Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen

Mice.Six-week-old BALB/cAnN mice, bred and maintained in microisolators in the animal facilities at the Biomedical Research Institute, Universidad Nacional Autónoma de México (UNAM), were employed for KETc7 immunization. All experiments reported herein were conducted in accordance with the principles set forth in the Guide for the Care and Use of Laboratory Animals (34). All protocols used in this study were approved by the Ethics Committee of the Biomedical Research Institute of UNAM.

Sequence analysis and phylogenetic analysis.The nTsKE7 sequence obtained here was compared with the already published sequence of the T. solium complete genome (23). Additionally, all KE7 orthologue protein sequences were identified in other helminth genomes available in the data set (http://www.genedb.org/Homepage ). The T. crassiceps and Taenia saginata sequences were reported in personal communications from J. P. Laclette and C. Maroni, respectively, and were based on unpublished results. Sequences were aligned by use of the ClustalW program (35). The alignment included the sequences of 28 taxa with 216 characters. A neighbor-joining tree was inferred with the software MEGA (v.6) (36) by the use of p distances and with the complete deletion option enabled. Tree nodes were supported with 10,000 bootstrap replicates.

Automated determination of protein features.Glycosylation patterns were predicted using the GPP prediction server (37). The TMHMM (v.2.0) server (38) was employed to search for transmembrane domains, and NetPhos (v.2) software was used to search for phosphorylation sites (39). Hydrophobicity and isoelectric points were predicted using the GPMAw-Lite server (http://www.alphalyse.com/gpmaw_lite.html ).

Protein domain automatic prediction.On the basis of the previously published nTsKE7 sequence from the T. solium genome (23), an automated protein BLAST search was performed, and the predicted conserved domains were confirmed by use of the NCBI Conserved Domain Database (40). Information regarding the predicted domains was manually retrieved (Fig. 1).

Cloning and expression of rTsKE7.The T. solium KE7 gene was synthesized by GenScript, optimizing the DNA sequence for its expression in Escherichia coli. Then, it was subcloned from pUC57-kan into the pET28b(+) plasmid and sequenced to confirm the identity of the construct. To this end, genes were amplified by PCR using the oligonucleotides 5′-GCCATACCATGGCAATCAATACGGCTCATACGCAAGAT-3′ with a NcoI restriction site (underlined) as a 5′ end primer and 5′-CTGCGAAAGCTTTTAGTGGTGGTGGTGGTGGTGCAGGTTCTTTTTGTCTTCCGGTTCCATC-3′ with an HindIII restriction site (underlined) as a 3′ end primer. The amplified product was purified and ligated into pET28b(+). The construct was sequenced entirely to exclude the possibility of the presence of inadvertent mutations. Overexpression was performed in E. coli Rosetta2 cells (Novagen) after transforming the pET28b(+) plasmid carrying the nTsKE7-encoding sequence. One liter of kanamycin-supplemented (50 μg/ml) LB medium was inoculated with a 5-ml preculture and incubated at 37°C. After an optical density at 600 nm (OD₆₀₀) value of 0.5 was reached, gene expression was induced by adding 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside), and growth was continued for another 16 h at 30°C. Cells were harvested by centrifugation at 4,000 rpm for 5 min at 4°C. Cell pellets were resuspended in 20 ml of 20 mM sodium phosphate buffer (pH 8), 150 mM NaCl, 0.5 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride (PMSF); then, the cells were lysed by sonication in a Branson Sonifier 450, applying six 50% pulses for 20 s at 30-s intervals at 4°C. The cells were centrifuged again (for 30 min at 10,000 rpm and 4°C) to separate the soluble cell extract from the insoluble fraction. nTsKE7 was purified from the soluble fraction in a nickel-Sepharose column (5 ml; HisTrap FF crude; GE Healthcare) that had previously been equilibrated with 20 mM sodium phosphate buffer (pH 8), 150 mM NaCl, 0.5 mM imidazole. The bound His6-tagged protein was eluted by applying a linear gradient from 1 mM to 500 mM imidazole. Fractions with the pure protein were loaded into a Superdex 200 column (GE Healthcare) that had previously been equilibrated with 20 mM sodium phosphate buffer (pH 8), 150 mM NaCl. Fractions with the pure protein were pooled, concentrated in an Amicon Ultra-15 system, and stored at 4°C until they were used.

AAR value for the nTsKE7 protein.The number of antigenic regions in the nTsKE7 protein was calculated by the BepiPred method (24, 41). The BepiPred (v.1.0) method predicts linear epitopes in a protein on the basis of the physicochemical profile of each amino acid (41). The threshold score was set to 0.35, and only those antigenic regions with 6 or more amino acids in length were selected. Then, an abundance of antigenic regions (AAR) value was calculated to normalize the number of antigenic regions according to the protein length, as previously reported (24).

Mouse antibodies.Anti-rTsKE7 antiserum was obtained by subcutaneously immunizing female BALB/cAnN mice with two 10-μg doses of rTsKE7 plus saponin (20 μg per mouse) every 10 days. Ten days after the last immunization, the mice were bled and serum was collected. The presence of specific antibodies was monitored by enzyme-linked immunosorbent assay (ELISA) during the immunization schedule, using the synthetic GK-1 peptide as the antigen source, according to a previously reported procedure with minor modifications (16, 42).

Parasite and protein extracts.The cyst tissue soluble fraction and vesicular fluid (VF) were obtained as previously described (43). T. solium cysticerci were excised from the skeletal muscle of naturally infected pigs and extensively washed with sterile 0.15 M phosphate-buffered saline (PBS; pH 7.3). Cysts were sectioned by using a scalpel, and the released VF was collected and diluted 1:2 using 50 mM Tris, pH 7.3, supplemented with protease inhibitors (0.5 M EDTA, 200 mM PMSF, 10 mM leupeptin, 1 mM pepstatin); VF was precipitated with acetone, and the pellet was solubilized in a conventional buffer (7 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}). Cyst tissue was washed several times using PBS supplemented with protease inhibitors and homogenized in 50 mM Tris, pH 7.3, with protease inhibitors using a Teflon pestle; tissue samples were subjected to three cycles of thaw-freezing. Then, the homogenized material was centrifuged at 14,000 × g for 60 min at 4°C. Soluble antigens in the supernatant were recovered and precipitated, and the pellets were solubilized in the same buffer. Protein fractions were quantified with a noninterfering (NI) protein assay kit with bovine serum albumin (BSA) as the standard (GBiosciences), aliquoted, and frozen at −70°C until use.

Two-dimensional polyacrylamide gel electrophoresis (PAGE).Isoelectric focusing (IEF) was performed using 7-cm immobilized pH gradient (IPG) strips (Bio-Rad) with a nonlinear 3 to 10 pH gradient and a Protean IEF cell (Bio-Rad) at a constant surface temperature of 20°C and a maximum current of 50 μA per strip. Samples of the protein extracts (150 μg) were diluted to a final volume of 125 μl using rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.2% IPG buffer for the respective pH gradient, bromophenol blue). These mixtures were used to rehydrate several IPG strips for at least 12 h at 20°C. IEF was performed for a total of 10,000 V-h. Then, the strips were reduced for 15 min in equilibration buffer (6 M urea, 0.05 M Tris pH 8.8, 2% SDS, 30% glycerol) containing 2% dithiothreitol and then alkylated using the same buffer supplemented with 2.5% iodoacetamide for 15 min. The second dimension was performed on 12% SDS-polyacrylamide precast gels (Bio-Rad) using a mini-Protean cell (Bio-Rad). After the gels were run at 200 V for about 60 min, the 2D gels were either silver stained or electroblotted onto nitrocellulose membranes for immunoblot analysis. The reproducibility of the proteomic pattern was verified by running the 2D gels in triplicate at different times. 2D gels were routinely stained with Coomassie brilliant blue R-250 (Bio-Rad) when used for mass spectrometry (MS) analysis. 2D gels were analyzed with PDQuest 2D analysis software (Bio-Rad).

Immunoblot analysis.Proteins were electrotransferred from the 2D gels to nitrocellulose membranes at 120 V for 70 min. The membranes were initially blocked by overnight incubation at 4°C with 10% dried skimmed milk in PBS buffer and then incubated for 2 h at room temperature with one of the pooled mouse serum samples obtained before and after immunization with the rTsKE7 protein (diluted 1:200 in the same buffer). The membranes were washed three times with PBS containing 0.1% Tween 20 (PBST) and incubated again for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse IgG antibody (Sigma) diluted 1:60,000 in PBST. After the immunoblots were washed as described above, they were developed by standard procedures using a West Femto chemiluminescence kit (Thermo). The reproducibility of immune recognition was verified by replicating the immunoblot at least three times.

Mass spectrometry (MALDI-TOF-TOF).Protein spots were cut from the gel and digested using proteomics-grade trypsin. Tryptic peptides were desalted and analyzed using a matrix-assisted laser desorption ionization–time of flight–time of flight (MALDI-TOF-TOF) 4800 instrument (AB Sciex). The instrument was operated in the positive-ion mode and externally calibrated using a mixture of different mass calibrators ranging from 900 to 3,600 Da (AB Sciex TOF/TOF). The laser source was set to 2,500 to 2,800 for MS spectrum acquisition and 3,500 to 3,800 for MS/MS spectrum acquisition. Readings in the MS-positive reflector mode were performed using 25 laser shots; the fragmentation of selected precursors was performed at a collision energy of 2 kV, using air as the collision gas (pressure, 2 × 10^–6 torr), with the accumulation of 400 shots for each spectrum. MS spectra were acquired at m/z 800 to 4,000. The parental ion of Glu1-fibrino-peptide B, diluted in the matrix (1.3 pmol/μl/spot), was used for internal calibration at m/z 1,570.690 Da. Up to 16 of the most intense spot signals with a signal-to-noise ratio of >20 were selected as precursors for MS/MS spectrum acquisition. Database searches were performed using Protein Pilot (v.2.0) software (AB Sciex) and the Paragon algorithm as the search engine. Each MS/MS spectrum was searched against the spectra for the T. solium and Sus scrofa proteins in protein databases. Searches were run using the cysteine-carbamidomethyl modification as a fixed modification and the methionine oxidation as a variable modification. The detected protein threshold unused postscore (confidence) was set to 0.47 to achieve a 66% confidence rate.

Identification of B-cell epitopes/mimotopes recognized by anti-rTsKE7 antibodies.Phages were selected by biopanning as previously described (44). A library of linear heptapeptides fused to the minor coat protein (pIII) of M13 bacteriophage was purchased from New England BioLabs (Beverly, MA). Polyclonal anti-rTsKE7 mouse hyperimmune serum was used for affinity selection. Microplate wells (MaxiSorp; Nunc, Roskilde, Denmark) were coated with 500 ng of mouse anti-IgG (Zymed, San Francisco, CA) diluted in 100 μl of PBS for 1 h at 37°C. After washing with PBS and blocking with 200 μl of 3.0% (wt/vol) BSA in PBS, 100 μl of anti-rTsKE7 antibody (1:100) was added, and the plates were incubated for 1 h at 37°C. After washing with 0.1% (vol/vol) Tween 20 in PBS, 1,011 phage particles from the library were added and the plates were incubated for 2 h at 4°C and for 1 h at 37°C. Then, nonbound phages were discarded and the wells were washed with PBS. Bound phages were eluted with glycine-HCl (pH 3). Eluates were transferred to microcentrifuge tubes, neutralized with 35 μl of 2 M Tris base, and amplified by infection of E. coli ER2537 cells in 2× YT medium (Gibco BRL, Gaithersburg, MD). Phages were purified by double precipitation with polyethylene glycol (USB, Cleveland, OH), and 1,011 PFU was used for the next biopanning round. After three rounds, individual phage clones were isolated. Phage single-stranded DNA was purified as described by Sambrook et al. (45) and used for DNA sequencing, performed with a T7 Sequenase (v.2.0) quick-denature plasmid sequencing kit (Amersham Biosciences, Cleveland, OH), the 96gIII sequencing primer (New England BioLabs), and [α-³⁵S]dATP (Amersham Biosciences) following the manufacturers' instructions. Both the DNA and the amino acid sequences of the inserts were analyzed with the ExPASy molecular biology server (https://www.expasy.org/tools/ ). Consensus sequences were determined by multiple-sequence alignment with the Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/ ).

Reactivity of phage-displayed epitopes/mimotopes by ELISA.To assess the binding of anti-rTsKE7 antibodies to the selected phage-displayed peptides, MaxiSorp multiwell plates (Nunc) were coated with 100 μl of a 5-μg/ml solution of an anti-mouse IgG anti-Fc monoclonal antibody (MAb) (Zymed) in PBS for 1 h at 37°C. After washing with 0.1% Tween 20 in PBS and blocking the wells with 200 μl of 3% BSA in PBS for 1 h at 37°C, anti-rTsKE7 antiserum was added and the plates were incubated for 1 h at 37°C. Then, phage particles, resuspended in 1% BSA in PBS, were added to the wells (1,010 PFU/ml) and the plates were incubated for 3 h at 4°C and for 30 min at room temperature (23°C). Positive phages were detected using anti-M13 MAb conjugated to horseradish peroxidase (Zymed) and ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] solution (Life Technologies, Frederick, MD, USA). The reaction was read at 405 nm.

Antigenic capacity.Twelve serum samples from T. solium cysticercotic pigs and 12 samples from noncysticercotic pigs diluted 1:100 were assayed by ELISA to test their reactivity against rTsKE7, GK-1, and GK-1M following a procedure previously described (15). A nonrelated recombinant protein originally isolated from Mycobacterium tuberculosis (RV1818C) with a His tag (kindly donated by Clara Espitia) and a nonrelated 17-amino-acid peptide (AALSPGSSAYPSATVLA) were used as negative controls. T. solium cyst vesicular fluid was employed as a source of antigen for the positive control. The accuracy of the antigen ELISA was evaluated by receiver operating characteristic (ROC) analysis. This was done by comparing GK-1-immunized pigs with nonimmunized animals. The optimal cutoff ratio value was selected as the point on the ROC curve (which displays the estimated percentages of sensitivity and specificity at a selected cutoff value) with the minimum distance to the (0, 1) coordinate. ROC curves were generated using the R software package (46). All sampled animals were previously evaluated by necropsy to ascertain their infection status, following the rules approved by the Ethical Committee of the Veterinary Medicine School (UNAM).

In silico prediction of an nTsKE7 cell epitope(s).To determine potential T-cell epitopes, the nTsKE7 protein sequence was submitted to the IEDB and SYFPEITHI web servers (http://tools.immuneepitope.org/main/ and http://www.syfpeithi.de/0-Home.htm , respectively), which measure the ligation strength of an amino acid sequence to a defined MHC haplotype. Analyses were run for MHC class I and II H-2 molecules of different haplotypes using the canonical peptide lengths (9 and 15 amino acids, respectively).

Protective capacity.The murine model of cysticercosis caused by T. crassiceps was employed to evaluate the protective capacity of rTsKE7, GK-1, and GK-1M. This experimental model has been extensively employed for this purpose and is based on the cross-reaction and cross-protection found between T. solium and T. crassiceps antigens (47, 48).

Mice were immunized with the recombinant rTsKE7 and synthetic GK-1 peptides, with saponin being used as the adjuvant. GK-1M was expressed in the filamentous phage M13. Mice were infected as previously reported (18) and sacrificed 30 days after infection; the parasites in the peritoneal cavity of control and vaccinated mice were counted. The protection level was estimated by the parasite load reduction in immunized mice with respect to the load in the controls, which received either saponin or M13 alone.

Statistical analysis.Data were collected in Excel (v.7.0) software (Microsoft, Redmond, WA) and analyzed with InStat software (GraphPad, La Jolla, CA).

The sensitivity and specificity of the assay for each tested antigen were compared using Fisher's exact test. Parasite loads between mouse groups were compared using the Mann-Whitney nonparametric test. A P value of <0.05 was considered statistically significant.