IL-7 Enhances Protective Immunity Induced by Eimeria tenella Elongation Factor-1? (EF-1?) DNA Vaccine Against Coccidiosis
Alfredo Panebraa and Hyun S. Lillehoja*
aAnimal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Service, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD, 20705, USA
*Corresponding author at:
Hyun S. Lillehoj
10300 Baltimore Avenue. Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Service, Agricultural Research Service-U.S. Department of Agriculture, BARC-East Bldg. 1043 Rm. 107, Beltsville, MD, 20705, USA.
Phone: +1 301-504-8771
e-mail address: [email protected]
The rising demand for poultry food products is challenged by factors including governmental limitations on the use of antibiotic growth promoters (AGP); high density production conditions; waste management; and emergence of drug-resistant infectious pathogens, particularly those causing intestinal diseases. Although the use of Eimeria sp. oocyst vaccines has been valuable in reducing the need for in-feed medication in chicken growth, experimental coccidiosis vaccines based on recombinant Eimeria genes have been shown to be effective in model systems of experimental coccidiosis. Our goal was to assess E. tenella antigen Elongation Factor-1? (EF-1?) and chicken IL-7 DNA vaccine against coccidiosis in broilers. After EF-1? and chIL-7 immunizations and later challenge; coccidiosis clinical parameters such as body weight gain, gut lesion score, oocyst shedding, humoral immune response, and intestinal proinflammatory cytokine expression were evaluated to determine the effectiveness of DNA vaccination against E. acervulina infection. Chickens immunized with EF-1? and chIL-7 showed overall improvements in clinical disease manifestation, possibly through cytotoxic effector cells. This study demonstrated the beneficial effects of combining EF-1? DNA vaccine with host cytokine chIL-7 DNA to improve T-cell-mediated effector function in coccidiosis-challenged broiler chickens.
Eimeria; DNA vaccine; EF-1?; chicken IL-7; Thymus; T-cell lymphocytes.
Avian coccidiosis is the most economically damaging infectious diseases affecting poultry. The etiologic agent Eimeria sp., is an obligate Apicomplexan intracellular parasite that infects chickens’ intestinal tracts and is transmitted through fecal-to-oral route (Shirley and Lillehoj, 2012; Chapman et. al., 2013). Infection is clinically displayed through intestinal epithelium damage, diminished nutrient absorption; affecting both feed utilization and animal growth that could eventually lead to high mortality rates (Williams 1999; Shirley et. al., 2004; Shirley et. al., 2005).
Coccidiosis is currently a big treat for poultry industry mainly due to the arisen of parasite drug resistance. For many years, infection was controlled with anti-coccidial drugs, but governments worldwide were engaged in withdrew use of antibiotic growth promoter (AGP) in poultry and increasingly in favors use of vaccines (McDonald and Shirley, 2009). Nowadays, there are several Coccidiosis vaccines in the market that contain live parasites (Coccivac-B), attenuated precocious (Paracox-5, -8), embryo-adapted (Livacox) or ionophores-tolerant lines (Coccivac-D) (Chapman et.al., 2005).
Avians hold an immune system that is poorly understood and they recently have been re-discovered (Lillehoj 1998; Lillehoj et. al., 2004; Dalloul and Lillehoj, 2006). Host-pathogen interaction in coccidiosis is complex and involves many facets of humoral and cell-mediated immunity (Gazzinelli and Denkers, 2006). While most efforts have been directed towards attenuated vaccine development, passive immunization with coccidial-specific egg-yolk antibodies spiked in feed resulted in production of parasite-specific antibodies that protect birds against coccidiosis (Wallach 1997; Gadde et. al., 2015). Furthermore, subunit vaccines based on recombinant antigens from merozoites and gametocytes (Wallach et. al., 1989), micronemes (Tomley et. al., 1991; 1996), rhoptries (Tomley 1994), refractile bodies (Vermeulen et. al., 1993), profilin (Jang et. al., 2011a, b), and more recently E. tenella elongation factor-1? (EF-1?) (Lin et. al., 2017) have been evaluated. DNA vaccines have received attention basically due to their ability to induce T helper (Th1) and activate CD8+ responses (Gurunathan et. al., 2000; Hoft et. al., 2007; Cherif et. al., 2011). Additionally, several studies have used cytokines as immunological adjuvants (Khan et. al., 1994; Kasper et. al., 1995, 1996). T lymphocytes are dependent on IL-7 for their development, survival, differentiation and homeostasis (Kasper et. al., 1995; Singh et. al., 2010), and has also been found to decrease morbidity and enhance immunogenicity of infectious bursal disease virus vaccine (Huo et. al., 2016 a, b; Cui et. al., 2018).
Elongation Factor 1-? (EF-1?), a ubiquitous expressed ribosomal protein is required for protein synthesis (Condeelis, 1995) and involved in parasite’s pathogenesis (Nandan et. al., 2005), invasiveness (Matsubayashi et. al., 2013, 2016), and immunity to Toxoplasma gondii and Eimeria sp. infections (Wang et. al., 2015; Lin et. al., 2017, respectively). T. gondii protein kinase 1 co-administered with host cytokines DNA vaccine enhanced immunity in mice to Toxoplasmosis (Chen et. al., 2016). Moreover, lack of IL-7 and IL-15 severely reduced cytotoxic response against Toxoplasmosis (Bhadra et. al., 2010). Other studies have reported that chicken IL-7 decreases morbidity and enhances immunogenicity in the presence of infectious bursal disease virus vaccine (Huo et. al., 2016 a, b; Cui et. al., 2018).
Our goal was to evaluate protection induced by EF-1? and cytotoxic memory response elicited by IL-7 DNA vaccine against Coccidiosis in broilers.
3.0 MATERIAL AND METHOD
2.1 Ethics statement
Trial procedures details were previously supported by Beltsville Agriculture Research Center Small Animal Care and Use Committee (BACUC, BA, ARS, USDA, Protocol Number: 15-022). Pain or experimental distress were minimized as possible and euthanasia used for humane endpoint.
2.2 Chickens, husbandry, adjuvant and immunizations
Broilers (Ross/Ross) were acquired from Longnecker’s Hatchery (Elizabethtown, PA) and allotted into nine groups (N = 25), fed with a basal diet (24% protein content) and water provided ad libitum trial wide.
Montanide Gel 01, a polymeric adjuvant designed to improve antigen immunogenicity, was supplied by Seppic (Puteaux, France) and administered at 10 % (Di Giacomo et. al. 2015).
Chickens were immunized with 50 or 100 µg E. tenella EF-1? and/or chicken IL-7 plasmids by intramuscular route twice at one week interval with 1ml tuberculin syringe with attached 27 G 3/4″ needle. A schematic representation of EF-1? and chIL-7 DNA vaccine against Coccidiosis trial protocol is shown in Figure 1.
2.2 Eimeria preparation
Eimeria acervulina Beltsville strain EA 12 sporulated oocysts were used for chicken infection. Prior to challenge, oocysts were washed, counted in McMaster chambers and used for infection at 1 × 104 oocysts per chicken by oral gavage.
2.3 Cloning and expression of E. tenella EF-1? and chicken IL-7, megapreps, and chIL-7 bioactivity
EF-1? (Accession Number KX900609) was isolated by PCR from E. tenella oocysts and cloned in pET32a (+) (Lin et. al., 2017). It was subsequently subcloned in pcDNA3.1(+) and expressed in COS7 cells by transient transfection using Fugene HD reagent (Promega, Madison, WI). Recombinant EF-1? was resolved on polyacrylamide gel, transfer to PDVF membrane and probed with EF-1?-specific rabbit polyclonal antibody (Pacific Immunology, Ramona, CA).
Chicken IL-7 (Accession Number KY020410) was isolated from E. maxima-infected pooled tissue cDNA by RT-PCR. It was subcloned in pcDNA3.1 (+) and a his-tag attached at its C-terminus to monitor expression and purification. Recombinant chIL-7 was purified from HEK-293T transfected cells’ conditioned media by affinity chromatography on Ni-NTA columns and immunodetected by anti-histidine-tag monoclonal antibody (EMD-Millipore, Burlington, MA).
For in vivo trials, EF-1?, chIL-7 and empty vector pcDNA3.1(+) high quality plasmid megapreps with low endotoxin level were prepared from Terrific Broth cultures using Pure Link™ Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo-Fisher Scientific, Frederick, MD) and endotoxin levels assessed with Pierce Limulus Amoebocyte Lysate Chromogenic Endotoxin Quantitation Kit (Pierce, Frederick, MD) and were ? 3 EU/mL.
Chicken IL-7’s biological activity was assessed by CCK 8 proliferation assays (Dojindo, Rockville, MD) on chicken thymocytes per Armitage et. al., 1990.
2.4 Clinical evidence of vaccine efficacy
Three parameters were chosen to monitor vaccine efficacy: body weight gain, fecal oocyst output, lesion scores. Additionally, we’ve analyzed humoral (ELISA) and cellular immune response (qPCR) in bloodstream and duodenum samples, respectively.
Regarding the effect of E. acervulina infection on body weight, it was assessed as the bird weight difference between 5 days’ post- minus pre-challenge of individually monitored bird (N = 10).
Fecal oocyst output was measured by counting oocysts present in fecal chicken samples (N = 10) collected from 4 to 9 days’ post-challenge using McMaster chambers (Ding et. al., 2004).
Recording lesion scores was assayed on 5 days’ post-challenge. Chickens (N = 6) were sacrificed and excised duodenum arranged for lesion score assessment in a blind fashion as reported by Johnson and Reid, 1970.
2.5 EF-1? serum antibody titer
Sera was obtained from blood collected by cardiac puncture and anti-EF-1? antibody levels assessed by an in-house developed ELISA protocol (Ding et. al. 2004). Recombinant EF-1? was coated on Nunc microtiter plates at 1 µg/well. Then, plates were washed and blocked with 3% Bovine Serum Albumin (Sigma-Aldrich, St. Louis, MO) and incubated for an hour. Plates were washed again and incubated with diluted sera samples (1 : 50) for 2 hours. Bound antibodies were detected with peroxidase-conjugated rabbit anti-chicken IgG and peroxidase-specific substrate (Sigma-Aldrich, St. Louis, MO). Optical density values were recorded on microplate reader EL-800 (Biotek, Winooski, VT).
2.6 Duodenum’s cytokine levels
On 2nd day post-challenge, chickens (N = 5) were sacrificed, duodenum excised and stored in RNAlater. Briefly, duodenum was washed with PBS, minced and homogenized in TRIzol reagent using TissueRuptor (Qiagen, Germantown, MD). Total RNA was isolated (Hong et. al., 2006) and treated with 1 U DNase I-RNase-free (DNase-free) (Ambion, Frederick, MD). RNA (1 µg) was converted into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Germantown, MD). Real Time Polymerase Chain Reaction amplification was performed using oligonucleotide primers for chicken IL-2, IFN-?, and GAPDH (Table 1) using Mx3000P system (Stratagene, San Diego, CA). Relative gene expression data were analyzed with the Q-Gene method (Simon 2003).
2.7 Statistical analysis
Data were analyzed by IBM SPSS 19.0 (SPSS Inc., Chicago, IL). Grouped data were compared by one-way ANOVA with Duncan’s post hoc test. Results were considered statistically significant if P ? 0.05.
3.1 Construction of chicken IL-7 and EF-1? DNA vaccine plasmids, protein expression, and chicken IL-7 bioactivity
We’ve assessed cloning of Eimeria EF-1? and chIL-7 by mega preps’ restriction endonuclease digestion. Cloned EF-1? had of an open reading frame (ORF) of approx.1.5 Kb., while chicken IL-7 had an ORF of approx. 550 bp as depicted in Figure 2A. Later, we have expressed recombinant EF-1? and chIL-7 in COS7 and HEK-293T, respectively by transient transfection using Fugene HD (Promega, Madison, WI).
Firstly, histidine-tagged chIL-7 was expressed in HEK-293T cells, released into conditioned media, purified and confirmed by immunoblot using anti-His-tag monoclonal antibody (Figure 2B). Chicken IL-7 predicted molecular weight was 25 KDa., HEK-293T-synthetized chIL-7 run in SDS-PAGE as several discrete bands ranging from 25 to 40 KDa. This may be due to post-translational modifications (own four putative N-glycosylation sites).
Likewise, rEF-1? synthetized by COS7 cells run as a discrete band of 47 KDa on SDS-PAGE (upper band in transfected lane). Meanwhile, common band of molecular weight 45 KDa. (lower band) present in empty vector- and EF-1?-transfected lanes corresponded to host EF-1?, since parasite’s peptide used for rabbit immunization shared ? 85 % identity with host EF-1? and shown in Figure 2C. Chicken IL-7 biological activity was assessed by thymocytes proliferation assay. A dose-dependent proliferation was observed when thymocytes were stimulated with increasing concentrations of chIL-7 (10 pg to 1 ng) and shown in Figure 3.
3.2 Body weight gain
Chickens of coccidiosis infected group showed a significant body weight loss compared to uninfected control. Immunization of 8-day-old chickens with 50 µg or 100 µg EF-1? plus 20 µg chIL-7 DNA vaccine outcome in a diminished body weight loss (Duncan post-test P ? 0.05) reaching up to 80 % and 95 % uninfected control weight, respectively (Figure 4). While, chickens immunized with 50 µg EF-1?, 100 µg EF-1?, or 20 µg chIL-7 showed a less significant decrease in body weight loss (P ? 0.05), reaching up to 30 – 40 % uninfected control weight.
3.3 Duodenal lesion scoring
E. acervulina infected chickens showed an average duodenal lesion scores of 2. Group immunized with DNA vaccine containing 20 µg chIL-7, or 100 µg EF-1? plus 20 µg chIL-7 showed significantly fewer duodenal lesions (average lesion score of 1.5), per Duncan’s multiple range test (P ? 0.05), as shown in Figure 5.
3.4 Oocyst output
Infected chickens shed higher oocysts than uninfected control. Chickens immunized with 20 µg chIL-7 or 50 µg EF-1? plus 20 µg chIL-7 showed significant less oocyst output reaching up to 40 % –50 % infected control (P < 0.05). Moreover, birds immunized with 50 µg or 100 µg EF-1? showed slightly, but still statistically significant reduction in oocyst output (P < 0.05) per Duncan post hoc test. Interestingly, group immunized 100 µg EF-1? plus 20 µg chIL-7 showed again a slight but significant (P < 0.05) greater oocyst output than infected controls (Figure 6).
3.5 Anti-EF-1? serum antibody titer
Only group immunized with 20 µg chIL-7 showed higher and statistically significant anti-EF-1? antibody levels than others groups including infected control (Figure 7). Interestingly, group immunized with 100 µg EF-1? showed lowest anti-EF-1? antibody levels reaching up 50 % of infected control (P ? 0.001).
3.6 Duodenum cytokine levels
As shown in Figure 8, significant upregulation of IFN-? (four-fold) was observed in chickens immunized with pcDNA3.1(+)-EF-1? plus 20 µg chIL-7, as compared with infected control (P ? 0.005), whereas other groups showed just barely IFN-?.
Furthermore, significantly higher chicken IL-2 upregulation was seen in group treated with 100 µg EF-1? plus 20 µg chIL-7, as compared with infected control (P ? 0.005). Similarly, barely IL-2 expression were detected in other groups as shown in Figure 9.
Results showed that EF-1? (50 and/or 100 µg) when co-administered with chicken IL-7 (20 µg), significantly improved body weight gain reaching levels close to uninfected control; ameliorated the intestinal damage produced by parasite multiplication; and decreased oocyst shedding after E. acervulina challenge. Furthermore, immunization of young chickens with EF-1?/chIL-7 DNA vaccine elicited high anti-EF-1? antibody titers and induced duodenal upregulation of proinflammatory cytokines (IL-2 and IFN-?), thus enhancing humoral and cellular immune response.
Although, there were few reports on chicken IL-7 role in viral pathogenesis and immune homeostasis (Huo et al., 2016 a, b; Cui et. al., 2018), we’ve found that chIL-7 stimulated thymocyte proliferation to increasing chIL-7 concentrations (10 pg to 2 ng) (Figure 4), but did not proliferate when thymocytes were stimulated with human or mouse recombinant IL-7, even at higher concentrations (2 µg) (data not shown). Furthermore, chIL-7 did not stimulate chicken lymphocytes isolated from bloodstream (PBMC), bursa, spleen, or mouse immature B-lymphocytes (2E8), a cell line exhibiting IL-7-dependent proliferation (data not shown); these results contradicted those reported by Huo et. al., 2016a.
Moreover, DNA vaccination with Leishmania major antigen co-administered with murine IL-12 induced protective and long-term immunity (Gurunathan et. al., 2000).
Meanwhile, human rIL-7 administration has protected mice against T. gondii by stimulating IFN-? production and augmenting cytotoxic T-lymphocyte response (Kasper et. al., 1995).
In eukaryotes, EF-1? constituted 1 – 2 % of total protein synthesized by normal cells (Dharmawardhane et al., 1991; Slobin 1980; Derventzi et. al., 1993). Furthermore, Wang et al., 2015 have reported that vaccination with pVAX-EF-1? triggered strong immune response and induced protection against Toxoplasmosis in mice. Likewise, EF-1? has been involved in actin binding and bundling (Yang et. al., 1990).
Our findings corroborated that reported by Lin et. al., 2017 regarding body weight gain, oocyst shedding and the humoral immune response on EF-1? use as coccidiosis vaccine, even though that study used recombinant EF-1? protein, whereas our trial used DNA vaccine. Furthermore, EF-1? has also been associated with host invasion in Cryptosporidium parvum (Matsubayashi et. al., 2013), and in E. acervulina EF-1? has been related to parasite invasion (Matsubayashi et. al., 2016) and thus, a candidate vaccine antigen against cryptosporidiosis or coccidiosis. Recent evidence indicated EF-1? released inside exosomes into extracellular milieu and delivered to host cells (Del Cacho et. al., 2011, 2012, 2016). In Leishmaniasis through activating host protein-tyrosine phosphatases, thus negatively regulating IFN-? signaling and hindering macrophage microbicidal arsenal (Silverman and Reiner, 2012; Atayde et. al., 2015; Piedra-Quintero et. al., 2015; Yu et. al., 2006). Furthermore, in Plasmodium falciparum (Costa et. al., 2013), Giardia intestinalis (Skarin et. al., 2011), and Echinococcus granulosus (Siles-Lucas et. al., 2017), EF-1? promoted immunity against infection.
This report showed protective results due to EF-1? and IL-7 DNA vaccine. Further research would determine underlying immune response and protection against coccidiosis by EF-1? and IL-7 DNA vaccine. Furthermore, nature of memory and cell types contributing prolonged immune response to EF-1? should be better characterized.
5.0 CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.
In summary, the current study demonstrated that an E. tenella EF-1? and chicken IL-7 DNA vaccination protocol against coccidiosis in broiler chickens induced a protective immune response mainly due to stimulation and proliferation of CD8+ T-cells, thus mediating a cytotoxic response and possibly exerting substantial beneficial effects toward the clinical manifestation of coccidiosis. Additionally, EF-1? DNA vaccine elicited higher humoral response and upregulated proinflammatory cytokines (IFN-? and IL-2) gene expression in chicken duodenum.
Declaration of Interest: Authors declare do not have any conflict of interest.
This study was fully supported by a Trust Fund Cooperative Agreement (58-8042-8-002-F) established between ARS, USDA and Seppic, Inc. (Puteaux, France). Authors want to thank Stacy O’Donnell for her technical assistance.
All authors attest they meet ICMJE criteria for authorship.
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Figure 1. Schematic ilustration chicken IL-7 and E. tenella EF-1? DNA vaccine against Coccidiosis protocol.
Figure 2 Chicken IL-7 and E. tenella EF-1? sub-cloning and expression. Chicken IL-7 and E. tenella EF-1? were sub-cloned in pcDNA3.1(+). A. Endonuclease digestion of empty vector (EV); and chIL-7 and EF-1? recombinant plasmids. B. Recombinant chIL-7 expressed in HEK-293T cells. C. Recombinant EF-1? expressed in COS7 cells.
Figure 3 Chicken thymocyte proliferation by chicken IL-7. Chicken thymii were excised and thymocytes isolated. Microtiter plate was seeded with 2 x 106 thymocytes/well and stimulated with increasing concentration of chicken IL-7 (10 pg to 1 ng). Proliferation assay was measured by CCK8 kit. Samples were run in triplicate and each dot represent mean ± SEM. Data were analyzed and plotted as dose-response curve.
Figure 4. Body weight gain. Chickens (N = 10) from different groups were weighed before and 6 days post-challenge. Body weight gain were calculated and data analyzed by one-way ANOVA with Duncan post hoc test. Bars with different letters are significantly different per Duncan’s multiple range test (P < 0.05).
Figure 5 Lession scores. Chickens (N = 6) were sacrificed on day 6 post-challenge for lesion scoring on a scale from 0 (zero) to 4 basis according to Johnson and Reid, 1970. Each number represent mean ± SEM. Bars with different letters are significantly different per Duncan’s multiple range test (P < 0.05).
Figure 6 Oocyst shedding. Chicken (N = 10) fecal samples were collected, blended, and diluted in saturated salt solution prior to count in Mc Master chamber. Each bar represent mean ± SEM. Bars with different letters were significantly different per Duncan’s multiple range test (P < 0.05).
Figure 7 Anti-EF-1? antibody levels in chicken sera. Chickens (N = 5) were bled by cardiac puncture and sera collected by low speed centrifugation. Anti-EF-1? antibody levels were measured by in-house developed ELISA protocol. Samples were processed in triplicate and corresponded to difference between Post- Minus Pre-E. acervulina challenge OD450nm. Each bar represent mean ± SEM. Data were analyzed by one-way ANOVA and significantly different per Duncan’s multiple range test (P < 0.001).
Figure 8 Duodenum IFN-? gene expression. Chicken duodenum (N = 5) were stored in RNAlater at -70°C. RNA (1 µg) was isolated using TRIzol and later retro-transcribed by QuantiTect Reverse Transcription Kit. Quantitative RT-PCR were run using specified primers (Table 1) in Mx3000P qPCR System. Relative quantification was calculated using Q-gene method (Simon P, 2003). Each sample was analyzed in triplicate and each bar represent mean ± SEM. Data were analyzed by one-way ANOVA and were significantly different per Duncan’s multiple range test (P < 0.005).
Fig. 9 Duodenum IL-2 gene expression. Chicken duodenum (N = 5) were stores in RNAlater at -70°C. RNA (1 µg) once isolated using TRIzol was retro-transcribed by QuantiTect Reverse Transcription Kit. Quantitative RT-PCR were run using specified primers (Table 1) in Mx3000P qPCR System. Relative quantification was calculated using Q-gene method (Simon P, 2003). Each sample was analyzed in triplicate and each bar represent mean ± SEM. Data were analyzed by one-way ANOVA and were significantly different per Du