Veterinary Parasitology 210 (2015) 19–24
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Immunogenicity and protective efficacy of an Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and chicken CD40 ligand Guangwen Yin a,b , Qian Lin a , Jianhan Qiu a , Mei Qin b , Xinming Tang b , Xun Suo b,c , Zhijian Huang a,∗ , Xianyong Liu b,c,∗∗ a Engineering Laboratory of Animal Pharmaceuticals and College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province 350002, China b National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193, China c Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
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Article history: Received 10 January 2015 Received in revised form 7 March 2015 Accepted 15 March 2015 Keywords: Eimeria tenella Immune mapped protein 1 CD40L Vaccine
a b s t r a c t The CD40 ligand (CD40L) has shown potential as a powerful immunological adjuvant in various studies. Here, the efficacy of a chimeric subunit vaccine, consisting of Eimeria tenella immune mapped protein 1 (EtIMP1) and chicken CD40L, was evaluated against E. tenella infection. The recombinant EtIMP1-CD40L was purified from E. coli over-expressing this protein. Chickens were vaccinated with EtIMP1-CD40L without adjuvant or EtIMP1 with Freund’s adjuvant. Immunization of chickens with EtIMP1-CD40L fusion protein resulted in stronger IFN-␥ secretion and IgA response than that with only recombinant EtIMP1 with Freund’s adjuvant. The clinical effect (cecal lesions, body weights gain, and oocysts shedding) of the EtIMP1-CD40L without adjuvant was also better than that of the EtIMP1 with adjuvant, as evidenced by the difference between the two groups in the oocyst output of E. tenella-challenged chickens. The results suggest that the EtIMP1-CD40L fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Avian coccidiosis is caused by the infection with Eimeria spp., contributing to major economic losses worldwide in poultry industries (Shirley and Lillehoj, 2012; Shirley
∗ Corresponding author. Tel.: +86 591 83845697; fax: +86 591 83845697. ∗∗ Corresponding author at: National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Tel.: +86 10 62734355; fax: +86 10 62734325. E-mail addresses: [email protected], [email protected] (Z. Huang), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.vetpar.2015.03.012 0304-4017/© 2015 Elsevier B.V. All rights reserved.
et al., 2005). Anticoccidial drugs are used extensively to control the disease, but the increase of resistant parasite populations underlines the need to find alternative strategies (Blake and Tomley, 2014). One of the most effective approaches in the management of infectious diseases in veterinary practice is through the induction of protective immunity by vaccination. The CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated T cells, basophils, and mast cells, which belongs to the tumor necrosis factor family (TNF) superfamily. Studies have demonstrated that the CD40-CD40L interaction can upregulate co-stimulatory molecules, activate antigen-presenting cells, and influence
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T-cell-mediated effector functions (Grewal and Flavell, 1998; Miga et al., 2000). Recently it has further been shown that murine CD40L can function as a molecular adjuvant enhancing both immune response and disease protection (Hashem et al., 2014; Lauterbach et al., 2013; Lin et al., 2009; Ninomiya et al., 2002). The immunostimulatory capacity of CD40L has also been observed in chickens and ducks (Layton et al., 2009; Pose et al., 2011; Sanchez Ramos et al., 2011; Tregaskes et al., 2005; Yao et al., 2010). Immune mapped protein-1 (IMP1), a newly discovered protein in Eimeria maxima, 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 an immunoprotective antigen from other apicomplexan parasites, such as Toxoplasma, Neospora and E. tenella (Cui et al., 2012a,b; Yin et al., 2013b). Immunization of birds with the EtIMP1 protein reduces 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). In the present study, we hypothesized that chicken CD40L may enhance immunogenicity of the IMP1 protein thus providing greater immune protection of chickens against Eimeria infection. To test this hypothesis, we investigated the adjuvant properties of CD40L for the EtIMP1 protein and evaluated the protective efficacy of the recombinant EtIMP1-CD40L fusion protein in chickens.
2. Materials and methods 2.1. Chickens and parasites One-day-old Arbor Acre (AA) broiler chickens were purchased from Beijing Arbor Acres Poultry Breeding (Beijing, China). They were housed in isolators, and given feed and water ad libitum. E. tenella Beijing Strain (Suo et al., 2000) was maintained and propagated in 2–3-week-old and coccidia-free AA broilers in our laboratory (Yin et al., 2013b). Oocysts were collected from feces of chickens 6–9 days post-infection, and were isolated, purified, and sporulated as described previously (Huang et al., 2011).
2.2. Cloning of the EtIMP1 gene Pellets of unsporulated oocysts (1 × 107 ) were vortexed with 0.5-mm glass beads until the total destruction of oocyst walls. Total RNA was obtained by treatment of broken oocysts with the Trizol reagent (Invitrogen, USA) for 5 min at room temperature, extraction with 20% chloroform, and precipitation with 50% isopropanol. RNA samples were resuspended in nuclease-free water. cDNA was synthesized using random primers and a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). According to the EtIMP1 sequence of Beijing strain of E. tenella (GeneBank Accession number: KC215109), the open reading frame of EtIMP1 was amplified by PCR using the following primers with introduced EcoRI, XhoI and AvrII sites (underlined): 5 -GAATTCATGGGGGGGGCTTGCGGGA-3
and 5 -CTCGAGACCTAGGAGTTGCTGCCGCCACATTTC-3 . PCR products were confirmed by sequencing. 2.3. Expression and purification of recombinant proteins The extracellular domain of chicken CD40L was generated by PCR using the following primers with introduced AvrII and XhoI sites (underlined): 5 CCTAGGGGAGCGCCGGTGGATCCTGC-3 and 5 -CTCGAG AGCGTAATCTGGTACGTCG-3 . EtIMP1 was inserted into the EcoRI and XhoI digested expression vector pET-28a (Novagen, Germany) to create pET-28a-IMPI; and then CD40L was inserted into the AvrII and XhoI digested vector pET-28a-IMPI to create pET-28a-IMPI-CD40L. The vector pET-28a-CD40L for expression of CD40L was constructed in the similar way. Next, E. coli was transformed with the above vectors for protein expression. The recombinant His6 -tagged proteins were purified from the soluble fraction of the bacterial lysates using the Hi-Trap metal chelating column (GE Healthcare, USA). The identity and the purity of the recombinant proteins were evaluated by SDS-PAGE on 12% polyacrylamide gels and Western blot analysis. 2.4. Vaccination and parasite challenge infection To test whether EtIMP1-CD40L vaccination confers effective protection against E. tenella infection, 3-week-old AA broiler chickens were randomly divided into 7 groups. The first two groups were immunized intramuscularly in the thigh muscle with 100 g recombinant EtIMP1 emulsified in Freund’s complete adjuvant (FCA) (Group 1), and 100 g EtIMP1-CD40L without adjuvant (Group 2). The challenged control group (Group 3) and the unchallenged control group (Group 4) were injected with PBS. Groups 5 and 6 were, respectively, immunized intramuscularly with 100 g CD40L and 100 l FCA as the adjuvants controls. Group 7 were immunized intramuscularly with 100 g EtIMP1 without adjuvant. Fourteen days after the primary immunization, birds in each group were boosted once with the same dose. Fourteen days after the second immunization, birds in Groups 1, 2, 3, 5, 6 and 7 were challenged with 5000 virulent E. tenella oocysts. Sera were collected 14 days after the second immunization and stored at −20 ◦ C for further analysis. At Day 5 post-challenge, cecal lesions 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 Day 0 and 8 post-challenge. Feces from each group were collected separately at Days 6–8 post-challenge. Oocyst shedding per bird was determined using a McMaster chamber (Lee et al., 2007; Sun et al., 2014). 2.5. Determination of anti-IMP1 antibodies Chicken IgG and IgA were detected by ELISA as previously described (Yin et al., 2013b). Briefly, 96-well 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% dry milk in PBST
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Fig. 1. Expression of EtIMP1, EtIMP1-CD40L and CD40L. (A) Purified EtIMP1, EtIMP1-CD40L and CD40L were resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B) Recombinant EtIMP1, EtIMP1-CD40L and CD40L were confirmed by Western blotting with anti-His6 monoclonal antibody.
(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 or rabbit anti-chicken IgA (Bethyl Laboratories, USA) conjugated to horseradish peroxidase (HRP) (1:10,000 dilution), followed by development using TMB and H2 O2 as substrates. Finally, the optical density was read at 450 nm (A450) with an ELISA reader (Bio-Rad, Model 680, USA).
2.6. IFN- ELISPOT assay Chicken IFN-␥ was detected by ELISPOT assay as previously described (Yin et al., 2013a,b). Briefly, 14 days after the final immunization, chicken peripheral blood mononuclear cells (PBMCs) 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-chicken IFN-␥ capture antibody (Biosource 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 antibody against chicken IFN-␥ (Biosource International). Then streptavidin–HRP conjugate (Biosource International) 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.
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-CD40L The recombinant EtIMP1, EtIMP1-CD40L and CD40L proteins were expressed as a His6 -tagged fusion protein in E. coli and purified by Ni2+ -affinity chromatography. The purity of the proteins is more than 90%. Protein bands at approximately 60 kDa for EtIMP1, 105 kDa for EtIMP1CD40L and 36 kDa for CD40L were visualized by SDS-PAGE, as detected by Coomassie brilliant blue staining (Fig. 1A). Furthermore, the three proteins could be recognized by a mouse anti-His6 antibody (Fig. 1B). Although some nonspecific bands could also be observed, these contaminating proteins appeared to be minor constituents and the purity of specific proteins are sufficient for use in our immunogenicity trials. 3.2. Seroconversion in chickens for recombinant EtIMP1-CD40L Two weeks after the second immunization, compared to the control groups, significantly (p < 0.01) increased levels of EtIMP1-specific IgG were present in EtIMP1-FCA and EtIMP1-CD40L-immunized groups (Fig. 2A). There were significant differences between EtIMP1-FCA and EtIMP1CD40L groups (p < 0.05). And as for the IgA, chickens immunized with EtIMP1-CD40L had a significantly higher level of EtIMP1-specific IgA than that in other groups (p < 0.01) (Fig. 2B). However, no significant differences were observed in IgG and IgA between FCA, CD40L, EtIMP1, and PBS control groups (Fig. 2A and B).
2.7. Statistical analysis
3.3. Cellular immune response to recombinant EtIMP1-CD40L
All data were statistically analyzed by one-way analysis of variance (ANOVA), complimented by post hoc analysis using Tukey’s HSD test. All statistical analyses were processed by the SPSS13.0 Data Editor software (SPSS Inc.,
As shown in Fig. 3, chickens immunized with EtIMP1CD40L had a significantly higher number (50 spots/106 cells) of IFN-␥-producing T cells in response to EtIMP1 protein stimulation, relative to EtIMP1 emulsified in FCA group
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Fig. 2. EtIMP1-CD40L fusion protein induces potent EtIMP1-specific antibody responses. Specific anti-IMP1 IgG (A) and IgA (B) in the sera of chickens in 14 days after the second immunization with EtIMP1, EtIMP1-CD40L, CD40L or PBS at a 2-week interval under the same condition. Results are expressed as OD450 readings. Bars (mean ± SE, n = 6) denoted with different letters are significantly different by Tukey’s HSD test (p < 0.05).
(15 spots/106 cells) and FCA, CD40L, EtIMP1, and PBS control groups (2 spots/106 cells). 3.4. Protective efficacy of EtIMP1-CD40L protein vaccination against E. tenella in chickens To determine if responses to CD40L fusion proteins affect 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-CD40L gained significantly greater body weight (p < 0.05), had significantly decreased cecal lesions (p < 0.05), and had reduced numbers of oocysts compared with chickens in other groups (Table 1). However, no significant differences were observed in body weight and cecal lesions between
Fig. 3. EtIMP1-CD40L fusion protein induces potent EtIMP1-specific Tcell responses. Antigen-specific cellular responses were examined by the IFN-␥ ELISPOT assay. ELISPOT values represent the number of antigenspecific IFN-␥ positive spots per 106 PBMCs following stimulation with the EtIMP1 protein. Bars (mean ± SE, n = 6) denoted with different letters are significantly different by Tukey’s HSD test (p < 0.05).
EtIMP1-FCA and EtIMP1-CD40L groups (Table 1). And there were also no significant differences between FCA, CD40L, EtIMP1, and PBS control groups (Table 1). 4. Discussion In the present study, a recombinant chimeric subunit vaccine consisting of EtIMP1 and a molecular adjuvant, chicken CD40L, was generated, and its efficacy against E. tenella infection was evaluated. We found that immunization with EtIMP1-CD40L reduced oocyst output in chickens by 78%, more potently than that with EtIMP1 emulsified in FCA (by 66%). The findings support our hypothesis that CD40L, as an adjuvant for cell-mediated immunity, can improve the immunoprotective effect of IMPI against Eimeria infection. 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). The vaccination with a recombinant antigen is often not sufficient to elicit a protective immune response against E. tenella infection (Crane et al., 1991; Jenkins, 1998; Miller et al., 1989). In this study, we demonstrated that the chicken CD40L-EtIMP1, a fusion protein, reduced oocyst output by 78%, more potently than EtIMP1 alone emulsified in FCA. In addition to having the strong antibody responses, chickens immunized with EtIMP1-CD40L also elicited a specific T-cell response, as evidenced by a greater IFN-␥ production by T-cells in response to EtIMP1 stimulation ex vivo. Likely, CD40L, fused to EtIMP1, may increase the uptake and processing of EtIMP1 by antigen processing cells, thus resulting in more efficient stimulation of adaptive immune responses. This was consistent with the report that a genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo (Thacker et al., 2009). In addition, recent research has shown that murine CD154 can function as an adjuvant for viral or DNA vaccines by administering adenoviral vectors
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Table 1 Protective effects of EtIMP-CD40L against experimental infection of E. tenella in chickens. Group
Average body weight gain (g)
PBS-unchallenged PBS-challenged CD40L FCA Et-IMP1 Et-IMP1FCA Et-IMP1-CD40L
520.56 280.62 295.42 288.35 280.20 368.86 405.75
± ± ± ± ± ± ±
14.55a 17.82b 9.54b 7.36b 8.25b 16.80c 17.32c
Body weight loss (%)
Oocyst shedding per bird (×107 )
Oocyst decrease ratio (%)
Lesion score
0 46.09 43.25 44.61 46.15 29.14 22.06
0 9.15 ± 0.04a 8.76 ± 0.04a 8.84 ± 0.05a 8.74 ± 0.04a 3.07 ± 0.02b 2.05 ± 0.04c
100 0 4.30 3.39 4.48 66.45 77.60
0 3.7 ± 0.2a 3.3 ± 0.4a 3.3 ± 0.4a 3.2 ± 0.2a 1.7 ± 0.2b 1.5 ± 0.2b
Note: Values are expressed as mean ± SD. Means in the same column with different letters were significantly different between treatment groups (p < 0.05).
or plasmids that express a vaccine antigen and CD154, individually, or as a fusion protein (Mendoza et al., 1997; Tripp et al., 2000; Xiang et al., 2001). Recently it has also been demonstrated that the extracellular domain of duck’s CD154 could enhance DNA vaccine responses in ducks both by co-expression with viral antigens and as a fusion protein to target viral antigen to APCs (Yao et al., 2010). In this study we observed that chicken CD40L-fused EtIMP1 elicited a stronger protective immune response evidenced by reduced oocyst output than EtIMP1 with adjuvant did. This is also consistent with the previous studies on the use of recombinant CD40L as a vaccine adjuvant with influenza virus antigen, showing promising results in influenza vaccine development in chicken tests (Pose et al., 2011; Sanchez Ramos et al., 2011). Our findings demonstrate that CD40L is able to enhance the immunogenicity of antigens, and the fusion of CD40L to parasite antigens could also be a valuable strategy to improve the immunogenicity of parasite proteins in the stimulation of cell-mediated immunity in birds. 5. Conclusion Our results demonstrate the feasibility of producing an effective vaccine against Eimeria infection using the novel membrane protein, EtIMP1, fused to CD40L, a molecular adjuvant. This is evidenced by significant reduction in the oocyst output in chickens challenged with E. tenella. Therefore, EtIMP1-CD40L fusion protein can be used as a potent immunogen in the development of subunit vaccines against E. tenella infection. Conflict of interest statement No actual or potential conflict of interest was identified that could inappropriately influence, or be perceived to influence the outcome of this work. Acknowledgements This study was supported by the National Natural Science Foundation of China (31330076 and 31001060), Research and Innovation Project for College Students of Fujian Agriculture and Forestry University (201410389065 and 201410389154), and Key Project of the Science and Technology Department of Fujian Province (2011N0001). We thank Dr. Shile Huang (Louisiana State University Health Sciences Center, USA) for reading the manuscript.
All experimentation using chickens was carried out in compliance with the current Chinese legislation and due attention was paid to the welfare of experimental chickens. The animal protocol was approved by the Animal Care Committee of Fujian Agriculture and Forestry University. References Bhogal, B.S., Jacobson, E.B., Tse, H.Y., Schmatz, D.M., Ravino, O.J., 1989. Parasite exposure elicits a preferential T-cell response involved in protective immunity against Eimeria species in chickens primed by an internal-image anti-idiotypic antibody. Infect. Immun. 57, 2804–2810. Blake, D.P., Billington, K.J., Copestake, S.L., Oakes, R.D., Quail, M.A., Wan, K.L., Shirley, M.W., Smith, A.L., 2011. Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite. PLoS Pathog. 7, e1001279. Blake, D.P., Tomley, F.M., 2014. Securing poultry production from the everpresent Eimeria challenge. Trends Parasitol. 30, 12–19. Crane, M.S., Goggin, B., Pellegrino, R.M., Ravino, O.J., Lange, C., Karkhanis, Y.D., Kirk, K.E., Chakraborty, P.R., 1991. Cross-protection against four species of chicken coccidia with a single recombinant antigen. Infect. Immun. 59, 1271–1277. Cui, X., Lei, T., Yang, D., Hao, P., Li, B., Liu, Q., 2012a. Toxoplasma gondii immune mapped protein-1 (TgIMP1) is a novel vaccine candidate against toxoplasmosis. Vaccine 30, 2282–2287. Cui, X., Lei, T., Yang, D.Y., Hao, P., Liu, Q., 2012b. Identification and characterization of a novel Neospora caninum immune mapped protein 1. Parasitology 139, 998–1004. Grewal, I.S., Flavell, R.A., 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16, 111–135. Hashem, A.M., Gravel, C., Chen, Z., Yi, Y., Tocchi, M., Jaentschke, B., Fan, X., Li, C., Rosu-Myles, M., Pereboev, A., He, R., Wang, J., Li, X., 2014. CD40 ligand preferentially modulates immune response and enhances protection against influenza virus. J. Immunol. 193, 722–734. Huang, X., Zou, J., Xu, H., Ding, Y., Yin, G., Liu, X., Suo, X., 2011. Transgenic Eimeria tenella expressing enhanced yellow fluorescent protein targeted to different cellular compartments stimulated dichotomic immune responses in chickens. J. Immunol. 187, 3595–3602. Jenkins, M.C., 1998. Progress on developing a recombinant coccidiosis vaccine. Int. J. Parasitol. 28, 1111–1119. Johnson, J., Reid, W.M., 1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28, 30–36. Lauterbach, H., Patzold, J., Kassub, R., Bathke, B., Brinkmann, K., Chaplin, P., Suter, M., Hochrein, H., 2013. Genetic adjuvantation of recombinant MVA with CD40L potentiates CD8T cell mediated immunity. Front. Immunol. 4, 251. Layton, S.L., Kapczynski, D.R., Higgins, S., Higgins, J., Wolfenden, A.D., Liljebjelke, K.A., Bottje, W.G., Swayne, D., Berghman, L.R., Kwon, Y.M., Hargis, B.M., Cole, K., 2009. Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge. Poult. Sci. 88, 2244–2252. Lee, S.H., Lillehoj, H.S., Dalloul, R.A., Park, D.W., Hong, Y.H., Lin, J.J., 2007. Influence of Pediococcus-based probiotic on coccidiosis in broiler chickens. Poult. Sci. 86, 63–66. Lillehoj, H.S., Ding, X., Dalloul, R.A., Sato, T., Yasuda, A., Lillehoj, E.P., 2005. Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants. J. Parasitol. 91, 666–673.
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G. Yin et al. / Veterinary Parasitology 210 (2015) 19–24
Lillehoj, H.S., Trout, J.M., 1996. Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9, 349–360. Lin, F.C., Peng, Y., Jones, L.A., Verardi, P.H., Yilma, T.D., 2009. Incorporation of CD40 ligand into the envelope of pseudotyped single-cycle Simian immunodeficiency viruses enhances immunogenicity. J. Virol. 83, 1216–1227. Mendoza, R.B., Cantwell, M.J., Kipps, T.J., 1997. Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization. J. Immunol. 159, 5777–5781. Miga, A., Masters, S., Gonzalez, M., Noelle, R.J., 2000. The role of CD40–CD154 interactions in the regulation of cell mediated immunity. Immunol. Invest. 29, 111–114. Miller, G.A., Bhogal, B.S., McCandliss, R., Strausberg, R.L., Jessee, E.J., Anderson, A.C., Fuchs, C.K., Nagle, J., Likel, M.H., Strasser, J.M., et al., 1989. Characterization and vaccine potential of a novel recombinant coccidial antigen. Infect. Immun. 57, 2014–2020. Ninomiya, A., Ogasawara, K., Kajino, K., Takada, A., Kida, H., 2002. Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice. Vaccine 20, 3123–3129. Pose, A.G., Gomez, J.N., Sanchez, A.V., Redondo, A.V., Rodriguez, E.R., Segui, R.M., Ramos, E.M., Molto, M.P., Rodriguez, E.S., Cordero, L.R., Mallon, A.R., Nordelo, C.B., 2011. Subunit influenza vaccine candidate based on CD154 fused to HAH5 increases the antibody titers and cellular immune response in chickens. Vet. Microbiol. 152, 328–337. Rose, M.E., Hesketh, P., 1979. Immunity to coccidiosis: T-lymphocyte- or B-lymphocyte-deficient animals. Infect. Immun. 26, 630–637. Sanchez Ramos, O., Gonzalez Pose, A., Gomez-Puerta, S., Noda Gomez, J., Vega Redondo, A., Aguila Benites, J.C., Suarez Amaran, L., Parra, N.C., Toledo Alonso, J.R., 2011. Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens. Comp. Immunol. Microbiol. Infect. Dis. 34, 259–265. Sathish, K., Sriraman, R., Subramanian, B.M., Rao, N.H., Balaji, K., Narasu, M.L., Srinivasan, V.A., 2012. Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis. Vaccine 29, 9201–9208. Shirley, M.W., Lillehoj, H.S., 2012. The long view: a selective review of 40 years of coccidiosis research. Avian Pathol. 41, 111–121. Shirley, M.W., Smith, A.L., Tomley, F.M., 2005. The biology of avian Eimeria with an emphasis on their control by vaccination. Adv. Parasitol. 60, 285–330.
Subramanian, B.M., Sriraman, R., Rao, N.H., Raghul, J., Thiagarajan, D., Srinivasan, V.A., 2008. Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds. Vaccine 26, 3489–3496. Sun, H., Wang, L., Wang, T., Zhang, J., Liu, Q., Chen, P., Chen, Z., Wang, F., Li, H., Xiao, Y., Zhao, X., 2014. Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis. Vaccine 32, 1869–1876. Suo, X., Li, A., Jiang, J., Lv, Y., Wang, Z., Wu, W., Qv, H., 2000. Studies on the efficacy of Eimerivac Plus, a coccidiosis vaccine for chickens. Acta Vet. Zooltech. Sin. 31, 353–359. Thacker, E.E., Nakayama, M., Smith, B.F., Bird, R.C., Muminova, Z., Strong, T.V., Timares, L., Korokhov, N., O’Neill, A.M., de Gruijl, T.D., Glasgow, J.N., Tani, K., Curiel, D.T., 2009. A genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo. Vaccine 27, 7116–7124. Tregaskes, C.A., Glansbeek, H.L., Gill, A.C., Hunt, L.G., Burnside, J., Young, J.R., 2005. Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate. Dev. Comp. Immunol. 29, 361–374. Tripp, R.A., Jones, L., Anderson, L.J., Brown, M.P., 2000. CD40 ligand (CD154) enhances the Th1 and antibody responses to respiratory syncytial virus in the BALB/c mouse. J. Immunol. 164, 5913–5921. Xiang, R., Primus, F.J., Ruehlmann, J.M., Niethammer, A.G., Silletti, S., Lode, H.N., Dolman, C.S., Gillies, S.D., Reisfeld, R.A., 2001. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice. J. Immunol. 167, 4560–4565. Yao, Q., Fischer, K.P., Li, L., Agrawal, B., Berhane, Y., Tyrrell, D.L., Gutfreund, K.S., Pasick, J., 2010. Immunogenicity and protective efficacy of a DNA vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekin ducks. Vaccine 28, 8147–8156. Yin, G., Qin, M., Liu, X., Suo, J., Suo, X., 2013a. Interferon-gamma enzymelinked immunosorbent spot assay as a tool to study T cell responses to Eimeria tenella infection in chickens. Poult. Sci. 92, 1758–1763. Yin, G., Qin, M., Liu, X., Suo, J., Tang, X., Tao, G., Han, Q., Suo, X., Wu, W., 2013b. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin. Biochem. Biophys. Res. Commun. 440, 437–442.
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Immunogenicity and protective efficacy of an Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and chicken CD40 ligand Guangwen Yin a,b , Qian Lin a , Jianhan Qiu a , Mei Qin b , Xinming Tang b , Xun Suo b,c , Zhijian Huang a,∗ , Xianyong Liu b,c,∗∗ a Engineering Laboratory of Animal Pharmaceuticals and College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province 350002, China b National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193, China c Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
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Article history: Received 10 January 2015 Received in revised form 7 March 2015 Accepted 15 March 2015 Keywords: Eimeria tenella Immune mapped protein 1 CD40L Vaccine
a b s t r a c t The CD40 ligand (CD40L) has shown potential as a powerful immunological adjuvant in various studies. Here, the efficacy of a chimeric subunit vaccine, consisting of Eimeria tenella immune mapped protein 1 (EtIMP1) and chicken CD40L, was evaluated against E. tenella infection. The recombinant EtIMP1-CD40L was purified from E. coli over-expressing this protein. Chickens were vaccinated with EtIMP1-CD40L without adjuvant or EtIMP1 with Freund’s adjuvant. Immunization of chickens with EtIMP1-CD40L fusion protein resulted in stronger IFN-␥ secretion and IgA response than that with only recombinant EtIMP1 with Freund’s adjuvant. The clinical effect (cecal lesions, body weights gain, and oocysts shedding) of the EtIMP1-CD40L without adjuvant was also better than that of the EtIMP1 with adjuvant, as evidenced by the difference between the two groups in the oocyst output of E. tenella-challenged chickens. The results suggest that the EtIMP1-CD40L fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Avian coccidiosis is caused by the infection with Eimeria spp., contributing to major economic losses worldwide in poultry industries (Shirley and Lillehoj, 2012; Shirley
∗ Corresponding author. Tel.: +86 591 83845697; fax: +86 591 83845697. ∗∗ Corresponding author at: National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Tel.: +86 10 62734355; fax: +86 10 62734325. E-mail addresses: [email protected], [email protected] (Z. Huang), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.vetpar.2015.03.012 0304-4017/© 2015 Elsevier B.V. All rights reserved.
et al., 2005). Anticoccidial drugs are used extensively to control the disease, but the increase of resistant parasite populations underlines the need to find alternative strategies (Blake and Tomley, 2014). One of the most effective approaches in the management of infectious diseases in veterinary practice is through the induction of protective immunity by vaccination. The CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated T cells, basophils, and mast cells, which belongs to the tumor necrosis factor family (TNF) superfamily. Studies have demonstrated that the CD40-CD40L interaction can upregulate co-stimulatory molecules, activate antigen-presenting cells, and influence
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T-cell-mediated effector functions (Grewal and Flavell, 1998; Miga et al., 2000). Recently it has further been shown that murine CD40L can function as a molecular adjuvant enhancing both immune response and disease protection (Hashem et al., 2014; Lauterbach et al., 2013; Lin et al., 2009; Ninomiya et al., 2002). The immunostimulatory capacity of CD40L has also been observed in chickens and ducks (Layton et al., 2009; Pose et al., 2011; Sanchez Ramos et al., 2011; Tregaskes et al., 2005; Yao et al., 2010). Immune mapped protein-1 (IMP1), a newly discovered protein in Eimeria maxima, 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 an immunoprotective antigen from other apicomplexan parasites, such as Toxoplasma, Neospora and E. tenella (Cui et al., 2012a,b; Yin et al., 2013b). Immunization of birds with the EtIMP1 protein reduces 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). In the present study, we hypothesized that chicken CD40L may enhance immunogenicity of the IMP1 protein thus providing greater immune protection of chickens against Eimeria infection. To test this hypothesis, we investigated the adjuvant properties of CD40L for the EtIMP1 protein and evaluated the protective efficacy of the recombinant EtIMP1-CD40L fusion protein in chickens.
2. Materials and methods 2.1. Chickens and parasites One-day-old Arbor Acre (AA) broiler chickens were purchased from Beijing Arbor Acres Poultry Breeding (Beijing, China). They were housed in isolators, and given feed and water ad libitum. E. tenella Beijing Strain (Suo et al., 2000) was maintained and propagated in 2–3-week-old and coccidia-free AA broilers in our laboratory (Yin et al., 2013b). Oocysts were collected from feces of chickens 6–9 days post-infection, and were isolated, purified, and sporulated as described previously (Huang et al., 2011).
2.2. Cloning of the EtIMP1 gene Pellets of unsporulated oocysts (1 × 107 ) were vortexed with 0.5-mm glass beads until the total destruction of oocyst walls. Total RNA was obtained by treatment of broken oocysts with the Trizol reagent (Invitrogen, USA) for 5 min at room temperature, extraction with 20% chloroform, and precipitation with 50% isopropanol. RNA samples were resuspended in nuclease-free water. cDNA was synthesized using random primers and a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). According to the EtIMP1 sequence of Beijing strain of E. tenella (GeneBank Accession number: KC215109), the open reading frame of EtIMP1 was amplified by PCR using the following primers with introduced EcoRI, XhoI and AvrII sites (underlined): 5 -GAATTCATGGGGGGGGCTTGCGGGA-3
and 5 -CTCGAGACCTAGGAGTTGCTGCCGCCACATTTC-3 . PCR products were confirmed by sequencing. 2.3. Expression and purification of recombinant proteins The extracellular domain of chicken CD40L was generated by PCR using the following primers with introduced AvrII and XhoI sites (underlined): 5 CCTAGGGGAGCGCCGGTGGATCCTGC-3 and 5 -CTCGAG AGCGTAATCTGGTACGTCG-3 . EtIMP1 was inserted into the EcoRI and XhoI digested expression vector pET-28a (Novagen, Germany) to create pET-28a-IMPI; and then CD40L was inserted into the AvrII and XhoI digested vector pET-28a-IMPI to create pET-28a-IMPI-CD40L. The vector pET-28a-CD40L for expression of CD40L was constructed in the similar way. Next, E. coli was transformed with the above vectors for protein expression. The recombinant His6 -tagged proteins were purified from the soluble fraction of the bacterial lysates using the Hi-Trap metal chelating column (GE Healthcare, USA). The identity and the purity of the recombinant proteins were evaluated by SDS-PAGE on 12% polyacrylamide gels and Western blot analysis. 2.4. Vaccination and parasite challenge infection To test whether EtIMP1-CD40L vaccination confers effective protection against E. tenella infection, 3-week-old AA broiler chickens were randomly divided into 7 groups. The first two groups were immunized intramuscularly in the thigh muscle with 100 g recombinant EtIMP1 emulsified in Freund’s complete adjuvant (FCA) (Group 1), and 100 g EtIMP1-CD40L without adjuvant (Group 2). The challenged control group (Group 3) and the unchallenged control group (Group 4) were injected with PBS. Groups 5 and 6 were, respectively, immunized intramuscularly with 100 g CD40L and 100 l FCA as the adjuvants controls. Group 7 were immunized intramuscularly with 100 g EtIMP1 without adjuvant. Fourteen days after the primary immunization, birds in each group were boosted once with the same dose. Fourteen days after the second immunization, birds in Groups 1, 2, 3, 5, 6 and 7 were challenged with 5000 virulent E. tenella oocysts. Sera were collected 14 days after the second immunization and stored at −20 ◦ C for further analysis. At Day 5 post-challenge, cecal lesions 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 Day 0 and 8 post-challenge. Feces from each group were collected separately at Days 6–8 post-challenge. Oocyst shedding per bird was determined using a McMaster chamber (Lee et al., 2007; Sun et al., 2014). 2.5. Determination of anti-IMP1 antibodies Chicken IgG and IgA were detected by ELISA as previously described (Yin et al., 2013b). Briefly, 96-well 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% dry milk in PBST
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Fig. 1. Expression of EtIMP1, EtIMP1-CD40L and CD40L. (A) Purified EtIMP1, EtIMP1-CD40L and CD40L were resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B) Recombinant EtIMP1, EtIMP1-CD40L and CD40L were confirmed by Western blotting with anti-His6 monoclonal antibody.
(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 or rabbit anti-chicken IgA (Bethyl Laboratories, USA) conjugated to horseradish peroxidase (HRP) (1:10,000 dilution), followed by development using TMB and H2 O2 as substrates. Finally, the optical density was read at 450 nm (A450) with an ELISA reader (Bio-Rad, Model 680, USA).
2.6. IFN- ELISPOT assay Chicken IFN-␥ was detected by ELISPOT assay as previously described (Yin et al., 2013a,b). Briefly, 14 days after the final immunization, chicken peripheral blood mononuclear cells (PBMCs) 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-chicken IFN-␥ capture antibody (Biosource 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 antibody against chicken IFN-␥ (Biosource International). Then streptavidin–HRP conjugate (Biosource International) 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.
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-CD40L The recombinant EtIMP1, EtIMP1-CD40L and CD40L proteins were expressed as a His6 -tagged fusion protein in E. coli and purified by Ni2+ -affinity chromatography. The purity of the proteins is more than 90%. Protein bands at approximately 60 kDa for EtIMP1, 105 kDa for EtIMP1CD40L and 36 kDa for CD40L were visualized by SDS-PAGE, as detected by Coomassie brilliant blue staining (Fig. 1A). Furthermore, the three proteins could be recognized by a mouse anti-His6 antibody (Fig. 1B). Although some nonspecific bands could also be observed, these contaminating proteins appeared to be minor constituents and the purity of specific proteins are sufficient for use in our immunogenicity trials. 3.2. Seroconversion in chickens for recombinant EtIMP1-CD40L Two weeks after the second immunization, compared to the control groups, significantly (p < 0.01) increased levels of EtIMP1-specific IgG were present in EtIMP1-FCA and EtIMP1-CD40L-immunized groups (Fig. 2A). There were significant differences between EtIMP1-FCA and EtIMP1CD40L groups (p < 0.05). And as for the IgA, chickens immunized with EtIMP1-CD40L had a significantly higher level of EtIMP1-specific IgA than that in other groups (p < 0.01) (Fig. 2B). However, no significant differences were observed in IgG and IgA between FCA, CD40L, EtIMP1, and PBS control groups (Fig. 2A and B).
2.7. Statistical analysis
3.3. Cellular immune response to recombinant EtIMP1-CD40L
All data were statistically analyzed by one-way analysis of variance (ANOVA), complimented by post hoc analysis using Tukey’s HSD test. All statistical analyses were processed by the SPSS13.0 Data Editor software (SPSS Inc.,
As shown in Fig. 3, chickens immunized with EtIMP1CD40L had a significantly higher number (50 spots/106 cells) of IFN-␥-producing T cells in response to EtIMP1 protein stimulation, relative to EtIMP1 emulsified in FCA group
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Fig. 2. EtIMP1-CD40L fusion protein induces potent EtIMP1-specific antibody responses. Specific anti-IMP1 IgG (A) and IgA (B) in the sera of chickens in 14 days after the second immunization with EtIMP1, EtIMP1-CD40L, CD40L or PBS at a 2-week interval under the same condition. Results are expressed as OD450 readings. Bars (mean ± SE, n = 6) denoted with different letters are significantly different by Tukey’s HSD test (p < 0.05).
(15 spots/106 cells) and FCA, CD40L, EtIMP1, and PBS control groups (2 spots/106 cells). 3.4. Protective efficacy of EtIMP1-CD40L protein vaccination against E. tenella in chickens To determine if responses to CD40L fusion proteins affect 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-CD40L gained significantly greater body weight (p < 0.05), had significantly decreased cecal lesions (p < 0.05), and had reduced numbers of oocysts compared with chickens in other groups (Table 1). However, no significant differences were observed in body weight and cecal lesions between
Fig. 3. EtIMP1-CD40L fusion protein induces potent EtIMP1-specific Tcell responses. Antigen-specific cellular responses were examined by the IFN-␥ ELISPOT assay. ELISPOT values represent the number of antigenspecific IFN-␥ positive spots per 106 PBMCs following stimulation with the EtIMP1 protein. Bars (mean ± SE, n = 6) denoted with different letters are significantly different by Tukey’s HSD test (p < 0.05).
EtIMP1-FCA and EtIMP1-CD40L groups (Table 1). And there were also no significant differences between FCA, CD40L, EtIMP1, and PBS control groups (Table 1). 4. Discussion In the present study, a recombinant chimeric subunit vaccine consisting of EtIMP1 and a molecular adjuvant, chicken CD40L, was generated, and its efficacy against E. tenella infection was evaluated. We found that immunization with EtIMP1-CD40L reduced oocyst output in chickens by 78%, more potently than that with EtIMP1 emulsified in FCA (by 66%). The findings support our hypothesis that CD40L, as an adjuvant for cell-mediated immunity, can improve the immunoprotective effect of IMPI against Eimeria infection. 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). The vaccination with a recombinant antigen is often not sufficient to elicit a protective immune response against E. tenella infection (Crane et al., 1991; Jenkins, 1998; Miller et al., 1989). In this study, we demonstrated that the chicken CD40L-EtIMP1, a fusion protein, reduced oocyst output by 78%, more potently than EtIMP1 alone emulsified in FCA. In addition to having the strong antibody responses, chickens immunized with EtIMP1-CD40L also elicited a specific T-cell response, as evidenced by a greater IFN-␥ production by T-cells in response to EtIMP1 stimulation ex vivo. Likely, CD40L, fused to EtIMP1, may increase the uptake and processing of EtIMP1 by antigen processing cells, thus resulting in more efficient stimulation of adaptive immune responses. This was consistent with the report that a genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo (Thacker et al., 2009). In addition, recent research has shown that murine CD154 can function as an adjuvant for viral or DNA vaccines by administering adenoviral vectors
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Table 1 Protective effects of EtIMP-CD40L against experimental infection of E. tenella in chickens. Group
Average body weight gain (g)
PBS-unchallenged PBS-challenged CD40L FCA Et-IMP1 Et-IMP1FCA Et-IMP1-CD40L
520.56 280.62 295.42 288.35 280.20 368.86 405.75
± ± ± ± ± ± ±
14.55a 17.82b 9.54b 7.36b 8.25b 16.80c 17.32c
Body weight loss (%)
Oocyst shedding per bird (×107 )
Oocyst decrease ratio (%)
Lesion score
0 46.09 43.25 44.61 46.15 29.14 22.06
0 9.15 ± 0.04a 8.76 ± 0.04a 8.84 ± 0.05a 8.74 ± 0.04a 3.07 ± 0.02b 2.05 ± 0.04c
100 0 4.30 3.39 4.48 66.45 77.60
0 3.7 ± 0.2a 3.3 ± 0.4a 3.3 ± 0.4a 3.2 ± 0.2a 1.7 ± 0.2b 1.5 ± 0.2b
Note: Values are expressed as mean ± SD. Means in the same column with different letters were significantly different between treatment groups (p < 0.05).
or plasmids that express a vaccine antigen and CD154, individually, or as a fusion protein (Mendoza et al., 1997; Tripp et al., 2000; Xiang et al., 2001). Recently it has also been demonstrated that the extracellular domain of duck’s CD154 could enhance DNA vaccine responses in ducks both by co-expression with viral antigens and as a fusion protein to target viral antigen to APCs (Yao et al., 2010). In this study we observed that chicken CD40L-fused EtIMP1 elicited a stronger protective immune response evidenced by reduced oocyst output than EtIMP1 with adjuvant did. This is also consistent with the previous studies on the use of recombinant CD40L as a vaccine adjuvant with influenza virus antigen, showing promising results in influenza vaccine development in chicken tests (Pose et al., 2011; Sanchez Ramos et al., 2011). Our findings demonstrate that CD40L is able to enhance the immunogenicity of antigens, and the fusion of CD40L to parasite antigens could also be a valuable strategy to improve the immunogenicity of parasite proteins in the stimulation of cell-mediated immunity in birds. 5. Conclusion Our results demonstrate the feasibility of producing an effective vaccine against Eimeria infection using the novel membrane protein, EtIMP1, fused to CD40L, a molecular adjuvant. This is evidenced by significant reduction in the oocyst output in chickens challenged with E. tenella. Therefore, EtIMP1-CD40L fusion protein can be used as a potent immunogen in the development of subunit vaccines against E. tenella infection. Conflict of interest statement No actual or potential conflict of interest was identified that could inappropriately influence, or be perceived to influence the outcome of this work. Acknowledgements This study was supported by the National Natural Science Foundation of China (31330076 and 31001060), Research and Innovation Project for College Students of Fujian Agriculture and Forestry University (201410389065 and 201410389154), and Key Project of the Science and Technology Department of Fujian Province (2011N0001). We thank Dr. Shile Huang (Louisiana State University Health Sciences Center, USA) for reading the manuscript.
All experimentation using chickens was carried out in compliance with the current Chinese legislation and due attention was paid to the welfare of experimental chickens. The animal protocol was approved by the Animal Care Committee of Fujian Agriculture and Forestry University. References Bhogal, B.S., Jacobson, E.B., Tse, H.Y., Schmatz, D.M., Ravino, O.J., 1989. Parasite exposure elicits a preferential T-cell response involved in protective immunity against Eimeria species in chickens primed by an internal-image anti-idiotypic antibody. Infect. Immun. 57, 2804–2810. Blake, D.P., Billington, K.J., Copestake, S.L., Oakes, R.D., Quail, M.A., Wan, K.L., Shirley, M.W., Smith, A.L., 2011. Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite. PLoS Pathog. 7, e1001279. Blake, D.P., Tomley, F.M., 2014. Securing poultry production from the everpresent Eimeria challenge. Trends Parasitol. 30, 12–19. Crane, M.S., Goggin, B., Pellegrino, R.M., Ravino, O.J., Lange, C., Karkhanis, Y.D., Kirk, K.E., Chakraborty, P.R., 1991. Cross-protection against four species of chicken coccidia with a single recombinant antigen. Infect. Immun. 59, 1271–1277. Cui, X., Lei, T., Yang, D., Hao, P., Li, B., Liu, Q., 2012a. Toxoplasma gondii immune mapped protein-1 (TgIMP1) is a novel vaccine candidate against toxoplasmosis. Vaccine 30, 2282–2287. Cui, X., Lei, T., Yang, D.Y., Hao, P., Liu, Q., 2012b. Identification and characterization of a novel Neospora caninum immune mapped protein 1. Parasitology 139, 998–1004. Grewal, I.S., Flavell, R.A., 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16, 111–135. Hashem, A.M., Gravel, C., Chen, Z., Yi, Y., Tocchi, M., Jaentschke, B., Fan, X., Li, C., Rosu-Myles, M., Pereboev, A., He, R., Wang, J., Li, X., 2014. CD40 ligand preferentially modulates immune response and enhances protection against influenza virus. J. Immunol. 193, 722–734. Huang, X., Zou, J., Xu, H., Ding, Y., Yin, G., Liu, X., Suo, X., 2011. Transgenic Eimeria tenella expressing enhanced yellow fluorescent protein targeted to different cellular compartments stimulated dichotomic immune responses in chickens. J. Immunol. 187, 3595–3602. Jenkins, M.C., 1998. Progress on developing a recombinant coccidiosis vaccine. Int. J. Parasitol. 28, 1111–1119. Johnson, J., Reid, W.M., 1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28, 30–36. Lauterbach, H., Patzold, J., Kassub, R., Bathke, B., Brinkmann, K., Chaplin, P., Suter, M., Hochrein, H., 2013. Genetic adjuvantation of recombinant MVA with CD40L potentiates CD8T cell mediated immunity. Front. Immunol. 4, 251. Layton, S.L., Kapczynski, D.R., Higgins, S., Higgins, J., Wolfenden, A.D., Liljebjelke, K.A., Bottje, W.G., Swayne, D., Berghman, L.R., Kwon, Y.M., Hargis, B.M., Cole, K., 2009. Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge. Poult. Sci. 88, 2244–2252. Lee, S.H., Lillehoj, H.S., Dalloul, R.A., Park, D.W., Hong, Y.H., Lin, J.J., 2007. Influence of Pediococcus-based probiotic on coccidiosis in broiler chickens. Poult. Sci. 86, 63–66. Lillehoj, H.S., Ding, X., Dalloul, R.A., Sato, T., Yasuda, A., Lillehoj, E.P., 2005. Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants. J. Parasitol. 91, 666–673.
24
G. Yin et al. / Veterinary Parasitology 210 (2015) 19–24
Lillehoj, H.S., Trout, J.M., 1996. Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9, 349–360. Lin, F.C., Peng, Y., Jones, L.A., Verardi, P.H., Yilma, T.D., 2009. Incorporation of CD40 ligand into the envelope of pseudotyped single-cycle Simian immunodeficiency viruses enhances immunogenicity. J. Virol. 83, 1216–1227. Mendoza, R.B., Cantwell, M.J., Kipps, T.J., 1997. Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization. J. Immunol. 159, 5777–5781. Miga, A., Masters, S., Gonzalez, M., Noelle, R.J., 2000. The role of CD40–CD154 interactions in the regulation of cell mediated immunity. Immunol. Invest. 29, 111–114. Miller, G.A., Bhogal, B.S., McCandliss, R., Strausberg, R.L., Jessee, E.J., Anderson, A.C., Fuchs, C.K., Nagle, J., Likel, M.H., Strasser, J.M., et al., 1989. Characterization and vaccine potential of a novel recombinant coccidial antigen. Infect. Immun. 57, 2014–2020. Ninomiya, A., Ogasawara, K., Kajino, K., Takada, A., Kida, H., 2002. Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice. Vaccine 20, 3123–3129. Pose, A.G., Gomez, J.N., Sanchez, A.V., Redondo, A.V., Rodriguez, E.R., Segui, R.M., Ramos, E.M., Molto, M.P., Rodriguez, E.S., Cordero, L.R., Mallon, A.R., Nordelo, C.B., 2011. Subunit influenza vaccine candidate based on CD154 fused to HAH5 increases the antibody titers and cellular immune response in chickens. Vet. Microbiol. 152, 328–337. Rose, M.E., Hesketh, P., 1979. Immunity to coccidiosis: T-lymphocyte- or B-lymphocyte-deficient animals. Infect. Immun. 26, 630–637. Sanchez Ramos, O., Gonzalez Pose, A., Gomez-Puerta, S., Noda Gomez, J., Vega Redondo, A., Aguila Benites, J.C., Suarez Amaran, L., Parra, N.C., Toledo Alonso, J.R., 2011. Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens. Comp. Immunol. Microbiol. Infect. Dis. 34, 259–265. Sathish, K., Sriraman, R., Subramanian, B.M., Rao, N.H., Balaji, K., Narasu, M.L., Srinivasan, V.A., 2012. Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis. Vaccine 29, 9201–9208. Shirley, M.W., Lillehoj, H.S., 2012. The long view: a selective review of 40 years of coccidiosis research. Avian Pathol. 41, 111–121. Shirley, M.W., Smith, A.L., Tomley, F.M., 2005. The biology of avian Eimeria with an emphasis on their control by vaccination. Adv. Parasitol. 60, 285–330.
Subramanian, B.M., Sriraman, R., Rao, N.H., Raghul, J., Thiagarajan, D., Srinivasan, V.A., 2008. Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds. Vaccine 26, 3489–3496. Sun, H., Wang, L., Wang, T., Zhang, J., Liu, Q., Chen, P., Chen, Z., Wang, F., Li, H., Xiao, Y., Zhao, X., 2014. Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis. Vaccine 32, 1869–1876. Suo, X., Li, A., Jiang, J., Lv, Y., Wang, Z., Wu, W., Qv, H., 2000. Studies on the efficacy of Eimerivac Plus, a coccidiosis vaccine for chickens. Acta Vet. Zooltech. Sin. 31, 353–359. Thacker, E.E., Nakayama, M., Smith, B.F., Bird, R.C., Muminova, Z., Strong, T.V., Timares, L., Korokhov, N., O’Neill, A.M., de Gruijl, T.D., Glasgow, J.N., Tani, K., Curiel, D.T., 2009. A genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo. Vaccine 27, 7116–7124. Tregaskes, C.A., Glansbeek, H.L., Gill, A.C., Hunt, L.G., Burnside, J., Young, J.R., 2005. Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate. Dev. Comp. Immunol. 29, 361–374. Tripp, R.A., Jones, L., Anderson, L.J., Brown, M.P., 2000. CD40 ligand (CD154) enhances the Th1 and antibody responses to respiratory syncytial virus in the BALB/c mouse. J. Immunol. 164, 5913–5921. Xiang, R., Primus, F.J., Ruehlmann, J.M., Niethammer, A.G., Silletti, S., Lode, H.N., Dolman, C.S., Gillies, S.D., Reisfeld, R.A., 2001. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice. J. Immunol. 167, 4560–4565. Yao, Q., Fischer, K.P., Li, L., Agrawal, B., Berhane, Y., Tyrrell, D.L., Gutfreund, K.S., Pasick, J., 2010. Immunogenicity and protective efficacy of a DNA vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekin ducks. Vaccine 28, 8147–8156. Yin, G., Qin, M., Liu, X., Suo, J., Suo, X., 2013a. Interferon-gamma enzymelinked immunosorbent spot assay as a tool to study T cell responses to Eimeria tenella infection in chickens. Poult. Sci. 92, 1758–1763. Yin, G., Qin, M., Liu, X., Suo, J., Tang, X., Tao, G., Han, Q., Suo, X., Wu, W., 2013b. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin. Biochem. Biophys. Res. Commun. 440, 437–442.
