induced a predominant

gG1 response before parasite challenge following immuniza-ion (P < 0.05). After challenge, all experimental groups showed

significant increase in all antibody (total IgG, IgG1 and IgG2a)esponses, with higher IgG1 levels in relation to IgG2a in all groupsP < 0.05).

IFAT results showed a different N. caninum immunostaininghen probed with sera from animals immunized with NLA + CpG orLA alone (Fig. 3A), showing a bright and linear peripheral fluores-ence of parasite surfaces, when compared to NcESA + CpG or NcESAroups (Fig. 3B) that showed only a weak and apical end stainingf tachyzoites. Immunoblot results also showed a distinct IgG anti-ody reactivity profile exhibited by sera from immunized animals,ith a strong staining of a number of NLA antigenic bands (29–32,

5–37, 64–66 and 82–86 kDa) in NLA + CpG or NLA groups in com-arison with a lower recognition of antigenic components (64–66nd 82–86 kDa) revealed by sera of NcESA + CpG or NcESA groupsFig. 3D). In parallel, NcESA antigenic bands presented higher reac-ivity with sera from mice immunized with NLA + CpG or NLAlone in relation to NcESA + CpG or NcESA alone, although with alearly lower intensity than for NLA antigens. For comparison, NLAnd NcESA gels stained with silver nitrate are also demonstratedFig. 3C).

.2. Proliferative responses and cytokine production by spleenells from immunized mice

Proliferative responses of spleen cells were determined after
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2 h stimulation with ConA, NLA or medium (Fig. 4A). Asxpected, the mitogenic stimulus elicited a strong splenocyteroliferative response in all experimental groups. Upon antigentimulus, immunization with NLA or NcESA combined with CpGnduced a significantly higher proliferative response when com-

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late (black arrow). Blood samples were collected at 0, 14, 28, 42 and 56 days afterimmunization. Values are indicated as mean OD405nm ± SEM. *P < 0.05 as indicated.

pared to antigens alone (P < 0.05), and all antigen groups showedhigher stimulation index than control (CpG and PBS) groups(P < 0.05).

Cytokine production was assessed in supernatants of spleen cellcultures after stimulation with NLA antigen (Fig. 4B–D). High levelsof IL-12 were found in all experimental groups (Fig. 4B), although


NLA or NcESA combined with CpG groups induced significantly 362

higher IL-12 levels in relation to antigen alone (NLA or NcESA) 363

groups and control (CpG and PBS) groups (P < 0.05). IL-12 produc- 364

tion was especially higher in NcESA + CpG than NLA + CpG groups 365


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ig. 2. Serological results for total IgG, IgG1 and IgG2a to N. caninum determined bnd immunization/challenge procedures. Serum samples were analyzed at 56 dayndicated as mean OD405nm ± SEM. *P < 0.05 when comparing IgG1 and IgG2a levels

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P < 0.05). IFN-� levels were significantly higher in NcESA + CpGnd NcESA groups than NLA + CpG or NLA alone (P < 0.05), andhese two latter groups produced higher IFN-� levels in relationo control groups (CpG and PBS) (Fig. 4C). Low levels of IL-10 were

ig. 3. Immunostaining of N. caninum tachyzoites tested with sera from animals immunizntibody test. Original magnification, ×40. SDS-PAGE at 12% stained with silver nitrate (C)ach experimental group: NLA + CpG (lane 1); NLA (lane 2); NcESA + CpG (lane 3); NcESA (nfected mice. Molecular mass markers (Mr) and immunodominant antigens are indicate

FA in mouse sera of all experimental groups. See Fig. 1 for abbreviations of groupsimmunization before challenge (BC) and 32 days after challenge (AC). Values are

h group.

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detected in all experimental groups, with no significant differences 370

(Fig. 4D). IFN-�/IL-10 ratio (Fig. 4E) revealed significantly increased 371

IFN-� production in relation to IL-10 in NcESA + CpG and NcESA 372

groups (P < 0.05). IL-4 levels were below the detection limit for 373

ed with NLA + CpG or NLA (A) and NcESA + CpG or NcESA (B) in indirect fluorescentand immunoblot (D) of NLA and NcESA antigens probed with representative sera oflane 4); CpG (lane 5); PBS (lane 6). Lane 7 is representative of sera from N. caninumd in kiloDaltons (kDa).


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6 D.P. Ribeiro et al. / Vaccine xxx (2009) xxx–xxx

Fig. 4. Proliferative response (A) and cytokine production (B–E) of spleen cells from mice immunized with NLA alone or combined with CpG, NcESA alone or combined withCpG, CpG (adjuvant control) or PBS (infection control). Cells were collected 2 weeks after the 3rd booster and cultured in the presence of 5 �g/ml Concanavalin A (ConA),10 �g/ml NLA, or medium alone. Stimulation index (SI) was determined after 72 h stimulation. Supernatants of spleen cell cultures stimulated with antigen were analyzeda FN-�/i ine as

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REfter 48 h for IL-12 (B) or 72 h for IFN-� (C) and IL-10 (D) by sandwich ELISAs. The I

ndicate the threshold for positive SI values and the limit of detection for each cytok

ll experimental groups. Levels of cytokines were observed afteritogenic stimulation in all analyzed groups, with no significant

ifferences (data not shown).

.3. Clinical signs after parasite challenge following vaccination

After challenge with NC-1 tachyzoites, clinical signs were eval-ated by morbidity scores and body weight changes (Fig. 5). Mice

mmunized with NLA + CpG had significantly lower morbidity scoreFig. 5A) in comparison to other groups (P < 0.01). Morbidity scoreor mice immunized with NLA alone was also significantly lowerhan that for other groups but NLA + CpG (P < 0.01). Significant body
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eight changes from baseline were observed in mice vaccinatedith NcESA + CpG, NcESA alone or PBS (P < 0.01), with considerableeight losses from 10th to 20th days after challenge (Fig. 5B). As

hown in Fig. 5C, mice immunized with NcESA + CpG or NcESA aloneresented the lowest survival percentages, although significant dif-

IL-10 ratio is also shown (E). Values are indicated as mean ± SEM. The dashed linessay. Different letters indicate statistically significant differences (P < 0.05).

ferences were found only in relation to NLA or NLA + CpG groups(P < 0.05).

3.4. Histological changes and tissue parasitism in the brain afterchallenge

The inflammatory lesions in the brain were characterizedby mononucleated cell infiltrates, glial nodules, vascular cuffingby lymphocytes and focal mononucleated cell infiltrates in themeninges (Fig. 6A). The inflammatory scores were more severe inmice immunized with NcESA + CpG as well as in PBS group (P < 0.05)(Fig. 6B).

Brain parasite burden after challenge was determined by real-


time PCR (Fig. 7A). Mice immunized with NcESA + CpG showed the 400

highest brain parasite load in relation to other groups, followed 401

by NcESA and PBS groups (P < 0.05). In contrast, significantly lower 402

brain parasite load was found in mice immunized with NLA alone or 403

NLA combined with CpG or only CpG as compared to other groups 404


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Fig. 5. Mean morbidity score (A), body weight change from baseline (B) and survivalcurves (C) of C57BL/6 mice after challenge with N. caninum. See Fig. 1 for abbrevi-ations of groups and immunization/challenge procedures. Mice immunized withNLA + CpG had significantly lower mean morbidity score in comparison to othergroups (P < 0.01) and NcESA + CpG or NcESA groups showed the highest morbid-ity scores and body weight changes (P < 0.05). Mice immunized with NLA + CpG orNLA alone had significantly greater survival percentage than those immunized withNab

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RcESA + CpG or NcESA alone (P < 0.05). Negative controls included non-immunizednd unchallenged mice, with no alteration in morbidity scores, weight change fromaseline and mortality (data not shown).

P < 0.05). The cerebral parasitism was also assessed by immuno-istochemical assays and it was observed a higher parasite load inice immunized with NcESA + CpG, NcESA alone and PBS in related

o other groups, showing strongly stained tachyzoites within para-itophorous vacuoles (Fig. 7B).

. Discussion

The essential goal of vaccine development against neosporosis is
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o utilize specific N. caninum antigens that elicit protective immu-ity. A number of N. caninum recombinant surface antigens, rhoptry,icroneme and dense granule proteins have been evaluated as

otential vaccine candidates, associated with different formula-ions of adjuvants and new antigen delivery systems [11,45]. In this

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context, soluble N. caninum tachyzoite antigens entrapped in non-ionic surfactant vesicles or administered with Freund’s completeadjuvant (FCA) [46] as well as incorporated into immunostimulat-ing complexes (iscoms) or mixed with Quil A [12] have shown thatprotection depends on the type of adjuvant used. CpG-ODNs areknown to activate innate immunity and to enhance the immuneresponse to vaccination by activating Th1 cells and release of asso-ciated cytokines as IL-12, IFN-� and TNF-� [26]. However, little isknown on the use of CpG ODN as immunoadjuvant in neosporo-sis. A single study on N. caninum vaccination used a constructcontaining CpG-ODN with plasmid DNA coding for NcGRA7, andevaluated its potential in congenital transmission abrogation [21].To our knowledge, no studies have yet been conducted in orderto evaluate the potential of CpG combined with NLA or NcESA ininducing protective immune response against N. caninum infectionin mice.

On the other hand, the role of excreted-secreted antigens (ESA)from parasites as T. gondii and Leishmania sp. in the stimulation ofthe host immune system by inducing antibody and cell-mediatedimmune responses as well as by containing parasite virulence fac-tors has been widely investigated [47–50]. Thus, ESA componentsplay important functions in the host-cell interaction, mechanismsof cell invasion, pathogenesis and evasion of host immune response.Recently, the use of ESA has been suggested in vaccination strate-gies against intracellular parasites, particularly T. gondii [48,51].However, it is necessary to take into account that parasite crude sub-cellular fractions, such as secretory components may be unsuitedfor vaccination approaches, since they do not only contain theonly protective antigens, but also additional components that couldinduce the reverse effect, being rather non-protective or even anti-protective.

In the present study, serological responses after vaccinationshowed a considerably higher immunogenicity of NLA than NcESA,particularly when combined with CpG, as demonstrated by highlevels of specific IgG isotype, mainly due to IgG2a response. Incontrast, vaccination with either NLA or NcESA alone, markedlyfor NLA, induced a higher IgG1 production. These results indi-cate that CpG-adjuvanted antigens were able to induce a Th1-typeantibody immune response in contrast to preparations contain-ing antigen alone that showed a preferentially Th2-biased immuneresponse. This is consistent with the enhancement of IgG2a sub-class switching for CpG adjuvant as previously reported in othertherapeutic vaccines against parasitic infections [27–29,52]. How-ever, the differential immunostaining seen in IFAT and the distinctantigenic pattern exhibited in immunoblot suggest that the anti-gen choice, rather than the adjuvant, influenced the magnitudeof the immune response, confirming a stronger humoral immuneresponse induced by NLA in comparison to NcESA.

We also verified if the type of response induced by immuniza-tion with CpG-adjuvanted NLA or NcESA would be maintained afterparasite challenge. There was a considerable increment in both IgG1and IgG2a isotypes after challenge in antigen-immunized groups,indicating that the parasite was able to induce both Th2 and Th1immune responses, although a Th2-biased humoral response wasmore evident. This pattern of response can be explained due to highdose of NC-1 challenge that switched the overall immune responsetowards a stronger IgG1 isotype. In addition, mice immunized withCpG alone or PBS also showed high seroconversion and increasedIgG1 levels in response to challenge, supporting the notion thatthe NC-1 isolate was responsible for driving a stronger Th2 anti-body profile. Similar findings were found in mice vaccinated with


a CpG-adjuvanted temperature-sensitive mutant strain (ts4) and 478

challenge with the RH strain of T. gondii [53]. In contrast, studies 479

using CpG-ODN combined with T. cruzi antigens demonstrated no 480

changes in the type of immune response after parasite challenge 481

following vaccination [42]. 482


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Fig. 6. (A) Histological changes in the brain of NcESA + CpG (a), NcESA (b) and PBS (c) immunized mice on day 32 post-challenge with N. caninum. See Fig. 1 for abbreviationsof groups and immunization/challenge procedures. Inflammatory infiltrates are observed in the parenchyma (asterisks) and vascular cuffing (arrows) and in the meninges( on, ×2w fferenu own).

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arrowhead). Slides were stained with haematoxylin and eosin. Original magnificatiith N. caninum NC-1 isolate. Different letters indicate statistically significant dinchallenged mice, with no histological change or inflammatory score (data not sh

Proliferative responses and cytokine production confirmed thatoth NLA and NcESA alone and mostly combined with CpG-ODN

nduced a strong cellular immune response, manifested by high IL-2 levels upon antigenic stimulation and consequent significantlyreater stimulation indexes. CpG alone also induced increased IL-2 levels, indicating that the CpG-ODNs stimulate innate immunitys previously reported [28,52]. However, NcESA + CpG or NcESAlone was able to induce significantly higher IFN-� levels thanLA + CpG or NLA groups, reflecting a vigorous Th1-driven immune

esponse. Levels of IL-10 were similarly low in all experimentalroups, although a high IFN-�/IL-10 ratio was observed for thecESA + CpG or NcESA groups, supporting that the type of antigen

ather than adjuvant was responsible to induce a strong Th1-profileellular immune response.

After parasite challenge, it was surprisingly found thatcESA + CpG or NcESA groups presented the highest morbidity

cores, body weight changes and mortality rates, in additiono an important inflammatory reaction in the central nervousystem. These findings were afterward associated with signifi-antly higher brain parasite burden in these groups. It is possibleo speculate that immunization with NcESA over-induced IFN-�roduction, together with a strong cellular activation and weakumoral immune response. Similarly, it has been demonstratedhat NcMIC4 immunized mice or immunization procedures withNA encoding NcMIC1 showed increased mortality and cerebral
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nfection in mice upon challenge with N. caninum tachyzoites, asso-iated with a low antibody response prior to challenge [19,20].urther studies are required to determine the exact proteins and theechanisms by which NcESA induces the observed anti-protective

ffects.

D0. (B) Inflammatory score in the brain tissue from all surviving mice after challengeces (P < 0.05). Negative controls included brain tissue from non-immunized and

On the other hand, NLA + CpG or NLA groups exhibited thelowest morbidity scores and body weight changes, with no ani-mal succumbing to challenge infection. These parameters werealso associated with the lowest parasite load and mild or moder-ate inflammatory changes in the brain. Thus, the high protectionobserved in mice vaccinated with NLA + CpG or NLA could beassociated with an effective humoral immune response charac-terized by high levels of total IgG and IgG2a response and aprotective cellular immune response with an adequate balanceof pro-inflammatory and regulatory cytokines (IFN-�/IL-10 ratio).Accordingly, it has been well established that both humoral andcell-mediated immune responses are required for immunity to N.caninum infection [7]. Also, mice immunized with N. caninum lysateantigens incorporated into iscoms or mixed with Quil A followingchallenge infection exhibited mild or transient symptoms, reducedlesion scores and protection correlated with high IFN-� levels com-pared to mice immunized with parasite lysate only [12]. In contrast,BALB/c mice immunized with soluble N. caninum tachyzoite anti-gens entrapped in nonionic surfactant vesicles or administeredwith FCA had clinical neurological disease and increased numbersof brain lesions compared to groups of mice inoculated with adju-vants alone or non-immunized controls, following virulent parasitechallenge [46]. In addition, increased brain lesions were associatedwith increased IL-4 secretion and IL-4/IFN-� ratio in vitro as well asincreased IgG1/IgG2a ratio in vivo [46].


In the present study, control groups (CpG and PBS) presented 538

intermediate morbidity scores, body weight changes and mortality 539

percentages. Surprisingly, parasite load and inflammatory scores in 540

the brain tissue were higher in mice immunized with PBS in relation 541

to CpG group, which may indicate that the immune stimulating 542


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D.P. Ribeiro et al. / Vaccine xxx (2009) xxx–xxx 9

Fig. 7. (A) Cerebral parasite burden assessed by real-time PCR from all surviving mice after challenge with N. caninum NC-1 isolate. See Fig. 1 for abbreviations of groups andimmunization/challenge procedures. Negative controls included brain tissue from non-immunized and unchallenged mice, with no detectable parasite DNA (data not shown).D ative pN para× e sects

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ifferent letters indicate statistically significant differences (P < 0.05). (B) RepresentcESA + CpG (a), NcESA (b) and PBS (c) groups, showing strongly stained N. caninum100) represent circled areas in each panel. Negative controls included brain tissu

erum, and no staining was observed (data not shown).

ffect of TLR9 activation by itself may be a good target for therapiesgainst acute infections and clinical neosporosis.

Altogether, it may be concluded that CpG-ODN does promotehe resistance of immunized mice to high-load parasite challenge,hrough the induction of predominant IgG2a levels and effectiveh1-driven cellular immune responses, when combined with NLA.n contrast, vaccination with NcESA alone or combined with CpGesulted in a strong cellular immune response associated with highevels of IFN-� and inflammation, rendering mice more susceptibleo parasite challenge. Further studies focusing in the discovery of. caninum-specific TLR agonists need to be performed, since thelicited receptor recognition and triggered responses may lead toesign potential prophylactic and therapeutic vaccine candidatesgainst this pathogen.

cknowledgement

This study was supported by Brazilian Funding Agencies (CNPq,APEMIG and CAPES).

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