Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response

Abstract

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8⁺ cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-γ (mean ± s.e.m.: 1398 ± 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.

Introduction

Infection with parasites of the genus Leishmania causes a spectrum of clinical disease, including cutaneous, mucocutaneous and visceral leishmaniasis. These diseases affect approximately 12 million people world wide with 1·5–2 million new cases occurring each year [1]. Currently available drugs are either toxic, have limited efficacy or both and emerging drug resistance is a significant concern [2]. Given these circumstances, development of an effective vaccine against leishmaniasis is a priority of tropical disease research.

In recent years, there has been substantial progress in understanding the immunopathogenesis of cutaneous leishmaniasis. It has been shown that acquired resistance to L. major is dependent upon the preferential induction and development of Th1-type responses [3]. These responses include the production of interferon (IFN)-γ, which mediates protection by activation of macrophages for microbicidal activity [4]. In contrast, susceptibility to cutaneous leishmaniasis (CL) is related to the development of Th2 responses leading to production of type 2 cytokines [e.g. interleukin (IL)-4 and IL 5] and down-regulation of Th1-driven effector mechanisms [5–7]. These findings provide a basis for screening for potential vaccine candidates and two basic approaches have been used to identify such reagents. One involves immunization-challenge experiments in mouse models and the second examines the candidate immunogen*s capacity to elicit Th1-like response from human peripheral blood mononuclear cells (PBMC) from individuals recovered from leishmania infection. Specifically, elicitation of IFN-γ and IL-12 is considered to be predictive of potential vaccine efficacy.

Several leishmania proteins, either native or recombinant, have been evaluated as leishmania vaccine candidates including: GP63 [8], GP46/M2 [9], LeIF [10], PSA2 [11], LmST11 [12] and TSA [13]. Although some of these candidate antigens are also expressed in amastigotes, most of them are expressed preferentially in the promastigote stage of the parasite. Given that disease is related directly and exclusively to proliferation and persistence of amastigotes in macrophages of the vertebrate host, molecules that are up-regulated or selectively expressed in the amastigotes have the potential to be superior vaccine candidates. Recently, we isolated and characterized a class I nuclease gene from amastigotes of L. major[14]. The sequence of corresponding protein showed high sequence homology to the P4 nuclease of L. pifanoi[15] and the 3′-nucleotidase/nuclease enzymes previously characterized from different trypanosomatids [16–21]. It has been shown previously that the P4 nuclease of L. pifanoi – a causative agent of New World cutaneous leishmaniasis − is also an amastigote specific protein able to induce a protective immune response in a murine model. This protection was associated with a Th1 type response with high levels of IFN-γ and low levels of IL-4 [22]. More recently, it was shown that the P4 nuclease of L. amazonensis administered as a DNA vaccine protected mice against L. amazonensis[23]. Because of these promising results with native P4 protein and DNA against a New World leishmania species, we investigated the immune responses to rLmaCIN in patients recovered from Old World cutaneous leishmaniasis. The results show that this protein elicits strong Th1-like responses characterized by high levels of IFN-γ, low levels of IL-10 and little or negligible amount of IL-5 production. These findings provide a basis for further study of this recombinant protein as a candidate vaccine.

Materials and methods

Subjects

Twenty recovered subjects − with a history of cutaneous leishmaniasis in the past 3 years − and 10 healthy non-exposed controls were sources of PBMCs. Recovered individuals were from rural areas south-east of the city of Kashan, Iran, which is an endemic area for cutaneous leishmaniasis caused by L. major. Healthy controls were from a non-endemic area. After obtaining informed consent from all subjects, they were examined by leishmanin skin test as an indication of exposure to leishmania [24], and heparinized blood was collected, according to the acceptance and regulation of ethical committee in Pasteur Institute of Iran.

Expression and purification of recombinant LmaCIN

The cloning and expression of recombinant LmaCIN in Escherichia coli has been described in detail elsewhere [14]. Briefly, the coding region for LmaCIN was excised by digestion of the BSc-LmaCIN plasmid with Nde I/Xho I and subcloned into the expression vector pET 22b (Novagen, Madison, WI, USA). E. coli, BL21(DE3) transformed with this construct was grown with vigorous shaking at 37°C in 500 ml LB media supplemented with 100 µg/ml ampicillin to an optical density of 0·4 at 600 nm. Protein expression was then induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0·5 mm and cells were incubated for an additional 4 h. Cells were harvested by centrifugation (10000 g for 15 min) and the pellet was washed twice with phosphate buffered saline (PBS) containing 1% Triton X-100. For purification of His-tagged protein, the pellet was resuspended in 20 ml of lysis buffer (20 mm Tris, 100 mm NaCl, pH 8) and disrupted by sonication. Inclusion bodies were obtained by centrifugation, dissolved in buffer B (20 mm Tris, 500 mm NaCl, 8 m urea, pH 8) and then applied directly onto an affinity column containing Ni-NTA resin (Qiagen, Chatsworth, CA, USA) pre-equilibrated with buffer B. The column washed twice with 20 ml of buffer B containing 20 and 50 mm of imidazole and then bound proteins were eluted with buffer B containing 150 mm imidazole. The eluate was dialysed against PBS buffer overnight at 4°C and purity was examined by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). Purified recombinant protein was tested for the presence of endotoxin using a chromogenic Limulus amebocyte lysate assay according to the manufacturer*s instructions (BioWhittaker, Walkersville, MD, USA).

Lymphocyte proliferation assay

PBMC were separated from heparinized blood by density gradient centrifugation over Histopaque-1077 (Sigma Aldrich, St. Louis, MO, USA). Mononuclear cells were resuspended in RPMI-1640 (Sigma) supplemented with 10% heat-inactivated fetal calf serum, 20 mm l-glutamine and 50 µg/ml gentamicin. Cells (4 × 10⁵) were distributed in triplicate in 96-well round bottomed microtitre plates ((Maxisorb, Nunc A/S, Roskilde, Denmark) in a final volume of 200 µl, in the presence of either rLmaCIN (5 µg/ml), ALM (25 µg/ml, a candidate whole cell vaccine against leishmaniasis, Razi Vaccine and Serum Research Institute, Hesarak, Iran) or phytohaemagglutinin P (PHA, 15 µg/ml, Difco Laboratories, Detroit, MI, USA). Cultures were incubated for 5 days and processed for incorporation of [³H]-thymidine as described previously [25]. Stimulation indices ≥2·5 were considered positive.

Phenotypic analysis of proliferating cells

Phenotypic analysis was performed on eight patients who provided enough cells for analysis. PBMCs (1 × 10⁶ cell/ml) were cultured in 24-well plates in the presence of either rLmaCIN, ALM or without antigenic stimulation. After 5 days, cells were harvested, washed with PBS and incubated for 30 min at 4°C in the presence of 5 µl of monoclonal antibodies specific for human CD3⁺ (CD3-FITC, clone UCHT1), CD4⁺ (CD4-RPE, clone MT310) or CD8⁺ (CD8-RPE, clone DK25) (all from Dako, Denmark). Cells were then washed with PBS and pellets were resuspended in 250 µl of PBS and phenotype analysis was performed by FACScan (Becton Dickinson, Mountain View, CA, USA).

By forward light scatter, smaller cells with the least light scatter were gated in R1, and larger blast cells which were activated in response to antigen stimulation, scattered into the most forward region gated as R2 [26]. The proportional increase of responding cells of a particular type was calculated taking the total large cells in the R2 region into consideration using the formula previously suggested [27] as follows:

(% large cell of total × % large CD⁺ of stimulated culture) + 1/(% large cell of total × % large CD⁺ of unstimulated culture) + 1

Cytokine assay

To examine cytokine secretion, PBMCs (1 × 10⁶/ml) were cultured in 24-well plates in the presence of either rLmaCIN, ALM or without antigen as mentioned above. Supernatants from each well were collected for cytokine assay and stored at −70°C until used.

The concentrations of IFN-γ, IL-5 and IL-10 in cell-free culture supernatants were determined by standard sandwich enzyme-linked immunosorbent assay (ELISA). In brief, 96-well microtitre plates (Nunc, Maxisorb) were coated with unconjugated capture antibody (BD Pharmingen, San Diego, CA, USA) specific for the above cytokines diluted in 0·1 m carbonate buffer (pH 9·5) and blocked with PBS containing 0·05% Tween 20 and 10% fetal bovine serum (FBS). Cell supernatants and standards (BD Pharmingen) were incubated in PBS containing 10% FBS for 2 h at room temperature. Bound cytokines were detected using biotinylated mouse antihuman cytokine antibodies in PBS containing 10% FBS for 1 h. Plates were developed using HRP-conjugated streptavidin with tetramethylbenzidine (Sigma) as a substrate. Absorbances at 450 nm were read using a microtitre plate reader and the concentrations of cytokines were calculated from standard curves. Detection limits were 15 pg/ml for IFN-γ, 10 pg/ml for IL-10 and 7·5 pg/ml for IL-5.

Statistical analysis

One-way anova and Student*s t-test were used to analyse variances and to compare mean percentage of cells (CD4/CD8) and mean amount of cytokines between groups, respectively. Pearson correlation was used to evaluate the relationship between responses to different antigens.

Results

Preparation of recombinant LmaCIN protein

Expression of recombinant LmaCIN induced by IPTG resulted in high-level expression of the C-terminal His-tagged protein with a molecular mass of 35 kDa. Purification of recombinant LmaCIN, performed by affinity chromatography on Ni-NTA resin (Qiagen) resulted in highly pure protein as analysed by SDS-PAGE (Fig. 1). The amount of endotoxin detectable in the final dialysed material was less than 10 EU/mg of protein by LAL assay.

Fig. 1.

Expression and purification of rLmaCIN. E. coli (BL21/DE3) transformed with the expression vector PET 22b harbouring LmaCIN gene was grown and induced with IPTG. Lysates of non-induced (lane 1) and induced (lane 2) cultures of E. coli and recombinant LmaCIN protein purified by Ni-NTA affinity chromatography (lane 3) were separated by SDS-PAGE and stained with Coomassie blue.

Proliferative response to rLmaCIN

PBMC from all subjects (individuals that had recovered from L. major infection and non-exposed healthy controls) proliferated in response to PHA mitogen. Eighteen of the 20 (90%) recovered cases showed proliferative responses to rLmaCIN with stimulation indices (SI) ranging from 2·8 to 14·7 (Fig. 2). In comparison, control antigen (ALM) induced proliferation in 19 of the 20 (95%) recovered subjects with stimulation indices (SI) ranging from 3 to 38. The mean ± s.e.m. SI for rLmaCIN and ALM were 6·5 ± 0·83 and 19·6 ± 3·82, respectively. Proliferative responses induced by rLmaCIN and ALM in PBMC from 10 uninfected healthy subjects were all negative (SI < 2).

Fig. 2.

Proliferative responses of PBMC from individuals with a history of cutaneous leishmaniasis (R) and healthy controls (H) to recombinant LmaCIN, in comparison to ALM and PHA. Proliferative responses were determined by incubating 2 × 10⁵ cells for 3 days with either rLmaCIN (5 µg/ml), ALM (25 µg/ml) or PHA (15 µg/ml). Cultures were pulsed with [³H]thymidine for the last 18 h. The results shown are the mean of triplicate measurements at each data point and the s.d. was less than 10% of the mean.

Phenotype of expanded cells

Both CD4⁺ and CD8⁺ cells proliferated in response to either rLmaCIN or ALM. However, the percentage of proliferated CD8⁺ cells was significantly higher in cultures stimulated by rLmacIN (mean = 16·8% ± 1·5%) than ALM-induced cultures (mean = 12% ± 1·3%) (P < 0·05, using the t-test). Conversely, the percentage of CD4⁺ responder cells were higher in ALM induced cultures (mean = 64% ± 1·5%) compared to cultures stimulated with rLmaCIN (mean = 50·2% ± 3·5%, P < 0·05). Analysis of the data to calculate the proportional increase of CD4⁺ and CD8⁺ cells demonstrated that rLmaCIN stimulated a greater proportional increase in CD8⁺ population (2·1 ± 0·1) than CD4⁺ cells (1·7 ± 0·3), whereas ALM induced a higher proportional increase in CD4⁺ cells (3·5 ± 0·6) than CD8⁺ cells (2·8 ± 0·4) (Fig. 3).

Fig. 3.

Phenotype of proliferating T cells in response to rLmaCIN (a), and ALM (b) in cultures of peripheral blood mononuclear cells from recovered cases. PBMC (1 × 10⁶ cell/ml) were cultured with recombinant LmaCIN or ALM for 5 days and stained with monoclonal antibody for CD3, CD4 and CD8 markers prior to analysis by flow cytometry. The results shown are the mean of triplicate measurements at each data point and the s.d. was less than 10% of the mean.

Cytokine analysis

Concentrations of IFN-γ, IL-5 and IL10 in culture supernatant of PBMC stimulated with rLmaCIN or ALM were determined by ELISA (Fig. 4). rLmaCIN induced high levels of IFN-γ production in PBMC cultures of all 18 recovered subjects who had shown positive proliferative responses, ranging from 250 to 3190 pg/ml (mean ± s.e.m.: 1398 ± 179 pg/ml) (Fig. 4a). ALM also induced high levels of IFN-γ in culture supernatants of PBMCs ranging from 340 to 4400 pg/ml (mean ± s.e.m.: 2660 ± 283 pg/ml).

Fig. 4.

Levels of IFN-γ (a), IL-10 (b) and IL-5 (c) in culture supernatants of PBMC from recovered individuals in response to recombinant LmaCIN and ALM antigens. Cytokine production was determined by incubating 1 × 10⁶ cell/ml with rLmaCIN (5 µg/ml) or ALM (25 µg/ml) for 5 days by ELISA. The results shown are the mean of triplicate determinations at each data point and the s.d. was less than 10% of the mean.

PBMC obtained from most (70%) of the recovered cases secreted some IL-10 in response to rLmaCIN ranging from 25 to 400 pg/ml (mean ± s.e.m. = 117 ± 18 pg/ml) (Fig. 4b), which was higher than IL-10 secreted in response to ALM ranging from 5 to 105 (mean ± s.e.m. = 37 ± 6) (P < 0·05). Conversely, the level of IL-5 induced by rLmaCIN was lower than the detection limit in most cases (Fig. 4c), whereas ALM induced the secretion of more than 40 pg/ml IL-5 in eight of 20 (40%) cases (mean ± s.e.m. = 39 ± 8) (P < 0·05).

Discussion

Recovery from cutaneous leishmaniasis is almost always associated with immunity to subsequent infection [28–30]. This immunity is based on the generation of populations of specific memory T cells that upon re-exposure to leishmania antigens rapidly expand and activate host protective effector mechanisms including a Th1 type response and IFN-γ production. Antigens that are involved in the induction and recall of such memory T cells are of great interest in terms of vaccine design strategy.

LmaCIN is a nuclease enzyme that is highly expressed in the amastigote stage of L. major. Analysis of the amino acid sequence of this protein showed 86% homology to P4 protein of L. pifanoi[14], a single-strand specific nuclease also shown to be expressed in the amastigote stage of parasite. Evaluation of immunological reponses to the L. pifanoi P4 protein as well as the L. amazonensis P4 DNA vaccine in animal models identified this stage-specific molecule as an attractive vaccine candidate for New World (American) cutaneous leishmaniasis [22,31].

In the present study, immune responses to the LmaCIN, a L. major homologue of the P4 nuclease, were characterized in patients that had recovered from Old World cutaneous leishmaniasis. To our knowledge, this is the first report focusing on the immunological evaluation of a recombinant leishmania amastigote nuclease. Data obtained in this study indicate that rLmaCIN is recognized by nearly all (90%) individuals who had recovered from L. major infection. The results are comparable to responses of the same individuals to ALM, which is an inactivated whole cell immunogen produced from L. major. This finding is in agreement with a report of Maasho et al. [34], showing that patients with a history of L. aethiopica infection recognized the P4 protein of L. pifanoi.

Analysis of the phenotypes of proliferating T cells showed that both CD4⁺ and CD8⁺ cells responded to rLmaCIN; however, the proportional increase for CD8⁺ cells was higher than for CD4⁺ cells (Fig. 3). In contrast, stimulation with ALM induced preferential expansion of CD4⁺ cells compared to CD8⁺ cells. These findings are consistent with observation of Coutinho et al. [32], where the L. pifanoi P4 protein was reported to stimulate both CD4⁺ and CD8⁺T cells to proliferate with a CD4⁺/CD8⁺ ratio of about 1 : 1 in American cutaneous leishmaniasis patients. These findings suggest that preferential activation of CD8⁺ cells is a property of the P4 protein family. A similar preference for CD8⁺ T cell expansion was observed in the case of other protective antigens, such as LACK [33], P8 [34] and all leishmania DNA vaccine preparations examined thus far. Indeed, one of the reasons for the success of DNA vaccines against intracellular pathogens has been attributed to their capacity to induce antigen-specific CD8⁺ cell populations [35–37].

Evidence from murine models indicated that CD8⁺ T cells producing IFN-γ are involved in the control of murine leishmaniasis caused by L. major[38–41]. Experiments using T cell subset depletion indicated clearly that elicitation of CD8⁺ (as well as CD4⁺) effector responses are required for protection. Further, mice lacking beta (2)-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8⁺ T effector responses [42]. In humans with active cutaneous leishmaniasis caused by L. braziliensis, it was shown that CD4⁺ cells were the predominant responding cells, whereas an increase in responding CD8⁺ T cells was observed after cure [32,43]. It has also been reported that a CD8⁺ response is required for long-lasting protection in humans [35]. Unlike some leishmania proteins such as LACK that elicited responses in non-exposed healthy donors [33], rLmaCIN did not induce the proliferation of cells from healthy subjects.

With regard to cytokine production, rLmaCIN induced high levels of IFN-γ production in all subjects with positive proliferative responses. This IFN-γ response was associated with a low level of IL-10 without any IL-5 production. In comparison, ALM induced high levels of IFN-γ production, but in most cases this was associated with IL-10 and IL-5. It has been established unequivocally that IFN-γ is a key cytokine in resistance against cutaneous leishmaniasis due to its ability to induce killing of parasites by macrophages [4]. The role of IL-10 in L. major infection is still far from completely understood. While the contribution of IL-10 in pathogenesis of visceral leishmaniasis has well been documented [44–46], there are conflicting reports on the action of IL-10 in experimental L. major infection. At least two reports showed that a Th1-type response in C57BL/6 mouse model was not apparently altered by IL-10, suggesting that IL-10 had no major influence on the Th1/Th2 balance [47,48]. In contrast, another study suggested an important role for IL-10 in suppression of a type I immune responses including IL-12-dependent production of IFN-γ[49]. Recent works also demonstrated that IL-10 is required for L. major persistence, as evidenced by sterile cure of IL-10-deficient or wild-type (C57BL/6) mice treated with anti-IL-10 receptor [50]. It has been shown that IL-10 exerts its effect by suppressing IFN-γ responses and deactivating macrophages IFN-γ-mediated intracellular killing [51]. Although in our study, low level production of IL-10 was observed in response to rLmaCIN, the simultaneous production of high levels of IFN-γ indicated that the IL-10 produced was not sufficient to suppress IFN-γ production in response to rLmaCIN.

In summary, the results of this study show that recombinant L. major amastigote nuclease has the capacity to elicit both the expansion of CD4⁺ and CD8⁺ cells and the production of IFN-γ by lymphocytes from patients immune to Old World cutaneous leishmaniasis. Given the relevance of these properties to control of disease due to L. major, further study of the LmaCIN as a vaccine candidate is warranted.