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Induction of cellular and humoral responses by autoclaved and heat-killed antigen ofLeishmania donovani in experimental visceral leishmaniasis

Rajeev Nagill, Raman Mahajan, Meenakshi Sharma, Sukhbir Kaur ⁎Parasitology Laboratory, Department of Zoology, Panjab University, Chandigarh-160014, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 30 January 2009Received in revised form 14 July 2009Accepted 16 July 2009Available online 26 July 2009

Keywords:Autoclaved Leishmania donovaniHeat-killed Leishmania donovaniImmunizationVaccineAntigens

The potential of autoclaved and heat-killed antigen of Leishmania donovani to induce cell-mediated andhumoral response has been evaluated in the present study. The vaccines were delivered thricesubcutaneously at an interval of 2 weeks. Two weeks after second booster, BALB/c mice were challengedwith 107 stationary phase promastigotes of L. donovani. Significant protection was achieved in immunizedmice against L. donovani challenge with 69% to 76% and 59% to 64% reduction in parasite load in the liver andspleen respectively. Immunization induced significantly higher level of delayed type hypersensitivity (DTH)response in mice immunized with heat-killed antigen followed by autoclaved antigen. The immune responsewas assessed by quantifying Leishmania-specific antibodies and cytokine production. The antibody responsewas predominantly of IgG type with increased IgG2a production and lesser amount of IgM. Theimmunization preferentially stimulates the production of IFN-γ and IL-2 in splenocytes which suggests aTh1 type response with a concomitant down-regulation of IL-10 and IL-4. These results indicate a potentialfor the heat-killed and autoclaved antigen as a vaccine which could trigger cell-mediated immune response.

© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Leishmaniasis, caused by the parasitic protozoa Leishmania iscurrently endemic in 88 countries worldwide, including SouthernEurope, North Africa, the Middle East, Central and South America andthe Indian subcontinent, affecting 12 million people and threatening350 million more with an estimated 1.5–2.0 million new cases and80,000 deaths each year [1]. Several species of Leishmania causehuman diseases that range from self-healing cutaneous lesions to fatalvisceral leishmaniasis (VL), mucosal leishmaniasis and diffusecutaneous leishmaniasis. Cutaneous leishmaniasis (CL) is an anthro-ponotic or zoonotic disease with wild rodents, canids and marsupialsas reservoirs. On the other hand, VL, the most severe disease, is ananthroponose in India and Central Africa. VL is caused by Leishmaniadonovani (Africa, India, Asia), Leishmania chagasi (America) andLeishmania infantum (Mediterranean basin). CL is caused by L.mexicana and L. braziliensis complexes in Americas and L. major, L.tropica and L. aethiopica in the Old World [2,3]. The clinical spectrumranges from asymptomatic infections to highly fatal infections.Treatment is difficult and no acceptable vaccine exists against thisinfection. Thus, the development of an effective and affordable vaccineagainst leishmaniasis is one of the major goals of the World HealthOrganization [4].

The lack of effective and low-cost treatments and the irreversibilityof tissue damage during infection necessitate intensive efforts forvaccine development [5]. Although the current strategy for vaccinationagainst leishmaniasis is based on the use of purified fractions,recombinant antigens or from parasite cultures (excreted/secretedantigens) [6–15], whole killed parasite vaccines still remain a reliableperspective considering their biochemical composition, antigenicity,cost and safety, and a number of such vaccines have been tested [16,17].The use of these vaccines was usually accompanied by strong cellularreactivity, as observed by lymphocyte proliferation [18]. Successfulprotection against challenge infection has been achieved withautoclaved L. major in simians [19–22] and mice [23], with killed L.braziliensis in canine [24,25] and with autoclaved L. donovani inhamster models [26]. The use of nonliving vaccines in humans hasbeen confined to autoclaved and killed promastigotes with or withoutBacillus Calmette-Guerin as an adjuvant. Killed Leishmania vaccineshave been tested against human cutaneous and visceral leishmaniasis[27–31]. Vaccination with autoclaved L. major (ALM) along with BCGhas been found to be safe but does not induce significant protectiveimmune response in healthy volunteers against cutaneous leishma-niasis [30,32] and against visceral leishmaniasis [31,33]. However,immunization with killed L. amazonensis along with BCG showshealing rate of more than 95% against localized cutaneous leishma-niasis and cure is associated with a Th1 like immune response in thepatients [34–36]. A trivalent preparation consisting of L. braziliensis,L. guyanensis and L. amazonensis was evaluated in Ecuador whichshowed significant protection against cutaneous leishmaniasis[29,37,38]. The use of killed or pasteurized L. braziliensis along with

Parasitology International 58 (2009) 359–366

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BCG as vaccine candidate also conferred protective immune responseto dogs [18] and mucocutaneous patients [39] and more recently, avaccine comprising killed L. braziliensis, sand fly gland extract (SGE)and saponin elicited a strong antigenicity against L. chagasi infection,related to the increase of anti-Leishmania IgG isotypes and increasednitric oxide production after in vitro stimulationwith L. chagasi [24,40].The promising results of the above studies encouraged us to evaluateand compare the protective immune response induced by autoclavedL. donovani and heat-killed promastigotes against L. donovani in mice.

2. Materials and methods

2.1. Parasites

Promastigotes of L. donovani, strain Dd8, were grown at 22 °C inNNN medium supplemented with MEM (pH-7.2), 200 U of strepto-mycin, 200 U of benzyl penicillin and 40 μg of gentamycin per ml andsubcultured in the same medium after every 48–72 h.

2.2. Animals

Inbred BALB/c mice of either sex, weighing 20–25 g, were obtainedfrom the Central Animal House of Panjab University, Chandigarh. Theywere fed with water and mouse feed ad libitum.

2.3. Preparation of antigens

Promastigotes of L. donovani were grown to stationary phase inculture. Promastigotes were washed twice with PBS and suspended ata concentration of 108 parasites/ml.

Killed L. donovani antigen (KLD): heat-killed L. donovani antigen(KLD) was prepared by incubating the parasite suspension (108

parasites/ml) at 56 °C for 10 min. 0.1 ml of this suspension was usedfor immunization [41].

Autoclaved L. donovani antigen (ALD): the preparation of auto-claved L. donovani antigen was made by autoclaving the parasitesuspension at 15 psi for 30 min. Protein content was quantified byLowry method [42] and 100 μg of autoclaved antigen was used forimmunization [21].

2.4. Immunization and challenge infection

BALB/c mice (6–8 weeks old) were immunized with threesubcutaneous injections of 1×107 or 100 μg of autoclaved L. donovaniantigen (ALD) or heat-killed L. donovani antigen (KLD), at a 2-weekinterval. Animal receiving only PBS served as infected controls. 10animals per group were used for immunization. 15 days after the lastimmunization, 5 animals per group were challenged by intravenousinjections of 107 promastigotes and 5 animals were kept unchal-lenged. Serum samples were collected from unchallenged mice on 30,45 and 60 days of post immunization and from challenged mice on 15,30 and 45 days post-challenge. Sera were stored at −20 °C till furtheruse. Livers of all the mice were removed for assessment of parasiteload by Giemsa stained imprints and expressed as Leishman–DonovanUnits (i.e. [the number of amastigotes/200 cell nuclei]×the organweight in milligrams) [43].

2.5. Assessment of delayed type hypersensitivity (DTH) response

A delayed type hypersensitivity (DTH) test was performed ininfected and immunized and control animals by injecting 20 μl(2×108/ml) of leishmanin antigen in the left footpad and PBS in theright footpad. Thicknesses of both footpads were then measured bothbefore and 48 h after injection and compared. The results wereexpressed as percentage increase in thickness of left footpad incomparison to the right footpad [44].

2.6. Assessment of antibody response

The humoral immune response induced by the antigens wasevaluated by measuring total specific IgG, IgG1 and IgG2a levels byindirect ELISA from serum samples collected from different groups ofanimals. Briefly, 96 well plates were coated with antigen, andincubated overnight at 4 °C. After blocking with 4% bovine serumalbumin, plates were incubated with serum samples at 37 °C for 1 hfollowed by three washes and addition of specific secondaryantibodies (goat anti-mouse IgG, IgG1 or IgG2a) conjugated withhorse-radish peroxidase, after which the substrate and chromogenwere added and absorbance read on ELISA reader (Lisa Plus, USA) at492 nm.

2.7. Determination of vaccine-induced cytokine production

The lymphocytes were isolated from the spleens of immunized andinfected mice and then cultured. For this the spleens were removedand spleen cellswere isolated frommice immunizedwithALD andKLDantigen and counted in Neubauer's chamber [44]. 1×106 cells per wellwere cultured in RPMI-1640 containing20mMNaHCO3,10mMHEPES,100 U of penicillin per ml, 100 μg of streptomycin per ml, 2 mM l-glutamine, and 10% fetal calf serum (complete medium [CM]).Cultured cells were then stimulated with respective antigen (i.e.splenocytes from ALD immunized mice stimulated with ALD antigenand those from KLD immunized mice stimulated with KLD antigen).Splenocytes from infected control mice were cultured in completemedium only and not stimulated with any of the antigen. The super-natant of all cultureswas collected and stored at−20 °C till further use.Thesewere subsequently used for the determination of cytokine levelsusing commercially available kits from Bender MedSystems, Austria(IL-4 and IL-10) and Diaclone, France (IFN-γ and IL-2).

2.8. Statistical analysis

All the experiments were carried out three times independently.All data comparisons were tested for significance by using Student'st-test; p-values below 0.05 were considered significant. Results wereexpressed as mean±S.D. of one of three independent experiments.

3. Results

3.1. Parasite burden and protection

Progression of infection in L. donovani infectedmicewasmonitoredin the liver and spleen by microscopic examination of impressionsmears and represented as Leishman–Donovan Units. The parasiticload in the liver reachedmaximum at day 30 post-challenge, however,the splenic parasite load began to rise slightly later and reachedmaximumat day 45post-challenge. Significant reduction (pb0.001) inhepatic and splenic parasite load was observed in both immunizedgroups as compared to controls on all post-challenge days. Immuniza-tion with heat-killed (KLD) and autoclaved Leishmania antigen (ALD)induced significant resistance to challenge infection as observed inliver (66.3% and 58% reduction in parasite load in KLD and ALD antigengroup respectively) on day 15 post-challenge which further increased(76% and 69% reduction in parasite load) on day 45 post-challenge. Inspleen, parasite load was reduced by 59% and 49% in KLD and ALDantigen immunized animals respectively on day 15 post-challengewhich further reduced by 64% and 59% respectively on day 45 post-challenge. When both immunized groups were compared, heat-killed(KLD) antigen conferred significantly higher degree of protection inboth the liver and spleenof animals as against that by autoclaved (ALD)antigen (Fig. 1).

The progressive increase in the parasite burden in liver led tomoderate hepatomegaly with 1.4 fold increase in liver weight of

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infected control mice as compared to same-aged normal mice. A slightbut significant increase in liver weight was observed in immunizedmice as compared to normal mice. Splenomegaly was observed ininfected control mice (1.3 to 1.7 fold increase) as well as in immunizedmice (1.15 to 1.2 fold increase) (data not shown).

3.2. Delayed type hypersensitivity (intradermal reaction to promastigotelysate)

The intradermal reaction to leishmanin was measured as an indexof cell-mediated immune response. The footpad swelling wasmeasured after 48 h of injection. Immunization exhibited strongDTH responses even in unchallenged and challenged mice ascompared to control mice. It further increased to 1.6 to 1.9 fold afterchallenge infection in immunized mice. The heat-killed (KLD) antigen

conferred maximum intradermal response followed by autoclavedantigen, however, the difference between themwas not significant. At15 days post-challenge, a significant amount of DTH response wasseen in infected control mice but the level was comparable to normalmice on day 45 post-challenge. The strong DTH response elicited byimmunized groups correlated with the protection against infection inthis group (Fig. 2).

3.3. Anti-leishmanial antibody responses

Mice were immunized with three subcutaneous injections of heat-killed (KLD) antigen and autoclaved (ALD) antigen at an interval of2 weeks. 15 days after second booster, half the mice were challengedwith 1×107 promastigotes and half of them remained unchallenged.Serum samples from unchallenged immunized mice, challenged

Fig. 1. Parasite burden in liver (A) and spleen (B) in terms of LDU in BALB/c mice immunized with autoclaved Leishmania antigen (ALD), heat-killed Leishmania antigen (KLD)and infected control animals. The data are presented as mean±S.D. of five mice per group. ⁎p value: ALD vs Infected control. †,# p value: KLD vs ALD. ⁎ – (pb0.001), # – (pb0.01),† – (pb0.05).

Fig. 2. (A) Footpad swelling in unchallenged BALB/c mice immunized with autoclaved Leishmania antigen (ALD) and heat-killed Leishmania antigen (KLD) on different postimmunization days. Normal mice represent the footpad swelling in normal unchallenged mice. (B) Footpad swelling in BALB/c mice immunized with ALD and KLD antigen afterchallenge infection on different post-challenge days. The data are presented as mean±S.D. of five mice per group. ⁎p value: ALD (unchallenged) vs normal mice. # p value: ALD vsInfected control. ⁎,# – (pb0.001).

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immunized mice and control mice were collected and analyzed byELISA.

The Leishmania-specific IgM, IgG and isotypes of IgG (IgG1 andIgG2a) were studied in sera of unchallenged and challenged mice.Immunization of mice with autoclaved and heat-killed antigenbrought significant increase in serum levels of anti-leishmanial IgMand IgG antibody as compared to normal mice. After challengeinfection, the antibody level further increased at 15, 30 and 45 days ofpost-challenge. With the disease progression, levels of these anti-bodies were also increased in infected control mice. Further analysisdemonstrated that the production of anti-leishmanial IgG2a antibodyincreased significantly in immunized animals. Though mice immu-nized with heat-killed antigen produced maximum amount of IgG2aantibody followed by autoclaved antigen, it further increased onsubsequent post-challenge days. However, themaximum level of IgG1antibody was seen in infected control mice (Figs. 3 and 4).

3.4. Induction of Th1 cytokines in vaccinated group

The supernatants of heat-killed (KLD) and autoclaved (ALD)antigen stimulated spleen cell cultures were collected at differentpost immunization and post-challenge days and stored at−20 °C. Theimmune response was determined by analyzing the cytokine profilesof the stimulated spleen cells. The levels of IL-2, IFN-γ, IL-4 and IL-10secreted by cells were measured by using cytokine assay kitsaccording to the manufacturer instructions (Diaclone, France; BenderMedSystems, Austria).

Spleen cells from mice immunized with autoclaved (ALD) andheat-killed antigen (KLD), upon stimulation with respective antigen,produced significantly higher amount of IFN-γ and IL-2 as comparedto infected control mice which further enhanced after challengeinfection. However, the amount of IL-4 and IL-10 was seen higher inspleen cells of infected control mice. The peak level of Th1 cytokines

Fig. 3. Levels of Leishmania-specific IgM, IgG, IgG1 and IgG2a in sera of unchallenged BALB/c mice immunized with autoclaved Leishmania antigen (ALD) and heat-killedLeishmania antigen (KLD) on different post immunization days. Normal mice represent the values of IgM, IgG, IgG1 and IgG2a antibody in normal unchallenged mice. (A) IgM,(B) IgG, (C) IgG1, (D) IgG2a. The data are presented as mean±S.D. of five mice per group. ⁎ p value: ALD (unchallenged) vs normal mice. †,# p value: KLD (unchallenged) vs ALD(unchallenged). ⁎,# – (pb0.001), † – (pb0.01).

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i.e. IFN-γ and IL-2 production were observed in spleen cells of miceimmunized with heat-killed antigen at 30 days post-challenge (Figs. 5and 6).

4. Discussion

In the present study we have investigated the potential of heat-killed and autoclaved L. donovani antigens against challenge withmetacyclic promastigotes in BALB/c mice. Our results suggest that thevaccination with heat-killed (KLD) and autoclaved (ALD) antigenresulted in significant protection against a progressive infection withL. donovani. Immunization with KLD reduced the parasite load by66.3% in liver and 59% in spleen at day 15 after challenge infectionwhich further reduced to 76% and 64% in liver and spleen respectivelyover the control mice at 45 days post-challenge. LDU levels of miceimmunized with autoclaved antigen (ALD) showed reduction inparasite load by 58% and 49% at day 15 post-challenge and 69% and59% in liver and spleen respectively at day 45 post-challenge (Fig. 1).

Reproducible levels of protection in BALB/c mice by the antigensindicate the efficacy of these vaccine candidates.

The partial protection by L. donovani crude antigen against L. majorhas been previously reported [45–47] and cross-protection offered byautoclaved Leishmania major (ALM) against challengewith L. donovanihas been reported in monkey model [19]. Killed parasite preparationsi.e. L. mexicana mixed with BCG and L. amazonensis (combined withlow dose of antimonial), have shown efficacy in immunotherapy andimmuno-chemotherapy in humans, respectively [48]. SSG+Alum/ALM+BCG [49] have been shown to significantly improve the therapyfor PKDL patients. Srivastava et al. [26] studied the efficacy of ALD andALM and suggested that ALD along with BCG can offer substantialprotection against visceral leishmaniasis in hamsters.

The cell-mediated immune response is often related to antigeninduced DTH response in vivo, and T-cell stimulation with mitogenand antigen in vitro [29,50]. BALB/c mice immunized with KLD elicitedstrong DTH response in unchallenged as well as in challenged group.Simultaneously, these animals also displayed strong antigen recall

Fig. 4. Levels of Leishmania-specific IgM, IgG, IgG1 and IgG2a in sera of BALB/c mice immunized with ALD and KLD antigen after challenge infection on different post-challenge days.(A) IgM, (B) IgG, (C) IgG1, (D) IgG2a. The data are presented as mean±S.D. of five mice per group. ⁎,†,‡ p value: ALD vs Infected control. ⁎ – (pb0.001), † – (pb0.01), ‡ – (pb0.05).

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responses. A significant level of DTH responses was also seen inanimals immunized with ALD suggesting a correlation between cell-mediated immune responses, and immunity to infection in mice.Lower level of parasitic-specific DTH responses in infected controlscorrelated with the disease progression in mice.

The distribution of parasite-specific antibodies (IgM, IgG, IgG1 andIgG2a) further characterized the humoral responses. The responsewas predominantly of IgG type with lesser amount of IgM component.IgG antibody response following the parasite infection, reflected theparasite load, was observed to be highest in infected controls and lessin KLD and ALD vaccinated groups. Immunization with KLD and ALDstimulated the induction of IFN-γ dependent IgG2a anti-leishmanialantibody along with antigen-specific IL-4 dependent IgG1. Challengeinfection further boosted the production of IgG2awith a little increasein IgG1. These results suggest an initial induction of Th1/Th2 typeresponse which polarized towards Th1 response upon challengeinfection. Our results are in close consistent with the studies ofBretscher et al. [51] where susceptible mice infected with L. majormount a Th2 response and produce IgG1 antibodies, whereas resistantmice suppress this activity and show enhanced IgG2a responses. Our

data demonstrate the development of cell-mediated immuneresponse of Th1 type as a result of immunization with KLD and ALDwhich correlated with the resistance against the visceral disease.

The disease caused by L. donovani infection caused severedepression of cell-mediated immunity (assessed by proliferativeresponse as well as level of cytokines namely, IL-2 and IFN-γ) inmonkey model [52] and in the kala-azar patients [53,54]. Control ofvisceral leishmaniasis in mice is believed to require IFN-γ, producedby splenic cells, that drives the immune response towards a protectiveTh1 type [55–57] with low levels of IL-4 [58]. Similar trend of cytokinepattern was observed in our study as splenic T cells of immunizedanimals produced higher amount of IFN-γ and IL-2 with down-regulation of IL-4 and IL-10. However, some studies indicate that evenvaccines triggering high levels of IFN-γ do not protect in the presenceof high levels of regulatory cytokine IL-10 [58]. Disease progression ininfected control mice corresponded with IL-4 production and a lack ofIFN-γ by spleen cells. Moreover, IL-4 and IL-10 act together in thepresence of exacerbatory antigens [59]. Vaccination also induced IL-4secreting CD4+ T cells along with IFN-γ, but the arrest of challengeinfection corresponded with a dominant Th1 response and down-

Fig. 5. Cytokine levels in unchallenged BALB/c mice upon immunizationwith autoclaved Leishmania antigen (ALD) and heat-killed Leishmania antigen (KLD). Normal mice representthe values of IFN-γ, IL-2, IL-4 and IL-10 in normal unchallenged mice. (A) IFN-γ, (B) IL-2, (C) IL-4, (D) IL-10. The data are presented as mean±S.D. of five mice per group. ⁎p value:ALD (unchallenged) vs normal mice. †,# p value: KLD (unchallenged) vs ALD (unchallenged). ⁎,# – (pb0.001), † – (pb0.01).

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regulation of Th2. Thus, immunization with heat-killed antigen (KLD)and autoclaved L. donovani antigen (ALD) in the present study sig-nificantly enhanced the protective immune response against murinevisceral leishmaniasis.

Acknowledgement

The authors hereby declare that the experiments comply with thecurrent laws in India.

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