Veterinary Immunology and Immunopathology 162 (2014) 117–121

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Research paper

Protective immunity against Eimeria tenella infection in chickens induced by immunization with a recombinant C-terminal derivative of EtIMP1 Guangwen Yin a , Qian Lin a , Wenjun Wei a , Mei Qin b , Xianyong Liu b,c , Xun Suo b,c , Zhijian Huang a,∗ a Engineering Laboratory of Animal Pharmaceuticals, College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China b National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing 100193, China c Key Laboratory of Zoonosis, China Ministry of Agriculture & College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

a r t i c l e

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Article history: Received 16 July 2014 Received in revised form 17 October 2014 Accepted 21 October 2014 Keywords: Eimeria tenella Immune mapped protein1 Coccidiosis Vaccine

a b s t r a c t Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. Cloning and sequence analysis has predicted the antigen to be a novel membrane protein of apicomplexan parasites. In order to assess the immunogenicity of EtIMP1, a C-terminal derivative of EtIMP1 was expressed in a bacterial host system and was used to immunize chickens. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the subunit vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. The results suggested that the C-terminal derivative of EtIMP1 might be considered as a candidate in the development of subunit vaccines against Eimeria infection. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Coccidiosis in chickens is caused by seven species of apicomplexan parasites belonging to the genus Eimeria. These infectious diseases lead to major economic losses worldwide in poultry industries (Shirley et al., 2005; Williams, 2002). Chemotherapeutic treatment remains the most cost effective means of controlling the disease, however, the increase of resistant parasite populations and increasing public pressure to limit the use of chemicals in animal

∗ Corresponding author. Tel.: +86 591 83845697; fax: +86 591 83845697. E-mail addresses: [email protected], [email protected] (Z. Huang). http://dx.doi.org/10.1016/j.vetimm.2014.10.009 0165-2427/© 2014 Elsevier B.V. All rights reserved.

feed underlines the need to find alternative methods (Blake et al., 2011). And one of the most effective methods in the management of infectious diseases in veterinary practice is through the induction of protective immunity by vaccination. Immune mapped protein-1 (IMP1) is a newly discovered protein in Eimeria maxima, and has been demonstrated to be immunogenic and confer protection against E. maxima challenge in chickens (Blake et al., 2011). Recently, IMP1 has also been identified as immunoprotective antigens from other apicomplexan parasites, such as Toxoplasma and Neospora (Cui et al., 2012a,b). And the IMP1 gene was also identified to be a vaccine candidate against Eimeria tenella infections (Blake et al., 2011). In our previous study, the EtIMP1 gene has been cloned and its protein has been expressed in Escherichia coli cells, with further

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identification and investigation of protective effect as a vaccine candidate (Yin et al., 2013c). Immunization of birds with the recombinant EtIMP1 protein reduced the oocyst output by 60%, comparable with the protective effects of other antigens, such as EtMIC1, EtMIC11 and profilin (Lillehoj et al., 2005; Sathish et al., 2012; Subramanian et al., 2008; Yin et al., 2013c). Bioinformatic analysis of EtIMP1 revealed that it may be a disordered protein, the N-terminal part (amino acid residues 1–160) contains most of the predicted disordered regions, and the C-terminal part (residues 161–397) was predicted to be a flavin mononucleotide (FMN)-dependent reductase (Yin et al., 2013c). In the present study, we hypothesized that the C-terminal derivative of EtIMP1 may be the immunodominant region of the protein thus providing similar or greater immune protection of chickens from Eimeria infection compared with the full length of EtIMP1. To test our hypothesis, we expressed EtIMP1-C and EtIMP1, investigated the immunogenicity of them and evaluated the protective efficacy of the recombinant proteins against E. tenella infection in chickens. 2. Materials and methods 2.1. Chickens and parasites One-day-old male Hy-Line variety brown layer chickens were purchased from Fuzhou Hy-Line variety brown Poultry Breeding. They were housed in isolators and given feed and water ad libitum. The E. tenella (XJ strain) was maintained and propagated in coccidia-free, 2- to 3-wkold AA broilers in our laboratory (Huang et al., 2011; Yin et al., 2013a). Oocysts were collected from feces of chickens 6 to 9 d postinfection and were isolated, purified, and sporulated as described previously (Long et al., 1976; Yin et al., 2013c). 2.2. Cloning and expression of a recombinant C-terminal fragment of EtIMP1 The C-terminal 711 bp fragment of EtIMP1 (amino acid residues 161–397) was amplified by PCR using the following primers with introduced EcoRI and XhoI sites (underlined): 5 -GAATTCATGGGAAGCAACGCGAACCTG-3 and 5 -CTCGAGAGTTGCTGCC GCCACATTTC-3 . PCR was performed with the following program: 4 min at 94 ◦ C; 30 cycles of 40 s at 94 ◦ C, 40 s at 55 ◦ C and 1 min at 72 ◦ C, and 72 ◦ C for 10 min. After EcoR I/XhoI digestion, the amplified fragment was ligated into the EcoR I and XhoI sites of the expression vector pET-28a (Novagen, Germany) and the recombinant plasmid was propagated by transformation into E. coli strain DH5-a. Then the vectors were transformed into E. coli (BL21) for protein expression. The transformed E. coli was grown overnight to mid-log phase, induced with1.0 mM of IPTG for 6 h at 37 ◦ C, collected by centrifugation, and disrupted by sonication on ice. The recombinant protein EtIMP1-C was purified using the Hi-Trap metal chelating column (GE Healthcare, USA) according to the manufacturer’s instructions. Final purity was confirmed by SDS-PAGE and Western blotting with mouse anti-His6

monoclonal antibody (Transgen, Beijing, China) at a 1:2000 dilution. 2.3. Vaccination and parasite challenge infection To test whether EtIMP1-C vaccination confers effective protection against E. tenella infection, two-week-old male Hy-Line variety brown layer chickens were randomly divided into five groups, each group consist of 30 birds. The first two groups were immunized intramuscularly in the breast with 100 ␮g recombinant EtIMP1 emulsified in Freund’s complete adjuvant (FCA) (Group 1), and 100 ␮g EtIMP1-C in FCA (Group 2). The challenged control group (Group 3) and unchallenged control group (Group 4) were injected with PBS. The final group was immunized intramuscularly with 100 ␮l FCA (Group 5) as the adjuvants controls. Fourteen days after the primary immunization, birds in each group were boosted with the same dose as the primary immunization. Fourteen days after the final immunization, birds in Groups 1, 2, 3, and 5 were challenged with 5000 virulent E. tenella oocysts. Sera (10 birds per group) were collected 14 days after the final immunization and stored at −20 ◦ C for further analysis. At 5 days post-challenge, cecal lesions (10 birds per group) were scored on a graded scale from 0 (none) to 4 (high) in a blinded fashion by two independent examiners as previously described (Johnson and Reid, 1970). Body weights were measured at 0 and 8 days post-challenge (10 birds per group). For the determination of fecal oocyst shedding, 10 birds per group were placed in oocyst collection cages separately and fecal samples were collected between 6 and 8 dpi. Oocyst shedding per bird was determined using McMaster egg counting chamber (Lee et al., 2007; Sun et al., 2014). 2.4. Determination of anti-IMP1 antibodies Chicken IgG was detected by ELISA as previously described (Cui et al., 2012a; Huang et al., 2011). Briefly, 96-well microtiter plates were coated with the recombinant EtIMP1 (4 ␮g/ml) in 50 mM carbonate buffer (pH 9.6) overnight at 4 ◦ C and blocked for 1 h at 37 ◦ C with 5% milk powder (DifcoTM skim milk, BD) in PBST (PBS containing 0.05% Tween 20). After washing with PBST, sera were added in a dilution of 1:100 and incubated for 1 h at 37 ◦ C. Antigen specific antibodies were detected using rabbit anti-chicken IgG conjugated to horse-radish peroxidase (1:104 dilution). The ELISA was developed using TMB and H2 O2 as substrates, and optical density was read at 450 nm (A450) with an ELISA reader (Bio-TekEL 680, USA). 2.5. IFN- ELISPOT assay Chicken IFN-␥ was detected by ELISPOT assay as previously described (Ariaans et al., 2008; Yin et al., 2013b; Yin et al., 2013c). Briefly, 14 days after the final immunization, chicken peripheral blood mononuclear cells (PBMC) (10 birds per group) were used to determine the levels of IFN-␥ secretion. ELISPOT 96-well plates (Multiscreen Assay System, Millipore, USA) were coated with 5 ␮g/ml mouse-anti-Chichen IFN-␥ capture antibody (Biosource

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International, USA) in PBS (pH 7.4). The plates were blocked for 2 h with 1% BSA. 1 × 106 PBMCs were added to each well and stimulated overnight at 41 ◦ C in 5% CO2 in the presence of RPMI 1640 (negative control), phytohemagglutinin (PHA, positive control), or EtIMP1 (5 ␮g/ml). Subsequently, the cells were washed and incubated for 2 h at 37 ◦ C with 1 ␮g/ml biotinylated detector antibody (Biosource International, USA). Then streptavidin-HRP conjugate (Biosource International, USA) was added to each well and incubated for 1 h at 37 ◦ C. The plates were washed and treated with 100 ␮l of AEC substrate solution (Dakewei, China) and incubated at room temperature for 20 min in the dark. The plates were then rinsed with distilled water and dried at room temperature. Spots were counted by an automated ELISPOT reader (Bioreader 4000; Bio-sys, Germany). The results were expressed as the number of spot forming cells (SFC) per 106 PBMC cells in the ELISPOT experiment. 2.6. Statistical analysis All data were expressed as the mean ± SD of 10 birds per group, and all data were statistically analyzed by one-way analysis of variance (ANOVA), complimented by post hoc analysis using the Tukey’s HSD test. All statistical analyses were processed by the SPSS13.0 Data Editor software (SPSS Inc., Chicago, IL). The differences between groups were considered to be significant if p values were less than 0.05. 3. Results 3.1. Identification of recombinant EtIMP1 and EtIMP1-C The recombinant EtIMP1 and EtIMP1-C proteins were expressed as a His6 -tagged fusion protein in E. coli and purified by Ni2+ -affinity chromatography. Protein bands at approximately 55 kDa for EtIMP1 and 28 kDa for EtIMP1-C were visualized by SDS-PAGE (Fig. 1). Furthermore, the two proteins could be recognized by a mouse anti-His6 antibody (Transgen, Beijing, China) (Fig. 1).

Fig. 1. Expression of EtIMP1 and EtIMP1-C. Purified EtIMP1 (lane 2) and EtIMP1-C (lane 3) resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB). Recombinant EtIMP1 (lane 4) and EtIMP1-C (lane 5) were confirmed by Western blot, lane 1 is with pre-stained marker (Transgen). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Protective efficacy of EtIMP1-C protein vaccination against E. tenella in chickens To determine if responses to EtIMP1-C proteins could impart protective immunity, body weight gain, cecal lesion score and the oocyst output were evaluated following virulent E. tenella infection (Table 1). After challenge, chickens vaccinated with the EtIMP1 or EtIMP1-C gained significantly greater body weight (p < 0.05), had significantly decreased cecal lesion (p < 0.05), and had reduced numbers of oocystscompared with chickens in other groups (Table 1).

3.2. Seroconversion in chickens for recombinant EtIMP1-C Two weeks after the first immunization, significantly increased levels of EtIMP1-specific IgG were present in EtIMP1 and EtIMP1-C immunized groups compared to the control groups (p < 0.01), and two weeks after the second immunization, the levels of antibodies increased much more higher than that after the first immunization (p < 0.01). There were no differences between EtIMP1 and EtIMP1-C immunized groups (Fig. 2). 3.3. Cellular immunce responses to recombinant EtIMP1-C As shown in Fig. 3, chickens immunized with EtIMP1C or EtIMP1 emulsified in FCA group had a significantly higher number (18 or 15 spots/106 cells) of IFN-␥producing T cells in response to EtIMP1 protein stimulation, relative to other groups (2 spots/106 cells).

Fig. 2. EtIMP1-C induces potent EtIMP1-specific antibody responses. Specific anti-IMP1 IgG in the sera of chickens after immunization twice with EtIMP1, EtIMP1-C, FCA or PBS at a 2-week interval under the same condition. Results are expressed as OD450 readings (mean ± SD, n = 10). Means in the same row with different letters were significantly different between treatment groups (p < 0.05).

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Table 1 Protective effects of EtIMP-C against experimental infection of E. tenella in chickens. Group

Average body weight gain (g)

PBS-unchallenged PBS-challenged FCA Et-IMP1-C Et-IMP1

68.93 43.52 45.32 58.85 59.30

± ± ± ± ±

6.57a 3.52b 5.56b 6.80c 4.27c

Relative body weight gain (%)

Oocyst shedding per bird (×107 )

Oocyst decrease ratio (%)

Lesion score

100 63.13 65.75 85.37 86.03

0 6.28 ± 0.06a 6.06 ± 0.05a 2.17 ± 0.02b 2.35 ± 0.04b

100 0 3.50 65.45 62.58

0 3.6 ± 0.4a 3.4 ± 0.4a 1.6 ± 0.3b 1.4 ± 0.2b

Note: Values are expressed as mean ± SD. Means in the same row with different letters were significantly different between treatment groups (p < 0.05).

4. Discussion IMP1 was a newly discovered protein in E. maxima, and was recognized as a highly conserved antigen in apicomplexan parasites (Blake et al., 2011). IMP1 from E. tenella was also a candidate protective antigen, as shown in our precious study, chicken immunized with EtIMP1 reduced the oocyst output by 60% (Yin et al., 2013c). In the present study, a recombinant C-terminal derivative of EtIMP1was expressed in E. coli and its efficacy against E. tenella infection was evaluated. Chicken immunized with EtIMP1-C prior to E. tenella infection was associated with reduced fecal oocyst shedding and diminished intestinal pathology compared with non-vaccinated and infected controls. The efficacy of EtIMP1-C in chickens was almost the same as EtIMP1, confirming our hypothesis that the C-terminal derivative of EtIMP1 is the immunodominant region of the protein. The advantage to use C-terminal derivative of EtIMP1 over the EtIMP1 is that it is much more easier to express and purify the portion of EtIMP1 than the full length of the protein, and more importantly, it is useful for the subunit vaccine development, as we can easier

Fig. 3. EtIMP1-C induces potent EtIMP1-specific T-cell responses. Antigen-specific cellular responses were examined by the IFN-␥ ELISPOT assay. ELISPOT values denote the number of antigen-specific IFN-␥ positive spots per 106 PBMCs following stimulation with the EtIMP1 protein (mean ± SD, n = 10), means in the same row with different letters were significantly different between treatment groups (p < 0.05).

incorporate the C-terminal derivative of EtIMP1 into the molecular adjuvant, or other protective antigens. Infection with Eimeria parasites elicits humoral and cell mediated immune responses, the relative roles of each in conferring resistance to coccidiosis are debated. It was believed from early studies that cell-mediated immunity appears to be the major component of the immune response in eliminating the infection of apicomplexan parasites, including E. tenella (Bhogal et al., 1989; Lillehoj and Trout, 1996; Rose and Hesketh, 1979), humoral immunity plays a very minor role in resistance against infection (Rose and Long, 1971; Sathish et al., 2011a). However, recent results showed the ability of antibodies to inhibit parasite development in vitro and in vivo and demonstrated the role of antibody in protection against coccidiosis (Wallach, 2010; Wallach et al., 1990). And it is clear that passive immunity against Eimeria infection can be transferred by hyperimmune egg yolk immunoglobulin Y (Lee et al., 2009). To evaluate the effect of our subunit vaccine, we measured the antibody levels and the IFN-␥ levels. The results revealed that EtIMP1-specific antibodies were significantly higher (p < 0.05) in the EtIMP1-C immunized group and EtIMP1 immunized group than that in PBSimmuned group, and the IFN-␥ levels were significant higher in EtIMP1-C immunized group and EtIMP1 immunized group than that in PBS-immunized group(p < 0.05). The results suggest that EtIMP1-C can also stimulate the host to develop both humoral and cellular immunity against E. tenella infection. A homologous challenge experiment was performed in chickens after recombinant EtIMP1-C vaccination. The average weight gain in vaccinated birds was higher compared to unvaccinated but infected birds. The oocyst output upon challenge had considerably reduced in the vaccinated birds. Although the reduction in oocyst output is significantly high (66%), like many other subunit vaccines, the EtIMP1-C failed to induce sterile immunity. Levels of protection observed in the present study were similar to other subunit coccidiosis vaccines (Blake and Tomley, 2014; Chapman et al., 2013, 2002; Jenkins, 1998). Reduced oocysts output would help reducing the disease burden on farm by lowering the number of birds exposed to the pathogen. Moreover, the lesser number of re-circulating oocysts will further help in establishing immunity in the birds (Sathish et al., 2011b). In conclusion, our results demonstrate the possibility of feasibly producing an efficacious vaccine against Eimeria infection using the C-terminal derivative of EtIMP1, evidenced by significant reduction in the oocyst output and reduced gut pathology in chickens challenged with

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