Immune Responses of Iranian Patients with Visceral Leishmaniasis and Recovered Individuals to LCR1 of Leishmania infantum

Antigen.LCR1 recombinant protein was produced and characterized as reported (12). Briefly, the procedure was performed as follows: Escherichia coli strain BL21(DE3) plysS (Invitrogen) was transformed by a pRSETA plasmid, including the lcr1 insert. The transformed bacteria was cultured and precipitated by centrifugation, the supernatants were discarded, and pellets were stored frozen at −20°C until use. The cell pellet was then resuspended in phosphate-buffered saline (PBS) and passed through five cycles of freeze and thaw (liquid nitrogen and 37°C, respectively), then centrifuged. The pellet was discarded and the supernatant which contained LCR1 was aliquoted and stored at −70°C until use. Since the recombinant LCR1 was produced in Escherichia coli, lysate of the same bacteria transformed by the same plasmid but without lcr1 insert was used as negative control in immunoblotting.

Soluble Leishmania antigens (SLA) were prepared as follows. L. infantum (strain MHOM/04/IR/IPI-UN10) was grown in culture medium (RPMI 1640, 10% fetal bovine serum [FBS], 2 mM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin). The stationary-phase parasites were harvested, washed in PBS (2 times), freeze-thawed (6 times), and centrifuged (16,000 × g, 20 min, 4°C), and then the supernatant was collected, aliquoted, and stored in −70°C until use. The protein content of SLA was 500 μg/ml as determined by the Bradford method (13).

Study population.Six patients with a recent history of VL were selected. The disease was diagnosed in these patients 4 to 11 months before sampling for the present study. The patients were 5 females and 1 male, with ages of 3.8 ± 1.9 years, from Bojnoord in the North Khorasan province located in northeast Iran. VL was confirmed in all patients by observation of parasites in bone marrow smears. In one case, the titer of a direct agglutination test (DAT) was available, which was 1/102,400. All patients had received meglumine antimoniate (Glucantime) as therapy for 28 days. Parents of all patients gave written informed consent, and the study protocol was approved by the ethics committee of the Pasteur Institute of Iran. Thirteen VL-recovered individuals, 8 females and 5 males, with ages of 4.3 ± 1.7 years, were selected from Ahar in the East Azerbaijan Province in northwest Iran. These individuals had received meglumine antimoniate (for 28 days in 12 patients and 14 days in 1 patient). They had recovered from VL between 3 to 42 months (27.53 ± 12.59 months) before blood draw for our study. VL was confirmed in these individuals by documented compatible clinical presentation in addition to either isolation (or microscopic detection) of parasite from bone marrow aspirate or a positive direct agglutination test (DAT) test (titer, ≥1/6,400). A leishmanin skin test (LST) was carried out in recovered cases and all cases were LST positive (LST indurations were from 7 mm to 15 mm, and the average was 8.95 ± 2.37 mm).

Ten healthy individuals, 8 male and 2 female, with ages of 31.2 ± 9.2 years, and without any history of VL or exposure to Leishmania infection, were selected from Tehran, which is an area where leishmaniasis is not endemic. LST was done, and all results were negative (LST indurations of <5 mm). Three healthy individuals were used as negative controls in immunoblotting. These individuals were male adults with no history of leishmaniasis.

Sample collection.Peripheral blood was withdrawn from all subjects of the study population. For use in immunoblotting, serum samples were separated after clotting and stored at −70°C until use. Serum samples were used in immunoblotting, individually. Pooled serum was used in some experiments. Peripheral blood mononuclear cells (PBMCs) were isolated through Ficoll-based gradient separation for the lymphocyte proliferation assay and were used in this assay within 8 h from the time of blood withdrawal.

Lymphocyte proliferation assay.The lymphocyte proliferation assay was performed according to the reported procedure (14). PBMC samples of each individual were cultured in triplicates in the presence of phytohemagglutinin (PHA), SLA, and LCR1, with final concentrations of 5, 5, and 7.5 μg/ml, respectively. A wide range of LCR1 concentrations (5 to 40 μg/ml final concentrations) were used in cell culture of selected VL-recovered individuals. After 4 days of culture, about half of the culture supernatant was harvested for cytokine assay, then [³H]thymidine (0.5 μCi/well) (Amersham Pharmacia Biotech, Buckinghamshire, England) was added to each well, and after 16 to 18 h cells were harvested on filter paper suitable for cell harvesting. Filter papers containing the cells were dried overnight at room temperature or for a few hours at 37°C. Filter papers corresponding to each well were transferred into separate scintillation vials. Two milliliters of scintillation fluid (Ready Safe; Beckman Coulter, Fullerton, CA) was then added to tubes and the tubes were counted in liquid scintillation counter (Wallac 1410, Turku, Finland). The lymphoproliferative results are presented in counts per minute (cpm) or stimulation index (SI). SI was calculated by dividing the cpm of the stimulated well by the cpm of the unstimulated well of the same individual.

Cytokine assay.PBMCs from recovered individuals were cultured in the presence of PHA, SLA, and LCR1 as described above. Supernatant were collected after 4 days and cytokines were assayed in the supernatants. IFN-γ and IL-10 were assayed by kits from e-Bioscience (human IFN-γ enzyme-linked immunosorbent assay [ELISA] Ready-SET-Go, and human IL-10 ELISA Ready-SET-Go, respectively). The assay procedure is briefly described as follows. ELISA plates (Corning, Lowell, MA) were coated with pretitrated capture antibody in ELISA coating buffer, sealed, and incubated overnight at 4°C. The plates were washed and any residual buffer was removed. Wells were blocked with assay diluent. The wells were aspirated and washed. Six consecutive 2-fold serial dilutions of standard for IFN-γ or IL-10 were prepared in assay diluent. Standards and samples from cell culture supernatants were added to the appropriate wells and incubated. The detection antibodies (diluted in assay diluent) were added to wells and incubated. Plates were washed and Avidin-horseradish peroxidase (HRP) (diluted in assay diluent) was added and incubated. Tetramethylbenzidine (TMB) substrate solution was added to wells and incubated. Stop solution (2 N H₂SO₄) was added to wells and the plates were read by a microplate reader (Anthos 2020; Eugendorf, Austria) at 450 nm and 620 nm. The values at 620 nm were subtracted from the values at 450 nm and the data were analyzed.

Bacterial sample preparation.Escherichia coli strain BL21(DE3) plysS (Invitrogen), which was transformed by the pRSETA plasmid lacking the lcr1 insert (12), was used as the negative control. The bacteria were grown in Luria Bertani (LB) medium supplemented with 50 μg/ml ampicillin and 35 μg/ml chloramphenicol at 37°C overnight. The cultures were then subcultured in fresh LB medium (1:50, old/new medium) without antibiotics at 37°C with vigorous shaking for 5 h. The bacteria from 25 ml culture media were divided to 1-ml aliquots and precipitated by centrifugation (3,000 × g, 10 min, 4°C), and then the supernatants were discarded. The pellets were stored frozen at −20°C prior to use. The cell pellet of each aliquot was then resuspended in 100 μl phosphate-buffered saline (PBS) and passed through five cycles of freeze and thaw (liquid nitrogen and 37°C, respectively), then centrifuged at 16,000 × g, for 10 min at 4°C. Since LCR1 is soluble (12), the pellet was discarded and the supernatant was kept at 4°C until use.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.SDS-PAGE was performed using a 10% concentration of acrylamide (CinnaGen, Tehran, Iran) in the resolving gel. Samples were prepared by adding a protein loading buffer (Fermentas) (1:6, loading buffer/protein suspension) and boiling for 5 min before loading onto an SDS-PAGE gel. The running buffer consisted of 1g SDS (CinnaGen, Tehran, Iran), 3.03 g Tris base (Sigma, St. Louis, MO) and 14.04 g glycine (AppliChem, Darmstadt, Germany) in 1,000 ml double-distilled water (ddH₂O). The samples were subjected to electrophoresis at 15 mA and 10 mA in stacking and resolving gel, respectively. Finally, the gel was stained with Coomassie brilliant blue R250 (Acros Organics, New Jersey).

Immunoblot analysis.The reactivities of sera from each VL patient against LCR1 and SLA were studied through immunoblotting according to established methods (15). Briefly, proteins were transferred from unstained SDS-PAGE gel to nitrocellulose membranes (Sigma, St. Louis) by a semidry apparatus (Multiphore II electrophoresis system; Pharmacia, Sweden) at 14 V for 60 min. The transfer was confirmed by observing prestained protein ladder (Vivantis, Malaysia) bands on the membranes. The membranes were incubated in 50 ml of 3% bovine serum albumin (BSA) (Merck, Darmstadt, Germany) in Tris-buffered saline (TBS) (Tris-HCl 100 mM [pH 7.5] and NaCl 150 mM) overnight. The membranes were then washed three times (15 min each time) in 200 ml of Tween-Tris-buffered saline (TTBS) washing buffer (TBS and 1% Tween 20). Each diluted serum was then added to a membrane containing the following proteins: LCR1, SLA, BSA, and lysate of bacteria lacking the lcr1 insert (negative control). Sera were used at 1/500 in TBS containing 1% BSA. The membranes were incubated for 2 h and were then washed and incubated for 2 h in HRP-conjugated anti-human IgG (Bethyl) (diluted at 1/8,000 in TBS containing 1% BSA) as secondary antibody (10 ml for each membrane). The membranes were washed again and 3.3′diaminobenzidine (DAB) (Sigma, St. Louis) solution (15 ml of TBS, 9 mg DAB powder, and 27 μl 30% H₂O₂) was added to each membrane. The membranes were washed with ddH₂O after signal development, and results were recorded.

Statistical analysis.A t test was used for comparison between different groups, and differences were considered significant at P values of ≤0.05.