Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis...

Photodynamic vaccination of hamsters with induciblesuicidal mutants of Leishmania amazonensis elicitsimmunity against visceral leishmaniasis

Shraddha Kumari1, Mukesh Samant1, Prashant Khare1, Pragya Misra1,

Sujoy Dutta2, Bala Krishna Kolli2, Sharad Sharma3, Kwang Poo Chang2

and Anuradha Dube1

1 Division of Parasitology, Central Drug Research Institute, Lucknow, India2 Department of Microbiology/Immunology, Chicago Medical School, Rosalind Franklin

University, North Chicago, IL, USA3 Division of Toxicology, Central Drug Research Institute, Lucknow, India

Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier

for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to

partially rectify their defective heme-biosynthesis renders them inducible with d-amino-

levulinate to develop porphyria for selective photolysis, leaving infected host cells

unscathed. Delivery of released ‘‘vaccines’’ to antigen-presenting cells is thus expected to

enhance immune response, while their self-destruction presents added advantages of

safety. Such suicidal L. amazonensis was found to confer immunoprophylaxis and

immunotherapy on hamsters against L. donovani. Neither heat-killed nor live parasites

without suicidal induction were effective. Photodynamic vaccination of hamsters with the

suicidal mutants reduced the parasite loads by 99% and suppressed the development of

disease. These suppressions were accompanied by an increase in Leishmania-specific

delayed-type hypersensitivity and lymphoproliferation as well as in the levels of splenic

iNOS, IFN-c, and IL-12 expressions and of Leishmania-specific IgG2 in the serum. More-

over, a single intravenous administration of T cells from vaccinated hamsters was shown

to confer on naı̈ve animals an effective cellular immunity against L. donovani challenges.

The absence of lesion development at vaccination sites and parasites in the draining

lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe

platform for vaccine delivery against experimental visceral leishmaniasis.

Key words: Porphyrinogenic mutant . Suicidal vaccination . T-cell-adoptive transfer .

Visceral leishmaniasis

Introduction

World Health Organization (WHO) considers leishmaniasis to be

one of the most serious, epidemic-prone parasitic diseases of the

poor and disadvantaged. An estimated 350 million people are at

risk of Leishmania infection worldwide with �1.5 million new

cases of cutaneous leishmaniasis (CL) and 500 000 cases of

visceral leishmaniasis (VL) reported each year. Recent epidemics

of VL or kala-azar in Sudan and India have resulted in over

100 000 deaths [1]. With the advent of the HIV epidemics, VL has

emerged as an important opportunistic infection in AIDS patients

[2, 3]. In India, high incidence of kala-azar has been reportedCorrespondence: Dr. Anuradha Dubee-mail: [email protected]

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.200838389 Eur. J. Immunol. 2009. 39: 178–191Shraddha Kumari et al.178

from the states of Bihar, Assam, West Bengal and Eastern Uttar

Pradesh. A recent survey in Bihar has recorded an alarming

100 000 cases, of which 10 000 are unresponsive to antimonials.

The numerous relapses due to the increasing incidence of drug

resistance are also associated with the toxic anti-leishmanial

drugs in use [4]. Hence, the development of an effective vaccine

becomes all the more urgent in the absence of suitable anti-

leishmanial drugs to control this disease.

Effective vaccines for VL require their activity to elicit cell-

mediated immunity (CMI) capable of activating macrophages to

a microbicidal state [5]. Further, it is desirable to have a suitable

animal model for human kala-azar adequate for realistic

evaluation of vaccine safety and efficacy. Golden hamster

(Mesocricetus auratus) is the best animal model of human VL in

having the same clinico-pathological features, including a

relentless increase in visceral parasitic burdens, progressive

cachexia, hepatosplenomegaly, pancytopenia, hyper-gamma-

globulinemia and ultimately death [6, 7].

Several vaccination strategies using live, killed and defined

vaccines have been attempted with particular emphasis on their

efficacy against CL instead of VL [3]. The only successful immuni-

zation strategy in humans has been the ‘‘leishmanization’’, which is

based on the development of durable immunity after recovery from

infection at a chosen site, usually the arm, with viable non-attenuated

parasites [8, 9]. The use of live vaccines is very promising since they

most closely mimic the natural course of infection and, therefore,

may elicit similarly effective immunity after cure. However, its use

has been restricted or abandoned entirely due to the safety concerns.

Nevertheless, WHO/Tropical Disease Research (WHO/TDR) has very

recently revived the consideration of using live, but genetically atte-

nuated vaccines [10]. Other potentially usable live vaccines include

Leishmania administered at a low dose [11], drug-attenuated Leish-

mania [12], recombinant Leishmania expressing cytokines [13] or

suicide markers [14] and parasites with CpG-oligodeoxynucleotide

motifs [15]. However, these experimental vaccines have yet to reach

the clinical trial stage. While each of them produced some level of

protective efficacy, the completeness and longevity of this protection

has not been clearly demonstrated. Hence, no effective vaccine

currently exists against any form of human leishmaniasis.

Recently, a xenotransgenic-L. amazonensis was constructed by a

novel suicidal design, which was predicted to effect its specific and

selective destruction in infected macrophages [16]. This inducible

suicidal mutant was produced transgenically to partially rectify its

natural defects in heme-biosynthesis. The second and third enzymes

(ALAD: d-aminolevulinate dehydratase, PBGD: porphobilinogen

deaminase) of the heme-biosynthetic pathway were episomally

expressed in Leishmania. This transfectant was thus responsive to

d-aminolevulinate (ALA), the product of the first enzyme in this

pathway, for accumulation of photosensitive uroporphyrin. This

porphyrin can be excited by light to produce leishmanolytic oxidative

species. With a complete heme-metabolic pathway, the host cells

only develop a very low level of transient porphyria in response to

ALA. This parasite versus host-cell difference in accumulation of

porphyrins was exploited to achieve selective photolysis of Leishma-

nia in the phagolysosomes of the antigen-presenting cells. The basic

principle for using the suicidal mutants as vaccine carriers is that they

retain infection molecules for homing to phagolysosomes, thereby

delivering vaccines to the desirable destination. Induction of their

photolysis therein achieves not only vaccine release for appropriate

processing and presentation but also their self-destruction.

An ideal live vaccine should elicit long-term effective immune

responses without undue persistence in the vaccinated host, as

safety is an important criterion for the use of live vaccines in

humans. The potential utility of the suicidal mutants as a vaccine

candidate was explored here against Indian kala-azar in hamster

model. Results obtained demonstrated the efficacy of such

photodynamic vaccination. In addition, a long-lasting protective

immunity was found to develop by a single intravenous injection

of lymphnode T cells from porphyrinogenic vaccinated hamsters.

This adoptive transfer is capable of protecting naı̈ve hamsters to

L. donovani challenges.

ResultsAbsence of cutaneous lesion and amastigotes inporphyrinogenic vaccinated hamsters

The site of vaccination was inspected in all groups for evidence of

any cutaneous lesions – a characteristic of CL. The lesions of

0.3–0.65 cm in diameter appeared at the site of injection after

3 wk of vaccination, but only in animals of the control non-

porphyrinogenic groups, viz. DT�ALA and ST1ALA (see Materi-

als and methods). In contrast, porphyrinogenic (DT1ALA)

produced no visible lesion throughout the experiment period till

day 180 post-challenges (p.c.), indicative of an excellent safety

margin. This was further confirmed by histopathological studies

of the skin in separate groups of hamsters 3 wk after vaccination

(Fig. 1). In skin sections of porphyrinogenic vaccinated animals,

only uninfected macrophages were observed without inflamma-

tory cell infiltrates (Fig. 1A and B). In contrast, the control

groups, i.e. DT�ALA and ST1ALA, produced lesions, which

clearly resulted from inflammatory responses due to cell

infiltrates, consisting mainly of lymphocytes, plasma cells, and

Leishmania-infected macrophages (Fig. 1C–F). No infected cells

were seen in histological sections of spleens, livers, and

lymphnodes from DT1ALA hamsters. Cultures of tissues from

the cutaneous sites of vaccination, their draining lymphnodes,

spleens, and livers yielded no promastigotes after incubation for 1

month at 261C, providing further evidence for the absence of

detectable parasites in these animals.

Vaccination with porphyrinogenic-L. amazonensisinduced optimum protection against L. donovanichallenges

The porphyrinogenic-mutant (DT1ALA) vaccinated hamsters

were protected from the challenge infection of L. donovani, as

indicated by their weight gain with time, like non-vaccinated and

Eur. J. Immunol. 2009. 39: 178–191 Immunity to infection 179


non-challenged group (Fig. 2A, solid bar versus blank bar) in

contrast to the significant weight loss (po0.001) of all the control

groups, i.e. infected, ALA, ST1ALA, DT�ALA (bars in between

the solid and the blank bars). Hepatosplenomegaly, normally

associated with the challenge infection, was absent in the

porphyrinogenic vaccinated group (Fig. 2B and C, solid bar).

This phenotype was evident, albeit much less pronounced in the

beginning, in the two groups vaccinated with non-porphyrino-

genic controls, indicative of their ineffective protection (Fig. 2B

and C, stippled and hatched bars). Parasite loads were positively

correlated with splenomegaly and hepatomegaly observed among

different groups: an increase from �103 to 4104 in all the

groups, except the porphyrinogenic vaccinated groups (Fig. 2D,

solid bar versus the rest), as seen in Geimsa-stained splenic

smears, from day 45 to day 120 p.c. In this last group, parasite

loads decreased from �2� 102 on day 45 to a negligible level

(po0.001) of o10 subsequently, rendering them difficult to

discern by microscopy (not shown). Similarly, in liver and bone

marrow, parasite loads sharply decreased after day 45 p.c. and

parasites were essentially absent by day 180 p.c. in the same fully

vaccinated group in contrast to all the control groups (Fig. 2E and

F, solid bars versus the rest). Cultivation of the spleen, liver, and

lymphnode tissues from the fully vaccinated hamsters in vitro

yielded no promastigotes after prolonged incubation for 3 wk.

Porphyrinogenic-L. amazonensis (DT1ALA) vaccinated

hamsters survived the challenges of L. donovani and remained

healthy thereafter until the termination of the experiment 6

months p.c. In contrast, hamsters vaccinated with non-porphyr-

inogenic mutants, i.e. DT�ALA or ST1ALA survived for only 3–4

months, while all those untreated or treated with ALA alone died

of kala-azar within �2 months.

Immunization of hamsters with killed DT mutants under the

same vaccination conditions gave only limited prophylactic effi-

cacy. This was demonstrated in a separate set of experiments, in

which three groups of hamsters at five males each were vacci-

nated for comparison with autoclaved DT, DT1ALA, and

DT�ALA, respectively. Evaluation of splenic amastigote loads 45

days p.c. showed parasite suppression of 95, 43, and 31% for DT

1ALA, DT�ALA, and killed DT, respectively. Photodynamic

vaccination with live DT1ALA was shown again to give the most

complete protection that is unmatched by either live DT alone or

killed DT. Since the killed mutants were least effective, they were

not included for further investigation.

Porphyrinogenic vaccination stimulates delayed-typehypersensitivity, mitogenic, and Leishmania-specificcellular responses

Porphyrinogenic and non-porphyrinogenic immunizations

elicited different CMI, as determined by assessing the delayed-

type hypersensitivity (DTH) and lymphoblast proliferation

responses to specific and/or non-specific antigens in these

hamsters after L. donovani challenges at different time periods.

Porphyrinogenic vaccinated hamsters displayed significant DTH

response, which increased progressively throughout the entire

p.c. period (Fig. 3A, DT1ALA) and reached the levels that were

significantly higher than those of the control groups (po0.001) at

all time points for the duration of the experiments for up to 180

days. The DTH response increased in the non-porphyric control

groups (DT�ALA, ST1ALA), but this increase was rather modest.

In vitro stimulation of the lymphocytes with Concanavalin A

(Con A) showed comparable proliferative response at high levels

in all the groups when assayed before challenges (Fig. 3B, day 0).

Con A induced lymphoproliferative response remained elevated

in porphyrinogenic vaccinated animals as much as those of the

normal hamsters throughout the entire p.c. period (Fig. 3B,

normal and DT1ALA bars), but it decreased precipitously with

time in all the other control groups (bars between normal and DT

1ALA). In soluble leishmania donovani (SLD)-specific re-stimu-

lation assays, lymphoproliferative response was negative

for all groups on pre-vaccination day 0 (Fig. 3C, blank bars)

and for the non-vaccinated control groups throughout

the p.c. period (normal, infected, and ALA). Cells from

porphyrinogenic vaccinated hamsters produced significantly

higher response (po0.001), which reached almost to the maxi-

mum on day 45 post-vaccination and increased further thereafter

Figure 1. Histopathology of the skin of hamsters vaccinated withtransgenic-L. amazonensis. Tissues were collected 3 wk post-vaccina-tion from hamsters immunized with DT1ALA (A and B), DT�ALA(C and D), and ST1ALA (E and F). Sections were processed and stainedwith H&E. Micrographs taken under 40� and 100� objectives shownin the left panel (A, C, E) and the right panel (B, D, F), respectively. Note:the normal microarchitecture of the skin (A) and the absence ofamastigote-infected macrophages (B) in the porphyrinogenic DT1ALAvaccinated hamsters; inflammatory responses due to cell infiltrationof the skin (C, E) and abundant vacuolated macrophages containingLeishmania-amastigotes (D, F circles and arrows) in non-porphyrino-genic DT�ALA and ST1ALA vaccinated hamsters.

Eur. J. Immunol. 2009. 39: 178–191Shraddha Kumari et al.180


(Fig. 3C, DT1ALA). In contrast, those from non-porphyrinogenic

vaccinated hamsters registered only a slight increase of this

response on day 45 post-vaccination, but declined almost to the

baseline level thereafter on day 120 shortly before their death

(ST1ALA and DT�ALA).

Lymphocyte-mediated activation of macrophages to produce

NO for leishmanicidal activities was found to differ between

control and experimental groups of hamsters. Macrophages

isolated from naı̈ve hamsters were exposed to the supernatants of

SLD-stimulated lymphocytes from these animals. Porphyrino-

genic vaccinated group was found to elicit significantly higher

level (po0.001) of NO, which increased with the p.c. periods up

to day 180 (Fig. 3D, DT1ALA). Non-porphyrinogenic controls

(Fig. 3D, ST1ALA, DT�ALA) elicited a modest increase of NO

production, which was seen only on day 45 p.c. and declined

gradually thereafter to the baseline levels, as observed in the

other control groups (normal, infected control, and ALA).

Porphyrinogenic vaccination alters Leishmania-specificIgG and its isotypes

The serum levels of leishmanial antigen-specific IgG and its

isotypes (IgG1 and IgG2) from all groups were assessed by ELISA.

The anti-Leishmania IgG and IgG1 were elevated progressively

with time to a high level in all groups, except the porphyrino-

genic, in which case they remained essentially the background

levels of the non-immunized and unchallenged normal (Fig. 3E

and F). In contrast, the porphyrinogenic (DT1ALA) was the only

group (Fig. 3G), which showed a significant elevation by one- to

twofold over the others (po0.001) in the level of IgG2. As a

measure of CMI, the elevation of IgG2 is consistent with the

development of effective immune response.

Porphyrinogenic vaccination of hamsters elicit cyto-kine transcript profiles of protective response

Impairment of CMI response during active VL is reflected by

marked T-cell anergy specific to Leishmania antigens [17, 18].

Since optimum protective efficacy was observed in porphyrinogenic

vaccinated hamsters, the expression of Th1 and Th2 cytokines was

evaluated only in this group for comparison with infected and

normal control groups. A comparative RNA cytokine profile of

splenocytes, analyzed on days 45, 60, 120, and 180 p.c. (Fig. 4),

showed that among the three groups of hamsters, expression of

IFN-g and IL-12 transcripts was significantly higher in vaccinated

group than infected groups (po0.001) (Fig. 4A). These and iNOS

Figure 2. Photodynamic vaccination of hamsters with suicidal L. amazonensis protects them against VL. Shown are body weights (A), organ weights(gm) (B, C), and parasite burdens in spleen (D), liver (E), and bone marrow (F) of vaccinated hamsters, i.e. DT1ALA, DT�ALA, ST1ALA, and non-vaccinated controls, i.e. untreated/non-challenged (normal), untreated/challenged (infected), and ALA alone/challenged (ALA). Parasite loads weredetermined as the number of amastigotes/103 cell nuclei microscopically enumerated in Geimsa-stained tissue impression smears. Each barrepresents pooled data (mean7SD) from three independent experiments (10–15 hamsters per group). ��po0.001 and ��po0.01 compared withrespective normal (A–C) or infected (D–F) controls.



transcripts were all elevated by three- to fourfold after vaccination

(Fig. 4B). The expression levels of Th1-suppressive cytokines, i.e.

TGF-b, IL-10, and IL-4 were up-regulated in infected group, but not

in porphyrinogenic vaccinated hamsters throughout the p.c. periods

from day 45 to day 180 (Fig. 4B).

Adoptive transfer of T cells from porphyrinogenicvaccinated group protects naı̈ve hamsters againstL. donovani challenges by generating specific cell-mediated immune response

Protection of hamsters against L. donovani challenges by

porphyrinogenic vaccination (Figs. 1 and 2) was unequivocally

confirmed by adoptive transfer of T lymphocytes from immunized

animals to naı̈ve hamsters (Fig. 5, DT1ALA/R). The recipients of

immune T cells were observed to gain as much weight as the

normal hamsters, both groups being 2–3 times heavier than the

animals in the group, which received no T cells, but challenged

with L. donovani (Fig. 5A, LD/R). In the protected group

(DT1ALA/R), hepatosplenomegaly was totally absent through-

out the entire p.c. period; parasite loads were significantly

suppressed in all visceral organs from day 45 to day 180, i.e. a

decrease by 41 log in the spleen and to negligible levels in the

liver and bone marrow (Fig. 5D–F). All the hamsters in this group

survived and remained healthy for 1 year when the experiment

was terminated. On the other hand, the animals receiving cells

from non-vaccinated, but infected hamsters (LD/R) developed

visceral disease and died of the challenging infection in �2

months. Although some non-porphyrinogenic control groups

were precluded from this study due to their death, the results

obtained provide significant evidence, indicating that the

protective immunity elicited by photodynamic vaccination is

transferable and thus long lasting.

The transfer of protective immunity from vaccinated animals

to the recipients was indicated by their cellular responses

(Fig. 6A–D, DT1ALA/R), which were significantly higher than

those of the control groups (po0.001), i.e. DTH, lymphoproli-

ferative responses, and NO production against SLD throughout

the p.c. periods from day 0 to day 180.

As expected, while the Leishmania-specific IgG and IgG1 were

of the background levels in the DT1ALA/R group, its IgG2 level

Figure 3. Cellular and humoral immune responses of photodynamically vaccinated hamsters. These were assessed at the same time points fromthe same groups of hamsters, as described in the legend to Fig. 2. Shown are DTH response (mm) to SLD as footpad swelling at 24 h (A) andlymphoproliferative response (SI) of lymphnode cells to Con A (B) and SLD (C). NO production (mg/mL) of macrophages measured in samples (seetext) by the absorbance of the reaction products at 540 nm using Greiss reagent (D); anti-leishmanial IgG levels (OD) (E) and its isotypes, i.e. IgG1 (F)and IgG2 (G), determined by ELISA. Each bar represents the pooled data (mean7SD) of three independent experiments each with 10–15 hamsters.��po0.001 compared with respective normal group.



became increasingly elevated with the p.c. periods; the reverse

was observed in all the control groups, e.g. LD/R (Fig. 6E–G).

There was significant up-regulation of iNOS, IFN-g, and IL-12,

concomitant with down-regulation of Th2 cytokines (IL-10, IL-4,

and TGF-b) (po0.001) in DT1ALA/R versus the infected control

groups (Fig. 7). All these findings corroborate our earlier results

with porphyrinogenic vaccination (Fig. 4).

DiscussionMildly virulent Leishmania (from the lesions of diseased patients)

have been used for centuries to vaccinate children (known as

‘‘leishmanization’’) with proven effectiveness for immunoprophy-

laxis against human simple CL – a disease known to heal

spontaneously, resulting in the development of life-long immunity

[19]. ‘‘Leishmanization’’ becomes more feasible for consideration,

with the advances in Leishmania molecular genetics to knockout

virulence genes or to express transgenic suicidal genes [20–24]. In

the present study, a transgenic mutant line of L. amazonensis was

demonstrated to have the potential as a suicidal and thus safe

vaccine candidate to facilitate antigen presentation, thereby

enhancing the magnitude and quality of T-cell immune responses.

A single subcutaneous injection of such a mutant was reported

here to elicit a protective immune response of hamster against

otherwise fatal challenges of L. donovani.

The doubly transgenic-Leishmania used become porphyric

only when exposed to ALA [16] – a water soluble inexpensive

metabolite that is in clinical use for photodynamic therapy

against cancers [25], but not for vaccination using Leishmania in

the manner as presented in this study. The closest is the bacterial

mutants of Legionella whose DNA repair genes were obliterated,

thereby rendering them sensitive to psoralin-dependent and UV-

sensitive lysis [26]. The potential use of suicidal Leishmania for

live-cell vaccination finds support from previous reports on the

immunization of laboratory animals with drug or genetically

attenuated mutants for avirulence [22, 27, 28]. A major differ-

ence of the present suicidal design from the previous ones is the

use of two very different suicidal signals in our model, i.e. ALA

Figure 4. Th1/Th2 cytokine and iNOS mRNA in protected porphyrinogenic-L. amazonensis vaccinated hamsters. (A) Splenic iNOS and cytokinemRNA expression profile analysis of normal, infected, and porphyrinogenic vaccinated hamsters challenged with L. donovani (DD8). Spleens ofhamsters were analyzed on days 45, 60, 120, and 180 p.c. for RT-PCR. Experiments were carried out in triplicate for each of the three representativehamsters from individual experimental groups. (B) Densitometry analysis of the data in (A) showing the relative mean % change in iNOS andcytokine mRNA expression7SD normalized against control (HGPRT). The differences between various groups are significant, as indicated(��po0.01 and ��po0.001 compared with respective infected control).



and light. Both have long been used clinically for different

purposes with excellent margin of safety and therapeutic

values. The use of two different signals in combination, although

laborious and cumbersome in practice, offers the modality of

fine-tuning the strengths and timing to deliver adequate

cytolysis of the live vaccines. This is further amenable to further

regulation, if necessary, by application of an additional photo-

sensitizer externally [29]. We have found no evidence for

‘‘escape’’ of the live mutants from the ‘‘treatment’’ for cytolysis by

leaving the vaccination sites prematurely. This is further

preventable in the future by using strictly cutanotropic Leishma-

nia and/or by rendering them porphyric before injection.

The suicidal design is thus particularly suitable for assessing

challenging parasite ‘‘persistence’’ [27, 30, 31] versus inanimated

vaccines, e.g. killed-microorganisms, peptide antigens, and

cDNA [3, 32], which may be used in combination or separately

for comparison in their ability to elicit memory T cells for lasting

immunity.

An ‘‘ideal’’ anti-leishmanial vaccine is the one, which can be

effective against both CL and VL, considering their co-endemicity

in many places. Cross-species protection has been reported

previously in experimental leishmaniasis [19, 33, 34]. Here, we

demonstrated this for the first time that a suicidal mutant

L. amazonensis induces cross-protective immune response against

a virulent L. donovani challenge. This suicidal design for photo-

dynamic vaccination makes it possible not only to elicit protective

immunity against L. donovani but also to let the live vaccine

undergo self-destruction. Although our results demonstrated the

utility of the mutants for eliciting protection against leishma-

niasis, they have the potential to serve more universally as a

platform to receive add-on vaccines for immunization against

other infectious and non-infectious diseases.

Figure 5. Protection of naı̈ve hamsters by adoptive transfer of lymphnode cells from immunized animals. Shown are body weights (A), organweights (gm) (B, C), and parasite burdens in spleen (D), liver (E), and bone marrow (F) of the recipient hamsters, i.e. DT1ALA/R, LD/R, No/(-)R, andnormal animals determined on different p.c. days as shown. See text and the legend to Fig. 2 for the assessment of parasite burdens. Each barrepresents pooled data (mean7SD) from three independent experiments (10–15 hamsters per group). ��po0.001 compared with respective normal(A–C) or infected control (D–F).



Another important feature of the suicidal vaccine is its

excellent safety margin. No parasites were directly demonstrable

by microscopic observations of relevant tissues and by their

cultivation after ‘‘vaccination’’ for weeks, accounting apparently

for the absence of inflammatory response and cutaneous lesion

development at the injection sites, as noted during the subse-

quent observations for up to 180 days. The inoculated mutants

were either lysed completely or reduced by the suicidal design to

a number insufficient to produce lesions. Notably, the same

mutants used as non-suicidal controls produced lesions of

considerable size, i.e. up to �0.7 cm in diameter.

All hamsters in the porphyrinogenic vaccination group

remained viable and healthy, whereas those in the non-

porphyrinogenic groups succumbed to kala-azar after challenges

with L. donovani. Disease regression in porphyrinogenic vacci-

nated animals is correlated with the strong DTH response seen

after challenging infection, which was absent in unvaccinated

and L. donovani-infected hamsters. Effective immunity elicited in

this vaccine model clearly results from the development of strong

cell-mediated immunological, cytokine responses [35–37].

Lymphocytes from porphyrinogenic vaccinated hamsters in

response to SLD were found to mediate an increased production

of NO by macrophages, that may further up-regulate their

production of IFN-g [38]. This is consistent with the critical role

of NO-mediated macrophage effector mechanism, which is

known to control parasite replication in this animal model [36].

The protection of hamsters by porphyrinogenic vaccination

against L. donovani challenges is clearly associated predomi-

nantly with a Th1-cytokine response, as indicated by the up-

regulation of iNOS, IFN-g, and IL-12, concomitant with the down-

regulation of TGF-b, IL-4, and IL-10. The reverse is otherwise the

case in L. donovani infection alone. This is expected, since it is

known that IL-12 is a strong inducer of IFN-g production and

plays a major role in the development of the Th1 immune

response to control the infection in experimental leishmaniasis

[39, 40], whereas IL-10 and TGF-b antagonize Th1-cytokine

synthesis and inhibit macrophage activation necessary for killing

intracellular parasites [37, 41]. The significance of IL-10 in the

Figure 6. Cellular and humoral immune responses of naı̈ve hamsters after adoptive transfer of lymphnode cells from immunized animals. Seetext for DT1ALA/R, LD/R, No/(-)R, and normal animals. See text and the legends to Fig. 3 for experimental conditions for DTH, NO,lymphoproliferative, and antibody assessments. ��po0.001compared with respective normal group.



pathogenesis of human VL is supported by the observations that it

down-regulates parasite-specific T-cell responses [42] and inhi-

bits NO production of human macrophages to kill L. infantum

[43]. These observations corroborate our data presented here,

indicating that the progressive disease in this kala-azar model is

associated with a defect in the generation of NO.

Apart from diminished cellular responses, VL is associated

with the production of high levels of antibodies, which have been

observed prior to detection of parasite-specific T-cell response

[44]. Unlike mice where IL-4 and IL-12 direct IgG subclass

switching of IgG1 and IgG2a, respectively, such distinct IgG

classes remain obscure in hamsters [45, 46]. It is believed that

hamster IgG1 and IgG2 correspond to mouse IgG1 and IgG2a/

IgG2b, respectively. It has been well established that IgG and

IgG1 antibodies increase in titer with the L. donovani loads [47].

The virtual absence of these antibodies is thus consistent with the

decreasing parasite loads seen in porphyrinogenic vaccinated

group. The significant increase in the IgG2 levels only in

porphyrinogenic vaccinated animals is indicative of enhanced

CMI.

Protective immunity can be passively transferred to naı̈ve

recipients with T-cell enriched preparations from donor animals

that have resolved infection [48]. Evidence obtained in human

and in experimental murine leishmaniasis indicates that the

healing of cutaneous lesions is associated with the development

of specific T-cell-dependent cellular response [49, 50], the role of

which has been further established by experiments showing that

resistance to infection was achieved by the transfer of syngeneic

immune T lymphocytes [51]. Adoptive transfer of the splenocytes

from immunized animals has been shown previously to achieve a

certain level of protection against L. donovani infection [52–54].

Successful protection was achieved in the present study by a

single intravenous administration of T cells from porphyrinogenic

vaccinated hamsters, conferring an effective control of infection

on hamsters against L. donovani challenges, as indicated both at

the level of restricting parasite loads and induction of protective

Figure 7. Th1/Th2 cytokine and iNOS mRNA of hamsters adoptively transferred with lymphnode cells from porphyrinogenic-L. amazonensisvaccinated hamsters. See legend to Fig. 5 for group designation. See text and the legend to Fig. 4 for experimental and other details. (A) SpleniciNOS and cytokine mRNA expression profiles; (B) densitometric analyses of data in part A, as described in the legend to Fig. 4 (��po0.01 and��po0.001 compared with respective infected control).



cellular responses. In animals of the DT1ALA/R group, indeed,

the virtual absence of parasites on day 180 p.c. is accompanied by

a strong induction of favorable immune parameters, i.e. antigen-

specific T-cell proliferation, production of NO, increased mRNA

expression of iNOS, IL-12, IFN-g, robust DTH responses, and low

antibody titers. Clearly, donor T cells are skewed toward a

protective Th1-type response, indicative of durable immunity.

Interestingly, the overall protective and immunological profiles

are more pronounced in DT1ALA/R group than the porphyr-

inogenic vaccinated ones. Thus, passive transfer of immunity in

the absence of parasites may favor the development of central

memory T cells for long-lasting protection.

Evidence presented in this study clearly indicates that the

porphyrinogenically lysed L. amazonensis after delivery in vivo is

protective against experimental VL. Worthy of emphasis are two

major findings. Firstly, porphyrinogenic-L. amazonensis appears

to be a promising live candidate vector for development of an

effective vaccine against Leishmania infections. Secondly, transfer

of T cells from effectively immunized animals alone, in the

absence of parasites, to the naı̈ve animals protects the latter with

robust and lasting immunity, suggestive of the development of

central memory T cells. It will be highly advantageous to obtain

such lasting immunity against VL by using porphyrinogenic

mutants from strictly cutaneous Leishmania, as their residence in

the skin makes them readily accessible to suicidal induction.

The work presented offers a new avenue for photodynamic

immunoprophylaxis and immunotherapy of other infectious

diseases [16]. Further studies may be envisaged to simplify the

applications, for example, by one-time administration of the

mutants followed by inducing their porphyria using commercially

available ALA ointment and photolysis with bandaged light

emitting diodes (heatless light emitting diodes).

Materials and methods

Animal

Laboratory bred male golden hamsters (M. auratus, 45–50 g)

from the Institute’s Animal Facility were used as the experimental

host. They were housed in climatically controlled room and fed

with standard rodent food pellet (Lipton India, Mumbai, India)

and water ad libitum. The usage of the animals was approved by

the Institute’s Animal Ethical Committee.

Parasites

Leishmania donovani

Wild-type (MHOM/IN/80/DD8) has been maintained by amas-

tigote to amastigote serial passages every �2 months in

hamsters. The parasites were isolated and purified from the

spleen of infected hamsters for challenging infection.

For the preparation of SLD, promastigotes were grown in L-15

medium (Sigma, USA) for 3–4 days and processed as before [55].

The protein content of the SLD was estimated [56] and stored at

�701C.

Leishmania amazonensis mutants

The construction, selection, and maintenance of L. amazonensis

non-porphyrinogenic-single transfectants (ST) and porphyrino-

genic-double transfectants (DT), used for vaccination, were

described elsewhere [16]. Briefly, wild-type L. amazonensis

(LV78) promastigotes (clone 12-1) were grown at 251C in

Medium 199 HEPES-buffered to pH 7.4 and supplemented with

10% heat-inactivated fetal bovine serum. Transfectants were

grown under similar conditions with different concentrations of

selective pressures, i.e. G418 and/or tunicamycin at 100 and/or

20 mg/mL, respectively. Cultures were grown for one cycle in

drug-free medium before use. These mutants (109 cells/mL) were

also heat-killed by autoclaving for 15 min and used in a separate

set of experiments for comparison of their prophylactic potentials

with porphyrinogenic and non-porphyrinogenic cells.

Vaccination with transgenic-L. amazonensis inhamsters

In three separate experiments, hamsters were vaccinated on the

back on shaved areas to facilitate their exposure to white light

illumination from the top after ALA treatment (20mL of 100 mM

ALA in Hank’s Balanced Salt Solution, pH 7.4) to induce specific

uroporphyria of intracellular transgenic-Leishmania. A total of 90

hamsters were divided into six groups at 10–15 per group. Hamsters

of the main experimental group or porphyrinogenic vaccinated

group (DT1ALA) were each vaccinated first with DT and then

administered with ALA 2–3 days later. The hamsters of second

group (DT�ALA) were vaccinated with DT without ALA and those

of the third group were given ST and ALA (ST1ALA) similarly as

described for the first group. The hamsters of the fourth group were

treated with ALA alone and those of the fifth were kept as

unvaccinated control. All animals in above-mentioned five groups

were challenged with L. donovani amastigotes as described [38].

The sixth and the last group comprised of unvaccinated and

unchallenged control animals. The second and third groups were

designated as the non-porphyrinogenic control groups. The experi-

mental schedules for vaccination are given in Table 1.

For histological studies, five hamsters were ‘‘vaccinated’’ as

described for the first group and sacrificed 3 wk after the vacci-

nation, the skin tissues at the site of injection, draining lymph-

nodes, and spleens were removed for histology sections and

assessed microscopically after staining with H&E.

Peritoneal exudates, lymphnodes, spleens, and blood were

collected from the hamsters of all groups sacrificed at different

time points as mentioned in Table 1 to obtain cells and sera for

evaluation of cellular and antibody responses. The criterion for

prophylactic efficacy included the assessment of parasite loads as



the number of amastigotes/103 splenic cell nuclei in Geimsa-

stained touch-blots of spleens, livers, and bone marrows. The

percentage inhibition was assessed as described elsewhere [38].

Various tissues were also incubated in vitro in NNN tubes with

RPMI-1640 as an overlay to assess the presence or absence of

viable amastigotes therein based on their conversion into

promastigotes.

Adoptive transfer of T-cell immunity from porphyr-inogenic vaccinated hamsters

For this experiment, a total of 40–60 hamsters at 10–15 per group

were used. The experimental groups were categorized as follows:

DT1ALA/R (animals receiving lymphocytes from porphyrino-

genic vaccinated group sacrificed on day 180 p.c.); LD/R

(animals receiving lymphocytes from non-vaccinated but

L. donovani challenged group); No/(-)R (naı̈ve animals receiving

no lymphocytes). Lymphocytes used were isolated from lymph-

nodes (inguinal and mesenteric) of porphyrinogenic vaccinated

(DT1ALA) and/or L. donovani challenged animals (on day 180

p.c.), and the isolated cells (107/100 mL) were suspended in

complete RPMI medium [38]. Hamsters were injected with 107

lymphocytes per animal intracardially and were challenged 21

days later with L. donovani (DD8) amastigotes (107 per animal),

except the normal group, which received neither lymphocytes nor

L. donovani challenges. Progression of disease and immunological

responses were monitored periodically up to 180 days p.c. when

the experiment was terminated.

Post-challenge survival assessments

Animals of both the experimental and control groups were given

proper care and observed for their survival which lasted for more

than 12 months p.c. Survival of individual hamsters was recorded

and mean survival period was calculated.

Lymphoproliferative assays

Lymphnodes of hamsters were excised aseptically and processed

for the isolation of lymphocytes [38]. The lymphocyte were

suspended to 106/mL and cultured at 105 cells/well in 96-well flat

bottom tissue culture plates (Nunc, Denmark). One hundred

microliters of Con A (10mg/mL; Sigma) or SLD (10mg proteins/

mL) were added to each well in triplicate. Wells without stimulants

served as negative controls. Cultures were incubated at 371C in a

CO2 incubator for 3 days in case of mitogen and for 5 days in case

of SLD antigens. Eighteen hours prior to termination of culture,

0.5mCi of [3H] thymidine (BARC, India) was added to each well

and then cells were harvested on glass fiber mats (Whatman);

radioactivity counted in a liquid scintillation counter. Results were

expressed as stimulation index (SI), which was calculated as mean

cpm of stimulated culture/mean cpm of unstimulated control. SI

values of more than 2.5 were considered as positive response.

NO assays

The presence of NO was assessed in the culture supernatants of

peritoneal macrophages from naı̈ve hamsters after exposure to

the supernatants of stimulated lymphocyte’s cultures by using

Griess reagent [57]. Isolated peritoneal macrophages [55] were

suspended in culture medium for plating at 106 cells/well and

exposed to the supernatants of above-described stimulated

lymphocyte’s culture supernatants from all the study groups.

The supernatants (100 mL) collected from macrophage cultures

24 h after incubation were each mixed with an equal volume of

Griess reagent (Sigma) and left for 10 min at room temperature.

Table 1. Experimental schedules for vaccination

Step Day (p.v./p.c.) Group Treatment

1 0 1–3 Intradermal inoculation of a shaved area (�2 cm2) on the back of the animals, with

porphyrinogenic DT, non-porphyrinogenic ST and heat-killed DT (1.0–1.5�107 cells/100mL

each), respectively

4–6 No vaccination controls

2 4–8 p.v. 1,3,

and 4

ALA administration four times daily to the vaccination site of each animal for ‘‘induction of

porphyria’’ with 20 mL of 100 mM ALA in HBSS, pH 7.4

2,5,

and 6

No ALA treatment

3 8–12 p.v. 1–6 Illumination of caged animals for�8 h daily � 5 days under white light (four fluorescent bulbs

of 100 W (Phillips) 1 two fluorescent tube lights of 40 W) placed 2 ft above the cages, thereby

maintaining a constant temperature of 26–271C

4 26 p.v. 1–5 Challenge intracardially with 107 DD8 strain of L. donovani amastigotes/animal

6 No challenge

5 0, 45, 60, 120, and

180 p.c.

1–6 Examination of 2–3 hamsters for pathological, parasitological, and immunological evaluation

p.v., post-vaccination; p.c., post-challenge.



The absorbance of the reaction products was measured at 540 nm

in an ELISA reader [57].

Measurements of DTH

DTH was performed by injecting intradermally 50 mg/50 mL of

SLD in PBS into one footpad and PBS alone into the other footpad

of each of the vaccinated and unvaccinated controls. The

response was evaluated 24 h later by measuring the difference

in footpad swelling between the two with and without SLD for

each animal [45].

RT-PCR of mRNA for cytokines and iNOS

RT-PCR was performed to assess the expression of mRNA for

various cytokines and iNOS in splenic cells. Splenic tissues

were taken from each of the three individual animals randomly

chosen from different groups. Total RNA were isolated using Tri-

reagent (Sigma) on days 45, 60, 120, and 180 p.c., and quantified

by using Gene-quant (BioRad). The primer sequences of cytokine

and iNOS primers used to amplify their respective cDNA were as

described [37] (Table 2). Housekeeping hypoxanthine-guanine

phosphoribosyltransferase (HGPRT) was used as a control.

One microgram of total RNA was used for the synthesis of

cDNA using first strand cDNA synthesis kit (Fermentas). An

amount of 0.5 mg of cDNA was amplified by PCR under the

following condition: Initial denaturation at 951C for 2 min

followed by 40 cycles, each consisting of denaturation at 951C for

30 s, annealing at 551C for 40 s and extension at 721C for 40 s per

cycle. The final extension step was carried out at 721C for 10 min.

RT-PCR products were quantitatively assessed by densitometry of

ethidium bromide-stained bands using the software AlphaIma-

gerTM 2200 (Alfainnotech). The mean percentage expression

values were presented as the intensities of PCR products relative

to those of their corresponding HGPRT.

Determination of Leishmania-specific anti-leishmanialantibody levels

The levels of anti-leishmanial antibodies – IgG and its isotypes –

IgG1 and IgG2 in sera samples from hamsters of different

groups were measured as described [58]. The 96-well ELISA

plates (Nunc) were coated in each well with SLD (2 mg/mL in

PBS) overnight at 41C and blocked with 1% BSA at room

temperature for 1 h. The optimum dilution of sera was

standardized at 1:200 for IgG, and 1:100 for IgG1 and IgG2,

respectively for 2 h at room temperature. HRP-conjugated goat

anti-hamster IgG (H1L) (Serotec, USA) and biotin-conjugated

mouse anti-Armenian and anti-Syrian hamster IgG1 (for IgG1)

(BD, Pharmingen) as well as mouse anti-Syrian hamster IgG2

(BD, Pharmingen) was added for 1 h at room temperature. IgG1

and IgG2 plates were further incubated with sterptavidin-

conjugated peroxidase (Sigma) for 1 h. Finally, plates were

developed using O-phenylene diamine dihydrochloride as the

substrate (Sigma) and the reaction products read at 492 nm in an

ELISA reader (BioTek, USA).

Statistical analysis

Results were expressed as meanSD. The results (pooled data of

three independent experiments) were analyzed by one-way

ANOVA test and comparisons with control data were made with

Dunnett’s post-test using Graph Pad Prism software program. The

upper level of significance was chosen as po0.001.

Acknowledgements: We expressed our sincere gratitude to the

Director CDRI for his keen interest and for providing facilities for

the experiments. Financial support for senior research fellowship

to S.K. and M.S. from CSIR, New Delhi and technical support of

S. M. Verma, Division of Toxicology are gratefully acknowledged.

This has CDRI communication no. 7449. Supported in part by

USA-NIH AI-68835 to K.P.C.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Transcript

of

Primer sequence

(forward/reverse)

Expected

size (bp)

HGPRT 50-ATCACATTATGGCCCTCTGTG-30 125

50-CTGATAAAATCTACAGTYATGG-30a)

iNOS 50-GCAGAATGTGACCATCATGG-30 198

50-CTCGAYCTGGTAGTAGTAGAA-30

IFN-g 50-GGATATCTGGAGGAACTGGC-30 309

50-CGACTCCTTTTCCGCTTCCT-30

IL-12 50-GTACACCTGYCACAAAGGAG-30 430

50-GATGTCCCTGATGAAGAAGC-30

TGF-b 50-CCCTGGAYACCAACTATTGC-30 310

50-ATGTTGGACARCTGCTCCAC-30

IL-4 50-CATTGCATYGTTAGCRTCTC-30a) 463

50-TTCCAGGAAGTCTTTCAGTG-30

IL-10 50-ACAATAACTGCACCCACTTC-30 432

50-AGGCTTCTATGCAGTTGATG-30

a) Sequences with base degeneracy (Y 5 C and T; R 5 A and G).



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Abbreviations: ALA: d-aminolevulinate � CL: cutaneous leishmaniasis �CMI: cell-mediated immunity � DT: double transfectants � DTH:

delayed-type hypersensitivity � HGPRT: hypoxanthine-guanine

phosphoribosyltransferase � p.c.: post-challenges � SI: stimulation

index � ST: single transfectants � SLD: soluble leishmania donovani � VL:

visceral leishmaniasis � WHO: World Health Organization

Full correspondence: Dr. Anuradha Dube, Division of Parasitology,

Central Drug Research Institute, Chattar Manzil Palace, MG Road,

Lucknow 226001, India

Fax: 191-0522-2623938

e-mail: [email protected]

Received: 2/4/2008

Revised: 30/8/2008

Accepted: 1/10/2008



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