Parasitology International 64 (2015) 18–25

Contents lists available at ScienceDirect

Parasitology International journal homepage: www.elsevier.com/locate/parint

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis Pranati Das a, Ajay Amit a, Shubhankar Kumar Singh a, Rajesh Chaudhary a, Manas Ranjan Dikhit b, Anupam yadav a, Krishna Pandey c, Vidya Nand Rabi Das c, Shanty Sundram d, P. Das e, Sanjiva Bimal ⁎,a a

Division of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, 800007 India Department of Biomedical Informatics, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, 800007 India Department of Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, 800007 India d Department of Biotechnology, University of Allahabad, Allahabad, Uttar Pradesh, 211006 India e Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, 800007 India b c

a r t i c l e

i n f o

Article history: Received 22 January 2014 Received in revised form 7 August 2014 Accepted 30 August 2014 Available online 16 September 2014 Keywords: Phosphoproteins Leishmania donovani Nitric oxide Visceral leishmaniasis Interferon gamma

a b s t r a c t As phospho proteins are reported to be involved in virulence and survival, the ability of Leishmania to inhibit macrophage effector functions may result from a direct interference of leishmanial molecules with macrophage signal transduction pathways. Several such proteins such as pp63, pp41 and pp29 have also been identified as a Th1 stimulatory protein in the Leishmania donovani. In the present study, the immunogenicity of a cocktail of pp63 + pp41 + pp29 was assessed by estimation of serum antibody titre, nitric oxide(NO) production, estimation of Th1 cytokine(IFN-γ) as well as Th2 cytokines(IL-4), and determination of parasite load in L. donovani infected mice. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 which reduced considerably by Day 50. Groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10-fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes. The animal groups immunized with pp63, pp41, pp29 and the antigenic cocktail showed reduced parasite load in the liver and spleen, as well as increased IFN-gamma production in the spleen. Furthermore immunized animals remained with a normal hematological profile, whereas L. donovani in unimmunized mice lead to significant anemia. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Infection with Leishmania donovani, the causative agent of visceral leishmaniasis (VL), in humans may result in subclinical infection or progress to a fatal outcome [1]. The infection spreads when leishmanial parasites are transmitted to their vertebrate hosts by phlebotomine sandflies, which become infected while feeding on infected vertebrates and may transmit the parasite as they take a later blood meal [2]. There are clear indications that Phlebotomus argentipes is the only vector for L .donovani in India [3] but all attempts to limit VL in India via vector control have shown little, if any success. For many years, Sodium antimony gluconate (SAG) has been used as the first line treatment for VL in many countries including India [4]. Recently, the increasing likelihood that the L. donovani in Indian case of VL will be SAG resistant have led to explore alternative drugs, oral miltefosine and parental amphotericin-B as first line treatment [5–7].These drugs have been used with good results but even these drugs may not always be effective ⁎ Corresponding author at: Division of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, Pin-800007. E-mail addresses: [email protected], [email protected] (S. Bimal).

http://dx.doi.org/10.1016/j.parint.2014.08.004 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.

and prevent relapse of VL. One stumbling block in the path towards effective control of VL has been the problem of determining the immunogenic antigens in infected leishmanial parasites and thus the probable potential of leishmanial antigens in any vaccine study. On the other hand protective immunity observed in cured patients provides considerable impetus for vaccine development. Interferon-gamma(IFN-γ) is the pre-dominant cytokine in antileishmanial defense [8] however, the disease progresses under the influence of strong parasite induced immunosuppression due to over expression of, transforming growth factor β (TGF-β), interleukin (IL)-4 and IL-10 which diminishes release of free radical (super oxide and nitric oxide (iNOS) generation and IL-12 production in macrophages of the infected hosts [9]. As known, Leishmania species maintain sustained immune response polarized to either IL-12 to IL-10 ratio or the viceversa which ultimately determine their fate in macrophages of the infected hosts. Such ratio is primarily regulated by the reciprocal signalling through extracellular stress- regulated kinase (ERK) 1/2and p38 mitogen-activated protein kinase (MAPK) [10,11]. Therefore, as an alternative to chemotherapeutical intervention, the identification of major immunogenic components from Leishmania with vaccine potential that can modulate the cell- mediated response towards a protective

P. Das et al. / Parasitology International 64 (2015) 18–25

phenotype will also help to develop effective control measure [12]. Certain abundantly expressed proteins of the parasite such as LPG, gp63, LACK, gp46, ESAgs [13–15], or amastigote specific proteins like A2, P4, and P8 [16] or complete soluble antigens from attenuated parasites [17] have been identified. However the use of peptide alone as vaccine has shown to exacerbate the disease [18]. As such other vaccination protocols involving recombinant vaccine from a fusion of two Leishmania parasite proteins, Leish-F3 + GLA-SE, have been tried but these cases require to be tested in clinical trials [19]. Efforts were also made in developing genetically defined centrin knock out live attenuated Leishmania parasites as vaccine candidates with the goal of achieving a low level of parasite persistence without being virulent in the host and inducing protective immunity [20]. Vaccine protocols on KMP-11 DNA plasmid, a Leishmanial antigen and a sandfly (Lutzomia longipalpis) salivary protein, LJM19 have been formulated and was shown to induce protection in hamsters against visceral leishmaniasis [21]. Reports on haemoglobin receptor (HbR) of Leishmania which remains conserved across various strains of this parasite, and an immunization with HbR-DNA are also available which induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and hamsters [22]. In general, none of these protocols have been evaluated by measuring their ability to produce such response in primates and ultimately, human trials. It is known that Leishmania parasites overcome the protection provided by the immune system of the host by employing several strategies [23]. In particular, phosphorylation reactions were reported to participate in several ways in escape mechanisms, at different levels of the parasite-host interaction. For instance, a protein kinase isolated from L. major (LPK-1) is able to phosphorylate components of the human complement system (C3, C5 and C9) leading to its inactivation [24]. Intracellular Leishmania amastigotes, not only adapt to phagolysosomal low pH (5.5) and high temperature (37 °C) in order to survive in the host cells [25], but also induce functional modifications in macrophages. These include decrease in cytokine production, inhibition of oxidative burst activity, alteration of antigen presentation, and of expression of MHC class II molecules. This ability of Leishmania to inhibit macrophage effectors functions may result from a direct interference of Leishmania molecules with macrophage signal transduction pathways. In an attempt to identify new antigens to be used as vaccines, we focused on those potential antigens expressed associated with polypeptide phosphorylation/dephosphorylation during transition of the parasite from promastigote (in sandfly gut) to amastigote form (in human host cells) of parasites many of which could be Th1 stimulatory and could contribute in defence mechanism against Leishmania infection [26,27]. The pp63, pp41 and pp29 are the potential polypeptides of L. donovani which undergo phosphorylation/dephosphorylation occurring during transition of the parasite from promastigote (in sand fly gut) to amastigote (in human host cells) form which boost the parasite virulence [26,27]. Although, these proteins have been reported to have a role in preferential activation of IFN-γ [10], which is critical for controlling Leishmania survival in vivo [8] limited information is available regarding cocktail of such antigens of Leishmania donovani that are important for human infections. The characterization of these antigens obviously has important implications for the design of new vaccine against VL. The present study deals with the immune modulatory potential of a cocktail of pp63 + pp41 + pp29 against VL. Because use of an appropriate adjuvant is required in any peptide vaccine formulation for generation of effective cell- mediated immune response, we have combined the use of cocktail of L. donovani phosphoproteins and Bacille Calmette-Guerin (BCG) in experimental L .donovani infection [28]. The novelty in our approach lies in the fact that while the cocktail of L.donovani phosphoproteins will induce more collateral host-protective effects while adjuvant, BCG ensures fast antigen delivery and there by the parasite-induced immune suppression.

19

2. Materials and methods 2.1. Mice and parasites This study was carried out in strict accordance with the recommendations of the Institutional Animal Ethical Committee. All experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Rajendra Memorial Research Institute of Medical Sciences, Patna, India).Inbred BALB/c mice were from the National Centre for Laboratory Animal Sciences, Hyderabad, India. Five to eight week old male BALB/c mice were used for the study. Animals were kept under conventional conditions with free access to sterile food and water. Experiments were performed with stationary phase promastigotes. The WHO reference strain of L. donovani, Dd8, maintained by passage on Syrian hamsters, amastigotes were isolated from infected hamster spleens and allowed to transform to promastigotes by cultivation at 24 °C in Schneider’s medium (Sigma, St. Louis, MO) supplemented with 20% heat inactivated fetal calf serum (GIBCO Life Technology, India), 20 mM L-Glutamine, 100 units/ml of penicillin and 50 μg/ml of gentamycin at pH 7.4. 2.2. Immunogen preparation Soluble Leishmania antigen (SLA) was prepared from late log phase promastigotes (MHOM/IN/80/Dd8) after few passages in liquid culture. Briefly, 200 x 106 promastigotes per ml were washed thrice in 5 ml of cold sterile PBS. After 5 cycles of freeze and thaw, the suspension was centrifuged at 10,000 g for 20 min. at 4 °C and the supernatant containing soluble Leishmania antigens were collected and stored at -70 °C till further use. Protein concentration was measured by Lowry’s method. Antigen extract thus prepared were subjected to SDS-PAGE using 12.5% resolution under reducing conditions [29]. Gel slices containing the respective antigenic proteins were then placed into separate micro centrifuge tubes, grinded and incubated in extraction buffer. Gel slurry were transferred to Nanosep MF device that contained Bio-inert modified nylon with polypropylene filtrate receiver and spun. Lower chambers containing the respective proteins in buffer were collected, while upper chambers of gel were rinsed and spun again to get any unseparated specific protein left in the process. 500 μl samples of 100 μg/ml protein solutions containing pp63, pp41, pp39 and pp29 respectively were centrifuged at 5000 g in Nanosep device to a final volume of 50 μl. Resulting retentates were checked for size and concentrated samples were recovered in two washes of 20 μl de-ionized water for use into downstream application at later stages in the study. Before vaccination, batches were tested for functionally relevant LPS contamination, by assaying their ability to synergize with IFN-γ for the induction of inducible NO synthase [30]. No activity was detectable in such assays (sensitivity b 1 ng/ml LPS; data not shown). 2.3. Immunization experiments BALB/c mice were devided into six groups: (1) uninfected control (ii) infected control (iii) immunized with BCG as control 1 and infected (iv) immunized with pp63 and infected (v) immunized with pp 41 and infected (vi) immunized with pp 29 and infected (vii) immunized with pp antigen cocktail and infected. Mice (n = 40) were immunized with sub cutaneous injection with 20 μg of different phosphoprotein antigens along with BCG as adjuvant dissolved in sterile saline on days 1, 7 and 15. On day immunized mice were challenged with intra cardial injection with 15x 10 7 virulent strain of freshly transformed L.donovani (Dd8) early stationary phase promastigotes. Twenty animals from each group were sacrificed in batches (3-5 per experiment) at Day 20 and Day 50 post infection for measurement of weight, spleen size, antiLeishmania antibody titer, T-cell function (IFN-γ, IL-4 and IL-10) and anti-Leishmania macrophage function (measurement and nitric oxide

20

P. Das et al. / Parasitology International 64 (2015) 18–25

production). At each time point; splenic parasite burden was also determined by the limiting dilution assay [33]. 2.4. Estimation of serum antibody titre Blood was collected from the mice before sacrificing and smear slides were prepared for total white bold cell count. The smears were air dried and stained followed by microscopic examination A direct agglutination test (DAT) was also performed to determine the presence of anti-Leishmania antibodies in infected mice [52]. Blood spots from mice were collected on Whatman paper number 4 and air-dried. Circular sections of the paper were obtained from the blood spotted regions using ear punching device. The sections were submerged in normal saline (N/S). One circular piece was immersed in 750 μl of saline to obtain 1:50 diluted serum in a Direct Agglutination Test (DAT) plate. DAT was performed to detect the serum antibody titre as previously described [31]. 2.5. Splenic Mononuclear cell preparation Spleens were isolated from different experimental groups of mice 20 and 50 days after infection and single cell suspensions of splenocytes were prepared after Ficoll PaqueTM (Sigma, USA) density gradient centrifugation. The crude isolates of MNCs were purified by washing in phosphate Buffer saline (PBS, Bangalore Genei, India) and then centrifuged (800 g, 15 min) over Histopaque-1077 (Sigma, USA). The cells collected from the layer immediately above Histopaque were washed thrice in PBS before being used further. All cultures were carried out using RPMI 1640 supplemented with 2 mM L-glutamine, 10% Fetal Calf Serum (GIBCO, Life Technology, India), 5x105 mM 2-mercapthoethanol and antibiotics (100U ml- Penicillin, 50 μg ml-1 Streptomycin (Himedia Laboratories Pvt Ltd, India). 2.6. Multiparameter flow cytometry To determine phosphoproteins–induced generation of antigenspecific CD4+ T cell response, single-cell suspension of splenocytes from different groups of mice 20 and 50 days after infection was prepared as described previously (21). Subsequently, 1x106 cells per well in complete RPMI 1640 were plated in 96-well plates and incubated for 24 hours at 37 °C in a 5% CO2 incubator in the presence or absence of the indicated proteins (20 μg/ml). Brefeldin-A (1 μg/ml) was added during the last 4-hrs of culture to impair protein secretion by the golgi complex. The cells were later harvested using ice cold PBS plus 0.09% sodium azide. The harvested cells were consecutively co incubated with PE-conjugated anti-CD4/CD8 antibodies, cytofix/cytoperm solution, and FITC conjugated IL-10/IFN-γ/IL-4 (BD Pharmingen, USA) before each sample was resuspended in 500 µL stain buffer. Isotype control panels (FITC and PE-labeled immunoglobulin control antibodies) (BD Pharmingen) were run in parallel to all experiments. Unbound antibodies were removed by washing the cells twice with wash buffer before each sample was resuspended in 450 μl stain buffer (containing 0.09% sodium azide) for examination by flow cytometry using FACS Calibur (BD Biosciences) and CellQuest software. Cells were analyzed and data were collected for individual mice. Limits for the quadrant markers were always set based on negative populations and isotype controls. For analysis of CD4+ and CD8+ T-cell response, quadrants were always set for CD4/CD8-PE high populations so as not to include CD4/CD8+ low-positive T-cells.

Briefly, 100 μl of culture supernatants from macrophage culture were dispensed in each well of a 96 well microtiter plate which followed incubation with 100 μl of Griess reagent to each well at room temperature for 20 minutes. The optical density of the coloured product formed was measured on an ELISA reader (BIO-RAD) at 540 nm. The amount of nitric oxide formed M/10 by 106 cells was calculated by comparing the standard sodium nitrite concentration curve. The lower limit of sensitivity of the nitrite assay was 0.08 μM/ml. 2.8. L. donovaniinfection in mice (in vivo) and determination of parasite burden: The parasite burden was determined by limiting the dilution of tissue sample in a limiting dilution assay (LSA) which has been widely employed in many of the studies in leishmaniasis to check infectivity in mice [32–34]. Briefly, the infectivity was determined by homogenizing a weighed amount of tissue using microtissue grinder(Medimachine, BD,USA) in a microcentrifuge tube containing 200 μl of M199 medium. The tissue homogenate and cell suspension of the splenocytes were serially diluted upto 10 fold in a 96 well flat-button microtitre plate containing biphasic medium prepared using 50 μl of NNN medium with 30% defibrinated rabbit blood and overlaid with 50 μl of M199. The plate was incubated at 24 ± 2 °C and observed on the 7th day. The number of viable parasites was determined in the well containing cell from one mg spleen fragment/ml after 7 days and expressed as parasites per mg of tissue [33]. 2.9. Statistical analysis All data were expressed as mean ± SE (standard error of the mean). Statistical analysis was carried out using GraphPad Prism5, USA software, by one way analysis of variance with a post-test, only perform post-test if p b 0.05 (Tukey test compared all pairs of columns). A value of significance p b 0.05 was considered statistically significant. 3. Results 3.1. Impact of immunization on blood cell count and antibody response All the mice, experimental and control survived the course of experiment and remained healthy and active. The average WBC count ranged around 4000-5000 cells/μl with slight increase in counts along with an increase in lymphocyte percentage in the pp63 and pp41 immunized mice, This could be indicative of a specific lymphoproliferative response induced by antigen immunization. A little reduction was observed in the total WBC count in the pp29 immunized mice but the differential counts remained unaltered thereby indicating sufficient population of lymphocytes. However, major variations in the sera antibody titers was observed. The DAT titre observed in uninfected control mice group was 1:100.The unimmunized group of infected mice showed an increased antibody titer, 1:800 by Day 20 and 1:1600 by Day 50. The pp41 and pp29 immunized animals showed an increase in the antibody titer (Table 1). However, immunization with pp63 seemed to resist the increase in titer. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 (1:3200) which reduced considerably by Day 50 (1:400). 3.2. Phosphoproteins significantly reduces splenic parasite burden in BALB/c Mice

2.7. Measurement of NO. Alternations in nitric oxide generated in macrophage in the vaccinated mice was monitored by Griess reagent as described previously [32]. This method is based on colorimetric detection of nitrite (oxidation product of nitric oxide) as an azo dye product of the Griess reaction.

The maximum presence of Leishmania amastigotes was observed in fully established infected mice which accounted for 10.64 amastigotes/mg tissue weight by Day 20 to 47.16 amastigotes/mg tissue weight by Day 50(Fig. 1). An increase in size and weight of spleen was also noticed. From here the outcome of immunization with different

P. Das et al. / Parasitology International 64 (2015) 18–25

21

Table 1 Clinicopathological features in different groups of experimental mice. Mice were challenged with L.donovani after immunization with indicated phosphoproteins using BCG as adjuvant. Animals were sacrificed at 20 days or 50 days after infection and antiLeishmania antibody titre (DAT), total blood count and weight (gm) were determined. Mice Group Healthy(Control)

Non-immunised BCG immunized 63 kDa immunized. 41 kDa immunized. 29 kDa immunized. Ag-Cocktail immunized

Weight (gm)

Antibody Titre (DAT)

20.88(± 2.56)

WBC (total count) (cells/ml)

50

Day 0

Day 20

Day 50

35 (±2.05) 90 (±1.67) 70 (±1.98) 33 (±2.04) 17 (±3.07) 21.70 (±3.41)

25.94 (±2.56) 25.12 (±2.29) 16.14 (±2.16) 20.77 (±1.56) 19.50 (±2.24) 22.15 (±2.16)

30.25 (±3.09) 28.30 (±2.55) 23.9 (±2.79) 23.18 (±1.09) 22.45 (±2.26) 30.29 (±3.18)

phosphoproteins delivered before establishing infection in mice was assessed at two different time points(day 20 and day 50) based on comparative reduction in parasite load brought about by either different phspho proteins alone or when all phosphoproteins were combined as antigen cocktail. It was observed that pp63 immunized group of infected mice showed increased spleen size with parasite load from 5.31 amastigotes/mg tissue weight by Day 20 to 9.91 amastigotes/mg tissue weight by Day 50 (p b 0.0001). However, Immunization with pp41 showed a more rapid clearance of amastigotes from infected spleen which reduced significantly to 5.69 amastigotes/mg tissue weight by Day 50 compared to unimmunized group of vaccinated mice (p b 0.0001). Further results demonstrated that Immunization with pp29 also proved effective in higher parasite clearance in groups of mice infected with Leishmania donovani strain as shown through reduced splenic parasite load from 8.89 parasites/mg tissue weight by Day 20 to 7.36 parasites/mg tissue weight by Day 50) compared to unimmunized and Leishmania donovani infected group (p b 0.0001). Further results demonstrated that antigen cocktail vaccination in infected mice led to maximum reduction in spleen parasite load compared to all other vaccine formulation by day 50.

Fig. 1. Immunization with L. donovani phosphoproteins controlled splenic parasite load in the BALB/c mice. Mice were challenged with L.donovani after immunization with indicated phosphoproteins using BCG as adjuvant. Animals were sacrificed at 20 days or 50 days after infection and amount of nitric oxide (μM) as well as parasite load in the spleen from mice was determined. Results are expressed as parasite/mg tissue in spleen.

Day 20

4967(±345) Day 50

Day 20

Day 50

800

1600

1600

1600

1600

800

800

1600

200

100

3200

400

4700 (±199) 5100 (±183) 4200 (±175) 5800 (±500) 3600 (±200) 5300 (±755)

4350 (±150) 4980 (±780) 5700 (±900) 5400 (±300) 3900 (±500) 5320 (±1080)

3.3. Amount of nitric oxide produced by differentially immunized BALB/c mice Because reactive oxygen and nitrogen species are two important leishmanicidal molecules [35], we examined if immunization with phosphoproteins induced NO in infected mice. It was observed that immunization with phosphoproteins increased nitrite production by 10-25 fold in comparison with the unimmunized group of Leishmania donovani infected control. Immunization of the mice with. BCG showed a two-fold increase in the NO generation whereas groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes (Fig. 1).These data indicate to NO-mediated antileishmanial function of phosphoproteins of L.donovani.

3.4. Phoshoproteins Enhances Interferon-gamma promoting Cytokine production from CD4+ and CD8+ T cells Cellular immune response involving CD4+ and CD8+ T cells is important for controlling infection by intracellular pathogens [22]. To understand the nature of T-cell immune response generated by phosphoproteins vaccination, Two way analysis of variance was applied to compare response of CD4+ and CD8+ T cells for the production of IFN-γ, IL-4 and IL-10 in the immunized and infected mice exposed to L.donovani strains. As shown in histogram analysis of the entire results (Fig.2A-F), pp41 and pp29 immunized mice generated a significantly higher population of antigen specific CD4+ (Fig.2A) and CD8+ (Fig. 2 B) T cells with high percentage of IFN-γ cytokine and low percentage of IL-4 (Fig. 2 C and D) and IL-10(Fig. 2 E and F) compared to infected un immunized controls (p b 0.001). However, although immunization with pp63 appeared to stimulate IFN-γ production both from CD4+ and CD8+ T cells were also observed with significantly higher frequency of IL-4 and IL-10 compared to that of pp41, pp29 and antigen cocktail vaccinated mice (p b 0.001). The cytokine secretion pattern of lymphocytes from lymph nodes also presented a similar pattern (Fig. 3 A-F). There was an increase in extracellular IFN-γ secretion in the groups immunized with pp63, pp41, pp29 and the antigenic cocktail with a corresponding decrease in IL-4 levels in all groups. Thus, an IFN-γ dominant upregulated CD4+ and CD8+ T cell associated mechanism exists during VL infection and more CD4+ and CD8+ T-cells show decreased sensitivity for IL-4 and IL-10 production after vaccination with pp41 and pp29.

22

P. Das et al. / Parasitology International 64 (2015) 18–25

Fig. 2. T cell (CD4+ and CD8+) responses to Leishmania infection in the spleen of immunized mice. To determine phosphoproteins specific IFN-γ, IL-4 and IL-10 positive CD4+ and CD8+ responses, phosphoprotein stimulated and isolated splenocytes from different groups of mice (Day 20 and 50 after infection) were stained for different cytokines and CD4+ and CD8+ surface markers. Cells were analysed by 4-colour flowcytometry to determine the frequency of CD4+ and CD8+ T cells. Histogram showing (A) Frequency of IFN-γ producing CD4+ T cells. (B) Frequency of IFN-γ producing CD8+ T cells. (C) Frequency of IL-4 producing CD4+ T cells. (D) Frequency of IL-4 producing CD8+ T cells.(E) Frequency of IL-10 producing CD4+ T cells.(F) Frequency of IL-10 producing CD8+ T cells.

4. Discussion The current standard approach to identify prospective antigens for vaccine development is to identify critical factors of parasite which not only affect Leishmania virulence in macrophage but may also be immunogenic. As previously reported, Leishmania phosphoprotein molecules, particularly, pp63, pp41 and pp29 undergo phosphorylation/dephosphorylation during transition of the parasite from promastigote to amastigote and influence both the parasite virulence [26,27] as well as preferentially activate of IFN-γ [10], which has been identified as

essential for controlling Leishmania survival in vivo [8]. Because, these phosphoproteins satisfies both criteria, the goal of the present study was to evaluate the potential of pp63, pp41 and pp29 as a vaccine candidate alone or as antigen cocktail against VL using laboratory animal models [36,37]. This study showed clearly that controlled pilot vaccine trial with cocktail preparation of phosphoproteins of L.donovani given with BCG used as adjuvant accelerated the early parasitological response in infected hosts and recovery became faster than with these phosphoproteins (pp63, pp41 and pp29) alone. Unfortunately, a similar vaccination

P. Das et al. / Parasitology International 64 (2015) 18–25

23

Fig. 3. T cell (CD4+ and CD8+) responses to Leishmania infection in the Lymph node of immunized mice. To determine phosphoproteins specific IFN-γ, IL-4 and IL-10 positive CD4+ and CD8+ responses, phosphoprotein stimulated and isolated lymph node from different groups of mice (Day 20 and 50 after infection) were stained for different cytokines and CD4+ and CD8+ surface markers. Cells were analysed by 4-colour flowcytometry to determine the frequency of Cd4+ and CD8+ T cells. Histogram showing (A) Frequency of IFN-γ producing CD4+ T cells (B) Frequency of IFN-γ producing CD8+ T cells (C) Frequency of IL-4 producing CD4+ T cells (D) Frequency of IL-4 producing CD8+ T cells (E) Frequency of IL-10 producing CD4+ T cells (F) Frequency of IL-10 producing CD8+ T cells.

given with BCG produced unexpectedly discouraging results in infected animals. The outcome of experimental leishmaniasis in mice is driven by a balance of Th1 and Th2 cells [39,40,41]. In VL patients, CD4+ subpopulation of T cells fail to produce IFN-γ to activate macrophages whereas the increase in IL-4 and IL-10 responses of CD4+ cells leads to severe disseminated forms of the disease [8]. As this study further showed, the majority of the experimental mice receiving the cocktail vaccine preparation of phosphoproteins of L. donovani showed an up regulated IFN-γ production from their T-lymphocytes, This effect of phosphoproteins immunization together with inability of most vaccine

formulations to exert effects on T cells from patients with visceral infection to secrete host protective IFN-γ [53,54] can give a strong argument for researchers to test vaccination involving phosphoproteins of Leishmania in patients with VL. Recent studies have also shown that despite the huge significance reported earlier for involvement of CD4+ T cells, both CD4+ and CD8+ T cells provide better protection against intracellular infections (22). Therefore, we have compared the levels of Th1 and Th2 cytokines secreted by CD4+ and CD8+ T cells in splenocytes isolated from vaccinated and control groups of animals. It was demonstrated that immunization with pp41, pp29 and antigen cocktail of L.donovani in mice

24

P. Das et al. / Parasitology International 64 (2015) 18–25

generated a significantly higher population of antigen specific CD4+ and CD8+ T cells with high percentage of IFN-γ cytokine compared to infected unimmunized control. In contrast, vaccination with pp41, pp29 and antigen cocktail also significantly inhibited the production of disease-promoting, Th1-suppressive cytokine IL-10 and the Th2 signature cytokine IL-4. Thus, higher release of IFN-γ with a suppressed release of IL-4 and IL-10 in pp41, pp29 and antigen cocktail vaccinated animals demonstrate that the Th1-biased protective immune response triggered both by CD4+ T and CD8+ T cells were generated by these phosphoproteins. However, the frequency of IFN-γ CD8+ T cells was observed slightly higher than that of CD4+ T cells in pp41, pp29 and antigen cocktail–vaccinated mice. Thus, pp41, pp29 and antigen cocktail vaccination can provide protection by generating adequate protective IFN-γ cytokine both from CD4+ and CD8+ T cells. As parasites replicate in macrophages, we focussed our study on these cells. The ability of a leishmanial antigen to downregulate the production of nitric oxide has been reported earlier for infected macrophages [55]. Indeed, it is well established that macrophages do not produce NO at the active stage of the disease. We show here that vaccination with pp41, pp29 phosphoproteins and the antigen cocktail made of these phosphoproteins strongly increase expression NO production by macrophages resulting in Leishmania clearance from host cells in the course of experimental in vivo infection. Further, the NF-κB has been shown to be required for the production of IL-12 and IFN-γ as well as inducible nitric oxide synthase (iNOs) [8,9,53,56]. This report is supported by our previous finding in the present study that pp41, pp29 phosphoproteins and even the antigen cocktail preparation of these phosphoproteins can reduce the downregulation of IL-10 production from T-cells. Most clinical VL studies support the expansion of IL-10 producing cells during the disease [56]. It was shown that IL-10 creates major hindrance in the retardation of Lymphocyte proliferation and IFN-γ production and as such, may have direct role in the pathogenesis of VL [57]. At this point, given the fact that phosphoproteins were key candidate to induce a protective IFN-γ dominant response in CD4+ and CD8+ T cells, therefore, it is likely that phosphoprotein antigens of L. donovani parasite can cause an up regulation of NF-κB transcription. Our results further show that although vaccination of mice with both pp41 and pp29 inhibits splenic parasite burden in comparison to infected control animals but the vaccination with antigen cocktail conferred complete protection against VL in BALB/c mice. This is supported by the finding that all mice immunized with antigen cocktail had survived and remained healthy against the lethal challenge of virulent L. donovani during the experimental period. The survival of animals infected with L. donovani and the clearance of parasites when vaccinated with phosphoproteins plus BCG either alone or as cocktail of these phosphoproteins suggest a major role of these antigens in immunogenicity and protection. BCG derived from Mycobacterium bovis is known to augment a delayed type hypersensitivity reaction and Th1 cell mediated immunity, and thereby used as an adjuvant in the present study [50,51]. In previous vaccination studies against leishmaniasis, diverse forms of adjuvants ranging from BCG [42,43], alum [44], MPL, interleukin 12 [45], Montanide ISA 720 [46], CpG ODNs [38,47,48] and CD2 antibodies [49] were used. Finally, use of BCG alone failed to augment the protective immune system and was not instrumental in ensuring parasite clearance from infected macrophages. In summary, this study demonstrates for the first time the immunogenicity of phosphoproteins, eliciting a dominant Th1-type cytokine profile. In addition, the significant prophylactic efficacy of the phosphoproteins makes it a strong and promising prophylactic vaccine candidate against VL. Acknowledgements This work was supported by the Council of Scientific and Industrial research (CSIR), Government of India. We are indebted to CSIR for the

fellowship awarded to Pranati to pursue this study. We are also thankful to Prakash Chandra, Kumar Vaibhav and Manish Ranjan for their support to prepare this manuscript. References [1] Baker R, Chiodini P, Kaye PM. Leishmaniasis. In: Geraint James D, Zumla A, editors. The Granulomatous Disorders. Cambridge: Cambridge University Press; 1999. p. 212. [2] Killick-Kendrick R. Phlebotomine vectors of the leishmaniasis: a review. Med Vet Entomol 1990 Jan;4(1):1–24. [3] Swaminathan CS, Shortt HE, Anderson LA. Transmission of Indian kala-azar to man by the bites of Phlebotomus argentipes. Indian J Med Res 1942;30:473–7. [4] T.K Jha. Drug unresponsiveness & combination therapy for kala-azar. Indian J Med Res 2006;123:389–98. [5] Sundar S, Singh VP, Sharma S, Makharia MK, Murray HW. Response to interferon-γ plus pentavalent antimony in Indian visceral leishmaniasis. J Infect Dis 1997;176: 1117–9. [6] Abdo MG, Elamin WM, Khalil EA, Mukhtar MM. Antimony resistant Leishmania donovani in eastern Sudan: incidence and in vitro correlation. East Mediterr Health J 2003;9:837–43. [7] Das VNR, Ranjan A, Bimal S, Siddique NA, Pandey K, Kumar N, et al. Magnitude of unresponsiveness to Sodium Stibogluconate in the treatment of Visceral Leishmaniasis in Bihar. Natl Med J India 2005;18:131–3. [8] Bimal S, Sinha S, Singh SK, Narayan S, Kumar V, Verma N, et al. Leishmania donovani: CD2 biased immune response skews the SAG mediated therapy for a predominant Th1 response in experimental infection. Exp Parasitol 2012;131:274–82. [9] Liew FY, O'Donnell CA. Immunology of leishmaniasis. Adv Parasitol 1993;32: 161–259. [10] Mathur RK, Awasthi A, Wadhone P, Ramanamurthy B, Saha B. Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. Nat Med 2004;10:540–4. [11] Adhikari A, Gupta G, Majumder S, Banerjee S, Bhattacharjee S, Bhattacharya P, et al. Mycobacterium indicus pranii (Mw) Re-Establishes Host Protective Immune Response in Leishmania donovani Infected Macrophages: Critical Role of IL-12. PlosOne 2012;Volume 7(7):e40265. [12] World Health Organization. Leishmaniasis Update. WHO; 2007. [13] Marques-da-Silva EA, Coelho EA, Gomes DC, Vilela MC, MAsioli CZ, Tavares CA, et al. Intramuscular immunization with p36 (LACK) DNA vaccine induces IFN-γ production but does not protect BALB/c mice against Leishmania chagasi intravenous challenge. Parasitol Res 2005;98:67–74. [14] Paragaui deSouza E, Bernardo RR, Palatnmik M, Palatnik de Sousa CB. Vaccination of BALB/c mice against experimental visceral leishmaniasis with GP36 glycoprotein antigen of Leishmania donovani. Vaccine 2001;19:3104–15. [15] Fujiwara RT, Vale AM. franca da Silva JC et al. Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis. Vet Res 2005;36:827–38. [16] Gurunathan SD, Sacks DL, Brown DR, Reiner SL, Charest H, Glaichenhaus N, et al. Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. J Exp Med 1997;186:1137–47. [17] Bhaumik SK, Naskar K, De T. Complete protection against experimental visceral leishmaniasis with complete soluble antigen from attenuated Leishmania donovani promastigotes involves Th1- immunity and down-regulation of IL-10. Eur J Immunol 2009;39:2146–60. [18] Scott P, Pearce E, Cheever AW, Coffman RL, Sher A. Role of cytokines and CD4+ T-cell subsets in the regulation of parasite immunity and disease. Immunol Rev 1989 Dec; 112:161–82. [19] Visceral Leishmaniasis Vaccine Trial Commences. www.medicalnewtoday.com/ articles/242247.php. [20] Selvapandiyan A, Duncan R, Debrabant A, Lee N, Sreenivas G, Salotra P, et al. Genetically modified live attenuated parasites as vaccines for leishmaniasis. Indian J Med Res 2006 Mar;123(3):455–66. [21] da Silva RA, Tavares NM, Costa D, Pitombo M, Barbosa L, Fukutani K, et al. DNA vaccination with KMP-11 and Lutzomyia longipalpis salivary protein protects hamsters against visceral leishmaniasis. Acta Trop 2011;120(3):185–90. [22] Guha R, Gupta D, Rastogi R, Vikram R, Krishnamurthy G, Bimal S, et al. Vaccination with Leishmania Haemoglobin Receptor–Encoding DNA Protects Against Visceral Leishmaniasis. Sci Trans Med 2013;5(202):102–21 (Vol 5 issue202). [23] Mauel J. Intracellular survival of protozoan parasites with special reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. Adv Parasitol 1996; 38:1–51. [24] Hermoso T, Fishelson Z, Becker SI, Hirschberg K, Jaffe CL. Leishmanial protein kinases phosphorylate components of the complement system. EMBO J 1991;10:4061–7. [25] Bates PA. Axenic culture of Leishmania amastigotes. Parasitol Today 1993;9:143–6. [26] Salotra P, Ralhan R, Sreenivas G. Heat stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani. Int J Biochem Cell Biol 2000;32:309–16. [27] Das Pranati, Chaudhary Rajesh, Sundaram Shanti, Das P, Bimal S. Leishmania donovani promastigote phosphoproteins stimulate helper T-cell and macrophage functions for parasite killing. IJAR 2013;1(7):645–57. [28] Ravindran R, Bhowmick S, Das A, Ali N. Comparison of BCG, MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis. BMC Microbiol 2010;10:181.

P. Das et al. / Parasitology International 64 (2015) 18–25 [29] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [30] Proudfoot L, AV Nikolaev, GJ Feng, WQ Wei, MA Ferguson, JS Brimacombe, et al. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages. Proc Natl Acad Sci USA 1996;93:10984. [31] Bimal S, Das VN, Sinha PK, Gupta AK, Verma N, Ranjan A, et al. Usefulness of the direct agglutination test in the early detection of subclinical Leishmania donovani infection: a community-based study. Ann Trop Med Parasitol 2005;99(8):743–9. [32] Mookerjee Basu J, Mookerjee A, Sen P, Bhaumik S, Sen P, Banerjee S, et al. Sodium antimony gluconate induces generation of reactive oxygen species and nitricoxide via phosphoinositide 3-kinase and mitogen-activated protein kinase activation in Leishmania donovani-infected macrophages. Antimicrob Agents Chemother 2006; 50:1788–97. [33] Basu R, Bhaumik S, Basu JM, Naskar K, De T, Roy S. Kinetoplastid membrane protein11DNA vaccination induces complete protection against both pentavalent antimonial-sensitive and – resistant strains of Leishmania donovani that correlateswith inducible nitric oxide synthase activity andIL-4 generation: evidence or mixed Th1- and Th2-like responses in visceral leishmaniasis. J Immunol 2005; 174:7160–71. [34] Mookerjee Basu J, Roy S. Sodium antimony gluconate (SAG) mediates antileishmanial effect by stimulating innate and cellular arms of the immune system. Sci Cult 2007; 73:138–43. [35] Murray HW, Nathan CF. Macrophage microbicidal mechanisms in vivo: Reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani. J Exp Med 1999;189:741–6. [36] Hommel M, Jaffe CL, Travi B, Milon G. Experimental models for Leishmaniasis and for testinganti-leishmanial vaccines. Ann Trop Med Parasitol 1995;89:55–73. [37] Engwerda CR, Kaye PM. Organ-specific immune responses associated with infectious disease. Immunol Today 2000;21:73–8. [38] Iborra S, Carrion J, Anderson C, Alonso C, Sacks D, Soto M. Vaccination with the Leishmania infantum acidic ribosomal protein plus CpG oligodeoxynucleotides inducers protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice. Infect Immun 2005;73:5842–52. [39] Padigel UM, Alexander J, Farrell JP. The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis. J Immunol 2003;171:3705–10. [40] Jones DE, Ackermann MR, Wille U, Hunter CA, Scott P. Early enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 interleukin-10-deficient mice does not lead to resolution of infection. Infect Immun 2002;70:2151–8. [41] Engwerda CR, Ato M, Kaye PM. Macrophage pathology and parasite persistence in experimental visceral leishmaniasis. Trends Parasitol 2004;20:524–50. [42] Scott F, Romagnani S. The Th1 and Th2 cells in health and disease. , 63. Basel: karger; 1990. p. 115–37. [43] Srivastava JK, Misra A, Sharma P, Srivastava B, Naik S, Dube A. Prophylactic potential of autoclaved Leishmania donovani with BCG against experimental visceral leishmaniasis. Parasitology 2003;127:107–14.

25

[44] Baylor NW, Egan W, Richman P. Aluminum salts in vaccines – US perspective. Vaccines 2002;20:S18–23. [45] Kenney RT, Sacks DL, Sypek JP, Vilela L, Gam AA, Evans-Davis K. Protective immunity using recornbinant human IL- 12 and alum as adjuvants in a primate model of cutaneous leishmaniasis. J Immunol 1999;163:4481–8. [46] Aucouturier J, Dupuis L, Deville S, Ascarateil S, Ganne V. Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines. Expert Rev Vaccines 2002;1(1):111–8. [47] Mendez S, Tabbara K, Belkaid Y, Bertholet S, Verthelyi D, Skeiky YA, et al. Coinjection with CpG-containing immuno-stimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous leishmaniasis but maintains its potency and durability. Infect Immun 2003;71:5121–9. [48] Iborra S, Carrion J, Anderson C, Alonso C, Sacks D, Soto M. Vaccination with the Leishmania infantum acidic ribosomal protein plus CpG oligodeoxynucleotides inducers protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice. Infect Immun 2005;73:5842–52. [49] Bimal S, Bagchi AK, Das V, Sinha PK, Lal CS, Ranjan A, et al. Effect of immunization with lipid associated polysaccharide antigen and anti-CD2 antibodies on class II MHC expression and cellular immune response in BALB/c mice infected with Leishmania donovani. Ind J Exp Biol 2001;39:878–82. [50] Molano I, Alonso MG, Miron C, Redondo E, Requena JM, Soto M, et al. A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L.infantum. Vet Immunol Immunopathol 2003;92:1–13. [51] Connell ND, Acosta ME, McMaster WR, Bloom BR, David R. Effective immunization against cutaneous leishmaniasis with recombinant bacilli calmette-guerin expressing the Leishmania surface proteinase gp63. Proc Natl Acad Sci U S A 1993;90:11473. [52] Harith AE, Kolk AH, Kager PA, Leeuwenburg J, Muigai R, Kiugu S, et al. A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis. Trans R Soc Trop Med Hyg 1986;80(4):583–7. [53] Bimal S, Singh SK, Sinha S, Pandey K, Sinha PK, Ranjan A, et al. Leishmania donovani: Role of CD2 on CD4 + T-cell function in Visceral Leishmaniasis. Exp Parasitol 2008; 118:238–46. [54] Narayan S, Bimal S, Singh SK, Gupta AK, Singh VP, Sinha PK, et al. Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis. Exp Parasitol 2009;121(1):69–75. [55] Mookerjee B, Mookerjee A, Banerjee R, Saha M, Singh S, Naskar K, et al. Inhibition of ABC Transporters abolishes antimony resistance in Leishmania infection. Antimicrob Agents Chemother 2008;52(3):1080–93. [56] Chaudhary R, Amit A, Yadav A, Singh A, Kumar V, Singh SK, et al. Immunomodulation in human dendritic cells leads to induction of interferon-gamma production by Leishmania donovani derived KMP-11 antigen via activation of NF-κB in Indian kala-azar patients. Biomed Res Int 2014:947606. [57] Carvalho EM, Teixeira RS, Johnson WD Jr. Cell-mediated immunity in American visceral leishmaniasis: reversible immunosuppression during acute infection. Infect immune 1981;33:498–500.