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Veterinary Parasitology 179 (2011) 7–13

Contents lists available at ScienceDirect

Veterinary Parasitology

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

recombinant DNA vaccine encoding C. andersoni oocyst wall proteinnduces immunity against experimental C. parvum infection

un Zhenga,1, Wenzhi Renb,1, Qingshan Panc, Qiuyue Wangd, I.A. Elfaki elhage,ianhua Lia,∗, Mingying Lia, Pengtao Gonga, Yingli Liua, Xichen Zhanga,∗

College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi‘an Road, Changchun 130062, ChinaExperimental Animal Center, Jilin University, 5333 Xi‘an Road, Changchun 130062, ChinaChina Agriculture University, Hebei Normal University of Science and Technology, Qinhuangdao 066000, ChinaKey Laboratory of Preventive Veterinary Medicine of Hebei Province, Hebei Normal University of Science and Technology, Qinhuangdao 066000, ChinaFaculty of Science, Zoology Department, Kordofan University, P.O. Box 160, El obied 517, North Kordofan, Sudan

r t i c l e i n f o

rticle history:eceived 9 October 2010eceived in revised form 12 February 2011ccepted 17 February 2011

a b s t r a c t

Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a causeof reduction of milk production in dairy cow. In this study, a novel chimeric DNA vac-cine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum wasdetermined. BALB/c mice were divided into 3 groups and immunized with DNA vaccineexpressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid

eywords:ryptosporidium andersoniNA vaccineocyst wall proteinross protectionryptosporidium parvum

containing the AB gene, respectively. After inoculation of 1 × 106 oocysts of C. parvum, thehumoral and cellular immune responses were detected. Experimental results showed thatthe recombinant plasmid can induce corresponding specific antibody response, simultane-ously influenced cellular immune responses, and provided greater protection rate (48.6%)than the other groups. These results indicated that chimeric DNA vaccine has a potential inCryptosporidium vaccine development.

. Introduction

Apicomplexan parasites of the genus Cryptosporidiumnfect the microvillus border of the gastro-intestinal (GI)pithelium of a wide range of vertebrates including humansSpano et al., 1998). Cryptosporidium parvum, a major causef severe diarrhea in young livestock, has emerged as a

idespread enteric pathogen in humans. A logical pro-

ression from passive immunization using colostrum, serar administration of mAbs or rAbs, is to develop a vac-ine to actively stimulate the immune system and induce

∗ Corresponding author. Tel.: +86 431 87981355;ax: +86 431 87981355.

E-mail addresses: xi.chen2010@hotmail.com, zhengjundk@163.comX. Zhang).

1 These authors contributed equally to this work.

304-4017/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.vetpar.2011.02.016

Published by Elsevier B.V.

protection. However, the lack of an in vitro culture sys-tem that supports the propagation of C. parvum life-cyclestages and the inability to separate intracellular stagesfrom the host epithelial cells makes the parasite derivedantigen(s) approach is not feasible for large-scale immu-nization (Wang et al., 2010). Cryptosporidium andersonihas been associated with gastritis, reduced milk yield andpoor weight gain in adult cattle, as well as high morbid-ity rates (Anderson, 1998; Lindsay et al., 2000). Recently,C. andersoni oocysts detected in humans using conven-tional methods were reported (Leoni et al., 2006; Guyotet al., 2001). Thus, development of effective and safe waysfor controlling Cryptosporidium infection in these animals

which play important roles in disease transmission iscrucial to human health, and vaccine is one of the strate-gies. Recently, DNA immunization has been studied as anovel strategy to elicit protection against Cryptosporidio-sis (He et al., 2004; Yu et al., 2010). However, so far, there

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8 J. Zheng et al. / Veterinar

is no effective protective vaccine available against thispathogen and more reports concentrate on diagnosis of thisdisease.

Several (flagellates, amoebae and ciliates) of proto-zoa exhibit rather simple asexual life cycles while others(coccidia and microsporidia) exhibit complex life cycleswith a sexual phase. However, all of protozoa have com-mon life-cycle strategies that involve leaving one host asan encysted form to infect another. The protective wallsof these encysted protozoa are often formed of proteinsand polysaccharides (Gerwig et al., 2002; Eichinger, 1997,2001; Arroyo-Begovich et al., 1980; Sterling and Arrowood,1993). To compare the differences in genetic diversityamong Cryptosporidium species, the Cryptosporidiumoocyst wall protein (COWP) gene have been studied (Katoet al., 2003; Pedraza-Diaz et al., 2000; Putignani et al., 1999;Spano et al., 1997). In the present study, oocyst wall proteinantigen of C. andersoni is investigated as vaccine candidatesin BALB/c mice, which are susceptible to C. parvum infec-tion, and evaluate its biological activity, so as to controlcryptosporidiosis in the future.

2. Materials and methods

2.1. Parasite and experimental animals

C. parvum oocysts (Changchun isolate, China, geno-typed as C. parvum-cattle genotype) were concentratedfrom naturally infected calf faeces using a one-step ethylether method. Briefly, 50 ml of fresh faeces suspensionwas mixed with 20 ml of ethyl ether, mixed vigorouslyfor 15 min, and centrifuged at 1000 × g for 15 min at 4 ◦C.After three times washing in phosphate buffered saline(PBS), the oocysts were centrifugated at 1500 × g for 10 minat 4 ◦C. Oocysts were further purified using discontinu-ous Sheather’s gradient centrifugation and cesium chloridegradient centrifugation as previously described (Kvác et al.,2003) and stored at 4 ◦C in 2.5% potassium dichromatefor futher studies. Before inoculation, the dichromate con-taining oocyst was washed with water, and the oocystconcentrations were adjusted to the levels desired (desiredlevels).

Oocysts of C. andersoni were obtained from adult cattle(6-year-old), from Jilin, China. Oocysts were then purifiedfrom cattle faeces by two rounds of ficoll centrifugation,leach-treated (5% bleach for 20 min at room temperature)and washed. The oocysts harvested were stored at 4 ◦C in2.5% potassium dichromate.

The mice used in this study were 6–8 weeks old femaleBALB/c mice (Center of Experimental Animals, Jilin Univer-sity, Changchun, China).

2.2. Construction of recombinant plasmids

Total RNA was isolated from freshly sporulatedoocysts using Trizol (Invitrogen). The AB gene seg-

ment (GenBank accession no. AB089289) was generatedfrom the cDNA of C. andersoni strains by PCR withprimers QF (5′-CCGGGATCCGCCGCCACCATGTTTACATTTTCAGGGAAGC-3′) containing BamHI restric-tion site (underlined) and QR (5′-CGCCTCGAGT

tology 179 (2011) 7–13

TACCTTGCAGTGTAAAATTTG-3′) containing XhoI restric-tion site (underlined), and cloned into the pMD18-Tvector. The 429-bp AB gene fragment was subcloned intothe BamHI/XhoI sites of pVAX1 vector. The eukaryoticexpression plasmid pVAX1-AB was identified by doubledigestion with BamHI/XhoI and the gene segment wassequenced in Shanghai Biotechnology Co., Ltd.

2.3. Sequence analysis of C. parvum COWP ABp

The ABp gene segment was generated from C. parvumstrains by RT-PCR with same primers motioned above ofAB gene, and cloned into the pMD18-T vector. The segmentof gene ABp was sequenced in TaKaRa Biotechnology Co.,Ltd., and the amino acid sequence alignment of AB and ABpgenes were performed.

2.4. Preparation of polyclonal antibodies against oocystsof C. andersoni

The purified oocysts of C. andersoni were frozen down inliquid nitrogen and thawed on ice for three cycles, furtherprocessed by ultrasound sonication (300 W, 5 min). Aftercentrifugation at 10,000 × g for 15 min, the supernatantwas collected as the antigen solution for immunization.Each BALB/c mouse was injected with 0.1 ml of anti-gen emulsified with equal volume of Freund’s completeadjuvant, and boost injection antigen with Freund’s incom-plete adjuvant after 2-week intervals. ELISA titer wasmeasured.

2.5. Cell culture and transfection

HeLa cells, obtained from Shanghai Institute of Bio-chemistry and Cell Biology, Chinese Academy of Sciences,were incubated at 37 ◦C in a 5% CO2 incubator in RPMI1640 supplemented with 10% FCS, 10 mM HEPES, 2 mMl-glutamine, 100 U/ml penicillin, 100 �g/ml streptomycin.HeLa cells were plated in 6-well plates at a densityof 3 × 105 per well 1 day before transfection. The cellswere transfected with 5 �g of pVAX1-AB by 5 �l of Lipo-fectAMINE2000 (Invitrogen, Rockville, MD) according tothe manufacturer’s instructions. After 48 h, the trans-fected cells were selected in medium supplemented with500 �g/ml of G418 (Promega) for a period of 2 weeks untilthe mock-transfected controls had died. The survival cellswere maintained in complete RPMI 1640 medium contain-ing 300 �g/ml of G418 until colonies appeared.

2.6. Indirect immunofluorescence assay of thetransfected cells

Two days after transfection, cells grown on glass cover-slips were washed three times by phosphate buffer saline(PBS) and then incubated with 3% BSA for 1 h at roomtemperature. Then cells were incubated with mouse anti-

C. andersoni serum at a dilution of 1:200 at 4 ◦C for 16 h.The coverslips were maintained with goat anti-mouse IgGantibody labeled by FITC diluted at 1:1000 for 40 min with-out light. After being washed in the dark, the fluorescencemicroscope was employed to observe the transcription of

y Parasitology 179 (2011) 7–13 9

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Fig. 1. Restriction endonuclease BamHI/XhoI digestion patterns. (A) Char-acterization of pMD18-T-AB by restriction enzyme digestion and analyzed

J. Zheng et al. / Veterinar

VAX1-AB in transfected cells. HeLa cells transfected withVAX1 performed as negative control.

.7. Western blotting

The transfected HeLa cells (3 × 105) were seeded into-well plates. Cells were collected on ice after 48 h ofulture. The cells were first washed twice with ice-coldBS and then re-suspended in 40 �l lysis buffer (Shang-ai Biotechnology Co., Ltd.). The lysates were incubatedn ice for 30 min, and centrifuged (12,000 × g at 4 ◦C) formin to obtain the cytosolic fraction. Samples were sep-rated by SDS–PAGE electrophoresis and transferred topolyvinylidene difluoride (PVDF) membrane. The blotsere then washed in Tris–Tween-buffered saline (TTBS,

0 mM Tris–HCl buffer, pH 7.6, 137 mM NaCl and 0.05%ween 20), blocked with 5% (wt/vol) nonfat dry milkvernight at 4 ◦C, and probed with mouse anti-C. andersonierum (1:100) and goat anti-mouse IgG HRP conjugatedntibody. Signals were detected using DAB.

.8. DNA immunization protocol

Female BALB/c mice, 16 in each group, were inoculatedntramuscularly in the tibialis anterior muscle with 50 �glasmid pVAX1-AB, pVAX1 and PBS as control respectively.wo identical doses were given, with a 2-week interval.lood was collected from tail vein at 7-day interval. Seraere separated and stored at −20 ◦C.

.9. Serum antibody responses in DNA vaccinated mice

Sera samples were collected from the tail vein at var-ous timepoints for serological tests and stored at −20 ◦C.ntibody levels were measured by ELISA in flat-bottomed6-well ELISA plates (Corning Costar) coated overnight at◦C with 100 �l crude C. andersoni antigens at 10 �g/ml in.1 M carbonate buffer pH 9.6. Plates were washed withBST (PBS pH 7.4 containing 0.05% Tween 20) and thenlocked with 5% bovine serum albumin (BSA) in PBS atoom temperature (RT) for 2 h. After washing with PBSThree times, sera at 1:200 diluted in 1% BSA-PBS weredded to corresponding wells and incubated at 37 ◦C forh. After washed with PBST for three times, the platesere incubated with HRP-labeled Goat anti-mouse IgG

ntibody (Beijing Biosynthesis Biotechnology Co., Ltd.)1:1000) at 37 ◦C for 1 h and washed again. Finally, sub-trate solution (100 �l) containing 15 �l 30% H2O2, 10 mlitrate-phosphate and 4 mg O-phenylenediamine (OPD)ere added. The reaction was stopped by 2 M H2SO4 and

he optical density (OD) value was read at 490 nm in aicroplate reader.

.10. Cellular immune response induced by DNA vaccines

The mice were killed 20 days after the last immu-ization. Lymphocyte subsets were analyzed as describedreviously (Aleixo et al., 1995). Briefly, fresh isolatedplenocytes were washed and resuspended in PBS at aoncentration of 1 × 106 ml−1. Cells were stained with PE

by electrophoresis. M: DNA Marker 2000; 1: pMD18-T-AB digested byBamHI/XhoI. (B) Characterization of pVAX1-AB by restriction enzymedigestion and analyzed by electrophoresis. M: DNA Marker 2000; 1, 2:pVAX1-AB digested by BamHI/XhoI.

conjugated anti-CD4 antibody (5 �g/ml) and FITC conju-gated anti-CD8 antibody (12.5 �g/ml) (PharMingen, SanDiego, CA) for 20 min at 4 ◦C. After 2 washes with PBS,cell suspensions were finally detected by flow cytometry(FACSan; Becton-Dickinson Immunocytometry Systems,Mountain View, CA).

2.11. Protective effect of the DNA vaccines

Each mouse was orally inoculated with 1 × 106 C.parvum oocysts in 0.5 ml water two weeks after the lastimmunization. Faeces were collected separately between1th and 21th days post-inoculation. 0.25 g of faeces fromeach mouse was homogenized with 750 �l water. Then4 ml sheather sucrose solution was added to each sampleand mixed. Oocysts in each faeces sample were counted ina cell counter (Sagodira et al., 1999).

2.12. Statistical analysis

Statistical analysis was performed using SPSS 16.0 soft-ware by variance (ANOVA) and Duncan’s multiple ranges.P < 0.05 was considered statistically significant.

3. Results

3.1. Plasmid DNA construction

The COWP AB gene was cloned into both pMD18-Tvector and eukaryotic expression vector pVAX1. Restric-tion endonuclease BamHI/XhoI digestion patterns showed

that the segment was corresponded to the expected sizeof 429 bp and it was inserted correctly into the vector(Fig. 1). The AB gene segment was sequenced by ShanghaiBiotechnology Co., Ltd. and compared the homology withthe COWP gene of C. andersoni (AB089289) in GenBank.

10 J. Zheng et al. / Veterinary Parasitology 179 (2011) 7–13

Fig. 2. Sequence analysis of Cryptosporidium parvum ABp oocyst wall protein (ROM) sequences. In both alignments different amino acids are marked withasterisks.

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anti-mo

T cells in vaccinated group were higher than that in thegroups treated with pVAX1 vector and PBS (P < 0.05). Theratio of CD4+/CD8+ increased simultaneously (data notshown).

Fig. 3. Expression analyses of recombinant plasmid pVAX1-AB in transtransfected with pVAX1. At 48 h post-transfection, the cells were fixed aagainst C. andersoni. followed by fluorescein isocyanate-conjugated goat

3.2. Comparing protein sequence identities

Comparison of the oocyst wall protein sequencebetween C. andersoni and C. parvum revealed that theCOWPs were conserved in Cryptosporidium. The aminoacid sequence alignment of AB from C. andersoni, and ABpfrom C. parvum shown in Fig. 2. There are three differentresidues marked in the figure. This result indicated that theAB gene could be used to construct recombinant plasmidagainst C. parvum.

3.3. Immunofluorescence staining

HeLa cells were transfected with the recombinant plas-mid pVAX1-AB. The expression of the fusion proteins weredetected by Immunofluorescence staining with mouseanti-C. andersoni serum and fluorescence labeled goat anti-mouse IgG. Strong green fluorescence was observed undera fluorescence microscope for cells transfected with therecombinant plasmids, and no fluorescence observed oncells transfected with pVAX1 vector. The results suggestedthat plasmids pVAX1-AB had been successfully expressedin HeLa cells (Fig. 3).

3.4. Western blotting

Western blot analysis using mouse anti-C. andersoni

serum and AP-labeled goat anti-mouse IgG showed thatHela cells transfected with recombinant plasmid resultedin the expressions of the expected 14.4 kDa recombinantprotein bands, whereas no band was detected on cellstransfected with pVAX1 vector (Fig. 4).

eLa cells. (A) HeLa cells transfected with pVAX1-AB and (B) HeLa cellsunofluorescence assays (IFA) were performed using an immune serum

use IgG. Fluorescent images were examined.

3.5. Serum antibody responses in DNA vaccinated mice

Specific antibody against C. andersoni was detected inthe experimental mice after the last immunization. Signif-icantly higher level of IgG was detected in the pVAX1-ABgroup then the groups immunized with pVAX1 plasmid andPBS (P < 0.05) (Fig. 5).

3.6. Cellular immune response induced by DNA vaccines

Cell-mediated immune responses induced by pVAX1-AB immunized mice and PBS controls are shown in Table 1.The proportions of splenocyte subsets in immunized micewere determined by flow cytometry 2 weeks after theboosting immunization. The percentages of CD4+ and CD8+

Fig. 4. Western blot analysis of recombinant plasmid pVAX1-ABexpressed in HeLa cells using mouse anti-C. andersoni serum antibodies.(1) Cell culture supernatant from pVAX1-AB transfected HeLa cells after48 h expression and (2) cell culture supernatant from control HeLa cellstransfected with empty plasmid pVAX1.

J. Zheng et al. / Veterinary Parasitology 179 (2011) 7–13 11

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ig. 5. Antibody responses induced by pVAX1-AB. Mice were immunized iose under the same conditions after 2 weeks. Sera were collected andean OD (±S.E., n = 10), the asterisks indicate significantly increased seru

.7. Protective effect of DNA vaccines

Mice were monitored for 21 days after inoculated with. parvum oocysts by examining the numbers and durationf parasite oocyst excretion. Mice in pVAX1-AB experimen-al group excreted less oocysts than the groups of pVAX1ector and PBS control (P < 0.05). Mice in the pVAX1-ABroup have a 48.6% reduction in the level of oocysts shed-ing (Fig. 6).

. Discussion

Species within the genus Cryptosporidium are obligaterotozoan parasites and cause infections in humans, farmnimals, companion animals, laboratory animals as well asn wild mammals (Fayer, 2004). Previous studies demon-trated that cattle could be infected with 10 differentryptosporidium species or genotypes at least, neverthe-

ess, C. parvum and C. andersoni were commonly detectedn cattle worldwide (Kvác et al., 2006; Fayer et al., 2008);

. parvum is dominant in pre-weaned calves (<2 months)Santin et al., 2004; Fayer et al., 2006) while C. andersoni areonsidered to be predominantly infectious to post-weanedalves (2–11 months) (Santín and Trout, 2007). C. ander-oni also infects Bactrian camel (Camelus bactrianus), Bobak

able 1hanges of CD4+ and CD8+ T cells in splenocytes from immunized mice.a

Groups CD4+ (%,mean ± S.E.,n = 10)

CD8+ (%,mean ± S.E.,n = 10)

pVAX1-AB 30.52 ± 0.4431 13.92 ± 0.5502pVAX1 21.96 ± 0.9698 11.98 ± 1.1041PBS 18.76 ± 0.7726 10.65 ± 0.8871

a Mice were i.n. vaccinated pVAX1-AB, pVAX1 and PBS. FCM determinedhe percentage of splenocyte subsets in immunized mice 2 weeks after thenal immunization.

pVAX1-AB, pVAX1 and PBS. pVAX1 and PBS as control, receiving a boosterby ELISA. A490 of sera diluted 1:200 is shown. Each bar represents the

ody titers compared with the PBS control (P < 0.05).

marmot (Marmota bobac), European wisnet (Bison bona-sus) (Ryan et al., 2003) and Mongolian gerbils (Merionesunguiculatus) (Koudela et al., 1998). The infection has beenalso described in HIV-positive patients (Guyot et al., 2001)and humans without immunocompromising illness (Leoniet al., 2006). Lately some experimental rodent infectionswere reported with novel isolates of C. andersoni, but it isnot clear whether the establishment of the infection is dueto strain variation or due to natural property of the species(Satoh et al., 2003; Matsubayashi et al., 2004, 2005; Kvácet al., 2007). But in this study, C. andersoni isolated from acattle farm was not infected SCID mice and BALB/c mice.So protective effect of the DNA vaccine against C. andersonishould be explored further.

C. andersoni oocyst wall protein is main component ofoocyst wall which play an important role when the para-sites pass through the external environment to get to thenext host. Since relatively little is known of the detailsof protozoan encystment, the unknowns leave us won-dering about the validity of such a strategy. Disruptingthe encystment process could lead to eventual cell deathdue to nutrient depletion (Jarroll and Sener, 2003). So,in this study, C. andersoni oocyst wall protein was inves-tigated as vaccine candidates in BALB/c mice. Our datashowed that the DNA vaccines induced significant anti-body response, simultaneously influenced cellular immuneresponses compared with control mice. Furthermore, thereduction of oocysts shedding was also significant, indi-cating a partial protection against C. parvum infection inBALB/c mice.

The gene coding for the Cryptosporidium oocyst wallprotein is one of the commonly used targets of molecular

tools for genotyping Cryptosporidium parasites. Character-ization of the COWP gene revealed genetic differencesamong human and bovine Cryptosporidium isolates (Xiaoet al., 2000). It was interesting that the homology of ABp andAB gene was unexpectedly reached 97%. The result of cross

12 J. Zheng et al. / Veterinary Parasitology 179 (2011) 7–13

Fig. 6. Protective results of the mice immunized with recombinant plasmids against C. parvum challenge. All the mice were infected with 1 × 106 C. parvumion was

oocysts in the seventh week after immunization and their oocysts excret

mice could partially prevent C. parvum infection.

immunity indicated that serum IgG level, the percentagesof CD4+ T and CD8+ T cells were all elevated in the experi-mental group. The protective effect of the pVAX1-AB groupwas also better than the groups of pVAX1 and PBS. Mice inthe pVAX1-AB group have a 48.6% reduction in the levelof oocysts shedding. C. andersoni COWP antigen gene caninduce specific immune responses and partial protect micefrom challenge with C. parvum oocysts according presentstudy.

Even though the recombinant plasmid pVAX1-AB inour study did not provide complete protection against C.parvum challenge, we developed a novel candidate targetfor Cryptosporidium vaccine development. With the devel-opment of resistance of anti-Cryptosporidium drugs existsand is likely to increase and given that currently no reli-able treatments exist for some of these infections, effortsto find new targets for chemotherapy must be continued.This study can provide a new direction for research of anti-Cryptosporidium drugs.

Acknowledgements

This work was supported by High TechnologyResearch and Development Program (863) of China(No. 2006AA10A207), National Key Technology Researchand Development Program of China (No. 2007BAD40B05)and National Natural Science Foundation of China (No.30400324). We take many thanks to supervisors andothers who provided laboratory assistance and helpfulsuggestions and advice. We declare that the experimentscomply with the current laws of China where they wereperformed.

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