Eimeria tenella Elongation Factor-1a (EF-1a) Coadministered with Chicken IL-7 (chIL-7) DNA Vaccine Emulsified in Montanide Gel 01 Adjuvant Enhanced the Immune Response to E. acervulina Infection in Broiler Chickens

Avian coccidiosis is one of the most economically damaging infectious diseases affecting poultry. The etiologic agent is Eimeria spp., an obligate eukaryotic intracellular parasite belonging to the phylum Apicomplexa, which infects chickens’ intestinal tracts and is transmitted through a fecal-to-oral route (7,49). Clinical manifestations of infection include damage to the intestinal epithelium, decreased nutrient absorption, inefficient feed utilization, and impaired growth rate, which in severe cases may lead to mortality (48,50,65).

Coccidiosis is a major problem for the poultry industry, and for many years prophylactic chemotherapy with specific anticoccidial drugs has been the preferred method of preventing and controlling the disease. However, drug resistance is a severe problem, and legislation in various countries increasingly favors the use of an expanding portfolio of vaccines (43). Several coccidiosis vaccines are commercially available and contain parasites that are live, non-attenuated, and nontolerant to ionophore strains (Coccivac-B and -D), to attenuated strains (precocious [Paracox-5, -8] and Eimeria tenella egg-adapted [Livacox]), and the ionophore-tolerant strain Nobilis Cox ATM (Intervet [now MSD], Madison, NJ) (8).

Avians possess both innate and adaptive immunity involving humoral and cell-mediated immune responses (14,38,39). Host-pathogen interaction in coccidiosis is complex and involves many facets of humoral and cellular immunity (24). Although most efforts have been directed toward attenuated vaccine development based on precocious lines, another form of vaccination has been introduced in which coccidial-specific egg-yolk antibodies are passively introduced in feed, thus resulting in the production of parasite-specific antibodies that protect against coccidiosis (23,62). Furthermore, various subunit vaccines based on recombinant antigens from merozoites and gametocytes (63), proteins associated with invasion and apical complex such as micronemes (58,59), rhoptries (57), refractile bodies (60), profilin (30,31), and more recently E. tenella elongation factor-1α (EF-1α) (40), have been evaluated with varying degrees of success.

Eimeria tenella Elongation Factor-1a (EF-1a) Coadministered with Chicken IL-7 (chIL-7) DNA Vaccine Emulsified in Montanide Gel 01 Adjuvant Enhanced the Immune Response to E. acervulina Infection in Broiler Chickens - Image 1

DNA-delivered vaccines have received considerable attention because of their ability to induce T-helper type 1 (Th1) and activate CD8⁺ cytotoxic T-lymphocyte response (10,25,26). Additionally, several studies have used cytokines, including, IL-7, IL-12, and IL-15, as immunologic adjuvants (33,34,35).

EF-1α, which is highly conserved and ubiquitously expressed in all eukaryotic cells, plays a central role in protein synthesis and is responsible for aminoacyl-tRNA loading onto the ribosomal site A (11). In parasites, EF-1α has been implicated in pathogenesis (44), host cell invasion (41,42), and protective immunity against Toxoplasma gondii, E. tenella, and Eimeria maxima infections (40,64).

IL-7 plays a critical role in homeostatic regulation of na¨ive and memory T-cells (34,54); it has also been found to decrease morbidity and enhance immunogenicity of infectious bursal disease virus vaccine (13,28,29). In addition, IL-7 expression by enterocytes was enough for extrathymic development of TCR-γδ cells and was crucial for organization of mucosal lymphoid tissue (36,39).

Thus, T. gondii calcium-dependent protein kinase 1, coadmin-istered with IL-7 and IL-15 DNA vaccine in mice, has been found to improve protective immunity against T. gondii (9). Moreover, the absence of IL-7 and IL-15 has been found to severely impair the CD8⁺ T-cell response against T. gondii (4).

The current study was undertaken to assess the immune response induced by EF-1a and IL-7 as a DNA vaccine against Eimeria acervulina infection in broiler chickens.

MATERIALS AND METHODS

Chickens, husbandry, adjuvant, and immunizations. One-day-old broiler chickens (Ross/Ross) were purchased from Longnecker’s Hatchery (Elizabethtown, PA). Upon arrival, chickens were allotted into seven groups (n ¼ 25), fed with basal diet (24% protein content), and water was provided ad libitum trial wide. Chickens were immunized with E. tenella elongation factor (EF)-1α (50 or 100 μg) and/or chicken interleukin (chIL)-7 (20 μg) DNA vaccine emulsified in Montanidet Gel 01 PR (10%), twice by intramuscular route at each leg, every other week. Montanide^®Gel 01, a polymeric adjuvant designed to improve safety and efficacy of DNA vaccines, was provided by Seppic (Puteaux, France) and used at 10% as recommended by manufacturers (21). The vaccine study schedule is shown in Fig. 1.

Trial procedures and experimental details were previously approved by Beltsville Animal Care and Use Committee, Agricultural Research Center, U. S. Department of Agriculture (BACUC, ARS, USDA).

Eimeria preparation. Eimeria acervulina Beltsville strain EA12 sporulated oocysts were used for chicken infection. Prior to challenge, sporulated oocysts were washed with phosphate-buffered saline (PBS), counted in McMaster chambers, and used for infection at 1 x 10⁴ sporulated oocysts per chicken by oral gavage.

Cloning and expression of E. tenella EF-1α and chicken IL-7, mega preps, and chIL-7 bioactivity. EF-1a (GenBank Accession Number KX900609) was isolated by PCR from E. tenella oocysts and cloned in pET32a(+) (40). The recombinant EF-1α protein used to assess humoral immune response was produced by GenScript (Piscat-away, NJ) from Escherichia coli BL-21 cultures and purified by affinity chromatography on Ni-NTA columns. It was subcloned in pcDNA3.1(+) and expressed in COS7 cells (fibroblast-like cell lines derived from monkey kidney tissue) by transient transfection using Fugene HD reagent (Promega, Madison, WI). Cellular lysate was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotted, and probed with anti-EF-1α rabbit polyclonal antibody (Pacific Immunology, Ramona, CA).

Chicken IL-7 (GenBank Accession Number KY020410) was isolated from E. maxima-infected chicken pooled tissue cDNA (intraepithelial lymphocytes, thymus, brain, heart, liver, bursa, and spleen) by reverse-transcription PCR (RT-PCR). It was subcloned in pcDNA3.1(+) and a his-tag was attached at its C-terminus to monitor its expression and purification. Recombinant chIL-7 was purified by affinity chromatog-raphy on a Ni-NTA column from conditioned media of HEK-293T transfected cells containing released chIL-7-6xH into the media, run on SDS-PAGE, blotted, and probed with monoclonal anti-his-tag antibody (EMD-Millipore, Burlington, MA).

For the in vivo trial, high quality mega preps with low endotoxin level pcDNA3.1(+)-EF-1α and -chIL-7 were purified from Terrific Broth cultures using Pure Link™ Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo-Fisher Scientific, Frederick, MD). The mega preps endotoxin level was assessed using a Pierce Limulus Amoebocyte Lysate Chromogenic Endotoxin Quantitation Kit (Pierce, Frederick, MD) and quantified in both mega preps as ≤3 EU/ml. The biologic activity of chicken IL-7 was assessed by a CCK 8 proliferation assay (Dojindo, Rockville, MD) on freshly isolated chicken thymocytes (1).

Body-weight gain, fecal oocyst shedding, and lesion scores. The effect of DNA immunizations and E. acervulina challenge on body weight gain was calculated by subtracting the weight of 10 individually wing-banded chickens per group (n = 10) between 5 days postchallenge minus weight at prechallenge.

Ten birds of each group were placed into three hanging cages with collection trays, and fecal droppings from these cages were collected from 4 to 9 days postinfection. Fecal samples weighed approximately 150–200 gr/group on average.

Lesion scores were evaluated at 5 days postchallenge. Six chickens (n = 6) were sacrificed per group and the duodenum excised and arranged for a lesion score evaluation in a blind fashion by three independent observers. Duodenal lesions were scored on a scale from 0 to 4 (32).

Briefly, scores represented 0 (no lesions), 1 (mild lesions), 2 (moderate lesions), 3 (severe lesions), or 4 (extremely severe lesions or death due to coccidiosis).

EF-1α serum antibody level. Blood was collected by cardiac puncture from three birds (n = 3) per group at prechallenge and 9 days postchallenge. Sera were obtained by low speed centrifugation and anti-EF-1α antibody levels were assessed by ELISA (22). Briefly, microtiter plates were coated with recombinant EF-1α produced by GenScript at 10 μg/ml in 0.05 M carbonate buffer (pH 9.6) and incubated overnight at 4 C. The next day, plates were washed three times with 1 x PBS-0.05% Tween^® 20 and blocked with PBS-3% BSA (Sigma-Aldrich, St. Louis, MO) for 1 hr at room temperature with gentle shaking. Plates were washed again and incubated with diluted serum samples (1:50) in blocking buffer for 2 hr. Bound antibodies were detected with peroxidase-conjugated rabbit anti-chicken IgG and peroxidase-specific substrate (Sigma-Aldrich, St. Louis, MO). Optical density values were measured at 450 nm on a microplate reader EL-800 (Biotek, Winooski, VT). The effect of DNA immunizations and E. acervulina challenge on the anti-EF-1α antibody levels was calculated by subtracting 9 days postchallenge optical density (OD)_{450 nm} values minus prechallenge OD_{450 nm} values (ODD) as shown in the following formula: ODD ¼ OD_{450 nm} 9 days post-challenge – OD_{450 nm} pre-challenge.

Duodenum cytokine levels. At 5 days postchallenge, three birds (n = 3) were sacrificed, the duodenum excised and stored in RNAlater, and stored at –20 C for further processing. Briefly, duodenum samples were opened, washed with cold Hank’s Balanced Salt Solution (HBSS), minced, and homogenized in TRIzol reagent using TissueRuptor (Qiagen, Germantown, MD). Total RNA was isolated (27) and treated with DNase I-RNase-free (DNase-free; Ambion, Frederick, MD). RNA (1 μg) was reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR (qPCR) amplification was carried out using equivalent amounts of RNA and oligonucleotide primers for chicken IL-2, IFN-γ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Table 1) using the Mx3000P system (Stratagene, San Diego, CA) and RT² SYBR Green ROX qPCR Master Mix (Qiagen). Normalized chicken IL-2 and IFN-γ messenger RNA (mRNA) values were obtained by extrapolating chicken proinflammatory cytokine cycle threshold (Ct) values from a standard curve generated by increasing the amount of cDNA and normalized to GAPDH.

Statistical analysis. Statistical analyses were performed in IBM SPSS 15.0 Data Editor (SPSS Inc., Chicago, IL). Most data differences across all groups were compared by one-way ANOVA with a Duncan post hoc test. Lesion scores was analyzed by a statistical nonparametric Kruskal-Wallis rank test with a Dunn multiple comparison post hoc test using Prism GraphPad 5 software (GraphPad Software, La Jolla, CA). Differences between groups were considered statistically significant at P ≤ 0.05.

RESULTS

Construction of chicken IL-7 and EF-1α DNA vaccine plasmids, protein expression, and chicken IL-7 bioactivity. We assessed the in-frame cloning of Eimeria EF-1α and chIL-7 in eukaryotic expression vector pcDNA3.1(+) by restriction endonuclease digestion and sequencing. The EF-1α open reading frame (ORF) had a size of approximately 1.5 kB whereas the chicken IL-7 ORF size was approximately 550 bp, as shown in Figure 2A.

Meanwhile, recombinant proteins EF-1α and chIL-7 were expressed in COS7 and HEK-293T, respectively, by transient transfection using Fugene HD (Promega).

Thus, histidine-tagged chIL-7 was expressed in HEK-293T-cells, released into media (conditioned media), purified by affinity chromatography, run on SDS-PAGE, blotted, and developed using anti-his-tag monoclonal antibody (Fig. 2B). Chicken IL-7 had a predicted molecular weight of 25 KDa whereas HEK-293T-synthetized chIL-7 showed on western blot as several discrete bands ranging from 25 to 40 KDa This may be due to posttranslational modifications (own four putative N-glycosylation sites).

Likewise, rEF-1α synthesized by COS7 cells was run as a discrete band of 47 KDa on western blot (upper band in transfected lane). Meanwhile, a common band of molecular weight 45 KDa (lower band), present in empty vector- and EF-1α-transfected lanes, corresponded to host EF-1α because the Eimeria EF-1α-derived peptide used for rabbit immunization shared ≥ 85% identity with host EF-1α (shown in Fig. 2C).

Chicken IL-7 biologic activity was assessed by proliferation assay on freshly isolated chicken thymocytes. A dose-response proliferation curve was observed when thymocytes were stimulated with increasing concentrations of rchIL-7 (0.01–1 ng; Fig. 3)

Body weight gain. The group that received chIL-7 (20 μg) plus EF-1a (100 μg) plus Gel 01 PR adjuvant showed no statistical difference from the uninfected control group. However, chickens immunized with EF-1α (50 μg) plus chIL-7 DNA (20 μg) plus Gel 01 PR displayed an increased and statistically significant rate of weight gain, reaching up to 80% compared to the uninfected control (P < 0.05). Moreover, for chickens immunized with either EF-1α (50 μg), EF-1α (100 μg), or chIL-7 (20 μg), all of them plus Gel 01 PR showed a lower weight gain rate compared to the infected control (P < 0.05; Fig. 4).

Fecal oocyst shedding. Chickens immunized with chIL-7 (20 μg), and EF-1α (50 μg) plus chIL-7 (20 μg), showed a negative effect on oocyst multiplication and shedding (around 40%–50%) regarding the infected control (P < 0.05). Moreover, groups immunized with either EF-1α (50 μg or 100 μg) showed a slight (EF-1α, 100 μg) to moderate (EF-1α, 50 μg) reduction in oocyst output (P < 0.05). This reduction in parasite multiplication and oocyst shedding could be due to CD8⁺ cytotoxic T-cells or Th1-type cellular immunity (38). Additionally, chickens immunized with EF-1α (100 μg) plus chIL-7 (20 μg) showed just a slight increase in oocyst output over the infected control (P < 0.05; Fig. 5).

Duodenal lesion scoring. Chickens of the infected control group showed an average duodenal lesion score of approximately 1.5. Meanwhile, chickens immunized with either chIL-7 (20 μg) plus Gel 01 PR (P < 0.001), or EF-1α (100 μg) plus chIL-7 (20 μg) plus Gel 01 PR (P < 0.05), showed a significant reduction in duodenal lesion scores (average lesion score 1.0) per the Kruskal-Wallis test and Dunn post hoc test. Other groups, either EF-1a (50 μg or 100 μg) or EF-1α (50 μg) plus chIL-7 (20 μg), displayed higher, but not significant, lesion scores as compared to the infected control (Fig. 6).

Anti-EF-1α serum antibody. Chickens immunized with chIL-7 (20 μg) showed a higher anti-EF-1α antibody level than did other groups, including the infected control (P < 0.05), indicating that chIL-7 alone can induce a relevant humoral immune response. EF-1α (50 μg) alone or in combination at both concentrations (50 and 100 μg) with chIL-7 (20 μg) induced anti-EF-1α antibodies levels just below the infected controls, but was not significant (P < 0.05; Fig. 7). For an unknown reason, chickens which were immunized with EF-1α (100 μg) showed reduced the serum antibody titers (P 0.001) compared to the uninfected control group.

Duodenum cytokine levels. Only birds immunized with the combination EF-1α (100 μg) and chIL-7 (20 μg) responded with a fourfold up-regulation of IFN-c transcript regarding the infected controls (Fig. 8; P < 0.005). Other groups presented IFN-c levels close to or just above the uninfected control levels. Also, birds immunized with the combination of EF-1α (100 μg) and chIL-7 (20 μg) displayed around a fourfold up-regulation in host IL-2 cytokine as compared with the infected controls, except EF-1α (100 μg), which had a twofold increased up-regulation but was not significant different from other groups (P < 0.005; Fig. 9).

In summary, chickens immunized with EF-1α (100 μg) plus chIL-7 (20 μg) induced up-regulation (around fourfold) of IFN-c and IL-2 cytokines in the challenged chicken duodenum that corresponded with Th1-type cellular immune response.

DISCUSSION

In the present study, a DNA vaccine consisting of E. tenella EF-1α antigen and chicken IL-7, both cloned in eukaryotic expression plasmid pcDNA3.1(+), were used to immunize broiler chickens to induce protective humoral and cellular immune responses against coccidiosis. First, we’ve demonstrated that the Eimeria antigen (EF-1α) and chicken cytokine (chIL-7) were both cloned in-frame and expressed in eukaryotic cells in vitro (COS7 and HEK-293T-cells, respectively; Figs. 2A–C). Then, we’ve assessed that recombinant chicken IL-7 expressed in HEK-293T-cells is physiologically functional by a proliferation assay using freshly isolated chicken thymocytes. Although few reports have documented the role of chicken IL-7 in pathogenesis and immune homeostasis (13,28,29), our results showed a dose-dependent proliferative response of chicken thymocytes with increasing concentrations of rchIL-7 (0.01 to 1 ng), but not with human or mouse recombinant IL-7, even at higher concentrations (2 μg; data not shown). This might be due to low amino acid-identity between chicken and human IL-7 (around 29% identity; data not shown).

Furthermore, chIL-7 did not stimulate chicken lymphocytes freshly isolated from the peripheral blood mononuclear cell (PBMC), bursa, or spleen obtained from 2–4-wk-old birds. Similarly, chIL-7 did not stimulate mouse immature B-lymphocytes (2E8), a cell line exhibiting IL-7-dependent proliferation (data not shown); this finding contradicts that of Huo et al. (28). Also, recombinant chIL-7 produced in bacteria (BL-21) did not promote chicken thymocytes proliferation, inferring that posttranslational modifications are important for its functionality (data not shown), also contradicting the Cui et al. report (13).

Our study clearly demonstrated the vaccine efficacy of EF-1a/ chIL-7 DNA vaccine in vivo against E. acervulina-challenge infection in commercial broiler chickens. The vaccine formulation that we used in this study included EF-1α (100 μg), and chicken IL-7 (20 μg) in Gel 01 PR, and this mixture improved body weight gain to a statistically significant level compared to infected control and reaching levels close to uninfected control (P < 0.05; Fig. 4). It also reduced intestinal lesion scores (P < 0.05; Fig. 6), with significant regulation of pro-inflammatory cytokines (P < 0.05; Figs. 8,9). Additionally, chickens immunized with chIL-7 (20 μg) plus Gel 01 PR adjuvant reduced oocyst output and lesion scores and induced higher anti-EF-1α antibody levels (Figs. 5–7; data not shown). Furthermore, chickens immunized with EF-1α (50 μg) plus chIL-7 (20 μg) plus Gel 01 PR showed a significantly improved body weight gain, which was higher than the infected control but lower than the uninfected control (P < 0.05; Fig. 4). The serum antibody levels of chickens which were vaccinated with EF-1α (100 μg) in Gel 01 PR were lower than those of the uninfected control (P0.05; Fig. 7). The underlying mechanism for the beneficial effects of this vaccine formulation need to be studied.

On the other hand, we cannot discard the possibility that adjuvant Gel 01 PR was in some way responsible for optimizing the humoral and cellular immune response ascribed to chIL-7 because there were several reports that support the notion that Gel 01 PR induced strong infiltration of monocytes and macrophages, thus enhancing phagocytosis of antigen complex with increasing activity of antigen-presenting cells. Perhaps innate immune response, induced by Gel 01 PR, amplified robust adaptive immunity in a highly antigenic-specific manner (19,21,37,61,66,69).

In general, these results mostly agreed with what was already reported regarding major advantages of DNA vaccines and their ability to generate stronger cellular immunity, preferentially involving MHC I-restricted CD8⁺ cytotoxic T-cells and MHC II-restricted Th1 cellular responses (3,5). Indeed, IL-7 is known to be a central regulator of peripheral T-cell homeostasis important for the survival of na¨ive memory CD4⁺ and CD8⁺ T-cells (6,45,46).

Moreover, DNA vaccination with Leishmania major antigen LACK (Leishmania homologue of receptor for activated C kinase) plus IL-12 induced protective immunity mainly enhancing CD8⁺ IFN-c-producing T-cells with enhanced long-term immunity (25). In the murine T. gondii infection model, exogenous administration of human recombinant IL-7 can protect mice against acute challenge by stimulating IFN-c production and augmenting CD8⁺ T-cell-mediated cytotoxic T-lymphocyte response (34). Likewise, in leprosy for instance, IL-7 mRNA has been found to be more strongly expressed in the tuberculoid form of the disease with limited infection than in the progressive lepromatous form (51).

In eukaryotes, EF-1α is the second-most abundant protein after actin, constituting 1%–2% of total protein in normal growing cells (18,20,56). Furthermore, Wang et al., 2015 (64) have reported that DNA vaccination with pVAX-EF-1α triggers strong humoral and cellular responses that provided effective protection against acute T. gondii infection in mice. Likewise, EF-1α has been implicated in actin binding and bundling (67).

Our findings corroborated those reported by Lin et al. (40) regarding enhanced body weight gains and reduced gut lesion scores that they saw following recombinant EF-1alpha vaccination. Parasite EF-1α has also been associated with host invasion process in Cryptosporidium parvum (42), and E. acervulina infection models. Since EF-1α has been known to facilitate the invasion process of the cytoskeleton at the apical region of zoites (41), this antigen may be a good candidate vaccine against some aplicomplexan parasitic infections such as cryptosporidiosis or coccidiosis. Interestingly, some reports have indicated a role of EF-1α, when released as secreted microvesicles (exosomes) into the extracellular milieu, delivering an effector cargo to the host target cells (15,16,17). In Leishmaniasis, EF-1alpha was involved in macrophage-mediated microbicidal activity and IFN-c signaling following the activation of host protein-tyrosine phosphatases (2,47,53,68). Furthermore, in Plasmodium falciparum (12), Giardia intestinalis (55), and Echino-coccus granulosus (52) infections, EF-1α also induced protective immunity against experimental challenge infections.

To our knowledge, this is the first report showing a protective effect of DNA vaccine comprising EF-1α and IL-7 against coccidiosis challenge infection. Additional studies are needed to determine the underlying immune mechanisms involved in protection against coccidiosis by the EF-1α and IL-7 DNA vaccine. Furthermore, the nature of the memory response and cell types involved in the prolonged immune response to EF-1α should be better characterized.