Immunization with the DNA-Encoding N-Terminal Domain of Proteophosphoglycan of Leishmania donovani Generates Th1-Type Immunoprotective Response against Experimental Visceral Leishmaniasis

Cell culture

Baby hamster kidney cell line BHK-21was procured from the National Centre for Cell Science (Pune, India). Cells were grown in RPMI 1640 (Invitrogen Life Technologies) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM NaHCO₃, 20 mM HEPES, along with 10% FCS (Life Technologies at 37°C in the presence of a 5% CO₂ supply.

Animals

Laboratory inbred male golden hamsters (Mesocricetus auratus, 45–50 g) from the Institute’s animal house facility were used for experimental purposes with prior approval of the animal ethics committee of the Central Drug Research Institute (Lucknow, India). They were housed in climatically controlled rooms and fed with standard rodent food pellet (Lipton India) and water ad libitum.

Parasites

The L. donovani strains sodium stibogluconate-resistant 2039 (procured from a patient admitted to the Kala Azar Medical Research Centre, Muzzafarpur, Bihar) and WHO reference strain Dd8 were cultured in vitro as described elsewhere (30). The strains have also been maintained in hamsters through serial passage, i.e., from amastigote to amastigote. For bulk cultivation, promastigotes were grown in L-15 medium (Sigma-Aldrich) with l-glutamine, supplemented with 10% tryptose phosphate broth (Himedia), 0.1% gentamicin, and 10% FBS (Life Technologies). Parasites were harvested after 3–4 days of culture (31).

Cloning and expression of N-terminal domain of ppg (1.6 kb)

L. donovani strain 2039 genomic DNA was isolated from 10⁸ promastigotes, washed, and suspended in NET buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM EDTA) and incubated with proteinase K (1 mg/ml; Invitrogen Life Technologies) and 0.5% SDS at 50°C for 4 h. Nucleic acids were extracted by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Genomic DNA was spooled and subjected to RNase (100 μg/ml) treatment. N-terminal domain of the ppg gene was amplified using Taq Polymerase (Sigma-Aldrich) lacking a 3′–5′ exonuclease activity. PCR was performed using ppg-specific primers (based on the L. major ppg3 gene sequence): forward, 5′-GGATCCACCATGTCTTTTCATAGGCGCCGG-3′ and reverse, 5′-GGATCCATCTGGAATTGTGAGGTGCACCAATCG-3′(BamHI site underlined) in a Thermocycler (Bio-Rad) under conditions at one cycle of 94°C for 4 min, 30 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 30 s, and finally one cycle of 72°C for 10 min. Amplified PCR product was electrophoresed in agarose gel and eluted from the gel by GenElute Columns (Qiagen). Eluted product was cloned in pTZ57R/T (T/A) cloning vector (Fermentas) and transformed into competent DH5α cells. The transformants were screened for the presence of recombinant plasmids with the ppg insert by gene-specific PCR under similar conditions as previously mentioned. Isolated positive clones were sequenced from Delhi University (New Delhi) and submitted to the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/nuccore/119656394; accession no. EF141073). ppg was further subcloned at the BamHI site in bacterial pET28a (Novagen) and mammalian expression pcDNA3 (Invitrogen) vectors.

The expression of PPG was checked in bacterial cells by transforming the ppg+pET28a construct in Escherichia coli Rosetta strain. The transformed cells were inoculated into 5-ml test tube culture medium (Luria-Bertani) and allowed to grow at 37°C in a shaker at 220 rpm. Cultures in logarithmic phase (at OD₆₀₀ of ∼0.5–0.6) were induced for 3 h with 1.0 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C. After induction, cells were lysed in 5× sample buffer (0.313 M Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, and 0.05% bromphenol blue, with 100 mM DTT) and whole cell lysates (WCL) were analyzed by 12% SDS-PAGE (32). Uninduced control culture was analyzed in parallel. The separated proteins from the polyacrylamide gel were transferred onto a nitrocellulose membrane in a semidry blot apparatus (Amersham) as described earlier (33). Membrane was incubated for 1 h in blocking buffer followed by a 2-h incubation at room temperature with mouse anti-His Ab (Novagen) as primary Ab (1/2500 dilution) and then incubated with goat anti-mouse HRP conjugate Ab (1/10,000; Bangalore Genie) for 1 h at room temperature. The blot was developed using an ECL kit (GE Biosciences).

The expression of PPG was further checked in mammalian cells by transfecting the ppg+pcDNA3 construct in BHK cells. For transfection and vaccination studies, endotoxin-free plasmid DNA was isolated using an Endofree plasmid purification Maxi kit as per the manufacturer’s protocol (Qiagen). For confirmation and cellular localization of the proteins expressed by the ppg+pcDNA3 construct, 2 × 10⁵ BHK cells were grown in four-chamber slides and transfected with four different sets of plasmids, the blank pcDNA3 plasmid (as negative control), ppg+pcDNA3 construct, and gfp+pcDNA3 construct (as positive control) and cotransfected with ppg+gfp-containing plasmids in serum-free DMEM (Life Technologies) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Expression was confirmed by RT-PCR and fluorescence microscopy. For RT-PCR, RNA was isolated from the transfected BHK cell line using TRIzol (Sigma-Aldrich) and the cDNA was synthesized using Moloney murine leukemia virus- reverse transcriptase as per the manufacturer’s protocol (Fermentas). Resulting cDNA was treated with RNase (10 μg/μl; Fermentas) and used as template to amplify the ppg gene using gene-specific primers. The expression of ppg at the appropriate cellular localizations was also confirmed by immunofluorescence using polyclonal Ab raised in rabbit against deglycosylated and dephosphorylated native filamentous PPG of L. major (gift from Dr. E. Handman, The Walter and Eliza Hall Institute of Medical Sciences, Melbourne, Australia). BHK cells were plated in four-chamber slides and allowed to grow for 24 h. For detection of intracellular PPG, cells were fixed using 4% paraformaldehyde and permeabilized by 0.5% Triton X-100 for 10 min followed by incubation with the same primary anti-serum and secondary anti-rabbit FITC conjugate (Bangalore Genei) for 1 h at room temperature. After washing in PBS, cells were visualized under a fluorescence microscope (Eclipse 80i Nikon). Cells transfected with the gfp+pcDNA3 construct was visualized directly under a fluorescence microscope.

Vaccination with ppg+pcDNA3 construct in hamsters

For immunization, a total of 85 Syrian golden hamsters (40–45 g) were divided into the following four groups, each containing 20–25 animals, wherein groups 1–3 served as controls as described below and group 4 as the main experimental group: group 1, unvaccinated and unchallenged (normal control); group 2, unvaccinated and challenged (infected control); group 3, pcDNA3 vaccinated (vector control); and group 4, ppg+pcDNA3 vaccinated (vaccinated group). The experimental schedules for vaccination/immunization were conducted as described below:

Detection of ppg DNA levels in muscles of hamster immunized with ppg+pcDNA3 construct by Southern blotting

For detection of the ppg DNA level in hamsters, total DNA was extracted from the muscle of ppg+pcDNA3- immunized as well as pcDNA3 blank vector-immunized hamsters using a NucleoSpin nucleic acid purification kit (MACHEREY-NAGEL) as per the manufacturer’s protocol. PCR amplification of the ppg gene was conducted using muscle DNA of hamsters as template as described earlier. For probe synthesis, PCR was performed using digoxigenin-labeled dUTPs (Roche) and ppg+pcDNA3 plasmid as template. The amplified product was resolved in 1% agarose gel, transferred onto nylon membranes (Hybond-N+; Amersham Biosciences), and subjected to Southern blot analysis with a ppg-specific probe (34, 35).

Measurement of body, spleen, and liver weight of vaccinated hamsters

The body weight and that of the spleen and liver (on necropsy) of hamsters of all of the experimental groups were assessed at different time intervals, i.e., on days 0, 45, 60, 120, and 180 p.c.

Preparation of soluble L. donovani (SLD) promastigote Ag

SLD promastigote Ag was prepared according to the method described by Scott et al. (36) and modified by Choudhry et al. (37). Briefly, late log-phase promastigotes (10⁹) were harvested from 3- to 4-day cultures, washed four times in cold PBS, resuspended in PBS containing protease inhibitors mixture (Sigma-Aldrich), and sonicated (Soniprep-150) for two periods of 1.5 min each in ice, (separated by an interval of 3 min) at medium amplitude. The sonicated sample was subjected to rapid freeze-thawing four times using liquid nitrogen and left at 4°C for 1 h for complete extraction of soluble Ag. The suspension was centrifuged at 4,000 × g for 20 min at 4°C followed by ultracentrifugation at 40,000 × g for 30 min. After assessing protein contents, the Ag was aliquoted and stored at −70°C.

Measurement of delayed-type hypersensitivity (DTH) in hamsters

DTH was performed by injecting 50 μg/50 μl of SLD in PBS intradermally into one footpad and PBS alone into the other one of each of the vaccinated and unvaccinated controls. The response was evaluated 48 h later by measuring the difference in footpad swelling between the two with and without SLD for each animal (38).

Assessment of parasitic burden in vaccinated hamsters

The prophylactic efficacy of all of the experimental groups was assessed on necropsy at different time intervals, i.e., on days 0, 45, 60, 120, and 180 p.c. The impression smears/touch blots of different organs, namely, spleen liver and bone marrow (femur bone) of experimental animals were made and the criteria for the assessment of parasitic burden was based on the counting of the number of amastigotes/1000 cell nuclei in each organ. The percentage of inhibition (PI) of parasite multiplication was calculated in comparison to the unvaccinated control using the following formula: PI = No. of parasite count from infected control − no. of parasites from the vaccinated group/no. of parasite count from infected control × 100.

Immunological assays

For evaluation of cellular and Ab responses, peritoneal exudate cells, inguinal lymph nodes, and blood were collected from hamsters on necropsy at various time intervals, i.e., day 0, 45, 60, 120, and 180 p.c.

Assessment of lymphoproliferative responses in vaccinated hamsters

Lymph nodes of hamsters were excised aseptically and processed for the isolation of lymphocytes (39). The lymphocytes were suspended to 10⁶/ml and cultured at 10⁵ cells/well in 96-well flat-bottom tissue culture plates (Nunc). One hundred microliters of Con A (10 μg/ml; Sigma-Aldrich) or SLD (10 μg/ml) was added to each well in triplicate. Wells without stimulants served as negative controls. Cultures were incubated at 37°C in a CO₂ incubator for 3 days in the case of mitogens and for 5 days in the case of SLD Ags. Eighteen hours before termination of culture, 0.5 μCi of [³H]thymidine (BARC) was added to each well and cells were harvested on glass fiber mats (Whatman) and radioactivity was counted in a liquid scintillation counter. Results were expressed as stimulation index (SI), which was calculated as mean cpm of stimulated culture/mean cpm of unstimulated control. SI values of >2.5 were considered as positive response.

Assessment of level of NO activity in macrophages of vaccinated hamsters

The presence of nitrite (NO₂⁻) content was assessed using Griess reagent in the culture supernatants of naive hamster peritoneal macrophages after the exposure with supernatant of stimulated lymphocyte cultures. Briefly, isolated peritoneal macrophages (30) were suspended in culture medium and plated at 10⁶ cells/well and exposed to the supernatants of the above described 5-day-old Ag-stimulated lymphocyte cultures from all of the study groups. The supernatants (100 μl) collected from macrophage cultures 24 h after incubation were mixed with an equal volume of Griess reagent (Sigma-Aldrich) and left for 10 min at room temperature. The absorbance of the reaction was measured at 540 nm in an ELISA reader (40).

Determination of antileishmanial Ab responses in hamsters

The levels of antileishmanial Abs-IgG and its isotypes, IgG1 and IgG2, in sera samples from hamsters of different groups were measured as described earlier (41). The 96-well ELISA plates (Nunc) were coated with SLD (0.2 μg/100 μl/well) overnight at 4°C and blocked with 1% BSA at room temperature for 1 h. The optimum dilution of sera was standardized at 1/200 for IgG, IgG1, and IgG2 for 2 h at room temperature. HRP-conjugated goat anti-hamster IgG (H + L) (Serotec) and biotin-conjugated mouse anti-Armenian and anti-Syrian hamster IgG1 (for IgG1) (BD Pharmingen) as well as mouse anti-Syrian hamster IgG2 (BD Pharmingen) were added for 1 h at room temperature at 1/800 dilutions. IgG1 and IgG2 plates were further incubated with streptavidin-conjugated peroxidase (Sigma-Aldrich) for 1 h. Finally, the substrate O-phenylenediamine dihydrochloride (Sigma-Aldrich) was added and the plate was read at 492 nm.

Estimation of expression of mRNA cytokines by real-time PCR in vaccinated hamsters

Real-time PCR was performed to assess the expression of mRNAs for various cytokines and inducible NO synthase (iNOS) in splenic cells. Splenic tissues were taken from each of the three individual animals randomly chosen from different groups. Total RNA was isolated using Tri-reagent (Sigma-Aldrich) at different time intervals and quantified by using Gene-quant (Bio-Rad). One microgram of total RNA was used for the synthesis of cDNA using a first-strand cDNA synthesis kit (Fermentas).

For real-time PCR, primers were designed using Beacon Designer software (Bio-Rad) on the basis of cytokines and iNOS mRNA sequences available on PubMed (42) (Table-1⇓). Real-time quantitative PCR was conducted as per the protocol described earlier (43). Briefly, it was carried out with 12.5 μl of SYBR green PCR master mix (Bio-Rad), 1 μg of cDNA, and primers at a final concentration of 300 nM in a final volume of 25 μl. PCR was conducted under the following conditions: initial denaturation at 95°C for 2 min followed by 40 cycles, each consisting of denaturation at 95°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 40 s per cycle using the iQ5 multicolor real-time PCR system (Bio-Rad). cDNAs from infected hamsters were used as “comparator samples” for quantification of those corresponding to test samples. All quantifications were normalized to the housekeeping gene HGPRT. A no-template control c-DNA was included to eliminate contaminations or nonspecific reactions. The cycle threshold (C_T) value was defined as the number of PCR cycles required for the fluorescence signal to exceed the detection threshold value (background noise). Differences in gene expression were calculated by the comparative C_T method (43). This method compares test samples to a comparator sample and uses results obtained with a uniformly expressed control gene (HGPRT) to correct for differences in the amounts of RNA present in the two samples being compared to generate a ΔC_T value. Results are expressed as the degrees of difference between ΔC_T values of test and comparator samples.

Postchallenge survival

Survival of hamsters belonging to group 4 was checked until day 180 p.c. in comparison to the normal hamsters (group 1). Animals in all of the groups were given proper care and were observed for their physical conditions until their survival period. Survivals of individual hamsters were recorded and mean survival period was calculated.

Statistical analysis

Results are expressed as mean ± SD of three to five individual animals per group at designated time points. Three replicates were done. The results (pooled data of three experiments) were analyzed by one-way ANOVA followed by Dunnet’s or Tukey’s post test where appropriate. All of the analyses were done using GraphPad Prism (version 3.03) software.

PPG was cloned, sequenced, and expressed in vitro in E. coli Rosetta strain and in BHK cells as well as in vivo in hamsters

The ppg gene of L. donovani was successfully cloned and sequenced in the T/A cloning vector, which was 95% homologous with L. major ppg3. It was further cloned in right orientation under the T7 promoter in bacterial expression pET28a (Fig. 2⇓I) as well as mammalian expression vector pcDNA3 (Fig. 2⇓III, A). The expression status of the cloned gene when checked in vitro at the protein level in the E. coli Rosetta strain by Western blotting using anti-His Ab exhibited the predicted ∼65.120-kDa recombinant protein (Fig. 2⇓II, B). In BHK cells, the expression level was established at the RNA level by RT-PCR depicting a 1.6-kb band in ppg+gfp cotransfected and ppg transfected but not in vector as well as gfp-transfected cells (Fig. 2⇓III, B). The expression at the protein level was confirmed by fluorescence microscopy using Ab raised in rabbits against deglycosylated and dephosphorylated native filamentous PPG of L. major. The BHK cells transfected with gfp+pcDNA3, ppg+pcDNA3, as well as gfp- and ppg- containing plasmids exhibited bright green fluorescence, indicating the expression of PPG inside the cells. Blank vector-transfected BHK cells showed no fluorescence (Fig. 2⇓IV).

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FIGURE 2.

Cloning and expression of ppg. Clone confirmation of ppg in pET28a vector. M, 1-kb molecular mass marker; lane 1, BamHI-digested ppg+pET28a plasmid construct; lane 2, XhoI-digested plasmid; lane 3, undigested plasmid (I). Expression of rPPG in prokaryotic cells, WCL of transformed E. coli separated on 12% acrylamide gel and stained with Coomassie blue. II, A, Western blot analysis using anti-His mAb. M, Molecular mass markers; Lane 1, WCL before IPTG induction; lane 2, WCL after IPTG (1.0 mM) induction at 37°C (II, B). Clone confirmation of ppg in mammalian expression pcDNA3 vector. M, Molecular mass marker; lane 1, BamHI-digested ppg+ pCDNA3 plasmid construct; lane 2, NotI-digested plasmid; lane 3, XhoI-digested plasmid; lane 4, undigested plasmid (III, A). In vitro expression of ppg in mammalian BHK cell line by RT-PCR (III, B) and fluorescence microscopy (IV). Lanes 1–4 (IIIA) and A–D (IV) are pcDNA3 transfected; gfp+ pcDNA3 transfected; and ppg+pcDNA3 transfected and ppg+gfp cotransfected BHK cells, respectively. In vivo expression of ppg in the hamster, PCR amplification of genomic DNA isolated from muscle tissue of hamsters 3 wk after immunization with pcDNA3 and ppg+pcDNA3 plasmid using ppg-specific primers. M, 1-kb molecular mass marker. Lane 1, pcDNA3; lane 2, ppg+pcDNA3 (V, A). Southern blot analysis of PCR-amplified ppg from the genomic DNA isolated from the muscle tissue of the hamster using the ppg probe (V, B).

The expression level of PPG was also confirmed in vivo at the DNA level by PCR and Southern blotting from immunized hamster tissues. The ppg signal was clearly visible in tissue from hamsters injected with ppg+pcDNA3 construct but not in those hamsters injected with blank vector (Fig. 2⇑V, A). To show the sensitivity and specificity of PCR, the PCR products were detected by Southern blot hybridization using ppg sequence-specific probe. As shown in Fig. 2⇑V, B, hamsters immunized with the ppg+pcDNA3 construct showed a significant amount of ppg DNA in muscle tissue.

Vaccination with ppg DNA-induced optimum protection against L. donovani challenges

The ppg DNA-vaccinated hamsters were found to be protected from the challenge infection of L. donovani, as indicated by their weight gain with time like normal animals. In contrast, there was significant weight loss (p < 0.001) in any of the animals of the infected and vector control groups (Fig. 3⇓A). There was an absence of hepatosplenomegaly in the vaccinated group that is normally associated with the challenge infection (Fig. 3⇓, B and C). A positive correlation of parasite loads with splenomegaly and hepatomegaly was observed among the experimental and control groups. An increase from ∼10³ to >10⁴ parasites in all of the groups, except in the ppg DNA-vaccinated group, was seen in Giemsa-stained splenic smears from days 45 to 120 p.c. (Fig. 3⇓D). In the vaccinated group, parasite loads decreased from ∼2 × 10² on day 45 to a negligible level (<10 subsequently) (p < 0.001) by day 180 p.c., rendering them difficult to discern by microscopy (data not shown). Similarly, in liver and bone marrow, parasite loads decreased sharply after day 45 p.c. and parasites were essentially absent by day 180 p.c. in the same vaccinated group (Fig. 3⇓, E and F). Cultivation of the spleen, liver, and lymph node tissues from the vaccinated hamsters in vitro yielded no promastigotes after prolonged incubation for 3 wk.

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FIGURE 3.

Body weight (A), spleen weight (B), and liver weight (C) in g as well as parasite burden (no. of amastigotes per 1000 cell nuclei) in the spleen (D), liver (E), and bone marrow (F) on days 0, 45, 90, 120, and 180 p.c. Significance values indicate the difference between the vaccinated groups and infected group (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001).

The ppg DNA-vaccinated hamsters survived the challenges of L. donovani and remained healthy until the day of termination of the experiment, i.e., 6 mo p.c. In contrast, hamsters vaccinated with the pcDNA3 vector and infected control survived for only 2–3 mo.

ppg vaccination stimulates DTH, mitogenic, and Leishmania-specific cellular responses

ppg DNA and blank vector immunizations elicited different cell-mediated immunity (CMI), as determined by assessing the DTH and lymphoblast proliferation responses to specific and/or nonspecific Ags in these hamsters after L. donovani challenges at different time periods. ppg DNA-vaccinated hamsters displayed significant DTH responses, which increased progressively throughout the entire p. c. period (Fig. 4⇓A) and reached the levels that were significantly higher than those of the control groups (p < 0.001) at all time points for the duration of the experiments for up to 180 days.

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FIGURE 4.

DTH response (mm) (A), LTT response (SI value) to SLD (B) and Con A (C), and Leishmania-specific IgG (D) and its isotypes IgG1 (E) and IgG2 (F) in ppg DNA-vaccinated hamsters in comparison to the unimmunized infected controls, vector-immunized controls, and uninfected normal hamsters on days 0, 45, 60, 120, and 180 p.c. Significance values indicate the difference between the vaccinated groups and infected group (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001).

In vitro stimulation of the lymphocytes with Con A showed comparable proliferative response at high levels in all of the groups when assayed before challenges (Fig. 4⇑B, day 0). Con A-induced lymphoproliferative response remained elevated in ppg-vaccinated animals as much as those of the normal hamsters throughout the entire p.c. period (Fig. 4⇑B, normal and vaccinated bars), but it decreased precipitously with time in all of the other control groups. In SLD-specific restimulation assays, the lymphoproliferative response was negative for all groups on prevaccination day 0 (Fig. 4⇑C) and for the nonvaccinated control groups throughout the p.c. period (normal, infected, and vector). Cells from ppg DNA-vaccinated hamsters produced a significantly higher response (p < 0.001), which reached almost to the maximum on day 45 after vaccination and increased further thereafter (Fig. 4⇑C).

ppg DNA vaccination alters Leishmania-specific IgG and its isotypes

The serum levels of leishmanial Ag-specific IgG and its isotypes (IgG1 and IgG2) from all of the groups were assessed by ELISA. The anti-Leishmania IgG and IgG1 were elevated progressively with time to a high level in all groups, except the ppg DNA vaccinated, in which case they remained essentially the background levels of the nonimmunized and unchallenged normal and blank vector immunized (Fig. 4⇑, D and E). In contrast, ppg DNA-vaccinated animals were the only group (Fig. 3⇑F) that showed a significant elevation by 1- to 2-fold over the others (p < 0.01) in the level of IgG2. As a measure of CMI, the elevation of IgG2 was consistent with the development of effective immune responses.

ppg DNA immunization induces NO production in vaccinated hamsters

Lymphocyte-mediated activation of macrophages to produce NO for leishmanicidal activities was found to differ between control and experimental groups of hamsters. Supernatants from stimulated lymphocytes of hamsters vaccinated with the ppg DNA when incubated with naive macrophages produced significant (p < 0.001) amounts of nitrite (19.5 ± 1.94) which was ∼4-fold more than that of unvaccinated infected controls and ∼2.3-fold more than the vector control group on day 45 p.c. Furthermore, the level of nitrite was increased incredibly on days 60 (21.5 ± 2.63), 120, and 180 p.c. (24.13 ± 3.17). Comparatively, a very low amount of nitrite was produced by unvaccinated (5.4 ± 1.3) and vector (5.827 ± 0.6378) control groups on day 45 p.c. (Fig. 5⇓A). Similarly, LPS (100 μg/ml, used as positive control)-stimulated cells from the vaccinated and normal control groups demonstrated good and significant (p < 0.001) nitrite production until day 180 p.c. while infected control and vaccinated with vector alone groups showed less nitrite production until they survived (Fig. 5⇓B).

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FIGURE 5.

NO production (μM) to LPS (A) and SLD (B) in the naive macrophages coincubated with supernatants of lymphocytes isolated from ppg DNA-immunized hamsters in comparison to the unimmunized infected controls, vector-immunized controls, and uninfected normal hamsters on days 0 45, 60, 120, and 180 p.c. Significance values indicate the difference between the vaccinated groups and infected group (∗∗, p < 0.01 and ∗∗∗, p < 0.001).

ppg DNA vaccination generates Th1-type cytokine profile as determined by quantitative real-time PCR

Impairment of the CMI response during active VL is reflected by marked T cell anergy-specific to Leishmania Ags (44, 45). Since optimum protective efficacy was observed in ppg DNA-vaccinated hamsters, the expression of Th1 and Th2 mRNA cytokines was further evaluated by real-time PCR on days 45 and 60 p.c. The expression of iNOS transcripts was observed to be significantly (p < 0.01 and p < 0.05, respectively, on days 45 and 60) elevated in ppg DNA-immunized hamsters (Fig. 6⇓) at both time points of the study. Similarly, the expression of TNF-α was also significantly higher on day 45 (p < 0.001) and day 60 (p < 0.05) in the vaccinated group (ΔC_T = 1.5 ± 0.4 and 2.3 ± 0.3, respectively) in comparison to the L. donovani-infected group (ΔC_T = 7.7 ± 0.8 and 5.6 ± 2.5, respectively, on days 45 and 60). The expression of IFN-γ, although variable at different time points, was suppressed in the infected group (ΔC_T = 6 ± 0.8, day 45 p.c.), but was significantly higher in the vaccinated group on day 45(p < 0.001) and day 60 (p < 0.05) p.c. and was at par with the normal ones. Similar was the case with IL-12 which was least expressed in the infected group on day 45 p.c. but was significantly (p < 0.01) expressed by 3.5- to 4.0-fold in vaccinated hamsters on days 45 and 60 p.c. On the other hand, the expression of Th2 type cytokines, i.e., TGF-β, IL-4, and IL-10, was significantly up-regulated (p < 0.05 to p < 0.01) in the infected groups compared with the vaccinated hamsters (Fig. 6⇓).

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FIGURE 6.

Splenic iNOS and cytokine mRNA expression profile analysis of normal, infected, and vaccinated hamsters on days 45 and 60 p.c. by quantitative real-time RT-PCR. Significance values indicate the difference between ΔC_T values of infected to normal and vaccinated groups (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001).