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Contents lists available at ScienceDirect

Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 6 7

Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine

3 4 5 8

Q1

Tran Duc Hoan a,⇑, Doan Thi Thao a, Javaid Ali Gadahi b a

9 10

b

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China Faculty of Animal Science and Veterinary Medicine, Bacgiang Agriculture and Forestry University, Vietyen District, Bacgiang Province, Viet Nam

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h i g h l i g h t s

1 5 316 0 17 18

 IFN-c concentration was increased to

19

 Levels of IL-10 and IL-17 were

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increased in experimental chickens.  TGF-b and IL-4 levels are higher in vaccinated than unvaccinated chickens.  pVAX1-EbAMA1 could increase serum IgG antibody and cytokine concentrations.  pVAX1-EbAMA1 could induce protection against E. brunetti infection.

g r a p h i c a l a b s t r a c t

the highest level against EbAMA1.

27 28 29

pVAX1 control pET-AMA1 pVAX1-AMA1

OD (450 nm)

0,4

0,3

0,2

0,1

0 Week 0

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Serum levels of EbAMA1 specific IgG in chickens immunized with DNA vaccines IFN-concentration Serum IgG c

3

b

400.00

b

350.00

2.5

300.00 2

(pg/ml)

b

1.5 1

250.00 200.00 150.00 100.00

0.5

a

a

50.00

0

a

a

TE control

pVAX1 control

0.00 TE control

pVAX1 control

Recombinant pETAMA1

pVAX1-AMA1

Recombinant pET-AMA1

pVAX1-AMA1

TGF - concentration 0.35

IL-4 concentration

b

b

Recombinant pETAMA1

pVAX1-AMA1

12.00

0.30

10.00

a

a

(pg/ml)

0.25 0.20 0.15 0.10

c

c

Recombinant pET-AMA1

pVAX1-AMA1

bc

ab

8.00 6.00 4.00 2.00

0.05

0.00

0.00 TE control

pVAX1 control

TE control

pVAX1 control

IL-17 concentration

IL-10 concentration 2500

70.00 b

60.00

d

b c

2000

50.00

(pg/ml)

26

Unchallenged control TE control

0,5

OD (450nm)

24 25

Epression of pVAX-EbAMA1 in chicken muscle by western blotting

0,6

(ng/ml)

23

Epression of pVAX-EbAMA1 in chicken muscle by RT-PCR

(pg/ml)

21 22

40.00 30.00 20.00

a

a

1500 1000

b a

500

10.00 0.00

0

TE contol

pVAX1 control Recombinant pET-AMA1

pVAX1-AMA1

TE control

pVAX1 control

Recombinant pET-AMA1

pVAX1-AMA1

Serum levels of EbAMA1 specific IgG and cytokine in chickens

32

a r t i c l e 3 4 4 6 35 36 37 38 39

i n f o

Article history: Received 3 December 2013 Received in revised form 9 March 2014 Accepted 23 April 2014 Available online xxxx

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Keywords: Eimeria brunetti Apical membrane antigen protein-1 gene Chicken DNA vaccine

a b s t r a c t This study aimed to determine the changes of cytokines, specific serum IgG and several parameters in chickens vaccinated with DNA vaccine encoding Eimeria brunetti apical membrane antigen-1 (EbAMA1) antigen. Two-week-old chickens were divided into five groups (four groups for experiment) randomly. Experimental groups of chickens were immunized with DNA vaccine while control group of chickens were injected with pVAX1 plasmid alone or TE buffer solution. All immunizations were boosted 2 weeks later. The EbAMA1 specific IgG antibody responses were measured at weeks 1–6 post-second immunizations and several parameters were also identified. The result showed that the antibody titers in chickens vaccinated with DNA vaccines were significantly different from those of the control groups 1 week after the second immunization and reached the maximum values 3 weeks post-second immunization. IFN-c concentration was increase the highest level against EbAMA1 of all chickens vaccinated with vaccines up to 56-fold, follow by the specific IgG antibody levels were increased 10–17-fold compared with those of TE solution and plasmid (pVAX1) control chickens 1–6 weeks post-second immunization. In case of the levels of IL-10 and IL-17 was increased in experimental chickens with 4–5-fold. Even though it was statistically significant, TGF-b and IL-4 levels are higher in vaccinated than unvaccinated chickens. The results suggested that DNA vaccines encoding E. brunetti apical membrane antigen-1 (EbAMA1) could increase serum specific IgG antibody and cytokines concentration and could give protection against E. brunetti infection. Ó 2014 Published by Elsevier Inc.

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⇑ Corresponding author. Fax: +84 2403 874 604. E-mail address: [email protected] (T.D. Hoan). http://dx.doi.org/10.1016/j.exppara.2014.04.015 0014-4894/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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1. Introduction Coccidia are protozoan unicellular organisms of the phylum apicomplexa that parasite most major lineages of vertebrates as well as invertebrates (Fayer, 1980). Which is an intestinal infection caused by apicomplexan protozoa possess of at least seven different species of Eimeria (Conway and Elizabeth, 2007; McDougald Q3 and Fitz-Coy, 2008). A variety of strategies have been used to reduce the negative impact of coccidiosis, including prophylaxis with anti-coccidial drugs, selection of disease resistant chicken strains, and augmentation of immunity (Caron et al., 1997; Lillehoj, 1994; Pinard-Van Der Laan et al., 1998; Yun et al., 2000b). None of these methods are without drawbacks. Coccidiosis has been estimated to be responsible for monetary losses greater than $3 billion annually (Williams, 1999). Approximately 80% of this cost is due to the direct effects on mortality, weight gain and feed conversion, and 20% is due to the cost of chemoprophylaxis. So far, relatively several chicken cytokines had been described. The first of these were isolation and characterization included interferon (IFN)-c, interleukin (IL)-2 and transforming growth factor (TGF-b). However, with the advent of the chicken genome project, a number of chicken cytokine and chemokine genes have been discovered (Hughes and Bumstead, 2000; Schneider et al., 2000; Sick et al., 2000; Avery et al., 2004; Degen et al., 2004; Hong et al., 2006a; Kaiser et al., 2005; Min and Lillehoj, 2002, 2004; Read et al., 2005; Rothwell et al., 2004; Wang et al., 2005). Cloning of these genes has led to the development of a comprehensive array of reagents for investigating avian innate and acquired immune responses at the molecular and cellular levels that was not possible only a few years ago (Swaggerty et al., 2000). In recent years, DNA vaccines have been used as a novel delivery system to achieve specific immune responses for its ability to provoke both humoral and cell-mediated immune responses (Gurunathan et al., 2000; Liljeqvist and Ståhl, 1999; Oshop et al., 2002; Whalen and Davis, 1995). Mean-while, considerable efforts have been made to develop DNA vaccines against coccidiosis (Ding et al., 2005; Min et al., 2001). Cytokines have the capacity to regulate host immune responses and co-delivery of cytokines as adjuvants can enhance the potential for DNA vaccines to induce broad and long-lasting humoral and cellular immunity (Egan and Israel, 2002). Interleukin (IL)-2 and interferon (IFN)-c are two of the extensively studied cytokine adjuvants (Kim et al., 2001; Wassef and Plaeger, 2002). Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells Apical membrane antigen-1 (AMA1), which is secreted by micronemes, was first identified as a conserved antigenic protein in the malaria parasite Plasmodium knowlesi (Thomas et al., 1984). In addition to rhoptry neck proteins (RONs), AMA1 is involved in the formation of the moving junction complex, which is a circumferential zone that moves backward and eventually pinches the PV from the host cell membrane (Alexander et al., 2005; Besteiro et al., 2005; Curtidor et al., 2011; Lamarque et al., 2011; Santos et al., 2011). Plasmodium falciparum AMA1 (PfAMA1) has been demonstrated to induce protective immunity against the parasite challenge in animal models (Remarque et al., 2008). Recently, AMA1 has been identified as immuno-protective proteins from other apicomplexan parasites, such as Babesia, Toxoplasma, Neospora and Theileria (Donahue et al., 2000; Gaffar et al., 2004; Tonukari, 2010; Zhang et al., 2007). No paper has been reported about the DNA vaccine encoding Q4 apical membrane antigen (AMA) in any parasite up to now. In this study, we described for the first time the analysis of specific serum IgG, cytokine levels and several parameters in chickens vaccinated with DNA vaccine encoding apical membrane antigen-1 (EbAMA1)

of Eimeria brunetti isolated from an outbreak sample in the Jiangsu Province, China.

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2. Materials and methods

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2.1. Animals and parasites

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New-hatched, coccidiosis free, white chickens (commercial breed) were obtained from Jiangsu, PR China and reared in clean brooder cages. Birds were screened periodically for their Eimeria infection status by microscopic examination of the feces and also by the polymerase chain reaction (PCR) method using specific primers (Haug et al., 2007). The birds were provided with coccidiostat-free feed and water ad libidum. Birds were shifted to an animal containment facility prior to challenge with virulent oocysts. Sporulated oocysts of E. brunetti JS strain were isolated from the Q5 Jiangsu Province of China and maintained in the Laboratory of Veterinary Molecular and Immunological Parasitology, stored in 2.5% potassium dichromate solution (K2Cr2O7) at 4 °C and passages through chickens every 3 months. For infections, sporulated oocysts were washed 3 times with phosphate buffered saline (300 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM NaH2PO4) to remove potassium dichromate (Shirley, 1995), then oocysts were purified by treatment with 5.57% sodium hypochlorite (NaClO) and incubated on ice for 30 min (Zhao. et al., 2001) followed by extensive washing several times with de-ionized water, the oocysts concentrations were adjusted to the desired levels and used immediately.

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2.2. Chicken immune sera against E. brunetti

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For raising polyclonal sera against E. brunetti, 2-week-old birds were inoculated with 1.0  105 sporulated oocysts orally and 1 week later 1.0  105 sporulated oocysts were boosted with oral inoculation. These oocysts were deposited directly into the bird’s crop using a catheter. Blood was collected 7 days after the booster dose. Serum was separated from the blood by centrifugation and stored at 20 °C until further use.

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2.3. Total RNA extraction

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Total RNA was isolated from sporulated oocysts of E. brunetti described (Carson et al., 2012; Maniatis et al., 1982; Sambrook et al., 2002). Briefly, the oocysts were grounded using a pre-chilled mortor and pestle, then Trizol (Invitrogen) was added and homogenized. Tri-chloromethane was added then RNA was precipitated from the supernatant by the addition of isopropyl alcohol. RNA pellets were dried, resuspended and washed by 70% ethanol. Pellet were suspended in DEPC water and used in subsequent cDNA preparations immediately.

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2.4. Reverse transcription and amplification of cDNA (RT-PCR)

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cDNA was prepared from total RNA using Thermoscript reverse transcriptase (Invitrogen, USA) according to the manufacturer’s instructions, and specific reverse primer. EbAMA1 cDNA was amplified using the following primer pair as follows:

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Forward primer, 50 -GAATTCATGTGTCGGTTGAGAACTGC-30 Reverse primer, 50 -AAGCTTTCAATAGTCTTGGTCTACAA-30

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PCR reaction and amplification were performed using the automated cycler (Bio-Rad, CA, USA). PCR products were separated by electrophoresis and photographed by a digital camera (CSE-0028, Cybertech, Berlin, Germany). The amplification products were

Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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recovered and purified using TaKaRa Agarose gel DNA purification Kit version 2.1, according to manufacturer’s instructions. DNA polymerase with proof reading activity (Proof Start; Qiagen, Germany) was used for the amplification of EbAMA1 cDNA. The RT-PCR amplified EbAMA1 cDNA was sequence verified and sub-cloned into bacterial expression vector pET28a (+) (Novagen, USA) using EcoRI and HindIII restriction enzymes (shown underlined in the primer sequences). The EbAMA1 clone in pET28a (+) was used to transform chemically competent BL21 DE3 Star Escherichia coli strain (Invitrogen, USA).

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2.5. Construction of the recombinant eukaryotic expression plasmids

3

(Millipore, USA) and then the Western blot was performed to detect the AIM proteins with a 1:30 dilution of E. brunetti chicken anti serum as primary antibody.

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2.8. Determination of serum antibody level by enzyme-linked Immuno Sorbent assay (ELISA)

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The 1656 bp ORF of EbAMA1 was first ligated with the eukaryotic expression vector to evaluate its immunogenicity. The pMD19T plasmid containing EbAMA1 ORF and the pVAX1 vector (Invitrogen, Life Technologies) were cleaved with EcoRI and the XhoI enzyme was then inserted into the pVAX1 vector. Recombinant vector pVAX1-EbAMA1 was digested with the same restriction enzymes above and sequenced by Invitrogen bio-tech (Shanghai Huajin Bio-tech Co. Ltd., PR China). The recombinant plasmids pVAX1-EbAMA1 acting as DNA vaccines were prepared using Qiagen Plasmid DNA Mid Kit (Qiagen, USA), according to the manufacturer’s instructions. The eluted products were dissolved in TE buffer and diluted up to a concentration of 1 lg/ll, and stored at 20 °C until required.

The IgG antibody levels against E. brunetti soluble antigen in the serum samples were determined by ELISA as described (Lillehoj et al., 2005a). Briefly, flat-bottomed 96 wells of plate (Marxi-Sorp, Nunc, Denmark) were coated overnight at 4 °C with 100 ll per well of the soluble antigens of E. brunetti in 0.1 M sodium bicarbonate buffer. The plates were washed with PBS containing 0.05% Tween20 (PBST) and blocked with 5% bovine serum antibody (BSA) in PBST for 2 h at 37 °C, followed by incubating for 2 h at 37 °C with 100 ll of the serum samples with a 1:100 dilution in PBST with 5% BSA in duplicate. After washing three times, the plates were incubated for 2 h at room temperature with a 100 ll/well of horseradish peroxidase-conjugated anti-chicken IgG antibody (Sigma) with a 1:2000 dilution with PBST in 5% BSA. ELISA was developed by chromagen 3,30 ,5,50 -tetramethylbenzidine (TMB, Sigma, USA). The plates were read at 450 gm wavelength in a micro-titer plate reader (ELx 808, Bio-Tek, UK). Antibody titers in the serum were reported as maximum sera dilution showing five times the average OD450 gm of pre-immune sera (Nardelli et al., 1997). The titer values were expressed as log10 values.

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2.6. Recombinant protein and DNA vaccine

2.9. Determination of serum cytokine concentration

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The recombinant EbAMA1 was prepared in the Laboratory of Veterinary Molecular and Immunological Parasitology, Nanjing Agricultural University, China as follows: the EbAMA1 gene was cloned into prokaryotic expression vector pET28a (+), and transformed into the E. coli BL21 (DE3) strain. The recombinant protein was expressed by inducing with IPTG (Sigma–Aldrich, USA). Purification of the recombinant protein was performed as previously described (Goto et al., 2003). Briefly, the inclusion body was washed with PBS 1% Triton X-100, solubilized in buffer containing 8 M urea and refolded by drop-wise dilution. The DNA vaccine pVAX-AMA1 was constructed using the eukaryotic expression vector pVAX1 as described (Song et al., 2010). The plasmid pVAX-EbAMA1 contained the open reading frame (ORF) of 1656 nucleotides of E. brunetti apical membrane antigen gene and encoded a protein of 64.7 kDa.

The concentrations of Immunoglobulin (IgG), interferon-c (IFN-c), tumor growth factor-b (TGF-b) interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-17 (IL-17) in serum were detected by utilizing an indirect ELISA with the ‘‘chick cytokine ELISA Quantitation Kits’’ of CUSABIO life science, USA) in duplicate, according to manufacturer’s instructions.

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2.10. Immunization and challenge experiment

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Fourteen-day-old chickens were weighed and randomly distributed into five groups of 30 each as shown in Table 1. Chickens in the unchallenged control group and challenged control group were injected with TE buffer (10 mM Tris–HCl pH 8.0 and 1 mM EDTA) at the same injection site. Experimental groups were respectively injected by intramuscular leg with 100 lg of plasmids pVAX1EbAMA1 and pET28a-EbAMA1 at 14 and 21 days of age. The pVAX1 plasmid was also used as the vector control group. At 28 days of age, all chickens except the unchallenged control group were inoculated orally with 1.0  105 sporulated oocysts of E. brunetti Jiangsu strain. The challenge dose was originally determined by preliminary testing. Seven days after challenge, all chickens were weighted and slaughtered for ileum and cecum collection. To determine the cytokine and antibody levels in serum, 10 chickens from each group were bled by cardiac puncture at 7 days after the second immunization.

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2.11. Evaluation of immune protection

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The efficacy of immunization was evaluated on the basis of lesion score, body weight gain, oocyst output, oocyst decrease ratio and anti-coccidial index (ACI). Body weight gain of chickens in each group was determined by the body weight of the chickens at the end of the experiments subtracting the body weight at the time of challenge. Lesion scores were evaluated as described previously (Johnson and Reid, 1970). Additionally, the ileum and cecum content for each group was collected separately and oocysts per gram of content (OPG) were determined using the McMaster’s

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2.7. Detection of the expression of proteins encoded by plasmids DNA in vivo by reverse transcription polymerase chain reaction (RT-PCR) assay and Western blot analysis Experimental chickens were injected intramuscularly in the leg muscle with 100 lg of recombinant plasmid pVAX1-EbAMA1. After Q7 one week post immunization , injected tissues were collected and extraction of total RNA was done. The contaminating genomic DNA or plasmid injected was removed by treating with RNase-free DNase I (TaKaRa, China). RT-PCR assays were performed with cloning primer pairs of the EbAMA1 gene as above. The PCR products were identified by electrophoresis onto 1% agarose gel. Western blot detection was performed as described by (Xu et al., 2008). Briefly, seven days after challenge, injected muscles were ground and treated with ice-cold RIPA solution (0.1 M phenyl-methyl-sulfonyl fluoride, 150 mM sodium chloride, 1% Nonnidet P-40, 0.1% SDS, and 50 mM Tris–HCl). Meanwhile the same site muscles from non-injected and pVAX1 plasmid Q8 injected chickens were collected for controls. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a nitrocellulose membrane

Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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Table 1 Effects of EbAMA1 against E. brunetti challenge on different parameters.

Q18

Groups

Average body weight gain (g)

Mean lesion score (mean ± SD)

Oocyst output (105) (mean ± SD)

Oocyst decrease ratio (%)

Anti-coccidial index (ACI)

Unchallenged control Challenged control pVAX1 control pVAX1-EbAMA1 Recombinant EbAMA1 protein

132.43 ± 9.97a 78.28 ± 16.43c 89.71 ± 15.59c 123.82 ± 12.63a 106.88 ± 21.07b

0d 2.43 ± 0.62a 2.27 ± 0.53a 1.17 ± 0.79c 1.97 ± 0.72b

0e 5.07 ± 2.07a 3.86 ± 1.47b 1.29 ± 0.73d 2.65 ± 1.15c

100 0 23.89 74.55 47.74

200 131 142 178 160

Body weight gain, lesion score in ileum and cecum, fecal oocyst output, oocyst decrease ratio and anti-coccidial index (ACI) were expressed as means ± SD and were performed using the SPSS statistical package (SPSS for Window 20.0, SPSS Inc., Chicago, IL, USA). Differences among groups were tested with the one-way ANOVA Duncan test, mean values (p-value of less than 0.05 was considered significant).

EbAMA1 gene (Fig. 2). In contrast, no corresponding band was detected in the muscle of chickens injected with TE solution and pVAX1 plasmid control.

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counting technique (Subramanian et al., 2008). Oocyst decrease ratio was calculated as follows: the number of oocysts from the challenged control chickens  vaccinated chickens/the challenged control chickens  100%. ACI was calculated as follows: (relative rate of weight gain + survival rate)  (lesion value + oocyst value).

3.2. IgG and cytokine levels in sera of immunized chickens

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2.12. Statistical analysis

334 The serum EbAMA1-specific IgG levels in chickens following 335 vaccination are shown in Fig. 3. The anti-EbAMA1 antibody titers 336 were higher in chickens vaccinated with DNA vaccines as com337 pared to TE control and plasmid (pVAX1) control chickens 1– 338 6 week post second immunization (P < 0.05). Antibody titers 339 increased highly in week 2 and started reducing at week 5 post 340 second immunization. Chickens vaccinated with pVAX-EbAMA1 341 developed the highest antibody titer compared with the other 342 three groups. Non-specific antibody was detected in TE control 343 and pVAX1 plasmid control chickens throughout the experiment 344 (Fig. 4). 345 As depicted in Fig. 4, serum from chickens immunized with 346 recombinant pVAX-EbAMA1 plasmid and recombinant EbAMA1 347 protein showed significantly high levels of IgG antibody 348 (p < 0.05) compared to those of controls, IFN-c concentration was 349 increased to the highest level against EbAMA1 of chickens immu350 nized with vaccines. On the other hand, significantly higher levels 351 of IL-10 and IL-17 were observed in chickens immunized with 352 recombinant pVAX-EbAMA1 plasmid and recombinant EbAMA1 protein compared to the control groups. In case of the levels of Q10353 354 TGF-b and IL-4 levels are higher in vaccinated than unvaccinated 355 chickens but between two groups recombinant pVAX-EbAMA1

305 306 307 308

311 312 313 314 315 316

Body weight gain, lesion score, fecal oocyst output, oocyst decrease ratio and anti-coccidial index (ACI) were expressed as means ± SD and were performed using the SPSS statistical package (SPSS for Window 20.0, SPSS Inc., Chicago, IL, USA). Differences among groups were tested with the one-way ANOVA Duncan test, Q9 mean values (p-value of less than 0.05 was considered significant).

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3. Results

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3.1. Identification of DNA vaccine and the expression of EbMIC2 gene in vivo

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A fragment of approximately 1656 bp of recombinant pVAX1EbAMA1 plasmid was identified by digestion with EcoRI and XhoI. This revealed that DNA vaccine pVAX1-EbAMA1 was successfully constructed. Results of RT-PCR indicated that the target fragment of EbAMA1 was determined from muscle RNA samples of chickens injected with pVAX1-EbAMA1 (Fig. 1). Non-specific bands were detected in non-injected control and pVAX1 plasmid control samples. Western blot detection of the muscle of chickens injected with pVAX1-EbAMA1 indicated a prominent band of 64.7 kDa, which revealed the expression of

1

3

2

1

M

3

M 72 55

1656 bp

2

64.7 kDa

2000 1000 750 500 250 100

Fig. 1. Detection of expression of pVAX-EbAMA1 in chicken muscle by RT-PCR. Lane M – DL2000 DNA marker. Lane 1 – pVAX1-EbAMA1 injected muscles showing transcription of EbAMA1. Lane 2 – pVAX1 plasmid injected chicken muscle. Lane 3 – The negative control.

Fig. 2. Detection of expression of pVAX-EbAMA1 in chicken muscle by Western blotting analysis. Lane M – Protein molecular marker. Lane 1 – The TE control. Lane 2 – The pVAX1 control. Lane 3 – The muscle sample from chicken injected with pVAX-EbAMA1 and probed with serum from chickens experimentally infected with E. brunetti as primary antibody.

Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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Unchallenged control TE control

0,6

pVAX1 control pET-AMA1 pVAX1-AMA1

0,5

OD (450 nm)

0,4

0,3

0,2

0,1

0 Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Fig. 3. Serum levels of EbAMA1 specific IgG in chickens immunized with DNA vaccines. Chickens were immunized intramuscularly with TE solution (TE control), or pVAX1 plasmid (pVAX1 control) pET-EbAMA1 recombinant or pVAX-EbAMA1. At weeks 1–6 post-second immunization, blood was collected by cardiac puncture and anti-AMA1 serum antibody levels were determined by ELISA. Each bar represents mean ± S.D. (n = 6). Bars with different small letters are significantly different (P < 0.05).

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plasmid and recombinant EbAMA1 protein was not statistically significant (p < 0.05).

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3.3. Protective effects of DNA vaccination against E. brunetti challenge

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The immunizing efficacies of the vaccines are described in Table 1. In this study, no chicken died of coccidial challenge in any group. There were no significant differences among the 4 groups of the body weights of chickens at the days of immunization and challenge. The body weight gain of chickens immunized did not significantly differ with that of the unchallenged control group (p < 0.05), but was significantly reduced in challenge control and pVAX1 control groups compared with the unchallenged control group (p < 0.05). Chicken immunized with recombinant EbAMA1 and pVAX1-EbAMA1 did not show significantly enhanced weight gains. Immunized chickens with pVAX1-EbAMA1 were significant in lower oocyst output and higher oocysts decreased the ratio as compared with the pVAX1 vaccine alone (p < 0.05). Significant alleviations in ileum lesion score were displayed in immunized chickens as compared with those of the challenged control group and pVAX1 control group (p < 0.05). The result of the chicken group immunized with pVAX1-EbAMA1 in ACI was 178, higher than that of the recombinant EbAMA1 protein vaccinated group (160).

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4. Discussion

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Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites and is a transmembrane protein that is secreted from the microneme organelles during invasion of host cells onto the parasite surface from where it is proteolytically cleaved by a mutli-functional membrane-bound serine proteinase that is also responsible for the shedding of other parasite surface molecules (Jiang et al., 2012). In the current study, the immune parameters containing serum EbAMA1 specific IgG levels and the systemic and local cytokine mRNA expression were assessed to evaluate vaccination efficacy. DNA vaccines stimulated the production of serum EbAMA1 specific IgG levels and increased mRNA levels of chicken IFN-c, TGF-b, IL-4, IL-10 and IL-17. Taken together,

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380 381 382 383 384 385 386 387 388 389 390

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391 these results suggested that DNA vaccines constructed in our study 392 may induce immunity against coccidiosis by enhancing humoral 393 and cell-mediated immunity. 394 The role of humoral immunity during coccidiosis was debatable 395 with most evidence pointing to a minor function (Lillehoj and 396 Trout, 1996; Yun et al., 2000a). However, a number of recent stud397 ies have demonstrated that antibodies do play an important role in 398 immunity against coccidiosis (Belli et al., 2004; Constantinoiu 399 et al., 2008). Combined immunization with 3-1E protein with 400 immunity-related cytokine genes induced higher serum antibody 401 response and better protective immunity against coccidiosis than 402 3-1E alone (Ding et al., 2004). 403 Cytokines are a category of signaling molecules that mediate 404 and regulate immunity, inflammation, hematopoiesis, and many 405 other cellular processes, forming a cytokine network. Cytokines 406 were initially identified as products of immune cells that act as 407 mediators and regulators of immune processes but many cytokines 408 are now known to be produced by cells other than immune cells 409 and they can show effects on non-immune cells as well (Paige 410 and Jennifer, 2011). Cytokines play an important role in the com411 munication between cells of multicellular organisms. As intercellu412 lar mediators, they regulate survival, growth, differentiation and 413 effector functions of cells. 414 In this report, we demonstrated that chickens vaccinated with 415 DNA vaccine pVAX-AMA1 and recombinant protein pET28a-AMA1 416 exhibited significantly greater than enhanced anti-body levels compared with water control and plasmid control. The antibody Q11417 418 titers in groups vaccinated with pVAX-EbAMA1 and pET28a-AMA1 419 were significantly greater than those of group immunized with all 420 control groups (P < 0.05). The results indicated that immunization 421 with a recombinant E. brunetti AMA1 in conjunction with chicken 422 IFN-c enhanced the antibody levels of chickens and the antibody 423 response might play a role in the protection of chickens against 424 coccidiosis. Theoretically, IFN-c inhibits the proliferation of 425 Th2-type cells involved in B-cell activation. Moreover, the antibody 426 levels in groups vaccinated with pVAX-AMA1 and pET28a-AMA1 427 showed significant differences during weeks 1–6 post second 428 immunization and in the case of three control groups showed no 429 differences (P > 0.05). 430 In the present study, IFN-c transcript levels in chickens vacci431 nated with DNA vaccines were significantly increased compared 432 with the control groups (P < 0.05). Theoretically, the detected 433 mRNAs of IFN-c should be included that are induced and expressed 434 by the vaccines. The levels of IFN-c mRNA expressions increased 435 56-fold compare with the control group, there are no differences 436 between groups injected with pVAX-EbAMA1 and pET28a437 EbAMA1. These results indicated that the transcript levels of IFN438 c detected in the study were both induced and expressed by the 439 vaccines. However, how to differentiate the cytokines expressed 440 and induced by the vaccines needs to be further researched. These 441 results indicated that Th1 cells might be activated by the DNA vac442 cines and secrete increased cytokines. It was reported that the 443 responses of T-cells against Eimeria were partially controlled and 444 regulated by cytokines (Cornelissen et al., 2009; Dalloul and 445 Lillehoj, 2005; Lillehoj, 1998). These cytokines can be distinguished 446 into Th1-type or Th2-type according to their role in the immune 447 response (Hong et al., 2006b). The Th1-type cytokines, such as 448 IFN-c and IL-2, are responsible for classic cell-mediated functions 449 and seem to be dominant during coccidiosis (Cornelissen et al., 450 2009; Lowenthal et al., 1997). IFN-c production in chickens has 451 been used to measure immune responses to Eimeria antigens and 452 chicken IL-2 could promote the growth of cd TCR+ cells and 453 enhance NK cell activity (Lillehoj et al., 2000; Martin et al., 1994; 454 Sundick and Gill-Dixon, 1997). 455 The Th2-type cytokines, mainly IL-4 are known to regulate 456 humoral immunity and are more effective as a helper for B-cell

Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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Serum IgG

IFN-concentration

c

3

b

400.00

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pVAX1-AMA1

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pVAX1-AMA1

IL-4 concentration

TGF - concentration 0.35

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TE contol

pVAX1 control Recombinant pET-AMA1

pVAX1-AMA1

TE control

pVAX1 control

Recombinant pET-AMA1

pVAX1-AMA1

Fig. 4. Serum levels of EbAMA1 specific IgG and cytokine in chickens. Chickens were boosted with TE (negative control), pVAX1 plasmid (pVAX1 control), recombinant EbAMA1 protein and pVAX1-EbAMA1. The IgG titers are expressed as mean ± SD with respect to absorbance at 450 nm. The concentrations of IFNc, IL-4, IL-10 and IL-17 (mean ± SD) in qg/ml, TGF-b concentration (mean ± SD) in gg/ml. Bars with different lower-case letters are significantly different (p < 0.05). (a) IgG titers; The concentration of (b) IFNc; (c) TGF-b; (d) IL-4; (e) IL-10 and (f) IL-17, respectively.

activation (Inagaki-Ohara et al., 2006). Here, the levels of IL-4 mRNA showed no differences between other vaccinated groups 459 and control groups (P < 0.05). However, antibody response in this 460 study showed elevated serum IgG levels following DNA vaccina461 tion. One possible reason of this result was that the enhanced anti462 bodies were IgG2a and/or IgG3 induced by IFN-c or other 463 cytokines, but not IgG1 induced by IL-4. Usually, IL-4 is considered 464 to play an important role in the induction of humoral immune 465 reactions (Fallon et al., 2002). 466 Interleukin-10 (IL-10) is a 19-kDa non-glycosylated homodi467 meric protein. It is mainly produced by Th2 cells but also from 468 M2 cells, NK cells, and some dendritic cells. Its targets are Th1 cells, 469 B cells, macrophages, NK cells, and mast cells (Tizard, 2013). IL-10 470 was significantly increased with DNA vaccines compared with TE 471Q12 control and pVAX1 plasmid control (P < 0.05). In contrast there 472 was no significance between pVAX-EbAMA1 and pET-AMA1. These 473 results indicated that IL-10 might play a role in Eimeria infection 474 and immune response to immunization with the Eimeria antigen. 475 Our data showed that IL-17 mRNA was significantly increased 476 at 7 days post second immunization with DNA vaccines compared 477 with TE control and pVAX1 plasmid control (P < 0.05). These 478 results indicated that IL-17 also might play a role in Eimeria 458

b

500

0.00

457

1000 a

10.00

Q19 Q20

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479 infection and immune response to immunization with Eimeria 480 antigen. IL-17 is produced by Th17 cells and mainly modulates 481 the inflammatory response (Martinez et al., 2008). It was reported 482 that IL-17 mRNA was up regulated in the spleen and intestine fol483 lowing Eimeria maxima infection (Hong et al., 2008). 484 In this study, TGF-b4 transcript levels in chickens vaccinated 485 with DNA were increased compared with the control groups. This 486 indicated that TGF-b4 played a role not only in Eimeria infection 487 but also in the immune response to Eimeria antigen. TGF-b is pro488 duced by inducible regulatory T (iTreg) cells and three different 489 isoforms of TGF-b have been identified in chickens (TGF-b) 490 (Martinez et al., 2008; Yun et al., 2000a). 491 Overall, the mRNA levels of all the above cytokines of chickens 492 immunized with vaccines were higher than those of control chick493 ens injected with pVAX1 plasmid alone or TE solution. This indi494 cated that the cytokines were induced by the vaccines. Usually, 495 the cytokines elicit and regulate immune responses and the functions are thought to be not specific (Davison et al., 2008). Once Q13496 497 the cytokines are induced, they will function on their target cells, 498 whether how the cytokines are induced. The function plays roles 499 in an antigen independent manner. Many studies have demon500 strated that these cytokines played an important role in Eimeria

Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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infection (Cornelissen et al., 2009; Choi et al., 1999; Hong et al., 2008; Park et al., 2007). Taken together, DNA vaccines could induce 503 changes in the cytokine profile in chickens and modulate the 504Q14 immune responses against E. brunetti infection. Additionally, simi505 lar studies were also conducted by other researchers, for example, 506 Gam82 immunization and we also observed elevated IL-2 and 507 IL-15 mRNA levels in non-infected chickens collected from the 508 intestinal tissues of chickens (Jang et al., 2010). 509 A homologous challenge experiment was performed in chickens 510 after recombinant EbMIC2 vaccination. We observed a significant 511 difference in increase in weight gain, post challenge, among the 512 vaccinated group compared to unvaccinated group. The oocyst out513 put upon challenge was considerably reduced in the vaccinated 514 birds. Though the reduction in oocyst output is nowhere near the 515 complete protection level, the oocyst output upon challenge was 516 considerably reduced in the vaccinated chickens more than 77%, 517 reduction may help to significantly reduce the oocyst burden in a 518 chicken shed. Immunization with recombinant EbMIC2 was effec519 tive in imparting partial protection in a homologous challenge. The 520 level of protection observed by us is similar to other subunit coc521 cidiosis vaccines tested by various investigators (Jenkins, 1998; 522 Lillehoj et al., 2005b). Reducing the disease burden in a poultry 523 shed may have a considerable economic impact, ranging from 524 reduced disease burden, increased weight gain in the vaccinated 525 birds and better productivity per flock. In other studies conducted 526 in our lab, oocyst reduction was up to 75% in vaccine pcDNA-TA4527 IL2 following the homolog challenge infection parallel to this study 528 (Xu et al., 2008), in vaccine pVAX1-cSZ-2-IL-2, oocyst reduction 529 was even up to 80% (Shah, 2010) and pVAX1-cSZ-JN1 was more 530 than 77% (Zhu et al., 2012). Our study was also in close agreement 531 with findings at the assessment of another sub-unit vaccine in coc532Q15 cidiosis (Jenkins, 1998). Chickens injected with the pVAX1 plasmid 533 alone showed no significance compared to the challenged control 534 indicating that the effects produced by EbMIC2 gene were not 535 attributed to non-specific factors related to the plasmid DNA. 536 A marginal reduction in the lesion score in vaccinated birds was 537 seen while evaluating the potential of EtMIC1 invasion as a candi538 date sub-unit vaccine for poultry (Subramanian et al., 2008). But in 539 this study, all immunized chickens showed significantly less lesion 540 scores as compared with non-immunized chickens. However there 541 was no significant difference between pVAX1-EbMIC2 and recom542 binant EbMIC2 protein. 543 The ACI of chickens immunized with pVAX1-EbMIC2 was 178, 544 indicating a good protection. In the case of the chickens immu545 nized with recombinant EbMIC2 protein the ACI was 160, showing 546Q16 partial protection. However the ACIs of challenged chickens 547 showed no protection. 548 In this study, the DNA vaccines were prepared from the bacteria 549 and there was potential that they were contaminated by LPS (Lipo550 polysaccharide) in the bacteria. A group of chickens were given 551 pVAX1 plasmid alone as the plasmid control. The levels of antibody 552 and cytokines transcript showed no differences between groups 553 injected with pVAX1 plasmid and TE solution. These results ruled 554 out the potential of LPS contamination. This is the first study to 555 report that apical membrane antigen has a role as DNA vaccine 556 encoding Eimeria species. 557 In conclusion, the results of this study demonstrated that DNA 558 vaccine encoding E. brunetti AMA1 and its recombination with 559 IgG, IFN-c, TGF-b, IL-4, IL-10, IL-17 could enhance expression of a 560 panel of cytokines of chickens and we analyzed the antibody 561 response. Results suggested that this vaccine might increase the 562 IgG antibody level and induce the expression of cytokines. The 563 enhanced mRNA levels of different type of cytokines indicated that 564 DNA vaccines could activate various types of cells and effectors to 565 participate in the immune response to Eimeria. However, cytokines 501 502

7

transcript may not always correlate with protein expression. The protein level of cytokines should be verified in future studies.

566

Acknowledgment

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This work was supported by the National Natural Science Foundation.

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References

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Please cite this article in press as: Hoan, T.D., et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.04.015

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