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. 2005 Apr;140(1):149–156. doi: 10.1111/j.1365-2249.2005.02745.x

Immunostimulatory cellular responses of cured Leishmania-infected patients and hamsters against the integral membrane proteins and non-membranous soluble proteins of a recent clinical isolate of Leishmania donovani

R Garg *, S K Gupta *, P Tripathi †, S Naik †, S Sundar ‡, A Dube *
PMCID: PMC1809348  PMID: 15762886

Abstract

Development of an effective immunoprophylactic agent for visceral leishmaniasis (VL) has become imperative due to the increasing number of cases of drug resistance and relapse. Live and killed whole parasites as well as fractionated and recombinant preparations have been evaluated for vaccine potential. However, a successful vaccine against the disease has been elusive. Because protective immunity in human and experimental leishmaniasis is predominantly of the Th1 type, immunogens with Th1 stimulatory potential would make good vaccine candidates. In the present study, the integral membrane proteins (IMPs) and non-membranous soluble proteins (NSPs), purified from promastigotes of a recent field isolate, Leishmania donovani stain 2001, were evaluated for their ability to induce cellular responses in cured patients (n = 9), endemic controls (n = 5) of visceral leishmaniasis (VL) and treated hamsters (n = 10). IMPs and NSPs induced significant proliferative responses (SI 6·3 ± 4·1 and 5·6 ± 2·3, respectively; P < 0·01) and IFN-γ production (356·3 ± 213·4 and 294·29 ± 107·6 pg/ml, respectively) in lymphocytes isolated from cured VL patients. Significant lymphoproliferative responses against IMPs and NSPs were also noticed in cured Leishmania animals (SI 7·2 ± 4·7 & 6·4 ± 4·1, respectively; P < 0·01). In addition, significant NO production in response both IMPs and NSPs was also noticed in macrophages of hamsters and different cell lines (J774A-1 and THP1). These results suggest that protective, immunostimulatory molecules are present in the IMP and NSP fractions, which may be exploited for development of a subunit vaccine for VL.

Keywords: cellular immune response, LTT, IFN-γ NO production, lymphocytes, macrophages, cured patients, cured hamsters

Introduction

Visceral leishmaniasis (VL or Kala-azar) is the most devastating type among a complex of leishmaniases and is caused by the invasion of the reticuloendothelial system (spleen, liver and bone marrow) by the haemoflagellate protozoan parasite Leishmania donovani. Though the disease is generally restricted to the areas, which are heavily infested by the sandfly (Phlebotomus spp.), the vector of this disease is widely distributed throughout the tropics, and is mostly rampant in the Indian subcontinent and south-west Asia [1,2]. In India, high incidence has been reported from the states of Bihar, Assam, West Bengal and eastern Uttar Pradesh where resistance and relapse are on the increase. A recent survey in Bihar has recorded an alarming 1000 000 cases with 10 000 unresponsive to antimonials, Pentamidine and Amphotericin B [1]. The available anti-leishmanial drugs are toxic, have serious side-effects and are associated with numerous relapses and there is an increasing incidence of drug resistance [1]. In the absence of suitable anti-leishmanial drugs, an alternative choice for the control of this disease is immunoprophylaxis.

The search for parasite antigens able to induce an immune response has been predominantly associated with the identification of proteins that may be used for vaccine development. Most studies aimed at identifying antigens from Leishmania spp. have searched for molecules with the ability to stimulate IL-2, IFN-γ and IL-12 [3–5], once the Th1 type immune response was known to be the major defense mechanism against Leishmania infection [6]. In fact, IFN-γ is the main cytokine implicated in the activation of macrophages for the killing of Leishmania[7]. The absence of a type 1 immune response to Leishmania antigen is documented in patients with visceral leishmaniasis disease characterized by parasite multiplication and dissemination [8,9].

Past studies indicated that leishmanial promastigote cell surface molecules are critical for recognition and establishment of infection in the mammalian host [10] and, therefore, offer the possibility for their use as potent immunizing agents against leishmanial infection. McConville et al. [11] and Murray et al. [10] have characterized the integral membrane proteins (IMPs) of L. major and successfully used them for vaccination against L. major in a mouse model. IMPs of L. donovani have also been characterized by Etges et al. [12] and Heath et al. [13] and their ability to provide protection against Leishmania challenge [14] has been investigated. Although a few studies have also focused on the protective potential of non-membranous or soluble fractions of Leishmania parasite against cutaneous disease [15–17], no significant investigations have been carried out against VL; however, attempts have been made to characterize L. donovani antigens from promastigote, discussing their possible use in serodiagnosis and immunoprophylaxis [18–22].

Further, it has been reported that some T cell epitopes that are protective in the murine host do not elicit an immune response in human [23], emphasizing the importance of evaluating leishmanial antigens for their cellular immune responses in humans. Since the prophylactic efficacy of IMPs has been evaluated in hamsters [14], we thought it would be worthwhile to assess the ability of this membranous antigen as well as the non-membranous soluble antigen to stimulate Th1 cellular responses in the macrophages and lymphocytes of both Leishmania infected and cured patients, as well as hamsters.

Materials and methods

Host

Laboratory bred male golden hamsters (Mesocricetus auratus, 45–50 g) from Central Drug Research Institute (CDRI) animal house facility were used as the experimental host. They were housed in climatically controlled rooms and fed with standard rodent food pellet (Lipton India Ltd) and water ad libitum.

Parasites

Isolation and cultivation of recent clinical isolates of L. donovani

L. donovani strain (2001) a recent field isolate was procured from a patient admitted to the Kala Azar Medical Research Centre of the Institute of Medical Sciences, BHU, Varanasi and was put into culture. The splenic aspirations of the patient was checked for the presence of LD bodies and graded as per standard criteria [24]. The patient was later cured with an antileishmanial drug sodium stibogluconate (SSG). The strain was also found sensitive to SSG in our experimental conditions (personal communication).

The biopsy material was cultivated initially at 26°C in triple NNN-agar tubes and subsequently promastigotes were passaged in HEPES-buffered (pH 7·4) Medium 199 (Sigma, USA) with 10–20% heat inactivated fetal bovine serum (HIFBS) at 25°C in 25 cm2 tissue culture flasks. The strain since then has been maintained in hamsters through serial passage, i.e. from amastigote to amastigote. For bulk cultivation promastigotes were grown in L-15 medium (Sigma) with l-glutamine, supplemented with 10% Tryptose phosphate broth (Himedia, India), 0·1% gentamycin and 10% fetal calf serum (Gibco, USA). Parasites were harvested after 3–4 days of culture [25].

Macrophage cultures

Peritoneal macrophages of hamster, mouse macrophage adherent cell line J774A.1 and human suspension cell line THP1 were maintained in RPMI-1640 medium (Gibco-Invitrogen, Grand Island, NJ, USA) supplemented with 10% FBS at 37°C in humidified atmosphere with 5% CO2.

Isolation of antigens from L. donovani promastigotes

Integral membrane proteins (IMP)

Triton X-114 solubilization and temperature-dependent phase separation of parasite antigens were performed essentially as described by Bordier [26], modified by [14,27]. Briefly, Triton X-114 was precondensed in phosphate-buffered saline (PBS; 137 mm NaCl/2·7 mm KCl/8·1 mm sodium phosphate, pH 7·2). In vitro cultured and PBS washed stationary phase promastigotes were subjected to the process. Promastigotes (1010) were solublized in 80 ml of 0·5% Tx-114 in PBS along with cocktail of Protease inhibitors and sonicated (Soniprep-150) at medium amplitude (10 KHz × 5 cycle, one minute each). Thus obtained promastigote suspension was kept on ice for 90 min with mild mixing at 10-min intervals. The suspension was then centrifuged at 10 000 × g for 15 min at 4°C to remove insoluble material. The supernatant was collected, and the centrifugation step was repeated. The insoluble pellet material was washed three times in 0·5% Triton X-114 and then frozen. The remaining detergent-soluble material was carefully layered over a cold sucrose cushion (6% sucrose and 0·06% Triton X-114) in a 50-ml tube (15 ml suspension on 10 ml sucrose cushion) with minimal disruption to the interface and placed in a 37°C incubator for 5 min. The tube was then transferred to a centrifuge in the warm room and spun at 500 g for 5 min. After centrifugation, the detergent-depleted upper layer was collected and chilled on ice. The sucrose cushion was discarded, and the detergent-enriched pellet (1–2 ml) was resuspended on ice with 10 ml of cold PBS. The resuspended detergent-enriched phase was again layered over a sucrose cushion, brought to 37°C for 5 min and repelleted by centrifugation. After this second precipitation, the detergent-enriched pellet, containing the putative integral membrane proteins, was resuspended to 5 ml in PBS and snap-frozen. After assessing protein contents [28] of integral membrane proteins were stored at −70°C until further use. To remove detergent, IMPs were precipitated with approximately 9 volumes of ice-cold methanol (−20°C) and left overnight at −20°C. Proteins were recovered by centrifugation at 10 000 r.p.m. for 30 min at −10°C.

Non-membranous soluble proteins (NSP)

NSP was prepared as per method described by Scott et al. [15] and modified by Choudhury et al. [17]. Briefly, promastigotes (109) were harvested from culture and washed four times in cold phosphate-buffered saline (PBS) and resuspended in PBS containing protease inhibitors cocktail (Sigma). The suspension was incubated for 10 min on ice and sonicated for 10 periods of 30 s. (separated by an interval of 1 min) at medium amplitude. The sonicate was left at 4°C for complete extraction of soluble antigen for 18 h. After incubation sonicated suspension was centrifuged at 4000 g for 30 min at 4°C. The supernatant so obtained was finally ultra centrifuged at 40 000 g for half an hour. After assessing the protein content of the supernatant, it was distributed in small aliquots and stored at −70°C.

Donors and isolation of mononuclear cells

The study groups were as follows:

  • Nine treated cured patients from hyper-endemic areas of Bihar who had recovered from VL (males 5, age range 5–30 years). Diagnosis was established in all cases by demonstration of parasites in splenic aspirates. All patients had received full course of either amphotericin B (n = 5) or miltefosine (n = 3) or Paromomycin (n = 1). Samples were collected six months after completion of treatment in four patients (splenic aspirate negative for parasites at time of study) and one month in the other five;

  • Five endemic household contacts (males 3, age range 6–33 years);

  • Ten normal healthy donors without a history of leishmaniasis from nonendemic areas (males 7, age range 20–30 years).

The study was approved by the Ethics committee of the Kala-azar Medical Research Centre, Muzaffarpur and CDRI.

Heparinized blood was collected from donors who had suffered from kala azar. Blood suspensions were loaded on Ficoll Hypaque density gradient centrifugation (Histopaque 1077) [30]. Interfaces containing mononuclear cells (PBMCs) were isolated and washed thrice with RPMI and a final suspension of 5 × 106cells/ml was made in complete RPMI medium (HEPES-buffered RPMI-1640 supplemented with streptomycin (100 µg/ml), Penicillin (1000 U/ml), l-glutamine (2 mm/ml), β-mercaptoethanol (5 × 10−5 M/ml) and 10% heat inactivated FCS after determining cell viability by trypan blue dye staining method.

Treatment of L. donovani infected hamsters with antileishmanial drug and isolation of mononuclear cells

Hamsters infected with 2001 strain were treated intraperitoneally with Sodium Stibogluconate (SSG), at 40 mg/kg body weight once a day for five days. The treated animals were rechecked for parasitic burden, if any, by splenic biopsy after one-month post treatment.

Lymph nodes (inguinal and mesenteric) from individual infected-treated as well as normal hamsters were removed aseptically and placed in a sterile Petri dish containing incomplete RPMI medium. After 3–4 washing with plain RPMI, lymph nodes were teased and crushed with sterile needles in sterile Petri dishes. The suspensions so obtained were transferred into the sterile centrifuge tubes and centrifuged at 49 g for 5 min. Supernatants were re-centrifuged in another tube at 778 g for 10 min to spin down the lymphocytes. The cells were washed thrice with RPMI and a final suspension of 1 × 106cells/ml was made in complete RPMI medium as mentioned above.

Immunological assays

Lymphocyte transformation test

100 µl of mononuclear cell-suspension of both patients (5 × 106/ml) and hamsters (1 × 106/ml) was cultured in 96 well flat bottom tissue culture plates (Nunc, Denmark). 100 µl of mitogens (PHA (10 µg/ml) Con A (10 µg/ml); Sigma) or antigen (IMP and NSP (10 µg/ml each)) were added to triplicate wells. Wells without stimulants served as controls. Cultures were incubated at 37°C in a C02 incubator with 5% C02 for 3 days in the case of the mitogens, and for 5 days in the case of the antigens. Eighteen hours prior to termination of culture, 0·5 µCi of 3H-thymidine (BARC, India) was added to each well. The cells were harvested on to glass fibre mats (Whatman) and counted in a liquid scintillation counter; results were expressed as mean counts of triplicate wells per minute. Results were expressed as stimulation Index (SI) which was calculated as mean cpm of stimulated culture/mean cpm of unstimulated control. SI values of unstimulated control group were compared with the values of stimulated (with antigen) groups and SI of more than 2·5 was considered as positive response.

Assessment of IFN-γ level

The PBMCs culture (isolated from human patients) was set up in 96 well culture plates and 5 × 104cells/well were dispensed and antigens/mitogen were added to triplicate wells. At the end of 24 h, supernatant was harvested carefully from each well for cytokine determination. IFN-γ in the supernatants was determined using an OptEIA set enzyme-linked immunosorbent assay kit (Pharmingen, San Diego, California) as recommended by the manufacturer. The assays were calibrated to detect IFN-γ within the range of 4·7–300 pg/ml.

Assessment of NO activity in macrophages

The presence of NO in culture supernatant of macrophages of hamsters and macrophage cell lines (J774A-1, THP1) was determined by Griess reagent [29]. Briefly, 1 × 106 peritoneal macrophage of cured hamsters as well as of cell lines was incubated with isolated antigens (IMP & NSP) in microtitre plate at 10 µg/ml conc. Supernatants was collected after 24 h 100 µl of sodium nitrite standards or macrophages culture supernatant samples were seeded in each well (in triplicates) of a 96 well microtitre plate except for the blank well in which only 100 µl of complete medium was placed. Subsequently 100 µl of freshly prepared Griess reagent (Sigma) containing 1 : 1 v/v mixture of 0·1% of N-1-napthyl-ethylenediamine in water and 1% of Sulfanilamide in 5% phosphoric acid were added to each well and the plate was incubated at room temperature for 10 min. The intensity of the colour developed was read at 550 nm in an ELISA reader with the blank well set as zero. The nitrite concentration in the macrophages culture supernatant samples was extrapolated from the standard curve plotted with sodium nitrite.

Prophylactic efficacy of IMP and NSP in vitro using GFP transfected parasite

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been introduced as a convenient reporter in many applications in eukaryotic organisms. This protein is intrinsically fluorescent, has a low toxicity, and allows easy imaging and quantification using fluorescence-activated cell sorting (FACS) or microscopy. In this study, we have utilized transgenic Leishmania donovani promastigotes [30], which constitutively express GFP in their cytoplasm, as target cells in macrophages for the evaluation of antigens in vitro.

Isolated macrophages from cured hamster were seeded in microtitre plates and were incubated with IMP and NSP (10 µg/ml) for 24 h at 37°C in CO2 incubator. After 24 h, 2 × 106 per ml (medium RPMI-1640 supplemented with 20% FCS) of GFP transfected Leishmania promastigotes from late log phase were inoculated into the macrophage cultures. After incubation for 24 h at 37°C, infected and noninfected macrophages were detached, washed and resuspended in 1 ml of PBS. The counting of infected and noninfected macrophages as well as fluorescent parasites was done by flow cytometry (excitation at 488 nm and emission at 520 nm).

Statistical analysis

Results were expressed as mean ± SE. In each experiment 6–8 animals were used in each group. Three sets of experiments were performed and the results were analysed by One-Way anova test using Sigma stat (version 2·0) software program. The upper level of significance was chosen as P < 0·001 (Highly significant).

Results

Evaluation of immune response in cured patients

Lymphoproliferative response to mitogens/antigens

The lymphocytes of nonendemic control, endemic control and cured patients were assessed for their proliferative responses (LTT) against the mitogen and leishmanial antigens at a predetermined concentration of 10 µg/ml. Endemic control and cured patients showed relatively higher SI index, i.e. 42·33 ± 13·4 and 46·15 ± 15·6 against the mitogens PHA (Fig. 1). No proliferative responses were observed either against IMPs and NSPs in the cells from nonendemic control group. However, significant proliferative response was observed against IMPs (5·8 ± 2·7 & 6·3 ± 3·4) and NSPs (4·6 ± 2·1 & 5·6 ± 2·3) in both the groups, i.e. Endemic control and cured patients (Fig. 1)

Fig. 1.

Fig. 1

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Proliferation of peripheral blood mononuclear cells (PBMC) from individuals of normal, cured VL patients and endemic controls in response to PHA and antigen (IMPs and NSPs) stimulation.

Level of IFN-γ in response to antigens

The supernatants of PHA, IMP and NSP stimulated PBMCs from nonendemic control, endemic control and cured patients, were assayed for IFN-γ concentration. IMP and NSP stimulated PBMCs showed low IFN-γ levels (47·0 ± 25·7 & 35·2 ± 14·3 pg/ml) in nonendemic controls groups, whereas endemic control and cured patients showed significantly (P < 0·01) good amounts of IFN-γ levels against IMP (315·24 ± 185·67 & 356·3 ± 213·4, respectively) and NSP (255·6 ± 94·7 & 294·29 ± 107·6, respectively) (Fig. 2).

Fig. 2.

Fig. 2

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IFN-γ production by peripheral blood mononuclear cells (PBMC) from individuals of normal, cured VL patients and endemic controls in response to antigen (IMPs and NSPs) stimulation.

Evaluation of immune response in cured hamsters

Lymphocyte transformation test

The proliferative responses of lymph node cells of hamsters of normal, L. donovani infected and SSG treated groups were assessed by lymphocyte transformation test (LTT) against the above stated antigens at a predetermined concentration of 10 µg/ml. Animals of normal group showed relatively higher SI index, i.e. 30·5 ± 11·6 against the mitogen Con-A. No proliferative responses were observed either against IMPs and NSPs or against mitogens in the cells from L. donovani infected group. However, considerably good proliferative response was observed against Con-A (27·8 ± 8·7) as well as IMPs (7·2 ± 4·7) and NSPs (6·4 ± 4·1) in the lymphocytes from SSG treated animals (Fig. 3)

Fig. 3.

Fig. 3

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Proliferation of mononuclear cells of lymph nodes from normal, and L. donovani infected but treated hamsters in response to PHA and antigen (IMPs and NSPs) stimulation.

Nitrite production in peritoneal macrophages of hamsters and different macrophage cell lines (J774A-1, THP1)

A significant NO production was observed in peritoneal macrophages of hamsters (17·42 ± 4·2 & 15·74 ± 3·2 µg/ml) and J774 (11·65 ± 5·2 & 10·25 ± 3·7 µg/ml), THP1 (12·45 ± 4·8 & 11·32 ± 3·6 µg/ml) cell lines after 24 h of incubation with IMP and NSP (P < 0·01) in comparison to unstimulated control (Fig. 4).

Fig. 4.

Fig. 4

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Nitric oxide production by macrophages of hamsters, J774 and THP-1 cell lines in response to LPS and antigen (IMPs and NSPs) stimulation.

Infectivity of L. donovani in different macrophages primed with IMPs and NSPs

GFP transfected promastigotes of isolates 2001 when inoculated into the macrophage cultures infect 70–80% macrophages 24 hours later with each macrophage harbouring on an average 4–8 amastigotes when observed under the fluorescent microscope. The flow cytrometric analysis revealed the significant (P < 0·01) prophylactic efficacies of IMP and NSP against L. donovani in peritoneal macrophages of hamster (7·53 ± 3·2 & 11·2 ± 5·4 Arbitory Florescence Unit (AFU), respectively) and J774A-1 (9·2 ± 3·6 & 13·5 ± 6·2 AFU, respectively), THP1 (8·42 ± 3·8 & 12·6 ± 5·7 AFU) cell lines (Fig. 5). The AFU value in unstimulated infected control of peritoneal macrophages of hamsters J774A-1 and THP1 was 54·2 ± 15·3, 45·6 ± 12·4 & 48·5 ± 13·4.

Fig. 5.

Fig. 5

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Assessment of infectivity of macrophages from cured hamsters, preincubated with IMPs and NSPs, with GFP transfected L. donovani promastigotes by flow cytometry.

Discussion

In human and experimental leishmaniasis immunity is predominantly mediated by T lymphocyte [31]. T lymphocytes participate in the immune response to L. donovani infection by producing different cytokines. Th1 and Th2 cells can be distinguished by the cytokines they secrete: Th1 cells secrete activators of cell-mediated immunity such as interferon IFN-γ, while Th2 cells secrete cytokines such as interleukin IL-4, which promote antibody responses. Further, IFN-γ induces production of nitric oxide (NO) in phagocytic cells that harbour Leishmania parasite (principally macrophages), which leads to destruction of parasite.

Considering the invasion strategy of the Leishmania parasite the molecules present on the cell surface of promastigotes are of paramount importance as the parasite membrane antigens are the interface between the parasite and its vertebrate and insect hosts. These are the proteins that first come in the contact of host cell. Their topography and receptors interactions with the host cell decide the acceptability, survival, multiplication, pathogenesis or killing of the parasite in the host [32]. Vice-versa the membrane proteins would be important for designing the vaccines also as these vaccines would check the entry of parasite by inactivating the parasitic invasion tools and also by triggering the cell mediated immune response. Non-membranous antigen on the other hand is a crude preparation of L. donovani soluble antigens of parasite, it contains a mixture of different antigens, and therefore it could stimulate different clones of memory T cells. Although there has been considerable interest in antigens stimulating immune responses to other Leishmania species, for example L. major[33–35] and L. amazonensis[36], few investigators have explored the potential of L. donovani antigens. Those studied include 80-, 72-, 70- and 63-kDa antigens [13,37,38] of which the former two confer partial protection upon immunization of mice [37,38]. Further, the integral membrane proteins (IMPs) have already shown their worth against L. major infection in mice [10,11] and L. donovani in hamsters [14]. The Triton X-114 phase separation procedure for IMPs had been applied successfully to Plasmodium falciparum[27], Schistosoma japonicum[39], Trypanosoma brucei gambiense[40] and L. donovani[13]. In this study, the nonionic detergent Triton X-114 by the phase separation method of Bordier [26] later modified by Smyth et al. [27] had been used to separate IMPs of stationary phase promastigote of recent field isolate of L. donovani. These IMPs are characterized by a hydrophobic domain, which interacts directly with hydrophobic core of lipid bilayer [41].

Characterization of the cellular immune response was performed in endemic non immune donors (household contacts without any clinical symptoms)) and in immune patients of VL that were cured either with amphotericin B or miltefosine, since it is known that a T cell response develops when cells from these individuals are stimulated with soluble Leishmania antigen. Some of the recombinant antigens have been previously shown to induce lymphocyte proliferation and IFN-γ production in subjects cured of visceral and in patients with cutaneous or mucosal leishmaniasis [3,5]. Although lymphocyte proliferation has been widely used to analyse T cell function, the documentation that the CD4 + population is heterogeneous regarding to the cytokine profile secreted, indicates that cytokines should be measured to determine if an immune response could be protective or deleterious. In the present study, while the IMPs and NSPs antigens of L. donovani induce strong T cell proliferation in subjects with endemic control and cured patients of VL, the IFN-γ production mediated by these antigens was significantly higher than that observed in healthy controls. Of the antigens tested the IMPs was the one that induced the highest lymphocyte proliferation and IFN-γ production. These antigens did not induce proliferation or IFN-γ production in healthy subjects evaluated. This type of binding leads to the first step of antigen selection in experimental models and in human being, in order to evaluate ability to induce protection against Leishmania infection.

The existence of human analogue to the rodent Th1 and Th2 subsets has been disputed. The reason for the reservation in accepting human Th1 and Th2 is probably that the dichotomy in the human system is not as clear as in rodent particularly murine cells. Besides, the infection pattern also does not simulate the human profile, as it is self-limiting in murine VL. On the other hand, systemic infection of the hamster with L. donovani results in a relentlessly increasing visceral parasite burden, progressive cachexia, hepato-splenomegaly, pancytopenia, hypergammaglobulinaemia, and, ultimately, death. These clinicopathological features closely mimic active human VL [42]. Hence, analysis of cellular immune response of the two antigens was carried out using hamsters’ lymphocytes as well as macrophages that have been cured with SSG in order to correlate the observations made with the human lymphocytes.

In the case of protozoal infections, macrophages become activated by IFN-γ derived from parasite-specific T cells, and are able to destroy intracellular parasites through the production of several mediators, principal among which is NO [43–45]. It is well documented that IFN-γ induces production of nitric oxide (NO) in phagocytic cells that harbour Leishmania parasite (principally macrophages), which leads to destruction of parasite. It has been shown that killing of L. major, an intracellular parasite living in macrophages, depends on NO production [46]. In the absence of cytokine reagents against hamsters, in this study we have observed the effect of IMPs’ and NSPs’ on NO production by peritoneal macrophages of hamster. The effect of the two antigens on NO production was also validated in J774A-1 and THP1 cell lines.

From the above studies it has been inferred from the analysis of LTT, NO estimation and IFN-γ of cured patients/cured hamsters that (i) besides IMPs, NSPs has also the potential to induce immune response against the L. donovani infection, though; the efficacy was less marked as compared to that of IMPs’. (ii) The cellular responses to IMPs and NSPs were similar in endemic controls and in cured patients of VL as well as in hamsters indicating that the results so obtained with the hamster could be translated into humans. The identification of antigens that elicit human T cell responses is an important step towards understanding the immunology of L. donovani infection and ultimately in the development of a vaccine.

Acknowledgments

The technical assistance by Mr S. C. Bhar is gratefully acknowledged. We gratefully acknowledged Department of Biotechnology, New Delhi and Indian Council of Medical Research, New Delhi for financial support to this work as well as for senior research fellowship to Mr Ravendra Garg.

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