Prophylactic Efficacy of High-Molecular-WeightAntigenic Fractions of a Recent Clinical Isolate ofLeishmania donovani Against Visceral Leishmaniasis

P. Tripathi*, S. K. Gupta�, S. Sinha�, S. Sundar§, A. Dube� & S. Naik*

Introduction

Visceral leishmaniasis (VL) is the most serious form,among the spectrum of clinical diseases caused by para-sites of the Leishmania species. In VL, Leishmania donovani

parasites infect macrophages of the spleen, liver andlymph nodes leading to progressive enlargement of theseorgans followed by cachexia and death if left untreated[1]. The disease is an important public health problemworldwide and a tenth of the world population is at risk

*Department of Immunology, Sanjay Gandhi

Postgraduate Institute of Medical Sciences,

Lucknow, India; �Division of Parasitology;

�DTDD, Central Drug Research Institute,

Lucknow, India; and §Department of Medicine,

Institute of Medical Sciences, Banaras Hindu

University, Varanasi, India

Received 15 April 2008; Accepted 22 July2008

Correspondence to: Dr S. Naik, Department of

Immunology, Sanjay Gandhi Postgraduate

Institute of Medical Sciences, Raebareli Road,

Lucknow 226014, India. E-mail: sitanaik@

sgpgi.ac.in

Abstract

T-cell mediated immune responses are key determinants to the natural courseof infection caused by intracellular parasites such as Leishmania. Thus, T-cellactivating proteins of these microbes continue to generate active interest par-ticularly in view of their possible role in the design and development of newerand more effective vaccines. We have recently reported the presence of T-cellimmunostimulatory antigens with the high-molecular-weight (MW) fractions(134–64.2 kDa) of whole Leishmania donovani antigen (strain 2001), whichstimulated variable amounts of IFN-c, IL-12 and IL-10 in exposed immuneindividuals. The present study was undertaken to further evaluate these high-MW antigenic fractions (MW range >100–60 kDa) for potential protectiveefficacy. The high-MW region of the parasite was resolved into five antigenicfractions (Prep A–E) using continuous elution gel electrophoresis. Prior toin vivo protection studies in hamsters, these fractions were used to evaluatein vitro cellular responses in eight Leishmania-exposed individuals and treatedcured hamsters. The protective efficacy of prep (A + B), C, D and E in combi-nation with BCG was evaluated in inbred hamsters using standard immuniza-tion protocol. Proliferative responses were seen in all eight of eight exposedindividuals to prep D [median stimulation index (SI): 5.2 (range 3.9–7.1)] andE [median SI: 5.6 (range 4.4–8.2)], five of eight individuals to prep B andprep C and three of eight to prep A [median SI: 0.2 (range 0.1–7.2)]. Themedian proliferative responses to prep D and prep E were significantly higherthan to fraction prep A; (P < 0.05) but not to prep B and prep C. However,prep A–E induced equivalent levels of IFN-c, IL-10 and IL-12 cytokines. Frac-tions D and E also exhibited marked parasite inhibition in spleen (52.5% and73.7%) and liver (65% and 80.2%) as compared with prep (A + B) (23% inspleen and 24% in liver) and prep C (38% in spleen and 24% in liver). PrepD and prep E vaccinated animals showed higher in vitro stimulatory responses(mean SI: 6.6 and 8.8) and nitric oxide (NO) induction (mean NO levels: 6.4and 10.7 lg ⁄ ml) against whole cell extract as compared with other groups.The protection also correlated with presence of suppressed Leishmania-specificIgG levels in prep D and prep E immunized hamsters. These studies indicatethe presence of immunostimulatory and protective molecules in 60–80 kDaregion of L. donovani, which may be further exploited for developing a subunitvaccine.

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of infection with >100,000 deaths in the recent epidem-ics of Sudan and India. It has also emerged as an impor-tant opportunistic infection in AIDS patients in manyparts of the world [2]. Since leishmanicidal drugs areexpensive, have serious side effects and drug resistance iscommon in endemic areas, the development of a vaccinehas become a global emergency [3].

The observations that only a small proportion of indi-viduals develop active disease in endemic areas and suc-cessfully cured patients seldom get reinfected, suggestthat vaccination against leishmaniasis is feasible [4].Although, several vaccination strategies have been testedin experimental leishmaniasis and a number of vaccinetrials have been initiated. The focus of these efforts hasbeen against the cutaneous form of leishmaniasis, causedby Leishmania major. Relatively fewer efforts have beenmade against VL and include the evaluation of attenuatedor killed parasites, crude antigen fractions, purifiedL. donovani membrane proteins and DNA vaccines [5].These strategies have had variable degrees of successagainst parasite challenge with L. donovani in experimen-tal models; however, these molecules have not beenexploited for human trials.

The control of leishmanial infection is mediated byTh1 type immune response and experimental studies inmurine models of cutaneous leishmaniasis (CL) haveestablished a clear-cut dichotomy between Th1-mediatedprotection and Th2-mediated disease susceptibility [4, 6].Although, in human CL, disease is associated with a Th2cytokine profile and immunity with a Th1 profile,immune response defining disease versus protection is notso well established in VL. Low IFN-c and IL-12 responses,the signature cytokines for Th1 responses, are seen duringacute VL [7, 8]. However, following successful treatmentthese responses revert to normal and high levels of thesecytokines along with high IL-10 levels persist for longperiods. Recently, it has been suggested that IL-10 plays arole in counterbalancing the exacerbated polarizedresponse that may develop following cure [6].

Earlier investigators have used the Th1 and Th2paradigm as a strategy for selection of antigen in vac-cine development against leishmaniasis. Thus, leishman-ial antigens that predominantly stimulate Th1 responsesin patients’ samples or mice infected with the parasitehave been recognized as ‘potential protective antigens’and promising vaccine candidates. Conversely, antigensthat predominantly stimulate a Th2 response have gen-erated lesser interest as vaccine candidates because theyare likely to be associated with pathology. However, ithas been observed that several leishmanial antigens thatinduce Th1 response during infection are not necessar-ily protective in vivo. Hence, it may not be appropriateto use stimulation of Th1 responses as readout for anti-gen selection in vaccine development against leishmani-asis [9].

We have previously reported the presence of T-cellimmunostimulatory antigens in the high-MW fractionsranging from 134 to 64.2 kDa, of total antigen repertoireof L. donovani [10]. These fractions did not stimulate apure Th1 response and produced variable amounts ofIFN-c, IL-12 and the regulatory cytokine, IL-10. In thepresent study, the same parasite antigen has been resolvedon continuous elution SDS-PAGE and five high-MW frac-tions corresponding to the previous immunostimulatoryregion have been evaluated for their prophylactic abilityin the hamster model for VL. They were also evaluatedfor their ability to stimulate cellular responses in leish-mania-exposed individuals, as well as treated cured inbredhamsters.

Materials and methods

Animals. Laboratory bred male golden hamsters (Mesoc-ricetus auratus, 45–50 g) were maintained at CDRI, Luc-know animal house. The animals were kept in plasticcages (38 · 27 · 13 cm) and fed with standard rodentpellets diet (Lipton India Ltd., Chandigarh, India) andwater ad libitum.

Culture of parasites. Leishmania donovani strain 2001,originally isolated from an Indian kala-azar patient, wasmaintained in golden hamsters as described previously[11]. Amastigotes from spleen of infected animals werecultured in L-15 medium (Invitrogen, Grand Island, NJ,USA) at 25 �C with 10% heat inactivated foetal calfserum (Invitrogen), 0.3% tryptose phosphate broth(Himedia, Mumbai, India) and gentamycin (Sigma, StLouis, MO, USA; 40 mg ⁄ l). Promastigotes were grownto stationary phase, harvested by centrifugation andwashed in phosphate-buffered saline (PBS) before use.

Preparation of antigens from L. donovani promastigotes.(1) Whole cell extract (WE): Stationary-phase promastig-otes were harvested by centrifugation and the pellet wasdissolved in SDS extraction buffer, vortexed and centri-fuged at 1500 g for 5 min. An equal volume of cold ace-tone was then added drop by drop to precipitate theprotein. The mixture was kept on ice for 15 min andcentrifuged at 2000 g for 15 min at 4 �C. Protein con-centration was determined and aliquots of antigen werestored at )80 �C.

(2) Antigenic fractions: Antigenic fractions were pre-pared from metacyclic parasites, pelleted by centrifuga-tion at 600 g for 15 min at 4 �C. Briefly, promastigoteswere washed and suspended in PBS containing proteaseinhibitors cocktail (Sigma) and centrifuged again at600 g for 15 min at 4 �C. Protein concentration wasdetermined and aliquots of antigen were stored at)80 �C. The antigen was reconstituted with PBS imme-diately before use.

(3) Fractionation of high-MW antigenic fractions: Frac-tionation of total parasite protein was done by continuous

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elution SDS-PAGE using Lammeli’s buffer system in a‘Prep cell’ (model 491; BioRad, Herculus, CA, USA). Aresolving gel concentration suitable for the whole rangeof proteins was determined by running a series of SDS-PAGE on mini gel slabs as described elsewhere [12].

Accordingly, 8% resolving gel (height 8 cm) and 4%stacking gel (height 2 cm) were cast in a 27 mm diame-ter tube. The parasite pellet comprising �20 mg proteinwas subjected to electrophoresis and fractions (100 ·3 ml) were collected starting immediately after the dyefront. UV absorbance at 280 nm was not used for moni-toring the elution profile as it failed to show individualpeaks, probably due to close association of proteins. Allthe fractions were analysed on mini slab gels and silverstained to visualize eluted proteins.

The fractions of interest (corresponding to identicalbands) were pooled to produce five minimallyoverlapping protein fractions covering the MW range:>100–60 kDa and lyophilized. The fractions were thenreconstituted in Milli-Q water, electrophoresed on 12%SDS-PAGE and visualized using silver staining (Fig. 1).The fractions, labelled as prep A–E were processed forremoval of SDS to be used for immunological studies asdescribed elsewhere [13]. For prophylactic studies inhamsters, fractions were used without removal of SDS.The five prep cell eluted antigenic fractions with theircorresponding MW are given in Fig. 1.

Study groups. Frozen peripheral blood mononuclearcells (PBMC) of eight leishmania-exposed individuals

[four males; median age 29 (range 26–33 years)] werethawed and used for the experiments. These individualswere from a kala-azar hyperendemic region in Bihar andtheir cells had shown strong positive in vitro T-cell pro-liferative response to WE of L. donovani in our previousstudy [10]. Five healthy adult donors [three males; med-ian age 26 (range 25–30 years)] from non-endemic regionserved as controls. Blood from each subject was drawnafter informed consent was obtained.

The cells were rapidly thawed using incompleteRPMI, washed three times and eventually resuspended tothe appropriate concentration in RPMI medium (Invitro-gen) supplemented with HEPES, L-glutamine, sodiumbicarbonate, sodium pyruvate, antibiotic–antimycoticmix (Sigma) and 10% heat-inactivated foetal calf serum(Invitrogen). The viability was assessed by trypan blueexclusion to be more than 70% in all samples.

Immunological studies. (1) Effect of WE and prep cellresolved antigenic fractions of L. donovani on human mono-nuclear cells: PBMC were suspended at a concentration of5 · 104 cells per well in cRPMI-1640 medium. Cultureswere set up in triplicates with WE (at 10, 5 and2.5 lg ⁄ ml) and prep cell eluted fractions, prep A–E (at1:10, 1:100 and 1:000 dilutions). Cultures with PHA(Invitrogen) at a dose of 1:100 served as positive control;wells without any stimulants served as negative controls.At the end of 24 h, 120 ll of supernatant was harvestedcarefully from each well and stored at )80 �C for cytokinedetermination. The wells were replenished with 120 ll ofcRPMI and further incubated at 37 �C in 5% CO2

atmosphere for 2 days for PHA-stimulated cultures and4 days for cultures stimulated with antigens. As responsesto 1:10 dilution of the fractions was consistently higherthan to 1:100 and 1:1000 dilution (data not shown), onlydata for 1:10 dilutions were analysed and subsequentcytokine analysis was done at this dilution.

Proliferation was assessed by thymidine (0.5 lCi ⁄ well,BARC, Mumbai, India) incorporation. Stimulation index(SI) was obtained by dividing the counts per minute(cpm) of stimulated cultures by the cpm of unstimulatedcultures. SI of 2.5 and above was taken as a positiveresponse. Individual having a positive proliferativeresponse of ‡2.5 to one of the three doses of WE wastermed responder. When positive response was observedto more than one dose, the highest value of LTT responsewas considered for analysis and the correspondingsupernatant for cytokine levels.

IFN-c, IL-12 and IL-10 levels in the 24 h antigenstimulated supernatants was determined by enzyme-linkedimmunosorbent assay kit (OptEIA set; Pharmingen, SanJose, CA, USA). The results were expressed as picograms ofcytokine ⁄ ml, based on the standards provided in the kit.The lower detection limits for various cytokines were asfollows: 4.7 pg ⁄ ml for IFN-c, 31.3 pg ⁄ ml for IL-12 and7.8 pg ⁄ ml for IL-10.

Figure 1 Silver stained 12% SDS-PAGE elution profile of prep cell

resolved into high-MW immunostimulatory antigenic fractions (prep

A–E; MW range >100–60 kDa) using total cell protein of L. donovani(strain 2001) promastigotes. MW is molecular weight marker and LD

represents the reference that is total cell extract of the parasite. Prep A

(>100 kDa), Prep B (100–90 kDa), Prep C (90–80 kDa), Prep D (80–

70 kDa) and Prep E (70–60 kDa).

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(2) Effect of WE and prep cell fractions on mononuclearcells of normal and treated cured hamsters: L. donovani-infected hamsters were treated intraperitoneally withsodium stibogluconate (SSG), as per protocol describedelsewhere [14]. Mononuclear cells were separated fromlymph nodes (inguinal and mesenteric) from treatedcured as well as normal hamsters and suspended at a con-centration of 1 · 106 cells ⁄ ml in complete RPMI med-ium as described previously [14]. Cultures were set up intriplicates with 100 ll of WE and prep A–E as per pro-tocol described in section Immunological studies. Cultureswith Con A (Sigma, USA) at a dose of 10 lg ⁄ ml servedas positive control.

Protection studies in hamsters. (1) Immunization andchallenge infection: Four hamsters each were immunizedintradermally with 25 lg ⁄ 0.05 ml ⁄ animal of prep A–Ein combination with BCG (0.1 mg ⁄ 0.05 ml). Fifteendays later, a booster injection of same amount of anti-gen along with BCG was given intradermally. Twenty-one days after the booster dose, the vaccinated andunvaccinated control groups were challenged intracardi-ally with 2 · 107 metacyclic promastigotes of L. dono-vani.

On day 45 post-challenge, two animals from eachgroup were sacrificed and body weight, liver and spleenweights were recorded and lymph nodes were removed.As only two animals were sacrificed, no statistical analysiswas done. Small groups (four animals in each group) weretaken for protection studies because of the limited avail-ability of antigen. Control animals were injected withPBS only. Peritoneal exudates cells, inguinal lymphnodes and blood were collected from hamsters pre-challenge and on day 45 post-challenge for evaluation ofcellular and antibody responses.

Survival of vaccinated hamsters was checked up to day90 post-challenge. Animal of all the groups were givenproper care and were observed for their survival period.Survivals of individual hamster were recorded and meansurvival period was calculated.

(2) Evaluation of parasite burden: Imprint smears of cutliver and spleen were methanol fixed, stained withGiemsa stain and examined under microscope to countparasite burden. The parasite burden was calculated asthe number of amastigotes per 1000 nucleated cells.

(i) Measurement of cell-mediated immune responses: In vitroT-cell proliferation was performed with Con A(10 lg ⁄ ml; Sigma) and WE (10 lg ⁄ ml) using mono-nuclear cells from vaccinated and unvaccinated groups. Inbrief, lymph nodes from individual hamsters wereremoved aseptically, placed in sterile petri dishes contain-ing RPMI medium and minced. Cell suspensions wereloaded on Histopaque 1.077 density gradient to isolatemononuclear cells as described previously [14]. For exper-imental use, the mononuclear cells were washed twicewith RPMI, adjusted to a concentration of

1 · 106 cells ⁄ ml. Cultures were set up in triplicates asper protocol described in section 2.5.1.

Animals were given intradermal inoculation of 50 llof WE (800 mg ⁄ ml) of L. donovani in one footpad andequal volume of PBS in the other footpad. The footpadthickness was measured after 24 h using a vernier cali-per and difference in thickness between the two foot-pads was taken as delayed type hypersensitivity (DTH)response.

(ii) Assessment of nitric oxide (NO) activity in macrophagesof hamsters: Nitrite accumulation in culture supernatantsof peritoneal exudates cells (PEC) harvested fromhamsters was used as an indicator of NO production andwas determined by the Griess reaction as previouslydescribed [15]. Briefly, 1 · 106 peritoneal PEC of curedhamsters were incubated with WE at 10 lg ⁄ ml concen-tration in 16 well plates. Supernatants (100 ll) were col-lected after 24 h, mixed with an equal volume of Griessreagent (Sigma, USA) and left for 10 min at room tem-perature. The absorbance of the test samples was mea-sured at 540 nm in an ELISA reader.

(iii) Measurement of antibody responses: Sera from immu-nized and infected hamsters were analysed by ELISA forthe presence of anti-leishmania antibodies (WE) asdescribed previously [14]. The plates were developedusing the OPD substrate (Sigma) and read at 450 nmusing an ELISA reader (Labsystems Multiscan MS,Helsinki, Finland).

Statistical analysis. Statistical analysis was performedusing SPSS 10 software and Mann–Whitney U-test forintergroup comparison of quantitative variables. P < 0.05was considered statistically significant.

Results

Evaluation of immunostimulatory activity of prep A–E in

leishmania exposed responder individuals

The separated fractions, prep A–E were evaluated fortheir capacity to stimulate an immune response inexposed immune individuals and immune hamsters. Apositive proliferative response (S.I ‡ 2.5) to WE was seenin all eight exposed individuals; the response [median SI:5.7 (range 4.3–9.8)] was significantly higher than healthyindividuals [median SI: 1.4 (range 1–2.1); P < 0.0001]

none of whom showed response to WE. This confirmedthat these fractions had in vitro immunostimulatory activ-ity. Positive proliferative responses to PHA were presentin all samples.

WE also stimulated significantly higher levels of IFN-c [median 317 pg ⁄ ml (range 131–445)], IL-12 [median1150 pg ⁄ ml (range 800–2676)] and IL-10 [median714 pg ⁄ ml (range 330–1100)] among the exposed indi-viduals than healthy controls (P < 0.0001; Fig. 2). Asproliferative responses were seen mostly with 1:10

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dilution of prep A–E, all cytokine analysis was performedonly with 1:10 dilution.

Prep D- and prep E-stimulated proliferative responsesin eight of eight exposed individuals, prep B and prepC in five of eight individuals and prep A in three ofeight individuals at 1:10 dilution (Fig. 3). The responseswere less frequent with 1:100 dilution and only occa-sional responses were seen to 1:1000 dilution (data notshown). The median proliferative responses of the eightexposed individuals to prep D [median SI: 5.2 (range3.9–7.1)] and prep E [median SI: 5.6 (range 4.4–8.2)]were significantly higher than to prep A [median SI: 0.2(range 0.1–7.2); P < 0.05] but not to prep B [medianSI: 3.4 (range 0.1–7.7) and prep C median SI: 4.1(range 0.1–7.1)] at a dilution of 1:10. The median pro-liferative responses of prep D, prep E and prep C werecomparable with each other as were those of prep A andprep B at 1:10 dilutions. None of the five healthy indi-viduals exhibited any response to any of the fractions(data not shown).

Prep A–E induced equivalent levels of IFN-c, IL-10and IL-12p40 cytokines in exposed responders, that isthe levels of these cytokines induced by each of the frac-tions prep A–E were comparable to each other (prep Aversus prep B to prep E: P = ns; Table 1). All the fivenon-endemic healthy individuals exhibited basal levels ofIL-12, IL-10 and IFN-c in response to prep A–E (datanot shown). Positive cytokine responses to PHA werepresent in all samples, which were comparable in healthycontrols and responder individuals.

Evaluation of immune response in hamsters

Lymphocytes from all SSG treated cured animals showedpositive proliferative response to WE (mean S.I: 16.7)and prep A–E (mean S.I: 7.5, 6.7, 8.7, 9, 10.3, respec-tively; Fig. 4), which was significantly higher(P < 0.0001) than of normal animals, none of whomshowed response. Prep D and prep E induced marginallyhigher proliferative responses than prep A, prep B andprep C; however, this difference was not statistically sig-nificant. Treated cured and normal animals showedequivalent proliferative responses to Con A (mean SI:26.5 and 31.2, respectively; P = ns) that was set up as apositive control.

Figure 2 IFN-c, IL-12p40 and IL-10 levels

in supernatants of PBMC from healthy

controls and leishmania-exposed responder

individuals stimulated in vitro with WE of

L. donovani. Each data point represents one

individual; median is indicated by hori-

zontal line. P indicates level of significance.

Statistical analysis was performed by Mann–

Whitney U-test. ‘n’ represents number of

individuals.

Figure 3 Proliferative responses of PBMC from leishmania exposed

responders stimulated in vitro with prep cell antigenic fractions (prep

A–E; 1:10 dilution) of L. donovani. Values are given as stimulation indi-

ces (SI). Each data point represents one individual; median is indicated

by horizontal line. SI of ‡2.5 was taken as positive response and is indi-

cated by dotted line. c versus a: P < 0.05; c versus b: P = ns. Statistical

analysis was performed by Mann–Whitney U-test. ns, not significant.

Table 1 Cytokine levels in supernatants of PBMC from exposed respon-

der individuals stimulated in vitro with fractions prep A–E (1:10

dilution) of Leishmania donovani.

Stimulants IFN-c IL-12p40 IL-10

Prep A 147 (89–152) 297 (101–378) 274 (203–302)

Prep B 135.3 (101–156) 330 (97–401) 288 (206–330)

Prep C 183 (121–190) 311 (134–413) 301 (290–312)

Prep D 140 (103–199) 299 (109–467) 287 (201–301)

Prep E 204 (121–221) 256 (101–378) 256 (199–278)

Values are given as median (range) concentration in pg ⁄ ml; n = 8 where

n represents number of individuals.

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Prophylactic efficacy of antigenic fractions (prep A–E) in

hamsters

Body weight and liver and spleen weights were recordedon day 45 post-challenge. The infected control animalswere noticeably cachectic and their body weights werereduced to almost half of that of age-matched normals(Table 2). Infected hamsters also developed nearly 15-and 4-fold increase of spleen and liver weights, respec-tively, over that of normal animals. These features arecharacteristic of failure to control infection. Animalsimmunized with BCG alone showed similar results as thePBS-treated infected group with 51.9% loss of bodyweight and nearly 12- and 4-fold increase in spleen andliver weight, respectively, in comparison to normalanimals.

While prep (A + B) + BCG, prep C + BCG and prepD + BCG vaccinated groups showed 48.5%, 40.6% and32.5% reduction in body weights, respectively, comparedwith normal animals prep E + BCG vaccinated animalshad body weights comparable to that of normal animals.

Prep (A + B) + BCG and prep C + BCG vaccinatedgroups showed considerable increase in liver and spleenweights, which were similar to that of infected unvacci-nated controls. Prep E + BCG and prep D + BCG vacci-nated groups had less marked increase in liver and spleenweights with those of prep E + BCG vaccinated groupbeing similar to the normal animals.

Parasitic burden in spleen, liver and bone marrow

The parasite burden in BCG-alone vaccinated group andcontrol animals was comparable in spleen (4234, 4720amastigotes ⁄ 103 cell nuclei), liver (937, 768 amastig-otes ⁄ 103 cell nuclei) and bone marrow (404, 442 am-astigotes ⁄ 103 cell nuclei), respectively (Table 3).

The parasite burden in prep (A + B) + BCG and prepC + BCG vaccinated groups was decreased in liver by24% in both groups, in spleen by 23% and 38%, respec-tively, and in bone marrow by 24% and 21%, respectively.The decrease in parasite burden in prep D + BCG vacci-nated group was 52.5% in spleen, 64% in liver and 52.5%in bone marrow, as compared with animals receiving PBSalone. Prep E + BCG immunized group showed the mostimpressive level of protection with decrease in parasiteburden of 73.7% in spleen, 80.2% in liver and 74% inbone marrow in comparison to control animals (Table 3).

Lymphoproliferative responses

Control animals and BCG immunized group had poorproliferative responses to WE on day 45 post-challenge.In contrast, the vaccinated groups exhibited variableresponses to WE with prep D + BCG (mean SI 6.6) andprep E + BCG (mean SI 8.8) vaccinated groups showingimpressive proliferative responses when compared withunvaccinated infected control (mean SI = 0.8). Prep(A + B) + BCG and prep C + BCG (mean SI of 3.8 and4.1, respectively) vaccinated groups showed lowerresponse to WE in comparison to prep D + BCG andprep E + BCG vaccinated groups (Fig. 5).

Table 3 Parasite burden in different organs of study groups on day 45

post-challenge.

Vaccinated

groups Spleen Liver Bone marrow

Infected control 4720 ± 229 937 ± 55 442 ± 29

BCG only 4234 ± 485 (10.3) 768 ± 28 (18) 404 ± 5 (8.6)

Prep A + B +

BCG

3633 ± 473 (23) 713 ± 32 (24) 335 ± 15 (24.2)

Prep C + BCG 2917 ± 104 (38.1) 710 ± 66 (24) 348 ± 28 (21.3)

Prep D + BCG 2242 ± 119 (52.5) 342 ± 38 (64) 210 ± 8 (52.5)

Prep E + BCG 1240 ± 40 (73.7) 185 ± 5 (80.2) 115 ± 5 (74)

Values represent the pooled data (mean ± SD) of two animals; parasite

burden expressed as number of amastigotes ⁄ 1000 cell nuclei in dab

smears; values given in brackets represent the percent inhibition.

Figure 4 Proliferative response of lymph node mononuclear cells iso-

lated from treated cured hamsters in response to stimulation with WE

(10 lg ⁄ ml) and antigenic fractions prep A–E (dilution of 1:10) of

L. donovani. Each bar represents the pooled data (mean ± SD) of three

experiments from three animals.

Table 2 Body and organ weights of vaccinated and unvaccinated

hamsters on day 45 post-challenge.

Vaccinated groups

Body weight

(n = 4)

Spleen

(n = 2)

Liver

(n = 2)

Normal 147.5 ± 4.1 0.3 ± 0.0 1.4 ± 0.2

Infected control 73 ± 5.5 (50.3) 4.4 ± 0.3 5.8 ± 0.3

BCG only 76.5 ± 3.5 (48.1) 3.7 ± 0.2 5.0 ± 0.1#Prep A + B + BCG 76 ± 6.0 (48.5) 3.7 ± 0.3 5.2 ± 0.3

Prep C + BCG 87.5 ± 7.6 (40.6) 2.4 ± 0.4 4.3 ± 0.2

Prep D + BCG 99.5 ± 5.5 (32.5) 1.3 ± 0.3 3.0 ± 0.1

Prep E + BCG 122.5 ± 7.6 (16.9) 0.5 ± 0.1 2.0 ± 0.3

n, number of animals in each group.#In prep A + B, mean body weight of three animals was taken as one

animal died before challenge with parasites.

Values are given as weight in grams (mean ± SD); values given in

brackets represent percent decrease ⁄ increase in comparison to normal

animals.

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Normal control animals showed impressive prolifera-tive responses to Con A (mean SI: 35.1) while infectedcontrol animals (mean SI: 2.1) and animals vaccinatedwith BCG alone (mean SI: 2.9) failed to show any prolif-erative responses. The responses of prep D + BCG (meanSI: 30.8) and prep E + BCG (mean SI: 34.5) vaccinatedgroups to Con A was similar to that of normal animalsand greater than responses to prep (A + B) + BCG (meanSI: 13.8) and prep C + BCG (mean SI: 16.1) vaccinatedgroups.

Production of NO in vaccinated hamsters

The NO levels generated by PECs in BCG-alone vacci-nated group (mean 3.8 lg ⁄ ml) was comparable to that ofinfected control group (mean 3.7 lg ⁄ ml) on day 45 post-challenge; Prep E + BCG vaccinated group producedmaximum NO (mean 10.7 lg ⁄ ml) in comparison togroups immunized with prep (A + B) + BCG (mean4.7 lg ⁄ ml), prep C + BCG (mean 5.6 lg ⁄ ml) and prepD + BCG (mean 6.4 lg ⁄ ml; Fig. 6).

Humoral responses in vaccinated and non-vaccinated

hamsters

Pre-immunization antileishmanial antibody levels of theexperimental groups ranged from 0.14 ± 0.03 to0.20 ± 0.02 OD values. The levels showed a slight

increase on day 15 following immunization and werebetween 0.17 ± 0.02 to 0.22 ± 0.01 OD values. Themaximum rise (OD: 0.69) was noticed in unvaccinatedcontrol group and the OD values for BCG-only vacci-nated group was 0.55 (Fig. 7). The antibody levels onday 45 post-challenge was 0.34 for prep E + BCG vacci-nated group and 0.56, 0.62 and 0.43 for prep A + B,prep C, prep D + BCG vaccinated groups, respectively.

Delayed type hypersensitivity response

Prep E + BCG immunized animals showed the maxi-mum DTH response (mean thickness 10 mm) supportingthe level of protection seen in this group. Control ani-mals (0.2 mm) and BCG vaccinated group (0.4 mm) didnot show any DTH response, while prep (A + B) + BCG(0.3 mm), prep C + BCG (0.4 mm) and prep D + BCG(0.5 mm) vaccinated animals had intermediate levels ofDTH response (Fig. 8).

Post-challenge mean survival of hamsters vaccinated with

prep cell fractions

Both prep E + BCG vaccinated animals (two ⁄ two), sur-vived ‡100 days post-challenge whereas it was ‡70 daysin two of two of the prep D + BCG vaccinated animals.For prep (A + B) + BCG and prep C + BCG vaccinated

Figure 5 Lymphoproliferative response (SI value) of study groups to

Con A (10 lg ⁄ ml) and WE (10 lg ⁄ ml) on day 45 post-challenge. The

number of animals was two in each group. Each bar represents the

pooled data (mean ± SD) of four replicates.

Figure 6 Levels of NO (lg ⁄ ml) produced by WE-stimulated peritoneal

macrophages of study groups on day 45 post-challenge. The number of

animals was two in each group. Each bar represents the pooled data

(mean ± SD) of four replicates.

Figure 7 Antileishmanial antibody levels (OD values) in study groups

on day 45 post-challenge. The number of animals was two in each

group. Each bar represents the pooled data (mean ± SD) of four repli-

cates.

Figure 8 DTH response in study groups on day 45 post-challenge.

Each bar represents the mean ± SD for two hamsters.

498 Protective Immune Responses in Leishmaniasis P. Tripathi et al...................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 68, 492–501

group it was <60 days. The survival of the BCG-alonevaccinated group and infected control group was £44 and£41 days, respectively.

Discussion

In the present paper, we report that antigens in the MWrange of 60–80 kDa, separated from a recent field isolateof L. donovani parasite provided significant protectionagainst promastigote challenge in a hamster model forVL. We have recently reported that high MW region(MW range 139–64.2 kDa) of this parasite had signifi-cant in vitro T-cell immunostimulatory activity [14]. Thisimmunostimulatory activity was evaluated using NCbound antigens, which cannot be used for animal immu-nization. Hence, the whole parasite antigen was fraction-ated using a continuous elution gel electrophoresis bywhich the 139–64.2 kDa region was resolved into fivefractions (labelled as prep A, prep B, prep C, prep D andprep E). The immunostimulatory capacity of this regionwas reconfirmed by the ability of these fractions toinduce in vitro T-cell proliferation and to stimulate pro-duction of the cytokines, IFN-c, IL-12 and IL-10. Immu-nization of hamsters with prep A–E along with BCG asan adjuvant confirmed that these fractions had variabledegrees of protection against L. donovani challenge. Ofthe five fractions, prep E (MW range 60–70 kDa) gavethe best protection, indicating the presence of strongprotective epitopes.

The five prep cell eluted fractions, prep A–E, elicitedfrequent proliferative responses in exposed immune indi-viduals and treated cured hamsters, with maximumresponses to prep D and prep E (MW range of60–80 kDa). All fractions induced comparable levels ofcytokines IFN-c, IL-12 and IL-10 in exposed immuneindividuals. The cytokine levels in hamsters were not asses-sed since reagents are not available. The levels of responsesto prep A–E in these eight exposed individuals were simi-lar to the responses seen in the same individuals when theywere tested earlier [14]. Thus, the immunostimulatoryactivity of the high-MW region was reconfirmed.

Protection studies in hamsters showed that prepD + BCG and prep E + BCG exhibited greater protec-tion against rechallenge than prep (A + B) + BCG andprep C + BCG. This was evident from the better bodyweight, small spleen size, higher in vitro proliferativeresponse of mononuclear cells to antigen and mitogen,Con A and NO production by peritoneal macrophages,low leishmania-specific IgG levels and good DTHresponses in these animals, which was comparable tonormal animals. In contrast, infected control and BCG-alone immunized animals showed poor Con A responses,no evidence of antigen-specific cellular immune responsesor reduction in parasite burden, which correlated withdisease progression during the course of the study. These

observations indicate that both prep E and prep D, butprep E more than prep D, in combination with BCGhave potent protective molecules.

In experimental systems [16] and in patients [17],active VL is associated with parasite-specific T-cell unre-sponsiveness, which is restored following cure [18]. Theabsence of mitogen-activated T-cell response in the con-trol animals with progressive systemic disease in thepresent study clearly shows that generalized immuno-suppression seen in clinical VL also occurs in thehamsters. This supports the rationale for using hamstersfor evaluating possible vaccine candidates for VL.

An important leishmanicidal effector mechanism isthe production of IFN-c by leishmania-specific T cellsthat in turn activates macrophages to kill intracellularparasites. The high-MW fractions under study, stimu-lated high IFN-c levels in exposed immune individualswho were not harbouring parasites. The antigen-stimu-lated NO produced by prep E + BCG and prepD + BCG immunized hamsters was also significantlyhigher than with the other fractions. As these animalsalso had low parasite burden, these data support the roleof NO-mediated macrophage effector mechanisms in thecontrol of parasite replication [19].

Active VL is characterized by high antibody titres,which is detected before parasitic specific T-cell responsesdevelop [20]; however, their protective role is controver-sial. In the present study too, antibody titres showed aninverse relationship with the parasite load, with highertitres in BCG vaccinated and unvaccinated control groupsas compared with the other vaccinated groups. The anti-body levels were low in prep E + BCG and prepD + BCG vaccinated groups and were comparable to thatof controls, emphasizing that successful vaccination reori-ents the immune response to a cell-mediated type. Thepost-challenge mean survival of hamsters was ‡100 daysin prep E + BCG and more than 70 days in prepD + BCG in comparison to the prep (A + B) andC + BCG groups and unvaccinated controls.

A Brazilian vaccine cocktail composed of the prom-astigotes from five Leishmania strains has been shown tobe safe and possess immunogenic properties [21].Attempts to identify the protective antigens from Leishv-acin� by immunoprecipitation with homologous sera,revealed the presence of eight major immunogenic com-ponents with estimated MWs of 13.5, 25, 40, 63, 73,85, 97 and 160 kDa. The results in field trials in ende-mic areas have shown that the preparation protected 70%of vaccinated subjects with skin test conversion. Some ofthese, including the 80, 70, 72 and 63 kDa L. donovaniantigens, have also been shown to confer partial protec-tion upon immunization of mice and several other mod-els [22–26].

The recently cloned glucose-regulated protein 78(GRP78) of L. donovani is a new and promising

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Leishmania vaccine candidate [27]. Formmel et al. (1988)[28] found effective immunity in BALB ⁄ c mice againstL. mexicana and L. major by subcutaneous injection of64–97 kDa proteins of L. major and L. infantum togetherwith muramil dipeptide (MDP) adjuvant. Malekzadehet al. (1998) [29] isolated fractions of L. major promastig-otes in the MW range of 40–60, 60–80 and 80–100 kDaproteins and showed that 60–80 kDa proteins inducedprotection upon subcutaneous vaccination of BALB ⁄ cmice.

Monjour et al. (1988) [26] have reported that anti-gens in MW range of 94–67 kDa (LiF2) from lysatesof L. infantum promastigotes confer resistance in mousemodels. Our protective antigenic fractions (prep D andprep E; MW range 60–80 kDa) may contain similarmolecules or gp63, one of the major parasite antigens,which is of MW range 60–70 kDa. However, molecu-lar characterization of the reactive fractions which donot correspond to previously described proteins isrequired and may lead to identification of novel anti-gens important in the immune response in humanleishmaniasis.

Thus, in summary our data show that prep E in com-bination with BCG, when used in a vaccination protocolconsisting of primary dose and a booster, is able to pro-vide effective protection against parasite challenge. Asthere are few attempts at vaccine design against visceraldisease caused by L. donovani, these findings need to beextended by further resolving the proteins in this prepa-ration.

Acknowledgment

We acknowledge the financial support of the Departmentof Biotechnology, Government of India, New Delhi forthis work. We thank Mrs Amrita Mathias for experttechnical assistance. Parul Tripathi is a PhD studentsupported by SGPGIMS, Lucknow.

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