Download Intradermal Challenge Evaluated Using Low

This information is current as
of August 3, 2017.
The Potency and Durability of DNA- and
Protein-Based Vaccines Against Leishmania
major Evaluated Using Low-Dose,
Intradermal Challenge
Susana Méndez, Sanjay Gurunathan, Shaden Kamhawi,
Yasmine Belkaid, Michael A. Moga, Yasir A. W. Skeiky,
Antonio Campos-Neto, Steven Reed, Robert A. Seder and
David Sacks
References
Subscription
Permissions
Email Alerts
This article cites 31 articles, 21 of which you can access for free at:
http://www.jimmunol.org/content/166/8/5122.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
J Immunol 2001; 166:5122-5128; ;
doi: 10.4049/jimmunol.166.8.5122
http://www.jimmunol.org/content/166/8/5122
The Potency and Durability of DNA- and Protein-Based
Vaccines Against Leishmania major Evaluated Using
Low-Dose, Intradermal Challenge
Susana Méndez,* Sanjay Gurunathan,† Shaden Kamhawi,* Yasmine Belkaid,*
Michael A. Moga,† Yasir A. W. Skeiky,‡ Antonio Campos-Neto,§ Steven Reed,‡
Robert A. Seder,† and David Sacks1*
G
enetic immunization represents a novel approach for
achieving specific immune activation. During the last
decade, DNA vaccines have been developed against several viral, bacterial, and parasitic infections (1–7). The ability of
plasmid DNA encoding specific Ag to induce both CD4⫹ and
CD8⫹ T cells suggests that this approach will be of particular use
for protection against diseases that require cell-mediated immunity, such as leishmaniasis.
Leishmania major, the etiologic agent of zoonotic cutaneous
leishmaniasis in the Old World, has been extensively used in
mouse models to understand the requirements for effective vaccination against healing and nonhealing forms of leishmaniasis. Depending on the genotype of the mouse, L. major infection leads to
the development of polarized Th1 or Th2 responses that control
resistance or susceptibility, respectively, to this intracellular parasite (8, 9). There have been a number of studies involving murine
vaccination with DNA encoding parasite Ags, including gp63 (10,
11), LACK (12, 13) and PSA-2 (14), with varying results. Al-
*Laboratory of Parasitic Diseases and †Laboratory of Clinical Immunology, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD 20892; ‡Corixa Corp., Seattle, WA 98104; and §Infectious Disease Research
Institute, Seattle, WA 98104
Received for publication December 1, 2000. Accepted for publication February
8, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. David L. Sacks, Laboratory of
Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4,
Room 126, Center Drive MSC 0425, Bethesda, MD 20892-0425. E-mail address:
[email protected]
Copyright © 2001 by The American Association of Immunologists
though most of the studies involving DNA as well as protein-based
vaccines have been conducted in susceptible BALB/c mice, the
healing lesions produced in C57BL/6 mice provide a more relevant
model of L. major infection in natural reservoirs and in human
hosts. Furthermore, in almost every case the efficacy of Leishmania vaccines has been evaluated using a high dose of parasites
(105–107) inoculated into the footpad or other s.c. sites. Recently,
a natural infection model in resistant mice has been developed that
takes into account two main features of natural transmission: lowdose (100 metacyclic promastigotes) and intradermal inoculation
(the ear dermis) (15). In this model, the evolution of small, healing
dermal lesions occurs in three distinct phases: 1) an initial “silent”
phase, lasting 4 –5 wk, favoring the establishment of the peak load
of parasites in the dermis in the absence of lesion formation; 2) an
acute phase, lasting 5–10 wk, corresponding to the development
and resolution of a lesion that is associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis,
and is coincident with the onset of immunity and the killing of
parasites in the site; and 3) a chronic phase, lasting for the life of
the animal, during which a low number of parasites persists in the
skin in the absence of overt pathology. Adaptive immunity in this
model confirmed a role for Th1 cells, and in addition revealed a
requirement for CD8⫹ T cells, based on the results obtained in
␤2-microglobulin-deficient mice, CD8-deficient mice, and CD8depleted mice, which in each case failed to control infection in the
skin.2
Y. Belkaid, E. von Stebut, S. Mendez, R. Lira, M. C. Udey, and D. L. Sacks. CD8⫹
T cells are required for pathogenesis and immunity in C57BL/6 mice following lowdose, intradermal challenge with Leishmania major. Submitted for publication.
2
0022-1767/01/$02.00
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge
model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice
were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with
autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely
protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads
compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against
dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained
completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if
any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely
abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8ⴙ and CD4ⴙ
T cells to the site of intradermal challenge and with IFN-␥ production by CD8ⴙ T cells in lymph nodes draining the challenge site.
These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection
against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs. The Journal of Immunology, 2001, 166:
5122–5128.
The Journal of Immunology
In the present work, the natural challenge model was used to
evaluate the potency and durability of two vaccines, components
of which are currently being tested in preclinical and clinical trials:
1) a mixture of plasmid DNAs encoding the Leishmania Ags
LACK (12, 13, 16), LmSTI1 (17), and TSA (18); and 2) heatkilled promastigotes plus recombinant IL-12 (19 –22). Vaccine efficacy was evaluated in the context of all three phases of infection.
Although both vaccines conferred complete protection against the
development of dermal lesions, this complete protection lasted
longer in the DNA-vaccinated mice. Furthermore, only the DNA
vaccine reduced the parasitic burden in the skin during the acute
and chronic stages to the low levels achieved in healed mice, and
only the DNA vaccine eliminated the capacity of healed mice to
serve as infection reservoirs for vector sand flies. The powerful,
long-lasting protection conferred by AgDNA was associated with
the trafficking of CD8⫹ T cells to the site of challenge in the skin
and with the production of IFN-␥ by CD8⫹ T cells in draining
lymph nodes.
Mice
C57BL/6 (B6) and BALB/c mice were purchased from the Division of
Cancer Treatment, National Cancer Institute (Frederick, MD). All mice
were maintained in the National Institute of Allergy and Infectious Diseases Animal Care Facility under pathogen-free conditions.
Plasmid construction and purification
A cDNA encoding a truncated LACK protein (aa 143–312) was cloned
in-frame downstream to a Kozak consensus sequence and an initiation
codon into a pECE vector. The insert was excised using HindIII and ligated
into expression vector PcDNA-3 downstream to the CMV promoter (Invitrogen, San Diego, CA). The full-length sequences of LmSTI1 and TSA
were PCR amplified (⬃1.64 and 0.6 kb. respectively) from L. major
genomic DNA using sequence-specific primers and subcloned into
pcDNA3.1 (BamHI and EcoRI sites). Plasmid DNAs were purified by double-banding cesium chloride gradient ultracentrifugation. The 260:280 UV
absorption ratios ranged from 1.8 to 2.0.
Immunization
Mice were injected in the left hind footpad with a mixture of 100 ␮g of
each plasmid DNA encoding either LACK, LmSTI1, or TSA (300 ␮g of
total DNA) or 300 ␮g of control DNA (empty vector) suspended in 50 ␮l
of sterile PBS. The immunization with protein was conducted by the injection of 50 ␮g of heat-killed L. major promastigotes (ALM)3 with or
without 1.5 ␮g of rIL-12 (Genetics Institute, Cambridge, MA). The protein-based vaccine was prepared from whole-cell, heat-killed L. major
(ALM) and is identical to that being used with BCG as adjuvant in phase
III clinical trials in Iran and Sudan (21, 22). Each group was boosted 2 wk
later using the same regimen.
Infectious challenge
L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were grown at
26°C in medium 199 supplemented with 20% Hi-FCE (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 (M199/S). Infective-stage promastigotes (metacyclics) of L. major were isolated from stationary cultures (4to 5-day old) by negative selection using peanut agglutinin (Vector Laboratories, Burlingame, CA). Mice were challenged 2 or 12 wk postboost
using 100 metacyclic promastigotes. Parasites were inoculated intradermally into the ear dermis using a 27.5-gauge needle in a volume of 10 ␮l.
The evolution of the lesion was monitored by measuring the diameter of
the induration of the ear lesion with a direct reading Vernier caliper (Thomas, Swedesboro, NJ).
Parasite quantitation
Parasite loads in the ears were determined as previously described (15).
Briefly, the ventral and dorsal sheets of the infected ears were separated,
3
Abreviations used in this paper: ALM, autoclaved Leishmania major promastigotes;
SLA, soluble leishmanial Ag; DTH, delayed-type hypersensitivity.
deposited dermal side down in DMEM containing 100 U/ml penicillin, 100
␮g/ml streptomycin, and 1 mg/ml collagenase A (Sigma, St. Louis, MO),
and incubated for 2 h at 37°C. The sheets were cut into small pieces and
homogenized using a Teflon-coated microtissue grinder in a microfuge
tube containing 100 ␮l of M199/S. The tissue homogenates were filtered
using a 70-␮m cell strainer (Falcon Products, St. Louis, MO) and serially
diluted in a 96-well flat-bottom microtiter plate containing biphasic medium, prepared using 50 ␮l of NNN medium containing 30% of defibrinated rabbit blood and overlaid with 50 ␮l of M199/S. The number of
viable parasites in each ear was determined from the highest dilution at
which promastigotes could be grown out after 7 days of incubation at 26°C.
The number of parasites was also determined in the local draining lymph
nodes (retromaxilar). The lymph nodes were recovered and mechanically
dissociated using a pellet pestle and then serially diluted as above.
In vivo recall response
Mice were injected in both ears with a combination of living and killed Ags
comprised of 106 metacyclic L. major promastigotes and 12.5 ␮g of soluble leishmanial Ag (SLA) prepared from 3⫻ freeze-thawed stationary
phase L. major promastigotes. The increase of the ear thickness was measured 72 h later with a direct reading Vernier caliper. At this time, mice
were sacrificed and cells from the ear dermis and local draining lymph
nodes (three to four mice) were obtained. Briefly, the retromaxillar draining lymph nodes were recovered, mechanically dissociated using a pellet
pestle, and pooled. The ears were collected and the ventral and dorsal
dermal sheets were separated and incubated, dermal side down on RPMI
1640, NaHCO3, penicillin/streptomycin/gentamicin containing 1 mg/ml
collagenase A (Sigma) for 2 h or a mixture of collagenase A (1 mg/ml) and
liberase CI enzyme blend (0.28 Wünsch units/ml; Boehringer Mannheim,
Indianapolis, IN) for 1 h. The ears were pooled, cut in small pieces, and
filtered through a 70-␮m nylon cell strainer (Becton Dickinson, Mountain
View, CA) before being washed twice in RPMI 1640, NaHCO3, penicillin/
streptomycin/gentamicin, 10% FCS, and 0.05% DNase (Sigma).
Pooled cells from draining lymph nodes were resuspended in RPMI
1640 containing 10% FBS, 100 U/ml penicillin, and 100 ␮g/ml streptomycin at 2.5 ⫻ 106 cells/ml, and 0.2 ml was plated in U-bottom 96-well
plates. Cells were incubated at 37°C in 5% CO2 for 24 h with or without
addition of soluble L. major Ag (SLA, 25 ␮g/ml) or Con A (10 mg/ml).
IFN-␥ in 24-h culture supernatants was quantitated by ELISA. For the
analysis of surface markers and intracytoplasmic staining for IFN-␥, cells
were stimulated with 25 ␮g/ml SLA in the presence of anti-CD28 and
recombinant mouse IL-2 for 6 h, at which time brefeldin A was added (10
␮g/ml). The cells were cultured for an additional 18 h and then fixed in 4%
paraformaldehyde. Before staining, cells were incubated with an antiFc␥III/II (PharMingen, San Diego, CA) receptor and 10% normal mouse
serum in PBS containing 0.1% BSA and 0.01% NaN3. The staining of
surface and cytoplasmic markers was performed sequentially: the cells
were stained for the surface marker CD3 (145-2 C11, FITC labeled;
PharMingen), CD4 or CD8 (RM4-5 and 53-6.7, cychrome conjugated;
PharMingen) followed by a permeabilization step and staining with antiIFN-␥ conjugated to R-PE (JE56-5H4; PharMingen). Each incubation was
conducted for 30 min on ice. The isotype controls used were rat IgG2b
(A95-1; PharMingen) and rat IgG2a (R35-95; PharMingen). The frequency
of CD4⫹ and CD8⫹ T cells was determined by gating on CD3⫹ cells. For
each sample, at least 50,000 cells were analyzed. The data were collected
and analyzed using CellQuest software and a FACSCalibur flow cytometer
(Becton Dickinson).
Transmissibility of parasites from infected ears to sand flies
At 12 wk after challenge, the ability of the infected ears to provide a source
of parasites to sand flies was investigated. Two- to 4-day-old Phlebotomus
papatasi females were obtained from a colony initiated by field-caught
specimens from the Jordan Valley and were reared at the Department of
Entomology, Walter Reed Army Institute of Research (Silver Spring, MD).
Fifteen sand flies were placed in individual vials with meshed surfaces.
Mice were anesthetized i.p. with 200 ␮l of 20 mg/ml ketamine HCl (Phoenix Pharmaceuticals, St. Joseph, MO). Individual ears of anesthetized mice
were pressed flat against the meshed surface of the vials using clamps that
were specially designed for this purpose. The flies were allowed to feed in
the dark for a period of 30 min to 1 h. Blood-fed females from each vial
were separated and maintained in individual pots lined with plaster of
Paris, provided a 50% sucrose solution and water, and their midguts were
dissected 48 h later and examined microscopically for the presence of
promastigotes.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Materials and Methods
5123
5124
L. major VACCINES EVALUATED USING A NATURAL CHALLENGE MODEL
Results
AgDNA and ALM/rIL-12 confer complete protection against
dermal pathology when challenged at 2 wk postvaccination
Protection conferred by AgDNA or ALM/rIL-12 is durable in
resistant mice
To evaluate the durability of the immunity induced by the DNA
and killed vaccines, the animals were challenged 12 wk after
FIGURE 1. Lesion size (diameter of dermal lesion) in C57BL/6 mice
challenged 2 wk after immunization by intradermal inoculation of 100
metacyclic promastigotes of L. major. Mice were unvaccinated (⽧) or
vaccinated twice (2-wk interval) in the footpad with either 100 ␮g of each
AgDNA (f), 300 ␮g of control DNA (䡺), 50 ␮g of ALM ⫹ 1.5 ␮g of
rIL-12 (F), or 50 ␮g of ALM alone (E). Mice with previously healed
dermal lesions were included as immune controls (〫). Values represent
the mean lesion diameter ⫾ SEM of 4 –10 mice, 8 –20 ears/group. ⴱ, p ⬍
0.001 for ALM ⫹ rIL-12 (F) and AgDNA (f) compared with the unvaccinated control.
FIGURE 2. Parasite quantitation in the ear dermis (a) and in the local
draining lymph nodes (b) in mice challenged 2 wk postvaccination and
quantitated at 4 wk postinfection (䡺) and 10 wk post infection (p). Results
are expressed as geometric mean ⫾ SEM of six to eight ears per group.
Parasite loads in draining nodes were determined from the pool of six to
eight lymph nodes. ⴱ, p ⬍ 0.05, significant decrease compared with unvaccinated, ALM alone, and control DNA.
boosting. BALB/c mice were included for comparison. As shown
in Fig. 3, the protection against dermal pathology that was
achieved using ALM/rIL-12 was mouse strain dependent: BALB/c
mice showed no evidence of protection, confirming earlier observations (13), whereas B6 mice maintained significant, though partial protection. In contrast, both BALB/c and B6 mice vaccinated
with AgDNA were almost completely protected against the development of dermal lesions for at least 10 wk postchallenge. Only
one AgDNA-vaccinated animal of either strain showed any lesion,
which in each case remained small and rapidly resolved. Mice
vaccinated with ALM alone or control DNA again showed no
significant reduction in dermal pathology compared with unvaccinated animals.
Quantitation of parasites in ears and draining nodes of B6 mice
vaccinated with ALM/rIL-12 showed that the reduction in the dermal pathology correlated with a 10-fold reduction in the number of
parasites in the skin at week 4 and a 100-fold reduction in the
draining nodes (Fig. 4). The tissue parasite burdens in each of
these sites was actually increased when re-examined at week 10,
suggesting that the vaccine may have delayed but not significantly
reduced the peak parasite loads established in the inoculation site.
In contrast, the parasite loads in the mice vaccinated with AgDNA
were reduced to barely detectable levels in the skin (⬍10) and
undetectable levels in the draining nodes, even when examined at
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Mice vaccinated with either AgDNA or ALM/rIL-12 and challenged 2 wk later with 100 metacyclic promastigotes in the ear
dermis were almost completely protected against the development
of dermal lesions, comparable to the resistance displayed by healed
mice (Fig. 1). The mice vaccinated with ALM alone or control
DNA (empty plasmid) developed lesions similar in size and duration to the unvaccinated mice: the lesions appeared at week 4,
reached a peak at ⬃6 –7 wk, and were completely healed at 10 –12
wk. Since in the natural challenge model, the peak number of
parasites in the site occurs just before the development of the lesion, the parasite load in the ear was monitored at week 4. The
absence of dermal pathology in the vaccinated mice correlated
with an approximate 100-fold reduction in the number of parasites
in the skin (4.4 ⫻ 104 parasites in naive mice vs 3.5 ⫻ 102 in the
AgDNA group and 8 ⫻ 102 in ALM/rIL12 group) (Fig. 2). These
groups also had an approximate 10-fold reduction in the parasite
burden in the lymph nodes draining the inoculation site. Vaccination with ALM alone or with the empty vector did not reduce the
number of parasites found in the skin or in the draining nodes.
After healing (10 wk post infection), ⬃90% of the organisms had
been killed or cleared from the inoculation site in all of the groups,
although the number of parasites in the ears of AgDNA-vaccinated
mice (1.2 ⫻ 102) or ALM/rIL-12 vaccinated mice (1.7 ⫻ 102)
remained ⬃100-fold lower compared with the unvaccinated group
(4.2 ⫻ 104). Interestingly, no parasites were found in the local
draining nodes of the AgDNA-vaccinated animals at 10 wk postinfection, a level of control that was otherwise achieved only in the
mice that had healed their primary infections.
The Journal of Immunology
5125
FIGURE 3. Lesion size (diameter of dermal lesion) in C57BL/6 mice
(a) and BALB/c mice (b) challenged 12 wk after immunization by intradermal inoculation of 100 metacyclic promastigotes of L. major. Mice
were either unvaccinated (⽧), healed (〫), or vaccinated twice in the footpad with AgDNA (f), control DNA (䡺), ALM/rIL-12 (F), or ALM alone
(E). Values represent the mean lesion diameter ⫾ SEM, 4 –10 mice, 8 –20
ears/group. ⴱ, p ⬍ 0.001 significantly less than unvaccinated and controlvaccinated mice. Data are representative of two experiments.
control plasmid (50%). In contrast, the transmission in mice vaccinated with AgDNA was completely abrogated (0 of 12 ears), and
no transmission was observed in the rechallenged site of the healed
mice. Of the ears that were capable of transmitting L. major, the
week 4, when ⬎10,000 parasites were found in the skin of unvaccinated and control DNA-vaccinated mice. In terms of tissue parasite burdens, the immunity conferred by AgDNA was as effective
as the naturally acquired immunity operating in the healed mice,
and its potency was actually increased at 12 wk compared with 2
wk postvaccination.
Parasites are not transmissible to vector sand flies in mice
vaccinated using AgDNA
To determine whether the number of parasites in the skin during
the posthealing, chronic stage of infection (12 wk postchallenge)
was sufficiently high to be picked up by vector sand flies, the ears
of unvaccinated and vaccinated mice, challenged 12 wk after vaccination, were exposed to the bites of a natural vector, P. papatasi
(15 flies per ear). At least 70% of the flies in each group successfully obtained a blood meal. Blood-engorged flies were dissected
48 h later and scored for the presence or absence of parasites in
their midguts. In results pooled from two separate studies, 50% of
the ears of the unvaccinated mice transmitted parasites to the sand
flies (Fig. 5). Such efficient transmission was also found in the
mice vaccinated with ALM/rIL-12 (42%), ALM alone (50%), or
FIGURE 5. Transmission of L. major to P. papatasi after exposure of
sand flies to C57BL/6 mice 12 wk following challenge. Mice were challenged 12 wk postvaccination in the ear dermis using 100 metacyclic promastigotes, and flies exposed to the ears were scored for the presence of
midgut promastigotes 48 h after engorgement. Values represent the percentage of the ears in each group that were able to transmit L. major to
exposed flies. Results are pooled from two separate experiments.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 4. Parasite quantification in ears (a) and draining nodes (b) in
C57BL/6 mice challenged 12 wk postvaccination. 䡺, 4 wk postinfection;
p, 10 wk postinfection. Results are expressed as geometric mean ⫾ SEM
of six to eight ears per group. Parasite loads in draining nodes were determined from individual nodes, six to eight per group. ⴱ, p ⬍ 0.05 significantly decreased compared with unvaccinated and control-vaccinated
groups. Data are representative of two experiments.
5126
L. major VACCINES EVALUATED USING A NATURAL CHALLENGE MODEL
Table I. L. major-specific DTH responses 12 wk postvaccination
Specific DTH after 72 ha
Groups
Unvaccinated
ALM ⫹ IL-12
ALM
AgDNA
Control DNA
Healed
0
0.18 ⫾ 0.06
0.25 ⫾ 0.5
0.51 ⫾ 0.26
0.11 ⫾ 0.16
1.2 ⫾ 0.56
a
Increase in ear thickness 48 h after intradermal injection of 106 metacyclic promastigotes plus 12.5 ␮g of SLA, mean ⫾ SD, n ⫽ 6 – 8 ears.
efficiency of transmission, as determined by the percentage of
blood-fed flies that were positive for parasites, ranged between 10
and 50%, with no significant difference between the groups (data
not shown).
Surrogate markers of immunity in vaccinated mice
Discussion
A natural model of L. major infection in C57BL/6 mice, reproducing the low-dose, intradermal inoculation associated with sand
fly challenge, has been used to evaluate the potency and durability
of DNA- and protein-based vaccines that are currently being developed for use in clinical trials. In the present studies, the ALM,
which has been given with bacillus Calmette-Guérin as adjuvant in
phase III clinical trials (21, 22), was given with rIL-12, which has
been shown to be a powerful adjuvant for killed Leishmania or
recombinant protein vaccines in both mouse and monkey models
(13, 16, 19, 20). The DNA vaccine used was a mixture of plasmid
DNAs encoding the Ags LACK, LmSTI1, and TSA. As recombinant proteins, LACK and TSA have produced at least partial protection against L. major in BALB/c mice (12, 13, 16, 18), and
LmSTI1 was shown to induce high levels of IFN-␥ by lymph node
cells from infected mice (17). The DNAs encoding the Ags LACK
(12, 13) and LmSTI1 and TSA (S. Gurunathan, unpublished data)
have each also conferred some protection in BALB/c mice following high-dose footpad challenge.
The rationale for applying a model of natural challenge in a
resistant mouse strain to the evaluation of vaccines intended for
use against cutaneous leishmaniasis is based on the contention that
the model more accurately reproduces clinical-pathological findings associated with human disease. In particular, the model has
revealed that the development of dermal lesions occurs only after
Table II. T cell recruitment to the site of intradermal challenge in C57BL/6 mice 12 wk postvaccination
Expt. 1b
Unvaccinated
ALM/rIL-12
ALM
Ag DNA
Control DNA
Healed
Expt. 2c
Unvaccinated
Ag DNA
Control DNA
Healed
a
b
c
Total No. of Cells/Ear
CD3⫹ (%)a
CD4⫹ (%)a
CD8⫹ (%)a
␥␦ T Cells (%)a
0.5 ⫻ 106
0.6 ⫻ 106
0.6 ⫻ 106
1.2 ⫻ 106
1.0 ⫻ 106
3.9 ⫻ 106
5 ⫻ 104 (10.4)
6.7 ⫻ 104 (11.2)
6.1 ⫻ 104 (10.1)
14.4 ⫻ 104 (12.8)
9.2 ⫻ 104 (9.2)
58.5 ⫻ 104 (15.4)
0.6 ⫻ 104 (1.2)
1.4 ⫻ 104 (2.3)
1.4 ⫻ 104 (2.3)
3.2 ⫻ 104 (2.7)
1.5 ⫻ 104 (1.5)
23.0 ⫻ 104 (6.0)
0.3 ⫻ 104 (0.8)
0.2 ⫻ 104 (0.3)
0.3 ⫻ 104 (0.3)
1.2 ⫻ 104 (1.0)
0.7 ⫻ 104 (0.7)
7.2 ⫻ 104 (1.8)
ND
ND
ND
ND
ND
ND
4.9 ⫻ 106
9.3 ⫻ 106
2.8 ⫻ 106
13.7 ⫻ 106
5.3 ⫻ 105 (11.1)
14.8 ⫻ 105 (16.0)
2.5 ⫻ 105 (9.1)
27.4 ⫻ 105 (20.1)
0.6 ⫻ 105 (1.2)
2.5 ⫻ 105 (2.7)
0.2 ⫻ 105 (0.8)
10.2 ⫻ 105 (7.5)
0.1 ⫻ 105 (0.2)
1.2 ⫻ 105 (1.3)
0.2 ⫻ 105 (0.5)
3.0 ⫻ 105 (2.3)
0.1 ⫻ 105 (1.1)
0.2 ⫻ 105 (2.3)
0.1 ⫻ 105 (0.9)
0.1 ⫻ 105 (1.3)
Percentage of total number of extracted cells/ear calculated from cell preparations pooled from three to four mice, six to eight ears for each group.
Cells extracted from collagenase-digested ears 72 h after injection of 12.5 ␮g of SLA and 106 metacyclic promastigotes.
Cells extracted from collagenase- and liberase-digested ears 72 h after injection of 12.5 ␮g of SLA and 106 metacyclics.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Surrogate markers of immunity to leishmaniasis are thought to
include Ag-induced delayed-type hypersensitivity (DTH) responses in vivo and IFN-␥ production by T cells following restimulation with Ag in vitro. These recall responses were evaluated
12 wk after vaccination by injecting into the ear dermis a mixture
of L. major-soluble Ag (SLA, 12.5 ␮g) and living metacyclic promastigotes (106), and then measuring 1) the change in ear thickness at 48 h and 72 h, 2) the number and kinds of T cells in the
inflammatory dermis, and 3) IFN-␥ production by lymph node
cells draining the ear following restimulation in vitro using SLA,
anti-CD28, and IL-2. Table I shows that the mice receiving
AgDNA displayed a stronger DTH (0.51-mm increase in ear thickness) compared with the other vaccinated or unvaccinated groups
(0 – 0.25 mm). The healed mice showed the strongest DTH response (1.20-mm increase). The DTH response was associated
with the recruitment of CD4⫹ (3.2 ⫻ 104) and CD8⫹ cells (1.2 ⫻
104) in the ears of mice vaccinated with AgDNA that in each case
was 2- to 3-fold greater than the number of CD4⫹ (0.6 –1.5 ⫻ 104)
or CD8⫹ cells (0.3– 0.7 ⫻ 104) recovered from the ears of each of
the other vaccinated or unvaccinated groups (Table II, experiment
1). T cell recruitment was highest in the rechallenged site of the
healed mice (23 ⫻ 104 and 7.2 ⫻ 104 for CD4⫹ and CD8⫹, respectively). In a subsequent experiment involving DNA-vaccinated mice and more efficient extraction of cells from the inflammatory ear dermis, there were 2.5 ⫻ 105 CD4⫹ and 1.2 ⫻ 105
CD8⫹ T cells present in the inoculation site of mice immunized
with AgDNA, representing a 4- and 6- fold increase in the numbers
of CD4⫹ cells and a 12- and 6- fold increase in the number of
CD8⫹ cells compared with unvaccinated and control DNA-vaccinated groups, respectively (Table II, experiment 2). Furthermore,
examination of the frequency of ␥␦ T cells in the inflammatory
dermis revealed a 2-fold increase compared with each of the other
groups, including the healed mice.
The lymph node cells draining the ear were recovered 72 h after
injection of Ag, and after restimulation in vitro for 24 h, the culture
supernatants were assayed for IFN-␥ by ELISA. Cells from healed
mice and from both ALM/rIL-12- and AgDNA-vaccinated mice,
secreted high levels of IFN-␥ (Fig. 6). Unvaccinated and controlvaccinated groups secreted little or no IFN-␥ in response to Ag.
The frequency of lymph node cells staining for IFN-␥ following
restimulation in vitro was high in the healed mice for both CD4⫹
and CD8⫹ T cells (9.5 and 8.4%, respectively; Fig. 7). An increased frequency of CD4⫹-IFN-␥-producing cells was observed
in the ALM/rIL-12 group (6.2%). The only vaccine group with a
substantial increase in the number of CD8⫹ IFN-␥-producing cells
was the AgDNA group (7.2%).
The Journal of Immunology
a prolonged silent phase of parasite amplification in the skin, and
is due to an inflammatory infiltrate that is dependent on and coincident with the onset of acquired immunity and the killing of parasites in the inoculation site. In contrast, high-dose s.c. inocula,
particularly in BALB/c mice, produce rapidly evolving lesions that
are formed as a consequence of large numbers of infected macrophages accumulating in the inoculation site. Vaccines that moderate the development of these sorts of lesions may not necessarily
protect against, and could conceivably exacerbate, immune-mediated dermal pathology. In addition, the natural challenge model
has revealed a role for CD8⫹ T cells in the resolution of primary
infection in the skin,2 which again has not been appreciated as an
important component of adaptive immunity to primary infection
following conventional high-dose s.c. challenge (23–26).
When challenged 2 wk postvaccination, both ALM/rIL-12 and
AgDNA conferred striking immunity in C57BL/6 mice that in
most cases resulted in the complete absence of dermal lesions.
Such complete protection against clinical disease, which to our
knowledge has not been observed in other laboratory-based trials
of Leishmania vaccines, seems especially relevant to the field evaluation of vaccine efficacy, for which the primary measure is a
decrease in disease incidence. Both vaccines maintained significant protection in C57BL/6 mice up to 12 wk after vaccination. In
contrast, when challenged at 12 wk, only the DNA vaccine was
able to protect BALB/c mice, confirming previous results established using high-dose s.c. challenge (13). The relative durability
of ALM/rIL-12 vaccination in C57BL/6 mice, in which immune
responses to L. major infection appear to more accurately reproduce those associated with human disease, suggests that BALB/c
mice might in some cases undervalue the potential of vaccines
intended for use against cutaneous leishmaniasis. The durability of
Leishmania-specific effector and/or memory T cells in BALB/c
mice may be compromised by the same conditions that establish
their strong Th2 bias to L. major infection that is observed in
naive mice.
Even in the C57BL/6 mice, however, the AgDNA demonstrated
greater potency and durability than ALM/rIL-12. Whereas the immunity elicited by ALM/rIL-12 began to wane by 12 wk, both in
terms of its ability to prevent lesion development and to reduce
tissue parasite burden, the immunity elicited by AgDNA was if
anything more potent at 12 wk postvaccination than at 2 wk. Furthermore, only the AgDNA conferred protection that extended to
the chronic phase; at 10 wk postinfection, viable organisms were
reduced to extremely low numbers in the skin (⬍10), and they
were undetectable in draining nodes. The effect of vaccination on
the chronic stage of infection has not, to our knowledge, been
evaluated before. To establish the significance of this result in the
context of reservoir potential, the chronic infection sites were exposed to the bites of P. papatasi, a natural vector of L. major.
Whereas the ears of ALM/rIL-12-vaccinated mice transmitted parasites to sand flies as efficiently as the unvaccinated and controlvaccinated mice, the AgDNA-vaccinated mice failed to provide a
source of parasites for pick up by flies. Applied to a field setting,
these outcomes predict that DNA vaccination would have the capacity to reduce both the incidence of disease and, in foci involving anthroponotic species such as Leishmania tropica, the generation of infection reservoirs.
The more powerful, long-lasting immunity conferred by
AgDNA may be related to its ability to efficiently prime CD8⫹ as
wells as CD4⫹ T cells (6, 27–30), since both appear to be required
for immunity in the natural challenge model.2 Long-lived LACKresponsive CD8⫹ and CD4⫹ T cells are induced by LACK DNA
in BALB/c mice (13, 31). In the present analyses, only the AgDNA
induced a DTH response that could be elicited up to 12 wk postvaccination, and only the AgDNA generated and maintained a high
frequency of CD8⫹ cells that could traffic to the site of Ag challenge in the skin and produce IFN-␥ in response to reactivation
with Ag in vitro.
FIGURE 7. IFN-␥ response by CD4⫹ and CD8⫹ T cells in C57BL/6 mice 12 wk postvaccination. Staining for surface markers and intracytoplasmic
staining for IFN-␥ were analyzed on draining lymph node cells obtained 72 h after intradermal injection of metacyclic promastigotes and SLA and 24 h
after in vitro restimulation with SLA plus anti-CD28 and IL-2. Analyses are gated on CD3⫹ cells. Numbers represent the percentage of CD4⫹ or CD8⫹
T cells positive for IFN-␥. Positive signals were established using PE-isotype controls. Data are representative of two experiments.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 6. IFN-␥ response in C57BL/6 mice 12 wk postvaccination.
IFN-␥ was measured by ELISA in culture supernatants of draining lymph
node cells pooled from three to four mice 72 h after intradermal injection
of metacyclic promastigotes and SLA and 24 h after in vitro incubation
without Ag (䡺), with SLA (black bars), or with Con A (p). Data shown are
the mean concentrations of duplicate assays.
5127
5128
L. major VACCINES EVALUATED USING A NATURAL CHALLENGE MODEL
In summary, the natural challenge model has revealed that both
protein- and DNA-based vaccines have the capacity to completely
protect mice against dermal leishmaniasis, but that the DNA vaccine can better maintain this immunity as well as reduce the intensity of acute and chronic phase infections to levels that prevent
the generation of reservoirs of disease transmission.
Acknowledgments
We thank Sandra Cooper for help with the mouse maintenance and Govind
Modi and Ed Rowton for their invaluable help with the rearing and maintenance of the sand fly colony.
References
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
1. Tang, D. C., M. DeVit, and S. A. Johnston. 1992. Genetic immunization is a
simple method for eliciting an immune response. Nature 356:152.
2. Ulmer, J. B., J. J. Donnelly, S. E. Parker, G. H. Rhodes, P. L. Felgner,
V. J. Dwarki, S. H. Gromkowski, R. R. Deck, C. M. DeWitt, A. Friedman, et al.
1993. Heterologous protection against influenza by injection of DNA encoding a
viral protein. Science 259:1745.
3. Fynan, E. F., R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro, and
H. L. Robinson. 1993. DNA vaccines: protective immunizations by parenteral,
mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. USA 90:11478.
4. Davis, H. L., M. L. Michel, M. Mancini, M. Schleef, and R. G. Whalen. 1994.
Direct gene transfer in skeletal muscle: plasmid DNA-based immunization
against the hepatitis B virus surface antigen. Vaccine 12:1503.
5. - Lowrie, D. B., R. E. Tascon, M. J. Colston, and C. L. Silva. 1994. Towards a
DNA vaccine against tuberculosis. Vaccine 12:1537.
6. Mor, G., D. M. Klinman, S. Shapiro, E. Hagiwara, M. Sedegah, J. A. Norman,
S. L. Hoffman, and A. D. Steinberg. 1995. Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA. J. Immunol. 155:2039.
7. Angus, C. W., D. Klivington, J. Wyman, and J. A. Kovacs. 1996. Nucleic acid
vaccination against Toxoplasma gondii in mice. J. Eukaryotic Microbiol.
43:117S.
8. Heinzel, F. P., M. D. Sadick, S. S. Mutha, and R. M. Locksley. 1991. Production
of interferon gamma, interleukin 2, interleukin 4, and interleukin 10 by CD4⫹
lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc.
Natl. Acad. Sci. USA 88:7011.
9. Scott, P., P. Natovitz, R. L. Coffman, E. Pearce, and A. Sher. 1988. Immunoregulation of cutaneous leishmaniasis: T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct
parasite antigens. J. Exp. Med. 168:1675.
10. Xu, D., and F. Y. Liew. 1995. Protection against leishmaniasis by injection of
DNA encoding a major surface glycoprotein, gp63, of L. major. Immunology
84:173.
11. Walker, P. S., T. Scharton-Kersten, E. D. Rowton, U. Hengge, A. Bouloc,
M. C. Udey, and J. C. Vogel. 1998. Genetic immunization with glycoprotein 63
cDNA results in a helper T cell type 1 immune response and protection in a
murine model of leishmaniasis. Hum. Gene Ther. 9:1899.
12. 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.
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.
14. Sjolander, A., T. M. Baldwin, J. M. Curtis, and E. Handman. 1998. Induction of
a Th1 immune response and simultaneous lack of activation of a Th2 response are
required for generation of immunity to leishmaniasis. J. Immunol. 160:3949.
15. 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 preexposure on the
long-term outcome of Leishmania major infection in the mouse ear dermis.
J. Exp. Med. 188:1941.
16. Mougneau, E., F. Altare, A. E. Wakil, S. Zheng, T. Coppola, Z. E. Wang,
R. Waldmann, R. M. Locksley, and N. Glaichenhaus. 1995. Expression cloning
of a protective Leishmania antigen. Science 268:563.
17. Webb, J. R., D. Kaufmann, A. Campos-Neto, and S. G. Reed. 1996. Molecular
cloning of a novel protein antigen of Leishmania major that elicits a potent
immune response in experimental murine leishmaniasis. J. Immunol. 157:5034.
18. Webb, J. R., A. Campos-Neto, P. J. Ovendale, T. I. Martin, E. J. Stromberg,
R. Badaro, and S. G. Reed. 1998. Human and murine immune responses to a
novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect. Immun. 66:3279.
19. Afonso, L. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G. Trinchieri, and
P. Scott. 1994. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 263:235.
20. Kenney, R. T., D. L. Sacks, J. P. Sypek, L. Vilela, A. A. Gam, and
K. Evans-Davis. 1999. Protective immunity using recombinant human IL-12 and
alum as adjuvants in a primate model of cutaneous leishmaniasis. J. Immunol.
163:4481.
21. Sharifi, I., A. R. FeKri, M. R. Aflatonian, A. Khamesipour, A. Nadim,
M. R. Mousavi, A. Z. Momeni, Y. Dowlati, T. Godal, F. Zicker, et al. 1998.
Randomised vaccine trial of single dose of killed Leishmania major plus BCG
against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet 351:1540.
22. Momeni, A. Z., T. Jalayer, M. Emamjomeh, A. Khamesipour, F. Zicker,
R. L. Ghassemi, Y. Dowlati, I. Sharifi, M. Aminjavaheri, A. Shafiei, et al. 1999.
A randomised, double-blind, controlled trial of a killed L. major vaccine plus
BCG against zoonotic cutaneous leishmaniasis in Iran. Vaccine 17:466.
23. Wakil, A. E., Z. E. Wang, J. C. Ryan, D. J. Fowell, and R. M. Locksley. 1998.
Interferon ␥ derived from CD4⫹ T cells is sufficient to mediate T helper cell type
1 development. J. Exp. Med. 188:1651.
24. Erb, K., C. Blank, U. Ritter, H. Bluethmann, and H. Moll. 1996. Leishmania
major infection in major histocompatibility complex class II-deficient mice:
CD8⫹ T cells do not mediate a protective immune response. Immunobiology
195:243.
25. Wang, Z. E., S. L. Reiner, F. Hatam, F. P. Heinzel, J. Bouvier, C. W. Turck, and
R. M. Locksley. 1993. Targeted activation of CD8 cells and infection of ␤2microglobulin-deficient mice fail to confirm a primary protective role for CD8
cells in experimental leishmaniasis. J. Immunol. 151:2077.
26. Huber, M., E. Timms, T. W. Mak, M. Rollinghoff, and M. Lohoff. 1998. Effective
and long-lasting immunity against the parasite Leishmania major in CD8-deficient mice. Infect. Immun. 66:3968.
27. Chen, Y., R. G. Webster, and D. L. Woodland. 1998. Induction of CD8⫹ T cell
responses to dominant and subdominant epitopes and protective immunity to
Sendai virus infection by DNA vaccination. J. Immunol. 160:2425.
28. Fu, T. M., A. Friedman, J. B. Ulmer, M. A. Liu, and J. J. Donnelly. 1997.
Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA
immunization. J. Virol. 71:2715.
29. Martins, L. P., L. L. Lau, M. S. Asano, and R. Ahmed. 1995. DNA vaccination
against persistent viral infection. J. Virol. 69:2574.
30. Sedegah, M., R. Hedstrom, P. Hobart, and S. L. Hoffman. 1994. Protection
against malaria by immunization with plasmid DNA encoding circumsporozoite
protein. Proc. Natl. Acad. Sci. USA 91:9866.
31. Gurunathan, S., L. Stobie, C. Prussin, D. L. Sacks, N. Glaichenhaus, D. J. Fowell,
R. M. Locksley, J. T. Chang, C. Y. Wu, and R. A. Seder. 2000. Requirements for
the maintenance of Th1 immunity in vivo following DNA vaccination: a potential
immunoregulatory role for CD8⫹ T cells. J. Immunol. 165:915.