Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis

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Experimental Parasitology 130 (2012) 39–47

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Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis Sanchita Datta a, Rupchand Adak a, Priyanka Chakraborty a, Arun Kumar Haldar b, Surajit Bhattacharjee c,1, Anindita Chakraborty d, Syamal Roy b, Madhumita Manna a,⇑ a

Department of Zoology, Bethune College, 181, Bidhan Sarani, Kolkata 700 006, India Infectious Diseases and Immunology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India Division of Molecular Medicine, Bose Institute, P1/12, C.I.T. Scheme VIIM, Kolkata 700 054, India d UGC-DAE Consortium for Scientific Research, 3/LB-8, Salt Lake, Kolkata 700 098, India b c

a r t i c l e

i n f o

Article history: Received 15 November 2010 Received in revised form 25 September 2011 Accepted 3 October 2011 Available online 12 October 2011 Keywords: Visceral leishmaniasis Immunization Radio-attenuated parasites Th1 and Th2 cytokines

a b s t r a c t The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (c)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Visceral leishmaniasis (VL) or Kala-azar (KA) is caused by kinetoplastid protozoan parasites belonging to Leishmania donovani species complex. Ninty percent of VL cases occur in India, Bangladesh, Brazil, Iran, Nepal and Sudan (Desjeux, 2004; Sundar, 2001). The symptoms of VL include fever, weight loss, anemia, edema, and hepatosplenomegaly (Bittencourt and Barral-Netto, 1995) and is fatal if left untreated. Co-infection of HIV with VL (Desjeux and Alvar, 2003; WHO, 1998) has complicated the situation further. In India, over 60% patients do not respond to the first-line antimonials (Croft et al., 2006). The second line of treatment with pentamidine and amphotericin B has high toxicity (Amato et al., 1990; Jackson et al., 1990). To solve this problem a search for novel chemotherapeutic agents should be accompanied with attempts to develop safe, long lasting and affordable vaccines against the disease. To date, there is no vaccine against leishmaniasis. However, recovery from the disease is usually accompanied by immunity ⇑ Corresponding author. Address: Department of Zoology, West Bengal Educational Service, Bethune College, 181, Bidhan Sarani, Kolkata 700 006, India. Fax: +91 33 2219 2097. E-mail address: [email protected] (M. Manna). 1 Present address: Department of Molecular Biology and Bioinformatics, Tripura University, Suryamaninagar, Tripura 799022, India. 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.10.001

against re-infection which may encourage vaccine development approaches. One of such attempts known as ‘leishmanization’ has been licensed in Uzbekistan where mixture of dead and live L. major is used (Khamesipour et al., 2005). Live parasites may cause pathology and to avoid this, the focus was shifted to killed or attenuated organisms. Attenuated parasites behave like natural isolates the power of virulence and may therefore lead to similar immune responses in the host. Substantial immune protection has been achieved by attenuated strains produced by long-term in vitro culture (Handman, 1997), heterologous carrier systems that carried the gp63 gene of L. major (Xu and Liew, 1994), gene replacement (Titus et al., 1995), promastigote antigen entrapped in liposomes (Ali and Afrin, 1997; Afrin and Ali, 1997), recombinant stage regulated surface of L. donovani (Stager et al., 2000) and atypical avirulent strain of VL[MHOM/IN/78/IICB1-UR6] (Mukhopadhyay et al., 1998, 2000). Rachamim and Jaffe (1993) reported a pure protein, dp72 (72 kDa protein, isolated from L. donovani) that gives both homologous and heterologous protection in experimental mice. More recent works include released proteins (Rosa and Rodrigues, 2007), soluble antigen of L. donovani entrapped in liposomes (Bhowmick et al., 2007) and complete soluble antigen from attenuated L. donovani (Bhaumik et al., 2009). Use of gamma irradiation to reduce virulence has also been considered (Alexander, 1982; Howard et al., 1982, 1984; Lemma and Cole, 1974; Rivier et al., 1993, 1999). These radio-attenuated


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leishmania parasites cannot undergo replication but can survive for sufficient time in the host to induce a protective immune response. Vaccination using radio-attenuated L. enriettii parasites in the guinea pig model (Lemma and Cole, 1974) failed and in another case, the author suggested that radio-attenuated L. major vaccine could play a heterologous protective role in CBA mice (Alexander, 1982). In BALB/c mice, protective immunity has been studied using irradiated L. tropica promastigotes (Howard et al., 1984). Rivier et al. (1999) has proposed the use of live radio-attenuated parasites along with adjuvant to get better protection in CBA mice model. The immune response against different forms of leishmaniasis varies (Awasthi et al., 2004; Mathur et al., 2004). Though some recent reports suggested the role of B lymphocytes in Th2 response development for susceptibility to L. major and L. amzonensis infection (Ronet et al., 2008; Wanasen et al., 2008), CL showed a predominantly cell mediated immune response in experimental resistant and susceptible mouse models showing Th1/Th2 dichotomy (Alexander et al., 1999; Engwerda and Kaye, 2000). In the case of L. donovani infection, the response is quite different. The protective immunity depends on IL-12 driven IFN-c production by the Th1 cells (Engwerda and Kaye, 2000; Murray et al., 1997) and the expression of an exclusively Th1 response is not sufficient to clear the parasites (Melby et al., 2001). IL-10 is found to be the major immunosuppressive cytokine in VL (Murray et al., 2005) and interestingly, IL-4 plays a protective role in resistance (Basu et al., 2005; Mazumdar et al., 2004). After obtaining promising results in our therapeutic study (Datta et al., 2010), the present study reports the efficacy of radio-attenuated L. donovani parasites for immunoprophylaxis in experimental murine visceral leishmaniasis.

isolate by isozyme analysis and by RAPD PCR techniques (Manna et al., 2005) and was designated as MHOM/IN/1983/IICB2-AG83. This was cultured at 22 °C in medium M 199 in presence of 100 U/ml penicillin and 100 lg/ml streptomycin supplemented with 10% heat inactivated FBS and maintained in BALB/c mice or hamsters. Amastigotes were isolated from spleen and transformed to promastigotes in medium M199 containing 30% FBS. Freshly transformed promastigotes were maintained at 22 °C in medium M199 with 10% FBS. 2.4. Radiation sources and methods L. donovani promastigotes used for immunization were exposed to doses from 50 Gray (Gy) to 450 Gy absorbed dose of Gamma (c) irradiation respectively from a 60Co Gamma chamber at University Grant Commission – Department of Atomic Energy (UGC DAE) Consortium for Collaborative Research, Kolkata Centre, India. Three doses (50, 100 and 150 Gy) were ultimately chosen for the present study. The rate of gamma ray emission was 6.5 kGy/h [i.e. 10 Gy in 5.5 s] at the time of our experiments. So, for 50 Gy, exposure time was 27.5 s, for 100 Gy, 55 s and for 150 Gy, 82.5 s, respectively. The promastigotes were washed three times in PBS before irradiation. 2.5. Survival curves of the irradiated parasites Post irradiation, the promastigotes were cultured at 22 °C. Promastigote viability was assayed by Trypan blue exclusion (Strober, 2001). Stock of Trypan blue was diluted 1:10 in PBS. The diluted stock was added to parasites in a 1:1 ratio. Counts were taken using a haemocytometer under a light microscope.

2. Materials and methods 2.1. Chemicals and reagents

2.6. Priming and challenging the experimental animal groups

Tissue culture chemicals (medium M199, medium RPMI-1640, sodium bicarbonate, streptomycin, penicillin, Histopaque-1083, Trypan blue and other reagents) were obtained from Sigma Chemicals. ELISA reagents were purchased from BD Sciences, USA. FBS was purchased from Invitrogen, USA.

BALB/c mice were immunized by i.m. route with irradiated L. donovani [AG83] promastigotes. Each dose of immunization contained 2  106cells in 100 ll PBS. A second immunization was done after 15 days of first immunization with the same dose. Parasite challenge was given after 30 days of first immunization with 2X106 virulent parasites in 100 ll PBS.

2.2. Animals For each experimental group, 6–8 BALB/c mice (4–6 weeks old) were used, irrespective of sex. Five such groups were selected, A, B, C, D and E. Healthy control group (A) received no priming and no parasite challenge, infected group (B) has got infection with highly virulent L. donovani (AG83) parasites. The other experimental groups designated as C, D, and E received radio-attenuated leishmania parasites, doses of attenuation being 50, 100 and 150 Gy absorbed doses of c-radiation, respectively. Another set of experiment was organized to measure the cytokines released in the experimental groups where attenuated parasites were injected at the same regimes stated above but no challenge infection was given and sacrificed after 135 days of second immunization. All experiments were repeated three times. Use of mice was approved by the Institutional Animal Ethics Committees of Indian Institute of Chemical Biology, India. All animal experimentations were performed according to the National Regulatory Guidelines issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India. 2.3. Parasite culture A clinical isolate, named as AG83 isolated from a confirmed Indian Kala-azar patient was further characterized as L. donovani

2.7. Isolation of splenocytes from and culture Spleens of experimental groups 120 days post infection or no challenge infection were taken aseptically under laminar flow, and then were macerated using PBS with the help of a pair of frosted glass slides. The cell suspension was added along the side of a 15 ml centrifuge tube over a Histopaque-1083 layer at a 1:1 ratio and was centrifuged in a swinging bucket rotor at 250 g for 30 min. The second layer from the top (off-white in color) was taken and washed thrice with PBS. The splenocytes which formed a pellet at bottom of the tube were cultured in 24-well tissue culture plates at a cell concentration 2  106cells/well at 37 °C in medium RPMI 1640 with 10% FBS in a water jacket CO2 incubator (5% CO2). 2.8. Organomegaly and parasite load analysis The animals were sacrificed at 120 days post infection. The weight and parasitic burden of the spleen and liver from healthy control group (A), infected group (B) and three immunized groups, C, D and E were assessed. The parasite loads were calculated as number of amastigotes/number of nucleated cell  organ weight in mg  2  105 (Stauber, 1958) and expressed as mean parasite number ± SD.

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2.9. Assessment of superoxide anion generation and nitrite assay 2.9.1. Nitroblue tetrazolium assay Superoxide generation in splenocytes from all experimental groups was determined by sacrificing the animals at 120 days post infection. The nitroblue tetrazolium (NBT) reduction assay (Rook et al., 1985) was employed. Briefly, splenocytes from different groups of BALB/c mice taken were stimulated with soluble leishmania antigen (SLA) 5 lg/ml for 1 h. The cells were washed with RPM1 1640 and incubated with NBT (1 mg/ml). After 24-h of incubation at 37 °C in 5% CO2 in moist air cell pellets were obtained by spinning at 2500 rpm for 15 min. The pellet was washed in methanol and dissolved. The reading was taken at 630 nm. 2.9.2. Nitrite assay Splenocytes (2  106cells/well) from different groups of experimental mice were incubated with 5 lg/ml SLA in 5% CO2 incubator at 37 °C. Splenocytes (2  106cells/well) were then cultured in a 24-well culture plate for 48 h. A standard curve was generated using NaNO2.The release of nitrite was measured in cell free supernatants by the Griess assay (Green et al., 1982). In these experiments LPS at a concentration of 500 ng/ml is used as a positive control.

Fig. 1. Survival curves of the leishmania parasites after being attenuated at three different absorbed doses of c-radiation. Control represented normal non irradiated leishmania parasites grown in liquid culture media M199 supplemented with 10% foetal bovine serum (FBS), 50, 100 and 150 Gy represented leishmania parasites attenuated at 50, 100 and 150 absorbed doses of c-irradiation and cultured in medium M199 plus 10% FBS. The inoculum size was 1  106 ml 1 for each group. Number of viable parasites was determined by Trypan blue exclusion method. Paired two-tailed Student’s t-test was performed and P < 0.05 was considered significant.

2.10. Cytokine ELISA in experimental groups Interferon-gamma (IFN-c), IL-2, IL-12, TNF-a, IL-4, IL-10 release by the splenocytes was detected by sandwich ELISA. 2X106 splenocytes for each animal were plated in 24-well tissue culture plates with 5 lg/ml SLA (soluble leishmania antigen) with 10% FBS containing RPMI-1640 medium and incubated in 5%CO2 containing 37 °C incubator. The supernatant for IL-12 assay was collected within 16–18 h while for other cytokines supernatant was collected within 20–24 h. ELISA was performed on the supernatant following the standard protocol of the supplier, BD Sciences, USA. 2.11. Statistical analysis Paired two-tailed Student’s t-test was used for statistical analysis of the data and values of P < 0.05 were considered significant. Results were expressed as mean ± SD for individual sets of experiments. In each experiment, about 6–8 animals were used in each group. Each experiment was performed three times and the representative data from one set of these experiments are presented. 3. Results 3.1. Survival curves of the radio-attenuated parasites in liquid culture After exposure to different doses of c-rays, these L. donovani promastigotes were cultured in liquid medium for monitoring survival. Fig. 1 shows the growth patterns of parasites attenuated at 50, 100 and 150 Gy along with the growth curve of healthy un-irradiated parasites. Group E (150 Gy) had shown severe sensitivity towards radiation and the parasites had lost the power to multiply altogether though they survived for five days of culture as assessed by Trypan blue exclusion method. Group D showed some initial attempts to grow but failed to survive after seven days of culture and compared to the control group, it was significantly lowered (P < 0.05). Group C, on the contrary, showed significant growth up to eighth day and achieved higher population density compared to other two groups, if fresh medium was added. Irradiated promastigotes must remain viable in the host to elicit the maximum immune response (Rivier et al., 1993). However viability and ability to replicate depends on the radiation dose (Lemma and Cole),

and therefore choosing the correct dose is essential for maximal effect. 3.2. Cytokine profiles of immunized BALB/c mice which were not challenged Separate experiment were established in order to analyze the safety of using live attenuated parasites and to check the cytokine profiles of the immunized groups at the time when they would be challenged with virulent leishmania parasites in our subsequent protection experiments. In this study three groups of mice each with 6 animals were used and immunized i.m. twice at fifteen day intervals with radio-attenuated parasites, as in the immunization/challenge experiments. The mice were not challenged with virulent parasites and 15 days after the last immunization, animals were sacrificed for checking cytokine profiles and parasite burdens, in the spleen and liver. The live, attenuated parasites could revert to the infective stage causing infection instead of giving protection against the disease. The ratio of IFN-c to IL-4 (Table 1) was compared in all three groups of mice and in group C, it was 1.3 while the IFN-c milieu was prevalent in groups D and E as evidenced by ratios above 3.0. When the release of IFN-c to that of IL-10 was compared, the same trend was observed thou to a lesser extent (Table 1). Thus radio-attenuated parasites have created a protective ambience in the immunized groups of mice. Group C, from this point of view, was inferior to other two groups. The weights of spleen and liver as well as the parasite load in both the organs did not increase in the immunized groups other than group C at this time (data not shown) indicating the safe use of the radio-attenuated parasites. 3.3. Parasite burden and organomegaly study after immunization and challenge Studies on organomegaly of the livers and spleens of experimental BALB/c mice with i.m. immunization revealed significant protection by gamma irradiated parasites. The spleen weight of the groups D and E was decreased by 35% and 39% with respect to the infected group (group B).The decrease in weight of liver in


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Table 1 Comparison of Th1:Th2 cytokines ratio in the primed with that of primed and challenged BALB/c mice. Groupsa

Cytokine ratio (Intramuscular route)b Primedc

Group C Group D Group E

Primed and challengedd





1.332 3.998 3.094

0.607 1.634 1.444

5.768 15.048 16.512

1.328 4.299 6.016

a Groups were C, D and E [BALB/c mice receiving parasites radio-attenuated at three different absorbed doses [50 , 100 and 150 Gy, respectively] of gamma irradiation]. b Th1 and Th2 cytokine ratio in the intramuscular primed groups of mice were presented here. c Primed group of mice were sacrificed after two vaccinations. Vaccinations were at fifteen days interval. The mice were not given the challenge infection with virulent parasites and their cytokine profiles were measured. d Primed and then challenge infected group of mice were sacrificed 120 days post infection. After second immunization, they were challenged with virulent parasites. e The IFN-c:IL-4. f IFN-c:IL-10 are the ratio of IFN-c to IL-4 and IFN-c to IL-10 of the groups of mice which has been vaccinated i.m. with radio-attenuated parasites and the groups of mice which has been vaccinated i.m. and then challenged with the virulent L. donovani parasites. The challenge infection further enhanced the Th1 cytokines release in the immunized and challenged groups and suppressed the disease progressing conditions as reflected by the decrease in Th2 cytokines release in the same groups.

groups D and E was 28% and 33%, respectively, in comparison to group B. Group C, on the other hand, only showed spleen and liver weight decreases about 12% and 5%, respectively (Fig. 2A and B). The weight of spleen and liver of healthy uninfected mice (group A) were, respectively, about 42% and 40% of that of infected group B. Parasite load of liver and spleen were expressed as leishman donovan units (LDU). While group C showed only 4% reduction in load of parasites in spleen (Fig. 2C) when compared to that of infected group (B), groups D and E showed about 68% and 88% reduction in parasite load respectively. In liver, parasite load was reduced by about 34%, 88% and 87% in groups C, D and E, respectively, when compared with that of the infected group (Fig. 2D). 3.4. Superoxide anion generation and nitrite assay Data of organomegaly and parasite load studies clearly indicated protection against the disease using radio-attenuated parasites as immunizing agent. The release of these free radicals was enhanced between 100 and 130 days, maximum being at 120 days post infection. Superoxide anion generation in the five experimental groups have been analyzed by NBT reduction assay and it has been seen that release of superoxide was about 1.4 and 1.5 times higher in two immunized groups (D and E, respectively) compared to the infected control group (B) while goup C animals showed almost no increase in superoxide level compared to the infected control group (Fig. 3A). The nitrite assay in all experimental groups showed enhanced release in group D (2.5 times) and group E (2.4 times) in comparison with the infected control group (B) as well as with group C (Fig. 3B). This was more than five times for groups D and E and more than double for group C with respect to the healthy control group (A).The production of nitric oxide, which macrophages utilize during killing of the parasites, depends on the modulation of the Th1 and Th2 cytokines (Bhaumik et al., 2009). In 50 Gy group (C), this immune modulation may not be properly operative while other two groups (100 and 150 Gy) showed much promise in this regard.

3.5. Cytokine modulation in i.m. immunized groups after challenge infection Immunization with radio-attenuated parasites induced a Th1 type response in the BALB/c mice as evidenced from Table 1. IFNc release had been increased distinctively from group C to group E while IL-4 has been seen to decrease significantly in groups D and E (Fig. 4A and B). Release of IFN-c in primed and challenged group E, is about 4.4 times more than that of the infected group (B) and about 9.0 times higher than that of the healthy control group (A) (Fig. 4A). In case of primed and challenged group D, these values were 3.9 and 8.1, respectively. IL-4 release has been assayed and its release was also reduced to about 66–68% in primed and challenged groups D and E (Fig. 4B) compared to the infected mice (group B) (Fig. 4B). Primed and challenged group C showed mixed release of Th1 and Th2 (Fig. 4A and B). The release of IL-2 in all immunized and challenged groups were also about 2.2 times and 2.6 times higher for groups D and E respectively compared to the infected group (Fig. 4C) and like IL-4, IL-10 cytokine release (Fig. 4D) was reduced in these two immunized groups but group C again showed higher release of IL-10 and lower release of IL-2 [1.4 times increase only from the infected group]. About 3.5–3.9 times more release of TNF-a was seen in groups D and E animals (Fig. 4F) with respect to the infected group (group B) (P < 0.01, P < 0.001) and group C (P < 0.01) again showed less release compared to other two groups. While IFN-c release was found in all protected groups in a progressive manner reaching maximum in group E, release of IL-4 and IL-10 were down regulated in groups D and E. Group C showed production of IFN-c along with increased levels of IL-4 and IL-10. Th1:Th2 cytokine ratios in the primed but not challenged BALB/ c mice showed establishment of Th1 ambience in these groups as stated in Table 1 which became significantly higher when the experimental animals experienced the challenge infection. After being challenged with virulent parasites, the primed animals released more Th1 type cytokines than Th2 type cytokines. In Table 1, the ratio of IFN-gamma to IL-4 was increased from 1.332 to 5.768 [4.33 times] in group C, from 3.998 to 15.048 [3.76 times] in group D and from 3.094 to 16.512[5.34 times] in animals of group E, respectively. The ratio of IFN-c to IL-10 was also increased from 2.19 to 4.17 times more in the immunized and challenged groups. It indicated the initiation of establishment of Th1 ambience in the primed BALB/c mice that has been amplified when these primed mice groups experienced infection challenge.

4. Discussion The present study had been undertaken to assess the potential role of radio-attenuated leishmania parasites as immunoprophylaxis against Indian visceral leishmaniasis and to understand the mechanism of protection. Uses of c-irradiated leishmania parasites were successfully attempted against L. major infection (Rivier et al., 1993, 1999) but c-irradiated parasites have never been used before against visceral leishmaniasis. The essence of the present study is that the virulent radio-attenuated parasites survives in the immunized organisms for some time without replicating, mimicing the naturally virulent parasites and may therefore lead to similar immune responses in the immunized groups. Rivier et al. (1993) studied the immune response against L. major infection in CBA mice using radio-attenuated parasites and commented that to ensure full efficacy of the ‘vaccine’, live attenuated parasites did not have better alternatives. The requirement of living parasites may be associated with the need for transformation of the injected promastigotes to the amastigote forms. They suggested that protection might be dependent on development of immune response against

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Fig. 2. (A, B) Liver and spleen weight of the BALB/c mice of different experimental groups. Left to right, bars represent A, healthy control: BALB/c mice received no priming and no challenge infection; B, infected: mice received infection only; C, D, E represented three groups of mice received attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150 Gy absorbed doses of c-radiation respectively. Immunization was given twice @ 2  106 parasites/animal and challenged with virulent Leishmania parasites (2106 parasites/animal) after one month of immunization. Spleen (A) and Liver (B) weights of all groups were taken sacrificing animals at 120 dpi. Data represents mean ± SD of eight animals per group and are representative of three independent experiments; paired two-tailed Student’s t-test was performed and P < 0.05 was considered significant. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. (C, D) Parasite load assessment in liver and spleen of immunized BALB/c mice showed protective role of the immunizing agent. Left to right, bars represent B, infected: mice received infection only; C, D, E represented three groups of mice received attenuated Leishmania donovani parasites (doses of attenuation were 50, 100 and 150 Gy absorbed doses of c-radiation, respectively). Animals were immunized as stated in (A) and (B). Data represents mean ± SD of eight animals per group and are representative of three independent experiments; paired two-tailed Student’s t-test was performed and P < 0.05 was considered significant. ⁄ P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

Fig. 3. Generation of superoxide anion (A) and nitrite productions (B) are increased in the experimental immunized groups of BALB/c mice. Left to right, bars represent A, healthy control; B, infected: mice received infection only; C, D, E represented three groups of mice received attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150 Gy absorbed doses of c-radiation, respectively. Immunization was given twice @ 2  106 parasites/animal and challenged them with virulent Leishmania parasites (2  106 parasites/animal) after one month of immunization. NBT reduction and Griess assay were performed after sacrificing the animals 120 dpi. Data represents mean ± SD of six animals per group and are representative of three independent experiments; paired two-tailed Student’s t-test was performed and P < 0.05 was considered significant. LPS stimulation was used as positive control. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

antigens of leishmania that were metabolically active. It has also been demonstrated that the protection exhibited by purified

antigens with or without adjuvant, did not reach the level of protection obtained by radio-attenuated or virulent promastigotes


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Fig. 4. (A, B) IFN-c and IL-4 cytokine releases in immunized BALB/c mice groups. Left to right, bars represent A, healthy control; B, infected: mice received infection only; C, D, E represented three groups of mice received attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150 Gy absorbed doses of c-radiation, respectively. Immunized (i.m.) (twice @ 2  106 parasites/animal) and challenged (2  106 parasites/animal) mice were sacrificed at 120 dpi. Splenocytes were isolated and cultured in medium RPMI-1640 in 24-well plates @ 2  106 splenocytes/well. Supernatants were collected and assayed by sandwich ELISA method. Data represents mean ± SD of six animals per group and are representative of three independent experiments; paired two-tailed Student’s t-test was performed. P < 0.05 was considered significant. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. (C, D) IL-2 and IL-10 cytokine releases in immunized BALB/c mice groups. Left to right, bars represent A, healthy control; B, infected: mice received infection only; C, D, E represented three groups of mice received attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150 Gy absorbed doses of c-radiation, respectively. Experimental protocol was same as stated in (A) and (B). Data represents mean ± SD of six animals per group and are representative of three independent experiments; paired two-tailed Student’s t-test was done and P < 0.05 was considered significant. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. (E, F) IL-12 and TNF-a cytokine production in immunized BALB/c mice groups. Left to right, bars represent A, healthy control; B, infected: mice received infection only; C, D, E represented three groups of mice received attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150 Gy absorbed doses of c-radiation, respectively. Experimental protocol was same as stated in (A) and (B). Data represents mean ± SD of six animals per group and are representative of three independent experiments; paired two-tailed Student’s t-test was done and P < 0.05 was considered significant. ELISA for all cytokines assay was done as per standard protocol stated in Section 2. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

(Rivier et al., 1999). Nylen et al. (2003) dissected the differences in cellular induction achieved by live vs. dead parasites and argued in favor of live promastigote vaccination. Live promastigotes had the potential to induce various cells to produce interferon-gamma, a

property which dead promastigotes lacked. Ferrua et al. (2006) showed that low-dose imprinting of Leishmania infantum in BALB/c mice conferred substantial spleen resistance to high dose challenge. Selvapandiyan et al. (2006) discussed the observation

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that a complete cDNA expression library injected into mice was more protective than any subpools of the library plasmids and this would reinforce the idea that whole parasites would make the best vaccine. The vaccinee should be exposed to complex antigens at right time without experiencing pathology. However, this approach is considered to be unacceptable especially in immunodeficient cases (Alvar et al., 1997) because of chance of persistence of virulent parasites which might cause disease. In our preliminary study, we noticed very few amastigotes in the spleen and liver of immunized and challenged mice (groups D and E) compared to group C. Maximum protection was observed in these two groups making it apparent that amastigote-specific antigens are not absolutely required for induction of protective immunity (Howard et al., 1984).We employed an intra muscular route for immunization. Intraperitoneal and intravenous routes have been reported to have same efficacy (Rivier et al., 1999) while the subcutaneous route led to disease exacerbation in the case of experimental L. major infection in BALB/c mice (Liew et al., 1985). However, a subsequent study of cutaneous leishmaniasis in CBA mice model showed equal potency of the subcutaneous and intravenous routes of immunization (Rivier et al., 1999). In BALB/c, intra peritoneal or subcutaneous immunization does not work as reported by Handman et al. (1990) and Connell et al. (1993). It was observed that the spleen and liver remained protected on challenge with virulent parasites in groups D and E but was less effective in group C that received parasites attenuated at lowest dose of irradiation. Thus, when we evaluated the efficacy of the irradiation doses, both groups D and E showed better protection than group C. In liquid culture, group C parasites survived and replicated for sometime suggesting they could overcome the effect of radiation and revert to infective state. This was apparent from the Giemsa stained photomicrographs of spleens from group C animals (data not shown) that 50 Gy might be inadequate for protection. Earlier, Ali and Afrin (1997) showed leishmania antigen (Lag) in the neutral liposomal vesicles resulted in only 70–73% protection while later a similar almost complete protection conferred against visceral leishmaniasis in both liver and spleen has been observed (Mazumdar et al., 2004). In another study, immunization with soluble L. donovani antigen, along with IL-12, against L. major, could induce effective immunity only in the liver of mice (Gurunathan et al., 1998). Immunization with a recombinant stage regulated surface protein from L. donovani, hydrophilic acylated surface protein BI (rHASPBI) induced significant protection in both liver and spleen (Stager et al., 2000). However, parasitic load reduction through both the immunogens was close to 50%. In our case, no adjuvant was used and a great extent of protection has been achieved for doses 100 and 150 Gy, respectively (about 88%). Fifty Gray group, on contrary, showed presence of parasites in the spleen (Fig. 3 and 50 Gy panel) and also in liver (data not shown) to a great extent compared to other two groups where parasites were almost absent in spleen. In terms of the organomegaly study of the spleen and liver, these two experimental groups showed encouraging results. We have experienced in our study that parasite burden in spleen has reached its peak at around four months post challenge and all data that have been presented here were of 120 dpi only. This was in congruence with the study of Mukherjee et al. (2003) on the infection pattern and immune response in the BALB/c mice infected intra-cardially by L. donovani parasites. Protection from the disease is achieved by the macrophage (Ma) killing of the parasites. This killing by Ma is not non-specific (Alexander, 1982) and kills parasites either with reactive oxygen species or with reactive nitrogen intermediates or both (Basu et al.2005). Ma activation by lymphokines results in a number of


physiological and metabolic changes in the host cell. Some of which might contribute the antileishmanial effects (Mosmann and Sad, 1996). This effect is closely related with the ability of these activated Mas to secrete high levels of reactive nitrogen intermediates (Kane and Mosser, 2001). To evaluate our system, we have carried out experiments to understand the status of reactive oxygen species as well as reactive nitrogen intermediates in healthy control group (A), infected group (B), and different immunized groups (C, D and E) respectively. We have observed higher release of both the species in groups D and E than group C. The expression of mixed Th1/Th2 in all immunized groups has varied; while group C receiving parasites attenuated at 50 Gy dose has shown more Th2 than Th1, other two immunized groups (D and E) have shown the reverse picture. In these two groups, we have noticed more Th1cytokine than Th2 cytokine release. IL-10 is another important cytokine which contributes in promoting the disease progression and has now become the focus of attention in visceral leishmaniasis (Mathur et al., 2004; Murray et al., 2005; Murphy et al., 2001). For the protection to be realized, immunized groups of BALB/c mice must show skewing of MHC (major histocompatibility complex) Class II restricted CD4 + T cells toward Th1 type (Mazumdar et al., 2004). Studies on experimental CL model in mice indicated that T-cell mediated immune responses play a central role in the progression or regression of the disease. It is the shifting of paradigm between Th1 and Th2 cell subsets that are operative for the onset or failure of the onset of the disease state. It is not the presence or absence but the predominance of a particular subset that determine the fate of the disease (Bogdan and Rollinghoff, 1998). In case of L. donovani infection in susceptible BALB/c mice, mixed Th1/Th2 responses were reported by many groups (Basu et al., 2005; Mazumdar et al., 2004). Control of visceral leishmaniasis in mice seems to be dependent on the release of IFN-c from the spleen cells which expresses the Th1 phenotype by IL-12 (Murray et al., 1997). In our study, we have noticed an increase in release of IL-12 from the two protected groups but not from the group C (Fig. 4E) again indicating the importance of selection of right dose of attenuation for preparation of vaccine candidates by gamma ray irradiation. Basu et al. (2005) suggested the stoichiometric proportion of IFN-c with TNF- a and immunosuppressive cytokine IL-10 would decide the fate of the disease. The TNF- a cytokine helps in removal of parasites from the liver and spleen and is directly related to the nitrite production (Mukhopadhyay et al., 2000; Mukherjee et al., 2003). We also observed enhanced TNFa release from two protected groups. We have not seen the protective roles of IL-4 as suggested by several authors (Basu et al., 2005; Bhowmick et al., 2007). This might be due to our experiments carried out at late stage of exposure to challenge infection (four months) while others showed the expression of Th2 cytokine IL-4 at the early infective stage and down regulation at the later stage of infection (Bhowmick et al., 2007). Another point of interest is that we have designed our study without adjuvant and vaccine without it is a better option provided the vaccine is effective. Many vaccines are prepared along with adjuvants to achieve maximum immunogenicity and when these are used without adjuvants, protection was minimal. In our study, we have achieved about 88% reduction in parasitic load in spleen of immunized animals. We have also undertaken a therapeutic study employing the same radio-attenuated parasites in same animal model through i.p. route (Datta et al., 2010). We observed the removal of parasites from the infected spleen and liver of BALB/c mice receiving radio-attenuated L. donovani parasites after challenge infection (Datta et al., 2010). Further studies on i.m. and other routes in our therapeutic study are underway.


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