Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity

Vaccine 28 (2010) 2980–2985

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Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity Seung I. Jang a,1 , Hyun S. Lillehoj a,∗ , Sung Hyen Lee a , Kyung Woo Lee a , Myeong Seon Park a , Sung-Rok Cha a , Erik P. Lillehoj b , B. Mohana Subramanian c , R. Sriraman c , V.A. Srinivasan c a b c

Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, USA Indian Immunologicals Limited, Research and Development Center, Gachibowli, Hyderabad 500032, Andhra Pradesh, India

a r t i c l e

i n f o

Article history: Received 18 November 2009 Received in revised form 5 February 2010 Accepted 8 February 2010 Available online 21 February 2010 Keywords: Coccidiosis Gam82 E. maxima

a b s t r a c t Intestinal infection with Eimeria, the etiologic agent of avian coccidiosis, stimulates protective immunity to subsequent colonization by the homologous parasite, while cross-protection against heterologous species is poor. As a first step toward the development of a broad specificity Eimeria vaccine, this study was designed to assess a purified recombinant protein from Eimeria maxima gametocytes (Gam82) in stimulating immunity against experimental infection with live parasites. Following Gam82 intramuscular immunization and oral parasite challenge, body weight gain, fecal oocyst output, lesion scores, serum antibody response, and cytokine production were assessed to evaluate vaccination efficacy. Animals vaccinated with Gam82 and challenged with E. maxima showed lower oocyst shedding and reduced intestinal pathology compared with non-vaccinated and parasite-challenged animals. Gam82 vaccination also stimulated the production of antigen-specific serum antibodies and induced greater levels of IL-2 and IL-15 mRNAs compared with non-vaccinated controls. These results demonstrate that the Gam82 recombinant protein protects against E. maxima and augments humoral and cell-mediated immunity. Published by Elsevier Ltd.

1. Introduction Avian coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria which invade discrete regions of the intestinal epithelium causing reduced feed conversion efficiency leading to decreased body weight gain [1]. Coccidiosis is one of the most economically costly diseases of the poultry industry, with estimated annual losses greater than three billion US dollars [2]. Prophylactic feeding of coccidiostat drugs is the major disease control method used in commercial settings. However, with increasing demands for high-protein meats and heightened consumer concerns over the use of antibiotics in poultry production, the search for alternative strategies against avian coccidiosis have intensified [1,3]. While immunization against avian coccidiosis has engendered some measure of success, the existence of numerous, antigenically distinct Eimeria species and their complex life cycle of asexual and sexual, as well as intracellular and extracellular stages, hinder further vaccine development [4,5]. An immunogenic Eimeria protein

∗ Corresponding author. Tel.: +1 301 504 8771; fax: +1 301 504 5103. E-mail address: [email protected] (H.S. Lillehoj). 1 This work was carried out during sabbatical leave from the Institute of Health and Environment, Daejeon Metropolitan City, Daejeon 305-338, Korea. 0264-410X/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.vaccine.2010.02.011

designated as 3-1E (profilin) has been shown to engender protective immunity using various immunization strategies. This protein was identified from the merozoite stage of Eimeria acervulina and encodes Eimeria profilin which is highly conserved across different stages of Eimeria life cycle and Eimeria species [6,7]. Several proteins associated with the sexual stage of Eimeria maxima including 14, 30, 56, 82 and 230 kDa antigens have been identified as potential vaccine targets for inducing transmission-blocking immunity [8]. Immunization with the 56 and 230 kDa antigens promoted cellmediated immunity against experimental coccidiosis and reduced fecal oocyst shedding, an important component of the Eimeria life cycle [8]. Additionally, the 56 and 230 kDa antigens induced the production of antibodies that were transferred to the embryo via egg yolk [9–12]. In particular, Gam82, the 82 kDa tyrosine-rich sexual stage glycoprotein of E. maxima, has been implicated in oocyst wall formation [11]. These promising results led us to the hypothesis that other gametocyte proteins also may promote cell-mediated and transmission-blocking immunity against subsequent parasite infection. Therefore, current study was undertaken to evaluate the effects of Gam82 vaccination on resistance to experimental E. maxima infection and to correlate disease resistance with humoral and cellmediated immunity. In addition, we compared various aspects of host immune response to Gam82 to those induced by Eimeria profilin.

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2. Materials and methods 2.1. Experimental animals One-day-old Ross broiler chickens were obtained from the Longenecker’s Hatchery (Elizabethtown, PA) and housed in Petersime starter brooder units in an Eimeria-free facility. The animals were provided with feed and water ad libitum and moved to larger hanging cages (2 animals/cage) until use. All protocols were approved by the Beltsville Area Institutional Animal Care and Use Committee. 2.2. Parasites The wild type strain of E. maxima originally developed and maintained at the Animal and Natural Resources Institute (Beltsville, MD) was used. Oocysts were cleaned by floatation on 2.5% sodium hypochlorite, washed three times with PBS, and enumerated using a hemocytometer prior to infection. 2.3. RNA extraction and amplification of the Gam82 gene Total RNA was isolated from intestinal scrapings of E. maximainfected chickens using Trizol (Invitrogen, Carlsbad, CA) and cDNA was prepared using Thermoscript reverse transcriptase (Invitrogen) with a Gam82-specific reverse primer. The Gam82 coding sequence was amplified by PCR using Proof Start DNA polymerase (Qiagen, Valencia, CA) with the following primer sequences containing Bam HI and Not I restriction enzyme sites (underlined): forward, 5 -AGCTGGATCCACCAGCTCTGGCCAGGATCAGGTG-3 ; reverse: 5 -TCTAGAGCGGCCGCTGCCCACATCTCTGATTGTTC-3 . Amplicons were cloned into the pET28a (+) plasmid vector (Novagen/EMD Chemicals, Gibbstown, NJ) downstream from an NH2 -terminal His6 epitope tag, plasmid clones were verified by sequence analysis, and used to transform competent Escherichia coli BL21 Star (Invitrogen). 2.4. Bacterial expression and purification of Gam82 recombinant protein Recombinant E. coli were induced for 4 h with 0.75 mM IPTG (Bangalore Genei, Bangalore, India) at OD600 = 0.6, the cells harvested by centrifugation, and lysed with 10 ␮g/ml of lysozyme (Sigma–Aldrich, St. Louis, MO) and sonication (Vibra-Cell, Sonics & Materials, Inc., Newtown, CT). The lysate was applied to a Ni2+ -chelating affinity column (HiTrap, GE Healthcare, Piscataway, NJ), the column was washed with PBS, pH 7.0 to remove unbound proteins, and bound proteins were eluted step-wise with PBS, pH 7.0 containing 0.5 or 1.0 M imidazole (Sigma). The eluted protein fractions were visualized on 10% SDS-acrylamide gels stained with Coomassie brilliant blue and on Western blots probed with horseradish peroxidase-conjugated anti-His monoclonal antibody (1:3000; Qiagen), and stored at −20 ◦ C. 2.5. Gam82 immunization and parasite-challenge infection The experimental design is illustrated in Fig. 1. At 1 wk of age, chickens (30/group) were intramuscularly immunized with 100 ␮l of 300 or 600 ␮g/ml of recombinant Gam82 in Freund’s complete adjuvant (FCA). Control animals received PBS in FCA. At 1 wk postimmunization, animals were given a booster injection with 100 ␮l of 300 or 600 ␮g/ml of recombinant Gam82, 3-1E, or PBS in Freund’s incomplete adjuvant. At 7 d post-secondary immunization, animals were given PBS or 2.0 × 104 E. maxima sporulated oocysts by oral gavage using an 18-gauge needle. Body weight gains were

Fig. 1. Schematic outline of the experimental design.

determined between 0 and 10 d post-infection. Fecal oocysts numbers were determined between 5 and 10 d post-infection using a McMaster counting chamber as described [13]. Lesion scores were determined at 6 d post-infection on a scale of 0 (none) to 4 (high) in a blinded fashion by two independent observers as described by Johnson and Reid [14]. 2.6. Anti-Gam82 antibody response Blood was collected by venipuncture from the wing vein at 7 d following the primary and secondary immunizations, clotted for 4 h at 4 ◦ C, sera were collected by centrifugation, and anti-Gam82 antibodies were measured by ELISA [15]. Microtiter plates were coated overnight with 10 ␮g/well of purified Gam82 recombinant protein, washed with PBS containing 0.05% Tween 20, and blocked with PBS containing 1% BSA (Sigma). Diluted sera were added, incubated for 4 h with continuous shaking, the plates were washed, and bound antibody detected with horseradish peroxidase-conjugated rabbit anti-chicken IgG antibody (Sigma) and peroxidase-specific substrate. Optical density at 450 nm (OD450 ) was determined with a microplate reader (Bio-Rad, Richmond, CA). 2.7. Cytokine quantitative RT-PCR Intestinal tissues were collected between the jejunum and the ileum at 3, 6, and 10 d post-secondary immunization, and total RNA was isolated from the intestinal tissues using TrizolTM reagent (Invitrogen, USA). Five micrograms of total RNA from each sample were incubated for 15 min at room temperature with 1.0 U of DNase I and 1.0 ␮l of 10× reaction buffer (Sigma), 1.0 ␮l of stop solution was added, and the mixture was heated for 10 min at 70 ◦ C. RNAs were reverse-transcribed using the Affinity Script Multiple Temperature cDNA Synthesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. PCR amplification and detection were preformed using the Mx3000P system and Brilliant SYBR Green QPCR master mix (Stratagene) using oligonucleotide primers for interleukin-2 (IL-2) and IL-15 (Table 1) as described [16,17]. Standard curves were generated using log10 diluted standard RNA and the levels of individual transcripts were normalized to those of GAPDH using the Qgene program [18]. Each analysis was performed in triplicate. To normalize RNA levels between samples within an experiment, the mean threshold cycle (Ct ) values for the amplification products were calculated by pooling values from all samples in that experiment. 2.8. Statistical analysis All data were expressed as mean ± S.D. values and subjected to one-way analysis of variance using SPSS software (SPSS 15.0 for Windows, Chicago, IL). Duncan’s multiple range test was used to analyze differences between the mean values. Differences were considered statistically significant at P < 0.05.

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Table 1 Oligonucleotide primers used for quantitative RT-PCR. RNA target GAPDH Forward Reverse

Primer sequences

PCR product size (bp)

Accession no.

5 -GGTGGTGCTAAGCGTGTTAT-3 5 -ACCTCTGTCATCTCTCCACA-3

264

K01458

IL-2 Forward Reverse

5 -TCTGGGACCACTGTATGCTCT-3 5 -ACACCAGTGGGAAACAGTATCA-3

256

AF000631

IL-15 Forward Reverse

5 -TCTGTTCTTCTGTTCTGAGTGATG-3 5 -AGTGATTTGCTTCTGTCTTTGGTA-3

243

AF139097

3. Results 3.1. Cloning and expression of Gam82 recombinant protein The Gam82 coding sequence was amplified by RT-PCR from E. maxima-infected intestinal tissue. A PCR product of ∼1.7 kb was observed in an agarose gel (Fig. 2A). The amplified product was cloned into the pET28a(+) bacterial expression vector and sequenced. The predicted amino acid sequence was identical to the published full-length sequence of Gam82 from the Houghton strain of E. maxima (GenBank accession no. AAO47083) [19]. The Gam82 cDNA containing an NH2 -terminal His6 epitope tag was expressed in E. coli, and the encoded protein was purified by Ni2+ chelate affinity chromatography. A protein band of approximately 70 kDa was observed on a SDS-acrylamide gel (Fig. 2B). A similar band profile was detected by Western blotting using a monoclonal antibody against the His epitope tag (Fig. 2C). The final yield of the affinity purified protein was 4.0 mg/l. 3.2. Effect of Gam82 vaccination on body weight gain, fecal oocyst shedding, and intestinal lesion scores following E. maxima infection We evaluated the effect of immunization with Gam82 on these three parameters of disease resistance. Animals immunized with 30 or 60 ␮g of Gam82 and challenged with E. maxima showed no improvement in body weight gain between 0 and 10 d post-infection compared with the non-vaccinated (PBS) and

parasite-challenged group (Fig. 3A). By contrast, increased weight gains were observed in animals immunized with either dose of the 3-1E protein. Animals immunized with 60 ␮g of Gam82 did exhibit reduced fecal oocyst shedding by an average of 1.1 × 107 oocysts/chicken between 5 and 10 d post-infection compared with the non-immunized, coccidia-challenged group (Fig. 3B). Neither the lower dose of Gam82, nor either dose of 3-1E, were effective in decreasing the number of fecal oocysts. Interestingly, animals vaccinated with 30 ␮g of Gam82, but not the higher dose, demonstrated reduced intestinal lesion scores compared with nonimmunized, E. maxima-challenged animals (Fig. 3C). 3.3. Effect of Gam82 vaccination on anti-Gam82 antibody response Serum antibody levels against Gam82 antigen were measured in Gam82-vaccinated animals. Chickens immunized with 30 and 60 ␮g of Gam82 protein showed elevated serum antibody levels at 7 d following the primary and secondary immunizations compared with the non-vaccinated control group (Fig. 4). 3.4. Effect of Gam82 vaccination on intestinal IL-2 and IL-15 mRNA levels The levels of two avian cytokines, IL-2 and IL-15, were measured following Gam82 immunization in non-infected animals. Animals vaccinated with 60 ␮g of Gam82 had greater levels of IL-2 transcripts at 3 and 6 d post-secondary immunization compared with

Fig. 2. Cloning and expression of Gam82 recombinant protein. (A) Lane 1, RT-PCR amplified Gam82 DNA coding sequence visualized on a 1% agarose gel. Lane 2, DNA size markers. (B) His-tagged Gam82 recombinant protein was applied to a Ni2+ affinity column, eluted with 0.5 and 1.0 M imidazole in PBS, the eluted fractions were resolved on a 10% SDS-acrylamide gel, and stained with Coomassie brilliant blue. Lane 1, 0.5 M imidazole elution fraction. Lane 2, 1.0 M imidazole elution fraction. M, Protein size markers. (C) Purified His-tagged Gam82 recombinant protein was analyzed by Western blotting with horseradish peroxidase-conjugated anti-His monoclonal antibody. Lane 1, lysate of E. coli not expressing Gam82. Lane 2, Purified Gam82 from E. coli clone #1. Lane 3, Purified Gam82 from E. coli clone #2. M, Protein size markers.

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Fig. 4. Antibody responses of animals immunized with Gam82 or 3-1E recombinant protein. Animals were immunized intramuscularly with PBS, or 30 or 60 ␮g of Gam82 or 3-1E and anti-Gam82 antibodies were measured by ELISA at 7 d following primary immunization (A) or 7 d following secondary immunization (B). Each bar represents the mean ± S.D. value (N = 3). Within each graph, bars with different letters are significantly different (P < 0.05) according to the Duncan’s multiple range test. Fig. 3. Effect of Gam82 recombinant protein vaccination on resistance to experimental coccidiosis. Animals were immunized intramuscularly with PBS, or 30 or 60 ␮g of Gam82 or 3-1E and non-infected or infected with 2.0 × 104 E. maxima oocysts and body weight gains (A), fecal oocyst shedding (B), and intestinal lesion scores (C) were determined. Each bar represents the mean ± S.D. value (N = 5). Within each graph, bars with different letters are significantly different (P < 0.05) according to the Duncan’s multiple range test.

the non-vaccinated group (Fig. 5). Indeed, only the 60 ␮g Gam82 group exhibited increased IL-2 expression at the earliest time point examined. Increased IL-2 transcripts following vaccination with 30 ␮g of Gam82 did not occur until 10 d following secondary immunization. In addition, all vaccination groups tended to display progressively decreasing IL-2 mRNA levels from the 3 to 10 d time points. The patterns of IL-15 mRNA levels were similar to those of IL-2, particularly with regards to the early and dramatic increase in the 60 ␮g of Gam82 group (Fig. 6). 4. Discussion This study was conducted to evaluate the effects of in vivo vaccination with an E. maxima gametocyte recombinant protein, Gam82, on host immunity following live-challenge infection with E. maxima. The major findings of this study are: (1) immunization with the Gam82 protein prior to E. maxima infection was associated with reduced fecal oocyst shedding and diminished intestinal pathology compared with non-vaccinated and infected controls, and (2) vaccination with Gam82 induced a rapid and robust increase in IL-2 and IL-15 transcript levels in the intestine compared with nonvaccinated controls.

Several studies have documented that vaccination of chickens with immunogenic Eimeria recombinant proteins induced protective immunity to subsequent live parasite challenge as assessed by increased body weight gain, reduced fecal oocyst shedding, and/or decreased intestinal pathology [4,12,15,20]. Gam82 is a sexual stage antigen of Eimeria that has been speculated to participate in protective immunity against avian coccidiosis [10,12,19]. The purified Gam82 protein as probed using Gam82specific immune sera identified a protein band of approximately 70 kDa which is the expected protein size of Gam82. The faster migrating bands likely represent proteolytic degradation fragments of the intact protein and/or downstream AUG translation initiation codons in the cloned gene as previously observed with polypeptides expressed in E. coli and purified by Ni2+ chromatography [7]. Due to its involvement in oocyst wall formation, Belli et al. [19] hypothesized that a Gam82 vaccine would generate transmission-blocking immunity by inhibiting normal oocyst formation, thereby obstructing the normal parasite life cycle. This prediction was confirmed in a follow-up study which demonstrated that vaccination with Gam82 provided stage-specific immunity against coccidia [21]. Wallach et al. [12] reported that newly hatched chicks from hens immunized with purified gametocyte antigens, including Gam82, showed greatly reduced oocyst production compared with those from the non-vaccinated controls, presumably due to the transfer of maternally derived Gam82-reactive antibodies. Reduction in gut lesions of Eimeria-infected poultry that had been immunized with gametocyte antigens were also reported although this study failed to demonstrate any significant effect on the body weight gain [22]. Attenuation of oocyst shedding and lessening of intestinal

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Fig. 5. Effect of Gam82 or 3-1E recombinant protein vaccination on intestinal IL-2 mRNA levels. Animals were immunized intramuscularly with PBS, or 30 or 60 ␮g of Gam82 or 3-1E and infected with 2.0 × 104 E. maxima oocysts and IL-2 mRNA levels were measured by quantitative RT-PCR at 3 (A), 6 (B), or 10 d (C) following secondary immunization. IL-2 mRNA levels were normalized to GAPDH mRNA levels. Each bar represents the mean ± S.D. value (N = 9). Within each graph, bars with different letters are significantly different (P < 0.05) according to the Duncan’s multiple range test. NS indicates not significant difference between the unvaccinated and the vaccinated groups.

pathology, in the absence of body weight gain, following Gam82 vaccination is confirmed in this report. Infection with Eimeria parasites elicits humoral and cellmediated immune responses, although the relative roles of each in conferring resistance to coccidiosis are debated. Nevertheless, it is clear not only that passive immunity against Eimeria infection can be transferred by hyperimmune egg yolk immunoglobulin Y, but also that cellular responses are mediated by the production of proinflammatory, Th1-type, and Th2-type cytokines [7,16,23–26]. Among their diverse functions, cytokines are potent adjuvants that amplify protective immunity to microbial pathogens. For example, co-administration of Eimeria vaccines with cytokines enhanced host immunity in avian coccidiosis compared with vaccination without cytokines [20]. IL-2 and IL-15 are related cytokines that possess overlapping functions, including the ability to induce Tcell proliferation. We previously reported that IL-2, one of the first T-cell growth factors identified, has a practical importance in enhancing vaccine-induced immune responses against coccidiosis [6]. Furthermore, chickens infected with Eimeria produced IL-15 which stimulates the proliferation of antigen-specific T lympho-

Fig. 6. Effect of Gam82 or 3-1E recombinant protein vaccination on intestinal IL-15 mRNA levels. Animals were immunized intramuscularly with PBS, or 30 or 60 ␮g of Gam82 or 3-1E and infected with 2.0 × 104 E. maxima oocysts and IL-15 mRNA levels were measured by quantitative RT-PCR at 3 (A), 6 (B), or 10 d (C) following secondary immunization. IL-15 mRNA levels were normalized to GAPDH mRNA levels. Each bar represents the mean ± S.D. value (N = 9). Within each graph, bars with different letters are significantly different (P < 0.05) according to the Duncan’s multiple range test. NS indicates not significant difference between the unvaccinated and the vaccinated groups.

cytes and NK cells [25]. In the current study, expression of both cytokines was rapidly up-regulated at 3 and 6 d following Gam82 vaccination. In contrast, increased cytokine expression following 3-1E vaccination was not detected until 6 d post-immunization. Elevated IL-2 and IL-15 mRNA levels are a major indication that host T-lymphocyte response was augmented at the earlier time points following Gam82 protein vaccination. Interestingly, however, IL-2 and IL-15 mRNA levels were not increased at 10 d following immunization with either Gam82 or 3-1E compared with non-vaccinated controls. We suspect that down-regulation of these cytokines at the later time point represents normal immune homeostatic mechanisms. In summary, this report demonstrates that vaccination with the Gam82 recombinant protein induced protective intestinal immunity against E. maxima, as revealed by decreased oocyst shedding and reduced gut pathology, which was associated with increased humoral and cell-mediated immune responses. While the detailed molecular and cellular mechanisms induced by Gam82 immunization, including cross-protection against multiple Eimeria species, warrant further investigation, these results suggest that host immunity to Gam82 may play an important role in impeding host

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