Prediction of protein secondary structure and linear-B cell epitopes
Epitopes are the foundation of protein antigenicity that determines antigen specificity [27, 28]. There are many types of epitope prediction methods in use, including hydrophilicity, accessibility, antigenicity, flexibility, charge distribution and secondary structure [29–34]. Despite the lack of an infallible method to predict antigenic epitopes, several rules can be followed to determine which peptide fragments of a protein are likely to be antigenic. Firstly, antigenic epitopes should be located in solvent-accessible regions and contain both hydrophobic and hydrophilic residues. Secondly, peptides lying in long loops connecting secondary structure motifs should be selected preferably, while peptides located in helical regions should be avoided. Whenever possible, peptides that are in the N- and C-terminal regions of the protein should be chosen because they are usually solvent accessible and unstructured.
According to the rules outlined above, we analyzed the linear-B cell epitopes of TgCPB and TgCPL using DNAStar software and chose peptides that have good hydrophilicity, high accessibility, satisfactory flexibility and strong antigenicity. Thereafter, we used DNAMAN software to search for linear-B cell epitopes in the TgCPB and TgCPL amino acid sequences.
Prediction of Th-cell epitopes
T. gondii is an obligate intracellular parasite; hence, cellular immunity mediated by T cells plays an important role in T. gondii infection . To develop an effective vaccine against toxoplasmosis, it is necessary to elucidate which type of Th cell-mediated immune response is necessary. Predicting Th cell epitopes is currently rather complicated and the results are ambiguous; however, there are some rules that can be used to predict Th cell epitopes [36, 37]. Here, we used the Immune Epitope Database (http://tools.immuneepitope.org/analyze/html/mhc_II_binding.html) online service to predict the half maximal inhibitory concentration (IC50) values of peptides binding to the major histocompatibility complex (MHC) class II molecules of TgCPB and TgCPL. We also used SYFPEITHI (http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm) to determine the ligation strength to a defined HLA (or H2) type for TgCPB and TgCPL. Note that such binding to MHC is necessary but not sufficient for recognition by T cells.
Parasites and mice
Female 6-week-old BALB/c mice were purchased from Shandong University Laboratory Animal Center. All mice were maintained under specific pathogen-free conditions. All of the animal experiments were approved by the Ethics Committee on Animal Experiments of the Medical School of Shandong University.
The T. gondii RH strain was harvested from the peritoneal fluid of the BALB/c mice 72 h after infection, and was washed by centrifugation and resuspended in sterile PBS. Half of the T. gondii tachyzoite suspension was used to extract total RNA and genomic DNA, while the other half was used to prepare soluble tachyzoite antigens (STAg) using an ultrasonic disintegrator. STAg preparations were aliquoted and stored at −80°C until use.
Construction of expression plasmids
The whole TgCPB gene was amplified from T. gondii total RNA by reverse transcription polymerase chain reaction (PCR) using the two primer pairs shown below. TgCPB for prokaryotic expression used the following primers plasmid construction: 5′-cgGAATTCATGGAGGGGCGAAAGTC-3′ (forward) and 5′-ccgCTCGAGCTACATTTCTCTCTCCTCTTCTG-3′ (reverse), both of which contain EcoRI/XhoI restriction sites (underlined). Plasmid construction for eukaryotic expression of TgCPB consisted of 5′-ataagaat GCGGCCGCATGGAGGGGCGAAA-GTC-3′ (forward) and 5′-ccgCTCGAGCTACATTTCTCTCTCCTCTTCTG-3′ (reverse), both of which contain NotI/XhoI restriction sites (underlined).
The whole TgCPL gene was PCR amplified from of T. gondii genomic DNA using the two primer pairs described below. Prokaryotic expression plasmid construction for TgCPL used the following primers: 5′-cgGAATTCATGGACAGCAGCGAGACGC-A-3′ (forward) and 5′-ccgCTCGAGTCACATCACGGGGAAAGACG-3′ (reverse); EcoRI and XhoI restriction sites are underlined. Eukaryotic expression plasmid construction for TgCPL used the following primers: 5′-ccAAGCTTATGGACAGCA-GCGAGACGCA-3′ (forward) and 5′-gcTCTAGATCACATCACGGGGAAAGACG-3′ (reverse); HindIII and XbaI restriction sites are underlined.
The PCR products for both genes were cloned into a pEASY-T1 vector (TransGen Biotech, China) to generate a recombinant cloning plasmid. After sequencing, TgCPB and TgCPL were subcloned into a eukaryotic expression plasmid pET-30a(+) (Novagen, USA) and the prokaryotic expression plasmid, pBudCE4.1 (Invitrogen, USA) to produce pET30a-TgCPB, pET30a-TgCPL, pBudCE4.1-TgCPB and pBudCE4.1-TgCPL. Finally, TgCPB and TgCPL were concurrently subcloned into the prokaryotic expression plasmid pBudCE4.1 to produce pBudCE4.1-TgCPB-TgCPL.
TgCPB and TgCPL expression in Escherichia coli and antigen production
pET30a-TgCPB and pET30a-TgCPL constructs were used to transform E. coli BL21(DE3) cells, which were then grown in Luria-Bertani medium with kanamycin (25 μg/mL). Synthesis of recombinant TgCPB and TgCPL proteins was induced using 1mM isopropyl-β-D-thiogalactoside for 6 or 8 h at 25°C. The cells were then lysed and centrifuged at 4°C for 15 min at 10,000 × g. Recombinant proteins were then purified via binding of their carboxy terminal histidine tags to Ni-NTA resin (Sangon Biotech, China).
Experimental mice were subcutaneously immunized with 100 μg of purified rTgCPB or rTgCPL prepared in equal volumes of Freund’s complete adjuvant for the first immunization. The second and third immunizations contained 50 μg of the purified protein in Freund’s incomplete adjuvant. Samples of antisera were collected 2 weeks after the last immunization.
Examination of antibody specificity by western blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to investigate antibody specificity, as described previously . STAg preparations were removed from the ultra-low temperature freezer and 500 ng of each preparation was used for SDS-PAGE. The separated protein bands were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA), each of which was blocked with 5% w/v skimmed milk powder diluted in PBS for 2 h at room temperature before separate incubation with mouse anti-TgCPB or anti-TgCPL antibodies, or pre-immune mouse sera (dilution 1:600). After a wash in PBS-Tween 20, each of the membranes was incubated with diluted goat anti-mouse IgG horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000; Sigma, USA) for 1h. Parasite proteins were visualized using electrochemiluminescence reagents (Cowin Biotech, China).
TgCPB and TgCPL expression in mammalian cells
When the cell density reached 80–90%, HEK293 cells were transfected with pBudCE4.1-TgCPB or pBudCE4.1-TgCPL using Lipofectamine™ 2000 reagent (Invitrogen, USA). After 24-h incubation, the cells were fixed with cold methanol for 20 min and protein expression was evaluated using an indirect fluorescence antibody test as previously described . Briefly, anti-TgCPB or anti-TgCPL antibodies were used as primary antibodies and a FITC-labeled goat anti-mouse IgG antibody (ZSGB-Bio, China) was used as the secondary antibody. After rinsing three times with PBS, the coverslips were immediately observed under a fluorescence microscope (Carl Zeiss, Germany). The cells were then lysed with RIPA Lysis Buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1% sodium deoxycholate; 0.1% SDS) containing 1 mM of the protease inhibitor phenylmethanesulfonyl fluoride, after which they were centrifuged at 12,000 × g for 10 min, at either 24 h or 48 h post-transfection.
SDS-PAGE and western blot analysis
Protein production from HEK293 cells was monitored by SDS-PAGE and western blotting. About 500 ng of the purified rTgCPB or rTgCPL proteins were separated using SDS-PAGE. The separated protein bands were transferred onto PVDF membranes. The detailed procedures are the same as in the above section “Identification of the antibody specificity by western blotting”.
Five groups of BALB/c mice (n = 13 each) were individually injected 4 times at two-weekly intervals with one of the following: PBS, pBudCE4.1, pTgCPB, pBudCE4.1-TgCPL or pBudCE4.1-TgCPB-TgCPL. Two weeks after the final immunization, the mice were challenged by intraperitoneal (i.p.) injection of 100 μL of PBS containing 1 × 104
T. gondii tachyzoites and the survival time of each mouse was recorded.
The levels of IgG antibodies against T. gondii were analyzed using an enzyme-linked immunosorbent assay (ELISA) . The microtiter plates (Costar, USA) were coated with STAg (10 pmol/well) and incubated at 4°C overnight. After washing three times with PBS-T, the plates were blocked with 1% bovine serum albumin for 1 h at 37°C. The plates were washed a further three times and incubated with the mouse sera diluted in PBS for 1 h at 37°C. After three washes, secondary goat anti-mouse IgG, IgG1 or IgG2a conjugated with HRP (Sigma) was added and incubated at 37°C for 1 h. Immune complexes were revealed by incubating with ortho-phenylenediamine (Sigma) and 0.15% H2O2 for 30 min. Reactions were stopped by the addition of 2 M H2SO4 and read at 490 nm with an ELISA plate reader (EL800; Bio-Tek, USA). All samples were run in triplicate.
Cytokine levels were detected according to the previously described method . Briefly, three mice per group on week 4 after the final immunization were euthanized and their spleens removed under sterile conditions. Viable splenocytes were dispensed into 96-well plates at 37°C in 5% CO2 and the cell-free supernatants were harvested and assayed for IL-4 levels at 24 h, or at 72h for IL-10, or at 96 h for IFN-γ using an ELISA kit (R&D Systems, USA).
Statistical analyses were performed using SPSS software. Antibody production and cytokine levels among the different groups were determined using a one-way analysis of variance. Survival times in the mice were compared using the Kaplan-Meier method. Values of P < 0.05 were considered statistically significant.
All experimental procedures using animals in the present study had received prior approval by the Institutional Animal Care and Use Committee of Shandong University under Contract 2011–0015. Humane endpoints to reduce pain or distress in the experimental animals were employed via euthanasia. Mice were monitored daily over 8 weeks for signs of toxoplasmosis, which included difficulties with their food and water intake, lethargy, or severe ascites. Mice that showed signs of illness were sacrificed immediately using CO2 gas; this involved placing the mice in a chamber and administering CO2 at a concentration of 60% to 70% over a 5-minute exposure time, after which the cervical dislocation method was sometimes used to ensure that effective euthanasia had occurred.