The Potency and Durability of DNA- and. major Evaluated Using Low-Dose, Intradermal Challenge. This information is current as of February 21, 2013.

Transcription

1 This information is current as of February 21, References Subscriptions Permissions Alerts 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 J Immunol 2001; 166: ; ; This article cites 31 articles, 21 of which you can access for free at: Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD Copyright 2001 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 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 1 * 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 CD8 and CD4 T cells to the site of intradermal challenge and with IFN- production by CD8 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: *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 Received for publication December 1, Accepted for publication February 8, 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 address: dsacks@nih.gov Genetic 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. Although 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 ( ) 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 CD8- depleted mice, which in each case failed to control infection in the skin. 2 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. Copyright 2001 by The American Association of Immunologists /01/$02.00

3 The Journal of Immunology 5123 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. Materials and Methods 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 ) 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 (4- to 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 10 6 metacyclic L. major promastigotes and 12.5 g ofsoluble 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, NaHCO 3, penicillin/streptomycin/gentamicin containing 1 mg/ml collagenase A (Sigma) for2horamixture 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, NaHCO 3, 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 cells/ml, and 0.2 ml was plated in U-bottom 96-well plates. Cells were incubated at 37 C in 5% CO 2 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 anti- Fc III/II (PharMingen, San Diego, CA) receptor and 10% normal mouse serum in PBS containing 0.1% BSA and 0.01% NaN 3. 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 , cychrome conjugated; PharMingen) followed by a permeabilization step and staining with anti- IFN- 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.

4 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 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 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 ( parasites in naive mice vs in the AgDNA group and 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 ( ) or ALM/rIL-12 vaccinated mice ( ) remained 100-fold lower compared with the unvaccinated group ( ). 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. 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 ( ) or vaccinated twice (2-wk interval) in the footpad with either 100 g of each AgDNA (f), 300 g of control DNA ( ), 50 g ofalm 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., p 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 ( ) 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., 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

5 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 ( ), healed ( ), or vaccinated twice in the footpad with AgDNA (f), control DNA ( ), ALM/rIL-12 (F), or ALM alone (E). Values represent the mean lesion diameter SEM, 4 10 mice, 8 20 ears/group., p significantly less than unvaccinated and controlvaccinated mice. Data are representative of two experiments. 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 4. Parasite quantification in ears (a) and draining nodes (b) in C57BL/6 mice challenged 12 wk postvaccination., 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., p 0.05 significantly decreased compared with unvaccinated and control-vaccinated groups. 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 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.

6 5126 L. major VACCINES EVALUATED USING A NATURAL CHALLENGE MODEL Table I. L. major-specific DTH responses 12 wk postvaccination Groups Specific DTH after 72 h a Unvaccinated 0 ALM IL ALM AgDNA Control DNA Healed a Increase in ear thickness 48 h after intradermal injection of 10 6 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 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 (10 6 ), 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 ( mm). The healed mice showed the strongest DTH response (1.20-mm increase). The DTH response was associated with the recruitment of CD4 ( ) and CD8 cells ( ) in the ears of mice vaccinated with AgDNA that in each case was 2- to 3-fold greater than the number of CD4 ( ) or CD8 cells ( ) 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 ( and 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 CD4 and 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%). 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 Total No. of Cells/Ear CD3 (%) a CD4 (%) a CD8 (%) a T Cells (%) a Expt. 1 b Unvaccinated (10.4) (1.2) (0.8) ND ALM/rIL (11.2) (2.3) (0.3) ND ALM (10.1) (2.3) (0.3) ND Ag DNA (12.8) (2.7) (1.0) ND Control DNA (9.2) (1.5) (0.7) ND Healed (15.4) (6.0) (1.8) ND Expt. 2 c Unvaccinated (11.1) (1.2) (0.2) (1.1) Ag DNA (16.0) (2.7) (1.3) (2.3) Control DNA (9.1) (0.8) (0.5) (0.9) Healed (20.1) (7.5) (2.3) (1.3) a 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. b Cells extracted from collagenase-digested ears 72 h after injection of 12.5 g of SLA and 10 6 metacyclic promastigotes. c Cells extracted from collagenase- and liberase-digested ears 72 h after injection of 12.5 g of SLA and 10 6 metacyclics.

7 The Journal of Immunology 5127 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 ( ), with SLA (black bars), or with Con A (p). Data shown are the mean concentrations of duplicate assays. 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.

8 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 1. Tang, D. C., M. DeVit, and S. A. Johnston Genetic immunization is a simple method for eliciting an immune response. Nature 356: 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 Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259: Fynan, E. F., R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro, and H. L. Robinson DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. USA 90: Davis, H. L., M. L. Michel, M. Mancini, M. Schleef, and R. G. Whalen Direct gene transfer in skeletal muscle: plasmid DNA-based immunization against the hepatitis B virus surface antigen. Vaccine 12: Lowrie, D. B., R. E. Tascon, M. J. Colston, and C. L. Silva Towards a DNA vaccine against tuberculosis. Vaccine 12: Mor, G., D. M. Klinman, S. Shapiro, E. Hagiwara, M. Sedegah, J. A. Norman, S. L. Hoffman, and A. D. Steinberg Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA. J. Immunol. 155: Angus, C. W., D. Klivington, J. Wyman, and J. A. Kovacs 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 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: Scott, P., P. Natovitz, R. L. Coffman, E. Pearce, and A. Sher 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: Xu, D., and F. Y. Liew Protection against leishmaniasis by injection of DNA encoding a major surface glycoprotein, gp63, of L. major. Immunology 84: Walker, P. S., T. Scharton-Kersten, E. D. Rowton, U. Hengge, A. Bouloc, M. C. Udey, and J. C. Vogel 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: Gurunathan, S., D. L. Sacks, D. R. Brown, S. L. Reiner, H. Charest, N. Glaichenhaus, and R. A. Seder Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. J. Exp. Med. 186: Gurunathan, S., C. Prussin, D. L. Sacks, and R. A. Seder Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection. Nat. Med. 4: Sjolander, A., T. M. Baldwin, J. M. Curtis, and E. Handman 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: Belkaid, Y., S. Kamhawi, G. Modi, J. Valenzuela, N. Noben-Trauth, E. Rowton, J. Ribeiro, and D. L. Sacks 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: Mougneau, E., F. Altare, A. E. Wakil, S. Zheng, T. Coppola, Z. E. Wang, R. Waldmann, R. M. Locksley, and N. Glaichenhaus Expression cloning of a protective Leishmania antigen. Science 268: Webb, J. R., D. Kaufmann, A. Campos-Neto, and S. G. Reed Molecular cloning of a novel protein antigen of Leishmania major that elicits a potent immune response in experimental murine leishmaniasis. J. Immunol. 157: Webb, J. R., A. Campos-Neto, P. J. Ovendale, T. I. Martin, E. J. Stromberg, R. Badaro, and S. G. Reed Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect. Immun. 66: Afonso, L. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G. Trinchieri, and P. Scott The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 263: Kenney, R. T., D. L. Sacks, J. P. Sypek, L. Vilela, A. A. Gam, and K. Evans-Davis Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis. J. Immunol. 163: 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 Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet 351: Momeni, A. Z., T. Jalayer, M. Emamjomeh, A. Khamesipour, F. Zicker, R. L. Ghassemi, Y. Dowlati, I. Sharifi, M. Aminjavaheri, A. Shafiei, et al A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran. Vaccine 17: Wakil, A. E., Z. E. Wang, J. C. Ryan, D. J. Fowell, and R. M. Locksley Interferon derived from CD4 T cells is sufficient to mediate T helper cell type 1 development. J. Exp. Med. 188: Erb, K., C. Blank, U. Ritter, H. Bluethmann, and H. Moll Leishmania major infection in major histocompatibility complex class II-deficient mice: CD8 T cells do not mediate a protective immune response. Immunobiology 195: Wang, Z. E., S. L. Reiner, F. Hatam, F. P. Heinzel, J. Bouvier, C. W. Turck, and R. M. Locksley Targeted activation of CD8 cells and infection of 2 - microglobulin-deficient mice fail to confirm a primary protective role for CD8 cells in experimental leishmaniasis. J. Immunol. 151: Huber, M., E. Timms, T. W. Mak, M. Rollinghoff, and M. Lohoff Effective and long-lasting immunity against the parasite Leishmania major in CD8-deficient mice. Infect. Immun. 66: Chen, Y., R. G. Webster, and D. L. Woodland Induction of CD8 T cell responses to dominant and subdominant epitopes and protective immunity to Sendai virus infection by DNA vaccination. J. Immunol. 160: Fu, T. M., A. Friedman, J. B. Ulmer, M. A. Liu, and J. J. Donnelly Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization. J. Virol. 71: Martins, L. P., L. L. Lau, M. S. Asano, and R. Ahmed DNA vaccination against persistent viral infection. J. Virol. 69: Sedegah, M., R. Hedstrom, P. Hobart, and S. L. Hoffman Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proc. Natl. Acad. Sci. USA 91: 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 Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8 T cells. J. Immunol. 165:915.