Comparative immunoprophylactic efficacy of Haemonchus contortus recombinant enolase (rHcENO) and Con A purified native glycoproteins in sheep

Experimental Parasitology 154 (2015) 98–107

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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

Comparative immunoprophylactic efficacy of Haemonchus contortus recombinant enolase (rHcENO) and Con A purified native glycoproteins in sheep Aravindan Kalyanasundaram a,1, Shabnam Jawahar a,1, Manikkavasagan Ilangopathy a, Azahahianambi Palavesam a, Muthusamy Raman a,b,* a

Department of Veterinary Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai 600007, India Translational Research Platform for Veterinary Biologicals (TRPVB), TANUVAS, II Floor, Central University Laboratory Building, Madhavaram Milk Colony, Chennai 600051, India b

H I G H L I G H T S

• • •

G R A P H I C A L

A B S T R A C T

rHcENO and native glycoproteins conferred partial protection against L3 larvae. HcENO has 17% of sequence identity to sheep and use as a safe drug and vaccine target. Immunoblot detected anti-HcENO antibodies in the natural and experimental sheep.

A R T I C L E

I N F O

Article history: Received 18 September 2014 Received in revised form 7 April 2015 Accepted 19 April 2015 Available online 23 April 2015 Keywords: Haemonchus contortus Enolase Con A purified native glycoproteins rHcENO Vaccine Sheep

* Corresponding author. Fax: +91-44-2536 2787. E-mail address: [email protected] (M. Raman). 1 Equal contribution. http://dx.doi.org/10.1016/j.exppara.2015.04.016 0014-4894/© 2015 Elsevier Inc. All rights reserved.

A B S T R A C T

Haemonchus contortus is the most economically important blood feeding nematode parasite of sheep and goats all over the world. Enolase in helminth parasites is a multi-functional enzyme which involves in glycolysis and host tissue invasion. In this study, the recombinant H. contortus enolase (rHcENO) was evaluated for its immunoprophylactic efficacy in sheep along with Con A purified native glycoproteins in a vaccine challenge trial. Group I and Group II experimental sheep were immunized thrice with rHcENO and Con A purified native glycoproteins along with Montanide ISA 61 VG adjuvant. The animals were challenged with 5000 L3 stage active H. contortus larvae after 21 days of third immunization. A significant increase in the IgG titre was observed in rHcENO and Con A purified native glycoproteins immunized animals as compared to the control animals. Immunoprotective efficacy of Con A purified native glycoproteins was comparatively higher than rHcENO antigen. © 2015 Elsevier Inc. All rights reserved.

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1. Introduction

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2. Materials and methods 2.1. Parasite source

Helminth parasites are a worldwide problem in causing economic losses to livestock industry. Haemonchus contortus is a blood feeding gastrointestinal nematode of small ruminants that causes anaemia and severe morbidity in small ruminants. Current methods of control depend solely on the use of anthelmintics. However, the emergence and spread of drug resistant strains of H. contortus had led to alternate control strategies such as immunoprophylaxis (Gilleard, 2006). Immunization trials in sheep using native midgut antigens from adult H. contortus worms such as H11 (Newton and Munn, 1999) and H-gal-GP (Smith et al., 1999) demonstrated (90% and 93%) reduction in the egg output, respectively. Combination of these antigens resulted in 85% of faecal egg reduction in field trials (Smith et al., 2001). Though the use of native antigens had proven to be more effective, vaccine production on commercial scale is not possible. Recombinant DNA technology allows production of large amounts of parasite antigens in a heterologous system with high level of purity. Several attempts exploiting the recombinant versions of H11 (Reszka et al., 2007), H-gal-GP (Cachat et al., 2010; Smith et al., 2003) and Thiol separose binding proteins (TSBP) (Redmond and Knox, 2004) failed to induce a satisfactory immune response against H. contortus. This emphasizes the need to work for a more potential vaccine target against H. contortus. Enolase or 2-phosphoglycerate hydratase is a glycolytic enzyme that catalyzes the penultimate step of glycolysis involving the conversion of 2-phosphoglycerate to phosphoenolpyruvate. Glycolysis is the prime source of energy for parasitic species within the vertebrate host (Velanker et al., 1997). In the past decade enolase received a lot of attention as a multifunctional enzyme (Pancholi, 2001). The possibility of exploiting enolase as vaccine candidate had been speculated in several parasitic species (Avilan et al., 2011; Ghosh et al., 2011). It had been shown to be located on the surface of several pathogenic bacteria such as Sterptococcus mutans (Jones and Holt, 2007), Streptococcus suis (Esgleas et al., 2008), Bacillus anthracis (Agarwal et al., 2008), Mycoplasma gallisepticum (Chen et al., 2011), and parasites such as Fasciola hepatica (Bernal et al., 2004), Leishmania mexicana (Quinones et al., 2007; Vanegas et al., 2007), Schistosoma bovis (de la Torre-Escudero et al., 2010) and Clonorchis sinensis (Wang et al., 2011) where it had been shown to bind host plasminogen. This property is essential for the degradation of the extracellular matrix and invasion of host tissue. Thus, enolase could play an essential role in the initiation of disease. The role of enolase in the development of larval stages in Ascaris suum was demonstrated using RNAi technology (Chen et al., 2010). Thus, parasite enolase could play an essential role in energy metabolism of parasites, growth and development of the parasite stages and virulence of the parasite. Enolase was found to be immunogenic in Streptococcus suis (Feng et al., 2009), Candida albicans (Eroles et al., 1997), Brugia malayi (Wongkamchai et al., 2011), Trichomonas vaginalis (Mundodi et al., 2008) and Plasmodium falciparum (Pal-Bhowmick et al., 2007). pVAXEnol, a DNA vaccine, triggered Th1/Th2 mixed response in mice against A. suum larvae (Chen et al., 2012). Native and recombinant form of H. contortus enolase proteins had elicited similar reactivity against experimentally infected goat sera (Han et al., 2012). This result indicates that the native and recombinant form of enolase is similar in eliciting immune response in goat. In the present study, an attempt was made to evaluate the recombinant enolase as an alternative vaccine candidate for control of H. contortus in sheep. Even though the immunogenic property of the H. contortus enolase was established, this is the first report on the immunoprophylactic efficacy of the recombinant H. contortus enolase antigen.

Adult Haemonchus contortus worms were collected from abomasum of sheep slaughtered in Chennai corporation slaughter house, Perambur. They were washed with 1× PBS, suspended in RNA later and stored at −80 °C for further use (Hartman et al., 2001). 2.2. Preparation of native glycoproteins from H. contortus Frozen adult H. contortus worms were thawed on ice and washed three times in 1× TBS (20 mM Tris, 150 mM NaCl, 100 μM CaCl2, 10 μM MnCl2, pH 7.4). The worms were homogenized with homogenization buffer containing 1% Triton X-100. The extract was centrifuged at 2500 × g for 20 mins and the supernatant was filtered with 0.22 μm syringe filter. The filtered homogenate was mixed with Con A lectin-agarose (Vector laboratories, UK). The mixer was then washed with wash buffer (20 mM Tris, 500 mM NaCl, 100 μM CaCl2, 10 μM MnCl2, 0.25% v/v Triton X-100, pH to 7.4). Finally, the elution was done with 0.2 M methyl-glucopyranoside and 0.2 M methyl-mannopyranoside (20 mM Tris, 500 mM NaCl, 100 μM CaCl2, 10 μM MnCl 2 , 0.25% v/v Triton X-100, 200 mM α methyl-Dmannopyranoside, α methyl-D-glucopyranoside, pH to 7.4). The concentration of the protein was estimated using bicinchoninic protein assay reagents according to the manufacturer’s instruction (Pierce, USA). 2.3. Cloning and expression of H. contortus enolase gene Total RNA was extracted from snap frozen H. contortus worms using Trizol as per manufacturer’s instructions (Invitrogen, USA) and the quality of intact RNA was checked by Biophotometer plus (Eppendrof, Germany) and cDNA was generated using iScript reverse transcription kit (Bio Rad, USA). Primers were designed for full length amplification of enolase coding sequence from H. contortus based on the previously published mRNA sequence (HM138086) (Han et al., 2012). A set of primers was designed for H. contortus enolase Open Reading Frame (ORF) incorporating the EcoRI and HindIII sites as restriction enzyme recognition sites (ENOL1 F 5′-CCG GAA TTCATG CCT ATC ACG AAA ATC CAC-3′ and ENOL1 R 5′-CCC AAG CTT TTA AAC TGG ATT GCG GAA GTT-3′) at 5′ and 3′ ends, respectively. Amplification was performed for 30 cycles involving 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min using the iCycler (BioRad). The PCR product was resolved in 1.5% agarose gel and the purified PCR product was digested with EcoRI and HindIII and cloned into pET32a vector (Novagen, USA). Clones were screened by colony PCR using T7 promoter primers and sequenced. 2.3.1. Sequence analysis of H. contortus enolase gene Sequence similarity was done by BLAST analysis in nucleotide and amino acid level (http://www.blast.ncbi.nlm.nih.gov/Blast). The predicted amino acid sequence of H. contortus enolase obtained in this study was aligned with enolase sequences of closely related parasites and sheep from GenBank using CLUSTAL W2 (http://www.ebi.ac.uk/ClustalW/). The alignment results were used for further analysis with SignalP (http://www.cbs.dtu.dk/services/ SignalP) to predict signal peptides of enolase protein. The functional motif and isoelectric point (pI) of H. contortus enolase were predicted using MOTIF FINDER and Compute pI/Mw online tools (http://web.expasy.org/compute_pi/). 2.3.2. Homology based model of H. contortus enolase In the study, sequence of H. contortus enolase gene sequence was used for homology modeling to predict the 3D model crystal structure.

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A 3D model of enolase protein has been built using the crystal structure of enolase 1α (2psn) molecule from human as the template. Templates were selected manually from the 5 enolase structures available in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org/pdb/), on the basis of the highest degree of homology to the specific enolase (70.1%). The predicted sequence of enolase protein was aligned with that of the human using CLUSTAL W2. Program Modeller (Sali and Blundell, 1993) was used for homology modelling (http://www.salilab.org/modeller/). The stereochemical quality of the model was checked using the program PROCHECK (www.ebi.ac.uk/thornton-srv/software/ PROCHECK/) and the Ramchandran plot was drawn. The model quality was assessed by What-Check (through Swiss-Model, http://swift.cmbi .ru.nl/gv/whatcheck/).

The animals were divided into three groups of six each. Group I was immunized with 1 ml of rHcENO antigen (200 μg/dose) and group II with 1 ml of Con A purified native glycoproteins (200 μg/dose). The animals were administered with one priming dose and two booster doses on day 0, 22nd and 43rd through intramuscular (0.5 ml) and subcutaneous route (0.5 ml). The adjuvant control group III animals were administered with 1 ml PBS emulsified with MontanideTM ISA 61 adjuvant on the corresponding days. All sheep were challenged orally with 5000 infective L3 H. contortus larvae at day 62 and slaughtered at day 99. The experimental animals were maintained as per the approved guidelines of Institutional Animal Ethical Committee (IAEC) of Tamil Nadu Veterinary and Animal Sciences University in Chennai, India (Approval#318/DFBS/IAEC/2010).

2.3.3. Expression and purification of H. contortus recombinant enolase H. contortus enolase was expressed as a fusion protein with thioredoxin in Escherichia coli BL21 (DE3) cells using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hrs at 37 °C. Bacterial lysate containing the recombinant enolase protein was purified by denaturing method with 8 M urea. Briefly, the IPTG induced bacterial cell pellet was resuspended in buffer A (8 M urea, 25 mM Na2HPO4, 1 mM Tris–HCl, pH 7.5), and the protein was purified from this lysate using Ni–NTA affinity column. The affinity column was washed with 20 volumes of buffer B (8 M urea, 25 mM Na2HPO4, 1 mM Tris–HCl, pH 6.5). The recombinant protein was eluted with buffer C containing 550 mM imidazole. Eluted recombinant enolase was dialyzed against 1× PBS with urea gradient (4 M, 2 M, 1 M and 0 M urea) and the protein concentration was estimated by BCA protein assay kit (Pierce, USA).

2.5. Parasitology

2.3.4. Immunoblot analysis of H. contortus enolase (rHcENO) with sheep sera Sero-reactivity of rHcENO was checked by immunoblot using sera collected from sheep experimentally and naturally infected with H. contortus (EPG >2000) and sera collected from sheep free of H. contortus infection was used as control. Briefly, 10 μg of rHcENO protein was separated on a 12% SDS–PAGE gel using a Mini Protean unit (Bio-Rad, USA) under reducing conditions. Protein was electrophoretically transferred onto PVDF membrane (Millipore, USA) using Semi dry-Blot Electrophoresis Transfer Cell (BioRad, USA). The membrane was then incubated with 5% skim-milk powder in PBST (PBS with 0.05% Tween 20) overnight at 4 °C for blocking. The membrane was incubated in sheep sera (1:100) for 1 hr at 37 °C and then incubated with of HRP-conjugated anti-sheep IgG (Sigma, 1:5000) for 1 hr at 37 °C. The signal was detected by incubating the membrane with DAB (3, 3′-diaminobenzidine) for 5–10 mins. 2.4. Immunization trial Eighteen, 6 month old Mecheri breed lambs were purchased from Mecheri Sheep Research Station (MSRS), Mecheri, Tamil Nadu, India. All the sheep were reared in clean and hygienic conditions in University farm as per the recommendation of institutional animal ethical committee. All the animals were dewormed with albendazole (10 mg/kg body weight) 21 days before the start of the trial. Faecal examination was done for all the experimental animals before starting the experiment and no strongyle eggs could be detected. The antigens were emulsified with Montanide ISA 61VG (Seppic, France) adjuvant. It comprises a specific enriched light mineral oil and allows the induction of both humoural and cell mediated immune response in cattle and sheep. MontanideTM ISA 61 VG is formulated with the antigen at a ratio of 60:40 (weight/weight) in the final vaccine. Vaccines were prepared by mixing the soluble antigen with the adjuvant under high shear mixing.

The faecal samples were collected thrice in a week and the worm egg counts were determined by Mc-Master method (HMSO, 1984) to monitor the status of H. contortus infection. Necropsy was done 42 days after challenge infection. The abomasums were examined for worm content.

2.6. Indirect ELISA The level of antibody response to recombinant enolase and Con A purified native glycoproteins was determined by indirect ELISA. Microtitre plate (MaxiSorb, Nunc, USA) wells were coated overnight at 4 °C with 2 μg/ml of rHcENO and Con A purified native glycoproteins in 0.06 M Bi-carbonate and carbonate buffer (pH 6.8). The wells were blocked with 3% (w/v) skim milk powder in 1× PBS and incubated for 1 hr at 37 °C. Serum collected from each animal was diluted 1:100 in 1× PBS containing 0.1% skim milk powder and100 μl of the diluted was added per well. The plates were incubated at 37 °C for 1 hr. Anti-sheep IgG secondary antibody diluted in 1× PBS (1:5000) was added and incubated at 37 °C. The calorimetric reaction was developed by Sigma-Fast OPD substrate in the dark room for 10 mins at room temperature. The reaction was stopped by the addition of 0.2 M sulphuric acid and OD values were measured spectrophotometrically at 492 nm. All the samples were added in duplicates.

2.7. Assessment of cell mediated immunity by FACS analysis Blood samples were collected from the jugular vein into heparin coated 4 ml vacutainer (BD, USA). Equal volume of whole blood was layered onto 3 ml of Histopaque (Sigma, USA) and centrifuged at 400 × g for 30 mins. Following centrifugation, the interface containing peripheral mononuclear lymphocytes were removed and washed twice with 1× PBS. Approximately, 1 × 107 of lymphocyte cells in 100 μl of optimal dilution of CD4+ and CD8+ antibodies marker (Serotec, USA) and incubated on ice for 1 hr. Then the stained cells were washed and resuspended with sheath fluid. Analysis was performed on the gated stained lymphocyte population. Both acquisition and analysis were undertaken on a FACSCalibur flow cytometer using CELLQuest Pro software (BD, USA).

2.8. Statistical analysis Statistical analysis was performed using SPSS statistical package (SPSS for Windows 18, SPSS Inc., Chicago, IL, USA). Statistical differences between groups were calculated by the ANOVA, one-way and Duncan’s test. Differences between groups were considered significant at P < 0.05.

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3. Results 3.1. SDS–PAGE profile of Con A purified native glycoproteins The SDS–PAGE profiles (reducing and non-reducing) of the native antigens used in the immunization trial are shown in Fig. 1. The glycoproteins of Con A eluted fraction was identical to the H-gal GP complex and H11 (Newton and Munn, 1999; Smith et al., 1999), it consists of >250 kDa, 150 kDa, 110 kDa, 55 kDa and 25 kDa proteins which are the family of H-gal GP complex and aminopeptidase (Smith et al., 1997). 3.2. Sequence analysis A pair of gene specific primer was successfully used to amplify a complete enolase ORF (1305 bp) from Chennai isolate of H. contortus (Fig. 5) and the PCR product was resolved at 1305 bp size. BLAST analysis of the sequence revealed the high identity (95%) to Chinese isolates of H. contortus enolase in nucleotide and amino acid level. Sequencing results revealed that the enolase nucleotide sequence with 1305 bp codes for the 434 amino acids with a predicted molecular weight of 46.8 kDa and an isoelectric point of 7.19 (Fig. 2). H. contortus enolase sequence of the present study was submitted to NCBI (JX968806). 3.2.1. Multiple alignments Sequence analysis results revealed that the Chennai isolate of H. contortus enolase had 95% identity to enolase of Chinese isolate. Multiple alignment results showed (Fig. 3) that the H. contortus enolase amino acid sequence shares the highest homology (95% identity) with Chinese H. contortus enolase (ADK47524), 83% identity with Caenorhabditis briggsae (XP_002631372), 82% identity with Caenorhabditis elegans (NP495900), 81% identity with Heterorhabditis indica (ADH95415), and Ascaris suum (ADQ00605), 77% identity with Anisakis simplex (CAD43170) and 74% identity with Brugia malayi (XP_001896281). Enolase signature peptide as a functional domain was predicted using motif finder (Fig. 3). The functional domain conserved across enolase of all parasites but valine was replaced

Fig. 2. Complete nucleotide and deduced amino acid sequence of H. contortus enolase. The deduced amino acid sequences are given below the nucleotide sequences. The predicted initiation (ATG) and stop codon (TAA) are underlined.

Fig. 1. SDS–PAGE profile of Con A purified native glycoproteins used in immunization experiments. M: molecular weight marker; lane 1: non reducing conditions; lane 2: reducing condition.

by isoleucine in Steinernema glaseri (Fig. 3). Enolase signature peptide is unique for enolase and is based on the much conserved region in the C-terminal of all the other enolases (Babbitt et al., 1996). Enolase is a dimeric enzyme that requires a magnesium ion (Mg2+) for catalysis

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Fig. 3. Comparison of the deduced amino acid sequences of H. contortus enolase with other parasite enolase sequences. The numbers at the right margin indicate the position of the amino acids. Identical (*), conserved (:) and semi conserved (·) residues are indicated.

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and to stabilize the dimer. A crystallography study demonstrated that the Mg2+ coordinates to the carboxylate side chains of Asp 246, and Glu 295, and Asp 320 with high affinity (Larsen et al., 1996). 3.2.2. 3D structure of H. contortus enolase Homology modelling of H. contortus enolase protein was built with Modeller (Fig. 4). Several molecular models for enolase were constructed based on already existing enolase structures. Sequence alignment results revealed that H. contortus enolase sequence had 70% identity with enolase 1α (2psn) crystal structure of human. Enolase three dimensional structure, consist of 20 helix, 23 β-pleated sheet, and 42 turns. Two Mg2+ ions were bound in the active site of the protein (Fig. 4). The 3D structure of this parasite antigenic protein was checked with PROCHECK for its stability and were found to be stable. There is no deviation in the peptide torsion angle in the Ramachandran plot. The predicted enolase signature functional motifs of enolase are shown in the box (Fig. 3). This functional domain consists of a fourteen residue loop (LLLKVNQIGSVTES) flanked on both sides by a fourteen residue alpha-helical domain. The highlighted peptide region in Fig. 3 (341–354) showed pattern similarity across other parasitic enolases.

3.3. Recombinant H. contortus enolase The enolase protein coding sequence was cloned into pET32a (Fig. 5a). Recombinant enolase expression was obtained in IPTG induced E. coli BL-21 (DE3) cells. Recombinant enolase production was assessed by SDS–PAGE and a high level expression was obtained in 3 hrs after IPTG induction. SDS–PAGE results revealed the presence of a 69.5 kDa band representing rHcENO including the 19.7 kDa thioredoxin fusion protein (Fig. 5b). Recombinant enolase

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was expressed as inclusion bodies and it was successfully purified under denaturing conditions. The purified rHcENO protein with a yield of 20 mg protein from 100 ml culture of bacterial cells was estimated.

3.4. Immunoblot Sera from naturally and experimentally infected sheep revealed the presence of sero-reactive bands at 69.5 kDa along with thioredoxin fusion protein (19.7 kDa) indicating the presence of rHcENO protein. Immunoblot results showed positive reaction with sera collected from sheep immunized with rHcENO. Fig. 5c summarizes the results of immunoblot with rHcENO.

3.5. Humoral immune response Results of groups I, II and III were plotted in Fig. 6. Values of preimmunization sera from all the groups were low. Group mean (±SE) serum IgG levels increased after the first immunization in rHcENO and Con A purified native glycoproteins immunized groups and reached peak values in the 8th week. These levels were maintained until the end of the 9th week after which a gradual decrease in the IgG titre was observed in both the immunized groups I and II. The antibody titre in the control group remained low throughout the course of the trial. Immunization with Con A purified native glycoproteins confers high level of IgG response in all the three doses compared to rHcENO. In contrast, a low level of antibody response was detected in sera collected from adjuvant control group III throughout the trial. There is no increase in IgG level observed after the L3 larval challenge in group I and group II animals.

Fig. 4. Molecular model of enolase, based on the crystal structure of enolase 1α (2psn) from Human was built with Modeller server. Arrows show that the α-helix, 23 β-pleated sheets, and highlighted Mg2+ ions.

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Fig. 5. (A) Amplification of complete ORFs of enolase gene from H. contortus by PCR using gene specific primers. Lane M: DNA ladder (100 bp), lanes 1 and 2: enolase (1305 bp). (B) Expression and purification of recombinant enolase induced by IPTG at a final concentration of 1 mM. Recombinant enolase (46.8 kDa) is expressed along with thioredoxin (TRX) fusion protein (19.7 kDa). Lane M: protein marker, lanes 1–6: purified recombinant TRX-enolase (69.5 kDa), lane 7: TRX-enolase in BL21 (DE3) cells. (C) Immunoblot analysis of H. contortus recombinant enolase using parasite infected sheep sera. The sero-reactivity of sera collected from naturally and experimentally infected sheep with H. contortus (EPG >2000) (lanes 1 and 2). Sera collected from sheep free of H. contortus infection were used as control (lane 3).

immunized groups showed (p < 0.05) significant difference from control group. 3.7. Lymphocyte subsets in peripheral blood mononuclear cells There were higher percentages of CD4+ T lymphocytes in group I and group II animals at days 27, 48, 61, 88 and 99 days (P < 0.05) compared to the control group (Table 1). There was no significant increase in the percentage of CD8+ T lymphocytes of all groups throughout the trial. There is an increase in CD4+ positive T lymphocytes level in all groups in the post challenge period due to the larval exposure. 4. Discussion

Fig. 6. ELISA titres (mean ± SE) of sera from sheep immunized with bacterially expressed recombinant H. contortus enolase (rHcENO) and Con A purified native glycoproteins of H. contortus. The assay was performed using indirect ELISA in a Maxisorp 96 well plate coated with E. coli expressed rHcENO and Con A purified native glycoproteins. Antibody titres in the serum were determined as maximum sera dilution showing OD 492.

3.6. Faecal egg counts and worm burden The reduction in faecal egg count was 50.3% (±15.3) and 84.35% (±11) in group I and group II, respectively, compare to the control animals (Fig. 7). The egg shedding began on the 22nd day post larval challenge and maintained the same level until necropsy in all experimental animals. Results indicated that the egg count did not increase of egg shedding in any of the immunized groups. In contrast, FEC values increased until slaughter in control animals. There was a reduction of 50% (±7.19) and 62.1% (±4.2) worm burden in group I and group II rHcENO immunized animals, respectively. P values of

Development of a safe and effective vaccine is essential to control parasites in small ruminants and would be a sustainable solution to tackle the anthelmintic resistance problem. Presently no recombinant vaccine has been shown to produce substantial production against H. contortus infection (Cachat et al., 2010; Redmond and Knox, 2004; Reszka et al., 2007). The recombinant form of these antigens failed to develop protective immunity in the host. The single subunit protein may not be effective against parasite like H. contortus. Enolase has proven to be a multifunctional enzyme (moonlighting protein) in both prokaryotes and eukaryotes (Avilan et al., 2011) that have non-catalytic functions apart from its normal catalytic activity. Enolase has been reported as a highly conserved protein in a wide variety of organisms (Pancholi and Fischetti, 1998). H. contortus enolase had been characterized recently (Han et al., 2012). In the quest to select promising candidate target antigens, we evaluated rHcENO for its protective efficacy against H. contortus infection. A proven subunit candidate vaccine containing Con A purified native antigens was used as positive standard. The H. contortus enolase gene of Chennai isolate had 95% identity in nucleotide and amino acid level to the enolase gene sequence of Chinese H. contortus isolate (Han et al., 2012) and other parasites to a lesser extent, confirming that it is a member of the enolase super family. This variation was mostly observed in the C-terminus

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Fig. 7. Faecal egg counts of immunized and control groups after challenge. Graph showing mean ± SE faecal egg counts from animals vaccinated with rHcENO (red), Con A purified native glycoproteins (blue), adjuvant control (black) and subsequently challenged with 5000 H. contortus L3 infective larvae. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 CD4+ and CD+ T cell subpopulations of sheep immunized with rHcENO, Con A purified native glycoproteins and adjuvant only. Days of experiment

Control

CD4+ lymphocyte Day 0 19.52 ± 0.199a Day 22 34.74 ± 2.859cd Day 27 27.66 ± 2.84b Day 35 23.96 ± 3.191ab Day 48 24.55 ± 2.07ab Day 56 28.47 ± 0.923cb Day 61 24.58 ± 1.152ab Day 86 38.18 ± 1.213d Day 99 23.96 ± 1.004ab Day 107 26.69 ± 3.73ab CD8+ lymphocyte Day 0 16.3 ± 1.186ab Day 22 11.14 ± 0.98a Day 27 17.03 ± 3.45ab Day 35 25.49 ± 0.34c Day 48 23.41 ± 2.39c Day 56 21.67 ± 2.08bc Day 61 15.95 ± 1.64ab Day 86 25.48 ± 3.63c Day 99 14.24 ± 0.75a Day 107 15.38 ± 0.55ab

rHcENO

Con A purified native glycoprotein

22.62 ± 1.46ab 27.456 ± 8.024abc 29.556 ± 3.517abcd 21.33 ± 4.79a 26.93 ± 2.48abcd 25.29 ± 1.94abc 35.47 ± 4.67bcd 37.88 ± 4.41cd 39.26 ± 3.68d 23.87 ± 1.63ab

20.87 ± 3.95a 30.61 ± 4.3ab 22.94 ± 5.45a 26.84 ± 2.84a 24.24 ± 2.7a 27.48 ± 2.29a 31.78 ± 7.22ab 41.08 ± 1.06b 34.06 ± 5.12ab 20.55 ± 2.2a

16.17 ± 3.01abc 9.4 ± 3.85a 13.66 ± 2.4ab 21.19 ± 4.09bc 20.28 ± 2.49bc 13.85 ± 1.39ab 19.24 ± 3.5bc 25.11 ± 1.98c 18.78 ± 1.7bc 19.7 ± 2.04bc

8.9 ± 2.75a 12.466 ± 0.86ab 10.766 ± 0.59a 18.54 ± 0.62c 18.64 ± 1.39c 13.58 ± 1.88acb 17.73 ± 2.19bc 24.61 ± 0.91d 17.58 ± 2.1bc 16.08 ± 1.48bc

Values are expressed as mean percentage (±SE) of the CD4+ and CD+ T lymphocyte population. a,b,c,d Values without a letter in common are significantly different (P < 0.05).

of the protein. Even though there is no variation in the functional domain of the enolase sequence. Amino acid replacements were observed in the C terminal region of the enolase and it is responsible for the protein function. These results suggest that enolase may have diverged within the α-enolase isoforms of H. contortus. However, the enolase amino acid sequence was highly conserved with several parasitic species (Fig. 3). Conservation of enolase signature sequence confirms that this gene is member of enolase family. These results revealed that all the parasitic enolases have similar functions in cellular metabolism. The 3D structure of H. contortus enolase has

70% identity with human enolase and 17% of sequence identity to sheep Ovis aries (AF233075) indicates that it could be a potential candidate antigen for haemonchosis infection in sheep. This is a good enough similarity to predict the structure of target protein by homology modelling. The Ramachandran plot analysis shows the amino acid residues within the most favoured region. Mg2+, a metal ion cofactor, was surrounded by amino acids in the active sites of enolase. Even though Mg2+ is known to be required for catalytic activity of the H. contortus enolase (Han et al., 2012), it may also play an important role in the stability of the enzyme structure. It was found as a plasminogen binding protein in several parasites (Wang et al., 2011). Despite the lack of signal sequences enolase has been shown to be located on the cell surface in several organisms (de la Torre-Escudero et al., 2010; Pancholi, 2001). Previous study suggested that the recombinant enolase was very similar to native form confirmed by SDS–PAGE profiles (Han et al., 2012). Immunoblotting with sera from naturally and experimentally infected animals confirmed the recombinant enolase protein and conservation of sequential epitopes. This indicates that H. contortus enolase is exposed to the host immune system during infection and potentially it can be used as diagnostic antigen. Previous studies have identified enolase in the excretory/secretory fractions of adult worms (Yatsuda et al., 2003). The key purpose of the present immunization trial was to evaluate the protective efficacy of rHcENO. A recent report indicated that native and recombinant enolase had a very similar pattern (Han et al., 2012). Even though several vaccine trials have suggested that the native antigens confer substantial reduction of haemonchosis (Cachat et al., 2010; LeJambre et al., 2008; Smith et al., 1999, 2003), no recombinant vaccine has been developed to date against H. contortus. In this study, both rHcENO and Con A purified native glycoproteins were found to provide partial protection against haemonchosis in the trial animals. Con A purified native glycoproteins gave better protection compared to rHcENO. This observation may be attributed to the fact that Con A purified native glycoproteins consist many number of glycoproteins to enhance the protection level in group II animals compared to group I animals.

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Even though single subunit of enolase conferred partial protection, the efficiency of the candidate vaccine can be improved by immunizing with cocktail of recombinant proteins. Sheep immunized with rHcENO antigen and Con A purified native glycoproteins showed 50.3% (±15.3) and 84.35% (±11) reduction in egg output upon H. contortus challenge. Similarly, worm burden had reduced by 50% (±7.19) and 62% (±4.2) respectively (Fig. 7). In this trial, the enolase immunized group animals showed significantly higher levels of IgG compared to control animals. Similar results were reported in recombinant fusion and non-fusion cysteine proteinaseenriched fraction (TSBP) vaccinated sheep (Redmond and Knox, 2001). This could be attributed to the difference in the number of female present in the abomasums. Increase in CD4+ positive T lymphocytes level in all groups in the post challenge period could be attributed to the immunomodulatory effect of H. contortus larvae infection and establishment. In sheep, it has been showed that CD4+ and CD8+ T cells play a critical role in regulating the immune response to H. contortus (Gill et al., 1993). High level of protection against haemonchosis was conferred in the Con A purified native antigen immunized group when compared to rHcENO and control groups could be due to numerous protective epitopes present in the multiple native antigen mixture. The rHcENO based vaccine conferred moderate level protection against H. contortus infection as a single molecule subunit vaccine. And this molecule is expected to give very high level of protection against H. contortus when combined with multiple protective antigens. In conclusion, rHcENO antigen was immunogenic and conferred partial protection against challenge with L3 infective larvae. Earlier tested vaccines with native antigens of H. contortus to control haemonchosis have limitation in large scale production and purity. Our results hold promise in developing an effective candidate recombinant vaccine. Further work is required to include few more potential candidate antigens to improve the protective efficacy.

Acknowledgement The corresponding author is grateful to Indian Council of Agricultural Research (ICAR) (Grant No. 3-5/GIP/2000), New Delhi for financial assistance under the AINP on GIP scheme and to Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) the Dean of Madras Veterinary College for providing the facility to carry out the research work. The authors would like to thank Dr. Mohana Subramaniam, Scientist, Translational Research Platform for Veterinary Biologicals (TRPVB), Chennai, India for the preparation of the manuscript.

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