INFECTION AND IMMUNITY, Aug. 2005, p. 4714–4722
Vol. 73, No. 8
Conditions Influencing the Efficacy of Vaccination with Live
Organisms against Leishmania major Infection
Khaled S. Tabbara,†‡ Nathan C. Peters,† Farhat Afrin,†§ Susana Mendez,
Sylvie Bertholet, Yasmine Belkaid, and David L. Sacks*
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892
Received 26 January 2005/Returned for modification 8 March 2005/Accepted 18 March 2005
Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated
immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major
remains the only successful vaccine in humans. We examined the expression of immunity at the site of
secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early
events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice.
Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and
durability of the memory response following subcutaneous vaccination with live parasites in the footpad are
even greater than previously appreciated. Antigen-specific gamma interferon (IFN-?)-producing T cells infil-
trate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following
rechallenge, followed by a rapid drop in IFN-??CD4?cell numbers in the site. In comparison, intradermal
vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the
rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated
with a rapid recruitment of interleukin-10 (IL-10)-producing CD4?T cells to the rechallenge site. Treatment
with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice,
indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing
the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to
optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens.
Currently, vaccines developed for use in humans that reli-
ably induce cell-mediated immunity, thereby providing protec-
tion against obligate intracellular pathogens such as Mycobac-
terium tuberculosis and Leishmania, have met with limited
success (10, 17, 25, 30). In the case of leishmaniasis, immuni-
zation with live, virulent Leishmania major, resulting in a lesion
that heals, is referred to as leishmanization and is the only
proven “vaccination” strategy in humans (12, 16, 28). While
highly effective, leishmanization has been largely abandoned
due to safety concerns, mostly surrounding the development of
ulcerating primary lesions that were slow to heal, or, in rare
cases, nonhealing lesions (24). In an attempt to emulate the
long-lasting cell-mediated immunity conferred by leishmaniza-
tion without the adverse effects of a primary infection, nonliv-
ing protein- or DNA-based vaccines or live vaccines employing
recombinant vectors or attenuated parasites have been devel-
oped that induce no, or very limited, primary disease (11, 15).
In the mouse model of leishmaniasis, antigen persistence is
a critical factor for the maintenance of protective immunity, as
both BALB/c and C57BL/6 mice that are manipulated to
achieve sterile cure following primary infection with L. major
also lose their resistance to reinfection (6, 33). Protective im-
munity appears to be optimized by the presence of at the time
of secondary challenge of both antigen-independent central
memory cells and antigen-dependent effector cells (34). The
requirement for persisting antigen appears, at least partially, to
be responsible for the fact that the Leishmania-specific immu-
nity generated by nonliving vaccines, either alone or in con-
junction with adjuvants, has the tendancy to wane, is nonpro-
tective over the long term, and has failed to match the potency
and durability of vaccination with live organisms in both mu-
rine and human disease (1, 13, 14, 17, 20, 22, 25, 29–32).
Despite its proven efficacy, the early events associated with
the expression of immunity in the rechallenge site and vari-
ables, such as route of inoculation, that might optimize or
compromise the efficacy of live vaccines have yet to be carefully
addressed. The route of inoculation seems especially relevant,
as the site of primary infection with Leishmania is known to
influence the immune response and outcome of disease (3, 5,
9, 23, 26). We were particularly interested to address how the
generation of interleukin-10 (IL-10)-producing CD4?CD25?
regulatory T (TREG) cells, which have been shown to control
parasite persistence in the skin and to compromise the expres-
sion of immunity to reinfection (6, 21), might be altered fol-
lowing primary infection/vaccination in different sites.
In the present report we attempt to more fully define the
protective secondary immune response generated by the use of
live vaccine and the influence of the site of vaccination on this
response. We give particular attention to the dynamic interplay
between effector T (TEFF) and TREGcells in the secondary
challenge site and find that the site of vaccination—subcuta-
* Corresponding author. Mailing address: NIAID, Laboratory of
Parasitic Diseases, Bldg. 4, Rm. 126, Center Dr. MSC 0425, Bethesda,
MD 20892-0425. Phone: (301) 496 0577. Fax: (301) 480-3708. E-mail:
† K.S.T., N.C.P., and F.A. contributed equally to this work.
‡ Present address: Arabian Gulf University, College of Medicine,
§ Present address: Centre for Biotechnology, Jamia Mamdard,
Hamdard University, New Delhi, India.
neous versus intradermal—with live parasites, can significantly
influence the cell types recruited to the secondary site, the
strength of the secondary immune response, and the efficiency
of parasite clearance upon rechallenge.
MATERIALS AND METHODS
Mice. C57BL/6 mice were purchased from the Division of Cancer Treatment,
National Cancer Institute (Frederick, MD) or Taconic (Rockville, MD). All mice
were maintained at the National Institute of Allergy and Infectious Diseases
animal care facility under pathogen-free conditions.
Parasite preparation and live vaccination protocol. L. major clone V1
(MHOM/IL/80/Friedlin) promastigotes were grown at 26°C in medium 199 sup-
plemented (M199/S) with 20% Hi-fetal calf serum (HyClone, Logan, UT), 100
U/ml penicillin, 100 ?g/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1
mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50% triethanolamine), and
1 mg/ml 6-biotin. Infective-stage promastigotes (metacyclics) of L. major were
isolated from stationary cultures (4- to 5-day-old cultures) by negative selection
of infective forms using peanut agglutinin (Vector Laboratories, Inc., Burlin-
game, CA). Mice were infected in the ear dermis by intradermal (i.d.) injection
using a 29.5-gauge needle in a volume of 10 ?l or in the footpad (FP) by
subcutaneous (s.c.) injection using a 27.5-gauge needle in a volume of 40 ?l. Care
was taken during footpad injection to avoid the intramuscular and/or intradermal
Lymphocyte preparation. The ventral and dorsal sheets of the infected ears
were separated, deposited dermal side down in Dulbecco’s modified Eagle’s
medium containing 100 U/ml penicillin, 100 ?g/ml streptomycin, and Liberase CI
enzyme blend (50 ?g/ml; Boehringer Mannheim). Ears were incubated for 2 h at
37°C. Footpads were similarly prepared following removal of the toes and bones.
Ear sheets and footpad tissues were ground in a Medimachine (Beckton Dick-
inson). Retromaxilar (ears) and popliteal (FP) lymph nodes were removed and
mechanically dissociated using a pellet pestle in 100 ?l of M199/S medium.
Tissue homogenates of both infected tissues and draining lymph nodes (DLN)
were filtered using a 70-?m-pore-size cell strainer (Falcon Products, Inc. St.
Estimation of parasite load. Parasite loads in the ear, FP, and DLN were
determined as previously described (4). Briefly, recovered lymphocytes were
serially diluted in a 96-well flat-bottom microtiter plate containing biphasic
medium prepared using 50 ?l of NNN medium containing 20% of defibrinated
rabbit blood and overlaid with 100 ?l of M199/S medium. The number of viable
parasites in each ear was determined from the highest dilution at which promas-
tigotes could be grown out after 7 to 10 days of incubation at 26°C.
In vitro recall response and fluorescence-activated cell sorting analysis. Mice
were sacrificed and single-cell suspensions from the ear dermis, FP, and local
DLN (pooled cells from 3 to 5 mice per time point) were obtained as described
above. For the analysis of surface markers and intracytoplasmic staining for
gamma interferon (IFN-?) and IL-10, cells were stimulated with L. major-in-
fected murine bone marrow-derived dendritic cells (BMDDC) as a source of
antigen for 16 h, at which time brefeldin A was added (10 ?g/ml) (7). The cells
were cultured for an additional 6 h and then fixed in 4% paraformaldehyde. Prior
to staining, cells were incubated with an anti-Fc? III/II (Pharmingen) receptor
and 10% normal mouse serum (NMS) in phosphate-buffered saline containing
0.1% bovine serum albumin and 0.01% NaN3. Cells were permeabilized and
stained for the surface markers T-cell receptor ? (145-2 C11; fluorescein iso-
thiocyanate) (Pharmingen), CD4 (RM4-5; Cychrome, Pharmingen), and CD25
(PC61; phycoerthrin; Pharmingen) and for the cytokines IFN-? (JE56-5H4;
Pharmingen) and IL-10 (JES5-16E3; Pharmingen) conjugated to R-PE. Incuba-
tions were carried out for 30 min on ice. The frequency of CD4?T cells was
determined by gating on T-cell receptor ??cells. For each sample 50,000 (see
Fig. 2 and 4) or 200,000 (see Fig. 6) cells were analyzed. The data were collected
and analyzed using CELLQuest software and a FACScalibur flow cytometer
(Becton Dickinson, San Jose, CA).
Measurement of IFN-? and IL-10 production. For cytokine measurement in
culture supernatants, pooled cells from DLN were resuspended in RPMI me-
dium containing fetal bovine serum-penicillin-streptomycin at 6 ? 106cells/ml,
and 0.1 ml was plated in triplicate in U-bottom 96-well plates. Cells were incu-
bated at 37°C in 5% CO2with uninfected or with L. major-infected BMDDC.
IFN-? and IL-10 production in 48-h culture supernatants was quantitated by
enzyme-linked immunosorbent assay (ELISA; Endogen, Woburn, MA).
Anti-CD25 and anti-IL-10R treatments. Mice were infected with 104metacy-
clic promastigotes in the FP or ear, and 10 weeks following infection, ear-
infected mice were injected intraperitoneally with 0.5 mg of anti-CD25 (PC61)
anti-IL-10 receptor (IL-10R; 1B1.3a) or control antibody (GL113) on days ?3,
0, and ?3. Antibodies were purified by ammonium sulfate precipitation and ion
exchange chromatography. On day 0, mice were challenged in both ears (FP-
immunized mice) or the contralateral ear (ear-immunized mice) with 500 to
1,000 metacyclic promastigotes. Parasite loads of ears, footpads, and DLN were
determined 3 weeks following challenge.
Statistical analysis. Statistical significance between means of various groups
was determined using a two-tailed t test for independent samples.
Protective immunity generated by s.c. vaccination with live
parasites in the footpad. In order to establish the efficacy of
using live vaccine, we immunized C57BL/6 mice with live par-
asites and assessed the early kinetics of parasite clearance and
the immune response at the site of secondary challenge. In
initial studies, we employed s.c. primary infection in the FP
with 104live L. major metacyclic promastigotes, as this inocu-
lum is consistent with the dose used for leishmanization in
FIG. 1. Protective immunity generated by FP vaccination with live
parasites is characterized by the rapid clearance of parasites from a site
of secondary challenge. Naive C57BL/6 mice (Na) or mice vaccinated
16 weeks prior in the RFP with 104L. major V1 metacyclic promas-
tigotes (FP) were rechallenged, i.d., in both ears with 500 metacyclic
promastigotes. Parasite loads were determined in the secondary ear
dermis site (A) or DLN (B) at the indicated time points following
rechallenge. Results shown represent parasite loads (per ear or DLN)
for individual ears or DLNs. Three mice were employed per group per
time point. ND, not done.
VOL. 73, 2005 VACCINATION WITH LIVE ORGANISMS AGAINST L. MAJOR4715
human trials. The FP was chosen in order to spare both ear
dermal sites for secondary challenge. Intradermal reinfection
in each ear with 500 to 1,000 L. major metacyclics was used to
more closely reproduce the tissue site and dose of natural
challenge. Secondary challenge was performed 16 weeks after
vaccination with live parasites as a stringent test of vaccine
durability. At this time point, the primary lesions have com-
pletely resolved, and a low number of parasites remain in the
primary site (data not shown). Vaccinated and naı ¨ve control
mice harbored similar numbers of viable parasites 1.5 weeks
after challenge (Fig. 1A). This number steadily increased in
naı ¨ve mice, attaining a geometric mean of 4.6 ? 104parasites
per ear in the fourth week. In contrast, parasites were sharply
controlled in FP-immunized mice, where, with the exception of
only one ear at 3 weeks, viable parasites were completely
cleared by 2.5 weeks postinfection. We also analyzed the DLN
for parasite dissemination from the injection site. Parasites
were detectable in the DLN of naı ¨ve mice as early as 2.5 weeks
following challenge (Fig. 1B), increasing in numbers in the
following weeks to a geometric mean of 144/DLN in the fourth
week of infection. In contrast, the dissemination of parasites
from the ear to the DLN of FP-immunized mice was not
detectable at any time following secondary challenge. These
data indicate that the immune state generated following the
use of live vaccine is extremely potent, leading to a rapid and
efficient clearance of parasites from the site of reinfection.
Shortly after reinfection (1.5 weeks), the secondary site in
FP-vaccinated mice was characterized by the infiltration of
large numbers of lymphocytes into the ear dermis (1.8 ?105/
ear). This infiltrate included a high proportion of antigen (Ag)-
specific IFN-?-producing CD4?T lymphocytes, representing
31% of total CD4?T cells (Fig. 2A). Over the course of
parasite clearance from the injection site, the number of IFN-
?-producing CD4?lymphocytes waned, but they did not en-
tirely disappear. Similarly, IFN-? production by DLN cells
from FP-vaccinated mice peaked sharply at 1.5 weeks following
rechallenge and waned quickly thereafter (Fig. 2B). In naı ¨ve
mice the immune response developed much more slowly, with
the appearance of infiltrating IFN-?-producing CD4?lympho-
cytes delayed until 4 weeks, eventually representing 28% of
total CD4?T cells. These findings were also reflected in the
lymph nodes of naı ¨ve mice, where IFN-? production was first
detected in low concentrations at 3.5 weeks following rechal-
lenge and increasing at 5 weeks, the last time point analyzed.
Comparison of protective immunity generated by s.c. versus
i.d. vaccination with live parasites. We were struck in these
studies by the dramatic protection achieved by FP vaccination,
especially in comparison with a number of previous studies
involving vaccination with live parasites in the ear dermis and
secondary challenge in the contralateral ear, in which parasite
clearance did not appear to be nearly so efficient (21, 22). To
directly address whether the site of primary infection can in-
fluence the efficacy of the use of live vaccine, mice were vac-
cinated with 104live metacyclic L. major promastigotes, either
i.d. in the right ear or s.c. in the right FP (RFP) and challenged
7 weeks later in the contralateral ear with 103metacyclics. At
1.5 weeks postchallenge there was no significant difference in
the ear parasite loads between the ear dermis-immunized mice
FIG. 2. Protective immunity generated by FP vaccination with live parasites is characterized by the rapid recruitment of IFN-?-producing CD4?
T cells to the site of secondary challenge. Naı ¨ve mice (Na) or mice vaccinated 16 weeks prior in the RFP with 104L. major metacyclic promastigotes
(FP) were rechallenged in both ears with 500 metacyclic promastigotes. (A) At various time points following rechallenge, cells from the ear dermis
were restimulated in vitro with L. major-infected dendritic cells and stained for surface markers and intracytoplasmic IFN-?. (B) IFN-? production
by pooled cells from the DLN of the secondary site following in vitro restimulation with L. major-infected BMDDC as determined by ELISA.
4716TABBARA ET AL.INFECT. IMMUN.
and the naı ¨ve controls, while in this experiment the mice vac-
cinated in the FP already displayed significant immunity in the
secondary challenge site (P ? 0.007) (Fig. 3A). By 3 weeks
postchallenge, the parasite loads in the FP-immunized mice
were further reduced (geometric mean, 19), whereas in the
ear-immunized mice the infections were still progressing (geo-
metric mean, 4,096), though not as rapidly as in the naı ¨ve
controls (geometric mean, 52,016). By 5 weeks, viable amasti-
gotes had been completely eliminated from FP-vaccinated
mice, whereas a reduction in the parasite burden in the ear-
vaccinated mice was only just becoming apparent. The dra-
matic control of parasite growth by FP-vaccinated mice was
again evident in the DLN (Fig. 3B), where the majority had no
detectable dissemination and all were negative at 5 weeks
postchallenge. In contrast, all of the ear-vaccinated mice
showed dissemination by 3 weeks, and mice still had parasites
in their DLN at 5 weeks postchallenge.
The delayed clearance of parasites by ear-vaccinated mice
compared to those vaccinated in the FP correlates with a de-
layed and weaker IFN-? response at the site of secondary
infection. In FP-vaccinated mice large numbers of IFN-?-stain-
ing CD4?lymphocytes quickly infiltrated the rechallenge site
1.5 weeks following challenge (Fig. 4). The frequency of IFN-
?-staining CD4?cells peaked at 33% in the third week, con-
comitant with clearance of parasites, and then declined to
about 14% at 5 weeks following complete elimination of par-
asites from the site. In contrast, IFN-?-staining CD4?cells
were not found in significant amounts at the secondary site in
ear-vaccinated mice until the third week of infection, where
they accounted for only 10% of the CD4?cells at both 3 and
These data confirm that while vaccination with live organ-
isms in the skin does confer immunity, the expression of im-
munity is delayed and less potent in comparison to that of the
s.c. FP-vaccinated mice, and this delay allows parasites to ex-
pand in the inoculation site and to disseminate to the local
draining node for up to 3 weeks postchallenge.
Analysis of the protective immunity expressed by s.c. versus
i.d. vaccinated mice. The dramatic difference between the pro-
tective immunity expressed by the ear (i.d.)- versus FP (s.c.)-
vaccinated animals prompted us to explore the possibility that
IL-10-producing CD4?CD25?TREGcells, known to regulate
the persistence of parasites in the primary site of infection in
the skin and to influence the expression of immunity to rein-
fection (6, 21), might be differentially activated in the vacci-
nated mice. Twenty weeks following vaccination with live par-
asites in the ear or FP, mice were rechallenged in the
contralateral ear, for ear-vaccinated mice, or both ears, in
FP-vaccinated mice. Two weeks following challenge, the sec-
ondary site was analyzed for parasite burden, numbers of
IFN-?- and IL-10-producing CD4?T cells, and total cytokine
production as assessed by ELISA. Both ear- and FP-vaccinated
animals had significantly reduced parasite burdens compared
to the naı ¨ve control, confirming the durable immunity (20
weeks) conferred by the use of live vaccine (Fig. 5A). Again,
however, the protection in the FP-vaccinated mice was signif-
icantly more potent, with 100 times fewer parasites in the
2-week rechallenge site compared to the ear-vaccinated mice.
Ear-vaccinated mice harbored both IFN-?- and IL-10-produc-
ing CD4?cells in the secondary site (Fig. 5B). There was,
however, a greater number of CD4?cells producing IL-10
compared to IFN-?, representing 12.5% versus 8.4% of total
CD4?T cells, respectively. In comparison, FP-vaccinated mice
harbored virtually no IL-10-producing cells (1.8% of all CD4?
cells) and considerably more IFN-?-producing cells (16.3%).
The decrease in total IFN-? production by dermal cells in the
rechallenge site in ear- versus FP-vaccinated mice was more
variable when analyzed by ELISA, with some experiments
showing comparable levels of total IFN-? being secreted by
cells in the rechallenge site following antigen restimulation in
vitro (Fig. 5C). We attribute at least part of this IFN-? secre-
tion to non-CD4?T cells, as indicated by the dot plot analyses
shown in Fig. 5B. Interestingly, ELISA analysis of IL-10 pro-
duction by dermal cells revealed increased levels of IL-10 in
ear-vaccinated mice, and, more importantly, this IL-10 produc-
FIG. 3. Parasite clearance from a site of secondary challenge is
delayed in mice vaccinated i.d. versus. s.c. Naı ¨ve mice (Na) or mice
immunized 7 weeks prior in the FP or ear with 104live L. major
metacyclic promastigotes were rechallenged in the contralateral ear
(ear-vaccinated mice) or both ears (naı ¨ve and FP-vaccinated mice)
with 103metacyclic promastigotes. At various intervals following in-
tradermal rechallenge the number of parasites in ears (A) and DLN
(B) of immune and naı ¨ve C57BL/6 mice was assessed. Results shown
represent parasite loads for individual ears or DLNs. Data are for six
mice per group per time point.
VOL. 73, 2005 VACCINATION WITH LIVE ORGANISMS AGAINST L. MAJOR4717
tion was antigen driven (Fig. 5D). The data suggest that anti-
gen-specific IL-10 production by CD4?T cells is more preva-
lent in the recall response of ear-immunized mice. Analysis of
the phenotype of CD4?cells at the secondary site revealed
that 10% of CD4?cells in FP-vaccinated mice also expressed
the CD25 marker, versus 7% in ear-vaccinated mice and 10%
in naı ¨ve mice (data not shown). These results indicate that, at
a site of active infection, the CD25 marker is not necessarily a
strong indicator of the presence of antigen-specific, IL-10 pro-
The role of IL-10 and CD4?CD25?TREGcells in the delayed
expression of immunity in i.d.-vaccinated mice. Our finding
that ear-vaccinated mice harbored increased numbers of IL-
10-producing CD4?cells that were rapidly recruited to the
rechallenge site suggested that these cells might be playing a
role in the suppression of immunity. To investigate whether
IL-10 and/or CD25?cells were modulating the immunity to
reinfection in the ear-immunized mice, these animals were
treated 10 weeks following primary infection with either anti-
CD25, anti-IL-10R, or control antibodies and challenged with
103metacyclics in the contralateral ear. Three weeks following
rechallenge, the parasite load at the secondary site was deter-
mined. Treatment of ear-vaccinated mice with anti-CD25 an-
tibodies significantly lowered the parasite burden at the sec-
ondary challenge site compared to control antibody treatment
(Fig. 6), as previously reported (21). Treatment with anti-IL-
10R antibody also significantly decreased the parasite load at
the secondary site of infection. Interestingly, anti-CD25 and
anti-IL-10R treatment did not convert ear-vaccinated mice to
an optimally immune, FP-vaccinated phenotype, as parasite
loads remained about 10-fold higher than those of FP-immu-
nized mice, suggesting that other factors are also involved.
Comparison of the antigen load and immune response at
the primary sites of infection. A possible explanation for the
difference in the efficacy of live vaccination s.c. in the footpad
versus. i.d. in the ear dermis is that different antigenic loads are
established during the acute or chronic phases of infection in
the primary sites, resulting in the differential generation, ex-
pansion, and/or maintenance of Leishmania-specific memory
cells. Two groups of mice were inoculated with 104metacyclic
promastigotes in the ear or FP, and the parasite loads at the
injection site and DLN were determined at various time points
following infection (Fig. 7). Three weeks following infection,
the parasite loads were identical in the two injection sites as
well as in the respective local lymph nodes draining these sites,
suggesting that either site is equally suitable for parasite via-
bility and growth during the acute phase of infection. At 7
weeks, during the peak of the adaptive immune response,
parasite loads were higher at the site of infection and the DLN
in FP- versus ear-vaccinated mice, although it is possible that
the peak parasite burdens were comparable, established some-
time between the 3- and 7-week time points chosen for anal-
ysis. At later time points, during the chronic phase of infection
when the parasite load reaches a steady state (5), the DLNs of
mice injected in the ear or FP showed comparable parasite
numbers. In contrast, and unlike the result seen at 7 weeks,
infected ears maintained a higher steady-state parasite load
than that observed in footpads. The bias toward higher parasite
loads at later time points in ear-vaccinated mice is reflected in
the immune state maintained in their DLNs, which is biased
slightly toward a “regulatory” phenotype, as shown by lower
levels of IFN-? and increased levels of IL-10 (Fig. 8). Levels of
Ag-driven IL-4 and the frequency of IL-4-producing CD4?T
cells in both the primary site and the DLN of the primary site
in ear- and FP-vaccinated mice were comparable and very low,
?150 pg/ml and ?1%, respectively, suggesting that the differ-
ence in the efficacy of vaccination is not controlled by a tissue-
specific Th2 bias (data not shown).
FIG. 4. Recruitment of IFN-?-producing CD4?T cells to a site of secondary challenge is delayed in mice vaccinated i.d. versus s.c. Mice
immunized 7 weeks prior in the FP or ear with 104live L. major metacyclic promastigotes were rechallenged in the contralateral ear or both ears
with 103metacyclic promastigotes. At various time points following intradermal rechallenge, cells from the ear dermis were restimulated in vitro
with L. major-infected BMDDC and stained for surface markers and intracytoplasmic IFN-?.
4718 TABBARA ET AL.INFECT. IMMUN.
Vaccination of humans with live L. major parasites, resulting
in a healed primary lesion, generates a highly protective im-
mune response, as demonstrated by a strong and long-lived
resistance to disease upon reexposure (12, 16, 28). Despite the
proven efficacy of live vaccines and the inability of other vac-
cination strategies to replicate the potency or durability of live
FIG. 5. Analysis of the rechallenge site in mice with chronic infection. Naı ¨ve mice (Na) or mice vaccinated 20 weeks prior with 104live L. major
metacyclic promastigotes in the FP or ear were rechallenged in the contralateral ear or both ears with 103metacyclic promastigotes. Fourteen days
following rechallenge, the secondary site was analyzed as follows: parasite load in the secondary ear dermis site (A); IFN-? or IL-10 intracyto-
plasmic staining of CD4?T cells isolated from the ear dermis following 24 h in vitro restimulation with BMDDC uninfected (DC) or infected with
L. major (iDC) (B); IFN-? production by dermal cells isolated from the secondary ear site measured by ELISA (C); and IL-10 production by
dermal cells prepared as in panel C (D). Five mice per group were used. Results are representative of three repeat experiments. *, P ? 0.0001
between iDC groups.
VOL. 73, 2005 VACCINATION WITH LIVE ORGANISMS AGAINST L. MAJOR4719
L. major vaccines, there does not exist a careful analysis of the
protective secondary response, including the early events as-
sociated with the control of infection in the secondary chal-
lenge site. Our analysis of the secondary immune response in
mice vaccinated up to 4 to 5 months previously s.c. in the FP
with live L. major parasites reveals a remarkably rapid immune
response, characterized by the accumulation of L. major-spe-
cific IFN-?-producing CD4?T cells within 1.5 weeks following
low-dose rechallenge in the ear dermis. The speed with which
this response was able to completely clear viable parasites from
the rechallenge site was surprising—as early as 2.5 weeks fol-
lowing reinfection—during which time there was little or no
parasite dissemination into the DLN. Importantly, this level of
protection conferred by vaccination with live parasites in a
subcutaneous site was not achieved when mice were immu-
nized with live L. major in the ear dermis, a compromise that
we attribute, at least in part, to a greater frequency of IL-10-
producing CD4?CD25?T cells that are generated by chronic
infection in the skin and that home early to the rechallenge
Prior studies have suggested that the site of primary infec-
tion can influence the outcome of disease and the Th1/Th2
balance of the primary immune response (3, 9, 26, 27). In
addition, Kirkpatrick et al. have shown that the site of primary
infection involving different intradermal sites on the back can
influence the severity of the primary lesion and the resolution
of a secondary lesion following reexposure to L. major (18).
These studies did not determine the nature of the immune
response at the secondary site, and no mechanistic studies that
might explain the different outcomes were carried out. In the
current studies, a comparison of the early events associated
with immunity in the rechallenge site revealed a stronger
IFN-? response and more rapid recruitment of IFN-?-produc-
ing CD4?cells in FP-vaccinated mice. In contrast, ear vacci-
nation resulted in a slower recruitment of IFN-?-producing
CD4?cells to the secondary challenge site, despite the fact
that a relatively strong IFN-? response was maintained in the
primary DLN at the time of rechallenge. The defect appeared
to be controlled not by a shift in the Th1/Th2 balance, as the
chronic infection in either site generated low or undetectable
levels of IL-4, but by a greater population of IL-10-producing
CD4?cells that were rapidly recruited to the secondary site.
Analysis of total IL-10 production by lesion-derived cells using
ELISA also demonstrated significantly higher levels of Ag-
driven IL-10 production.
It was previously reported that CD25?TREGcells are the
primary CD4?T-cell source of IL-10 in the chronically in-
fected ear dermis (6). It was also shown that adoptive transfer
of lesion-derived CD25?TREGcells can compromise the ex-
pression of immunity in the rechallenge site and that anti-
CD25 depleting antibody enhances parasite clearance (21). In
the current studies we found that treatment of ear-vaccinated
mice with either anti-CD25 or anti-IL-10R antibodies en-
hanced the early clearance of parasites from the secondary site.
These observations suggest that in comparison to s.c. infection,
chronic infection in the skin generates a greater population of
IL-10-producing CD25?TREGcells that rapidly home to a
FIG. 6. Treatment with anti-IL-10R or anti-CD25 antibodies leads
to reduced parasite loads at the secondary site of infection in i.d.
ear-vaccinated mice. C57BL/6 mice, vaccinated in the FP or ear 10
weeks prior with 103L. major metacyclic promastigotes were rechal-
lenged in the contralateral ear with 104L. major promastigotes. Ear-
vaccinated mice were treated with 0.5 mg of anti-CD25, anti-IL-10R,
or isotype control (GL113) antibodies i.p., 3 days prior to, on the day
of, and 3 days following rechallenge. Parasite load in the secondary ear
site was determined 3 weeks following rechallenge. Each data point
represents the secondary challenge site obtained from one mouse.
Results are representative of three repeat experiments.
FIG. 7. Analysis of the parasite load during the course of primary
infection in ear- and FP-immunized mice. The number of parasites at
the site of infection (A) and DLN (B) in C57BL/6 mice was deter-
mined at various time points following immunization with 104meta-
cyclic L. major promastigotes in the ear or FP. Each data point rep-
resents an individual animal. *, P ? 0.05; **, P ? 0.005.
4720 TABBARA ET AL.INFECT. IMMUN.
secondary site of infection, thus influencing immunity. The
more “regulatory” phenotype observed following intradermal
vaccination may be physiologically relevant. The skin and mu-
cosa represent the most likely sites of exposure to foreign
noninfectious antigens, infectious agents, and, perhaps most
importantly, normal flora. Thus, these tissues would potentially
benefit most from the presence of TREGcells, as evidenced by
the immunopathology associated with the absence of CD25?
TREGcells in colitis, antigen-specific contact sensitivity, and
dermatitis (2, 8, 19).
In our search for immune correlates of the powerful immu-
nity elicited by vaccination with live organisms in a subcutane-
ous site, we found that the number of IFN-?-producing CD4?
T cells diminished quickly after parasite clearance. Thus, the
association of this response with vaccine potency may be un-
derestimated in studies where the secondary immune response
is assessed at late time points. Our studies also more critically
evaluated vaccine potency by challenging up to 20 weeks post-
vaccination, with little or no loss in the powerful protection
conferred by vaccination with live parasites in a subcutaneous
Our observations in FP-vaccinated mice appear to reveal the
ideal immune state, one in which a low-level, chronic infection
at the primary site is established, presumably by a careful
balance of TEFFand TREGcells that provides persisting anti-
gen to maintain effector cells but does not compromise the full
expression of concomitant immunity at a secondary site. In
contrast, in ear-vaccinated mice, greater numbers of IL-10-
producing TREGcells are generated that maintain a higher
level of persistent infection in the primary site and that are able
to migrate into the secondary site of infection and suppress
immunity. The consideration of this ideal immune state is
important for the generation of an effective, safe L. major
vaccine and may also have implications for vaccination strate-
gies against other intracellular pathogens.
K. Tabbara was a recipient of a Fulbright Scholar grant. F. Afrin was
supported by a grant from the Department of Biotechnology, Govern-
ment of India.
1. Alexander, J., G. H. Coombs, and J. C. Mottram. 1998. Leishmania mexicana
cysteine proteinase-deficient mutants have attenuated virulence for mice and
potentiate a Th1 response. J. Immunol. 161:6794–6801.
2. Asseman, C., S. Read, and F. Powrie. 2003. Colitogenic Th1 cells are present
in the antigen-experienced T cell pool in normal mice: control by CD4?
regulatory T cells and IL-10. J. Immunol. 171:971–978.
3. Baldwin, T. M., C. Elso, J. Curtis, L. Buckingham, and E. Handman. 2003.
The site of Leishmania major infection determines disease severity and
immune responses. Infect. Immun. 71:6830–6834.
4. Belkaid, Y., S. Kamhawi, G. Modi, J. Valenzuela, N. Noben-Trauth, E.
Rowton, J. Ribeiro, and D. L. Sacks. 1998. Development of a natural model
of cutaneous leishmaniasis: powerful effects of vector saliva and saliva pre-
exposure on the long-term outcome of Leishmania major infection in the
mouse ear dermis. J. Exp. Med. 188:1941–1953.
5. Belkaid, Y., S. Mendez, R. Lira, N. Kadambi, G. Milon, and D. Sacks. 2000.
A natural model of Leishmania major infection reveals a prolonged “silent”
phase of parasite amplification in the skin before the onset of lesion forma-
tion and immunity. J. Immunol. 165:969–977.
6. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. L. Sacks.
2002. CD4? CD25? regulatory T cells control Leishmania major persistence
and immunity. Nature 420:502–507.
7. Belkaid, Y., E. Von Stebut, S. Mendez, R. Lira, E. Caler, S. Bertholet, M. C.
Udey, and D. Sacks. 2002. CD8? T cells are required for primary immunity
in C57BL/6 mice following low-dose, intradermal challenge with Leishmania
major. J. Immunol. 168:3992–4000.
8. Cavani, A., F. Nasorri, C. Ottaviani, S. Sebastiani, O. De Pita, and G.
Girolomoni. 2003. Human CD25? regulatory T cells maintain immune tol-
erance to nickel in healthy, nonallergic individuals. J. Immunol. 171:5760–
9. Constant, S. L., K. S. Lee, and K. Bottomly. 2000. Site of antigen delivery can
influence T cell priming: pulmonary environment promotes preferential
Th2-type differentiation. Eur. J. Immunol. 30:840–847.
10. Fine, P. E. 2001. BCG: the challenge continues. Scand. J. Infect. Dis. 33:
11. Ghosh, M., and S. Bandyopadhyay. 2003. Present status of antileishmanial
vaccines. Mol. Cell. Biochem. 253:199–205.
12. Greenblatt, C. L. 1980. The present and future of vaccination for cutaneous
leishmaniasis. Prog. Clin. Biol. Res. 47:259–285.
13. Gurunathan, S., C. Prussin, D. L. Sacks, and R. A. Seder. 1998. Vaccine
requirements for sustained cellular immunity to an intracellular parasitic
infection. Nat. Med. 4:1409–1415.
14. Gurunathan, S., D. L. Sacks, D. R. Brown, S. L. Reiner, H. Charest, N.
Glaichenhaus, and R. A. Seder. 1997. Vaccination with DNA encoding the
immunodominant LACK parasite antigen confers protective immunity to
mice infected with Leishmania major. J. Exp. Med. 186:1137–1147.
15. Handman, E. 2001. Leishmaniasis: current status of vaccine development.
Clin. Microbiol. Rev. 14:229–243.
16. Kellina, O. I. 1981. Problem and current lines in investigations on the
epidemiology of leishmaniasis and its control in the U.S.S.R. Bull. Soc.
Pathol. Exot. Filiales 74:306–318.
17. Khalil, E. A., A. M. El Hassan, E. E. Zijlstra, M. M. Mukhtar, H. W. Ghalib,
B. Musa, M. E. Ibrahim, A. A. Kamil, M. Elsheikh, A. Babiker, and F.
Modabber. 2000. Autoclaved Leishmania major vaccine for prevention of
visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in
Sudan. Lancet 356:1565–1569.
18. Kirkpatrick, C. E., T. J. Nolan, and J. P. Farrell. 1987. Rate of Leishmania-
induced skin-lesion development in rodents depends on the site of inocula-
tion. Parasitology 94:451–465.
19. Loughry, A., S. Fairchild, N. Athanasou, J. Edwards, and F. C. Hall. 2005.
Inflammatory arthritis and dermatitis in thymectomized, CD25? cell-de-
pleted adult mice. Rheumatology 44:299–308.
20. Mendez, S., S. Gurunathan, S. Kamhawi, Y. Belkaid, M. A. Moga, Y. A.
Skeiky, A. Campos-Neto, S. Reed, R. A. Seder, and D. Sacks. 2001. The
potency and durability of DNA- and protein-based vaccines against Leish-
mania major evaluated using low-dose, intradermal challenge. J. Immunol.
21. Mendez, S., S. K. Reckling, C. A. Piccirillo, D. Sacks, and Y. Belkaid. 2004.
Role for CD4(?) CD25(?) regulatory T cells in reactivation of persistent
leishmaniasis and control of concomitant immunity. J. Exp. Med. 200:201–
FIG. 8. Analysis of L. major Ag-specific cytokine production by
cells derived from the DLN of the site of chronic, primary infection.
Naı ¨ve C57BL/6 mice were infected in the FP or the ear 16 weeks prior
with 104L. major promastigotes. (A) IFN-? production by DLN cells
following in vitro restimulation with BMDDC infected (iDC) or unin-
fected (DC) with L. major as determined by ELISA. (B) IL-10 pro-
duction by DLN cells as in panel A. *, P ? 0.001 between iDC groups.
The results are representative of two repeat experiments.
VOL. 73, 2005VACCINATION WITH LIVE ORGANISMS AGAINST L. MAJOR4721
22. Mendez, S., K. Tabbara, Y. Belkaid, S. Bertholet, D. Verthelyi, D. Klinman,
R. A. Seder, and D. L. Sacks. 2003. Coinjection with CpG-containing immu-
nostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vac-
cine against cutaneous leishmaniasis but maintains its potency and durability.
Infect. Immun. 71:5121–5129.
23. Menon, J. N., and P. A. Bretscher. 1998. Parasite dose determines the
Th1/Th2 nature of the response to Leishmania major independently of in-
fection route and strain of host or parasite. Eur. J. Immunol. 28:4020–4028.
24. Modabber, F. 1995. Vaccines against leishmaniasis. Ann. Trop. Med. Para-
sitol. 89(Suppl. 1):83–88.
25. Momeni, A. Z., T. Jalayer, M. Emamjomeh, A. Khamesipour, F. Zicker, R. L.
Ghassemi, Y. Dowlati, I. Sharifi, M. Aminjavaheri, A. Shafiei, M. H. Alimo-
hammadian, R. Hashemi-Fesharki, K. Nasseri, T. Godal, P. G. Smith, and
F. Modabber. 1999. A randomised, double-blind, controlled trial of a killed
L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran.
26. Nabors, G. S., and J. P. Farrell. 1994. Site-specific immunity to Leishmania
major in SWR mice: the site of infection influences susceptibility and ex-
pression of the antileishmanial immune response. Infect. Immun. 62:3655–
27. Nabors, G. S., T. Nolan, W. Croop, J. Li, and J. P. Farrell. 1995. The
influence of the site of parasite inoculation on the development of Th1 and
Th2 type immune responses in (BALB/c ? C57BL/6) F1 mice infected with
Leishmania major. Parasite Immunol. 17:569–579.
28. Nadim, A., E. Javadian, G. Tahvildar-Bidruni, and M. Ghorbani. 1983.
Effectiveness of leishmanization in the control of cutaneous leishmaniasis.
Bull. Soc. Pathol. Exot. Filiales. 76:377–383.
29. Rhee, E. G., S. Mendez, J. A. Shah, C. Y. Wu, J. R. Kirman, T. N. Turon,
D. F. Davey, H. Davis, D. M. Klinman, R. N. Coler, D. L. Sacks, and R. A.
Seder. 2002. Vaccination with heat-killed leishmania antigen or recombinant
leishmanial protein and CpG oligodeoxynucleotides induces long-term
memory CD4? and CD8? T cell responses and protection against Leish-
mania major infection. J. Exp. Med. 195:1565–1573.
30. Sharifi, I., A. R. FeKri, M. R. Aflatonian, A. Khamesipour, A. Nadim, M. R.
Mousavi, A. Z. Momeni, Y. Dowlati, T. Godal, F. Zicker, P. G. Smith, and F.
Modabber. 1998. Randomised vaccine trial of single dose of killed Leishma-
nia major plus BCG against anthroponotic cutaneous leishmaniasis in Bam,
Iran. Lancet 351:1540–1543.
31. Titus, R. G., F. J. Gueiros-Filho, L. A. de Freitas, and S. M. Beverley. 1995.
Development of a safe live Leishmania vaccine line by gene replacement.
Proc. Natl. Acad. Sci. USA 92:10267–10271.
32. Uzonna, J. E., G. F. Spath, S. M. Beverley, and P. Scott. 2004. Vaccination
with phosphoglycan-deficient Leishmania major protects highly susceptible
mice from virulent challenge without inducing a strong Th1 response. J. Im-
33. Uzonna, J. E., G. Wei, D. Yurkowski, and P. Bretscher. 2001. Immune
elimination of Leishmania major in mice: implications for immune memory,
vaccination, and reactivation disease. J. Immunol. 167:6967–6974.
34. Zaph, C., J. Uzonna, S. M. Beverley, and P. Scott. 2004. Central memory T
cells mediate long-term immunity to Leishmania major in the absence of
persistent parasites. Nat. Med. 10:1104–1110.
Editor: W. A. Petri, Jr.
4722 TABBARA ET AL.INFECT. IMMUN.