Leishmania amazonensis:The Asian Rhesus Macaques (Macaca mulatta) as an Experimental Model for Study of Cutaneous Leishmaniasis

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EXPERIMENTAL PARASITOLOGY ARTICLE NO. 0005

82, 34–44 (1996)

Leishmania amazonensis: The Asian Rhesus Macaques (Macaca mulatta) as an Experimental Model for Study of Cutaneous Leishmaniasis VERONICA F. AMARAL,*,1 VANESSA A. O. RANSATTO,* FATIMA CONCEIÇA˜ O-SILVA,† ETELCIA MOLINARO,‡ VIRGILIO FERREIRA,‡ SERGIO G. COUTINHO,† DIANE MCMAHON-PRATT,§ ,2 AND GABRIEL GRIMALDI, JR.* Departamento de *Imunologia and †Departamento de Protozoologia, Instituto Oswaldo Cruz, and ‡Centro de Primatologia/FIOCRUZ, C.P. 926, Rio de Janeiro, RJ 21045-900, Brazil; and §Department of Epidemiology and Public Health, Yale University School of Medicine, P.O. Box 208034, New Haven, Connecticut 06520-8034, U.S.A. AMARAL, V. F., RANSATTO, V. A. O., CONCEIÇA˜ O-SILVA, F., MOLINARO, E., FERREIRA, V., COUTINHO, S. G., MCMAHON-PRATT, D., AND GRIMALDI, G., JR. 1996. Leishmania amazonensis: The asian rhesus macaques (Macaca mulatta) as an experimental model for study of cutaneous leishmaniasis. Experimental Parasitology 82, 34–44. As a means of assessing the usefulness of the Rhesus macaque (Macaca mulatta) as a nonhuman primate model for studying cutaneous leishmaniasis, monkeys were infected with Leishmania amazonensis. Variation in the level of susceptibility was found; however, animals inoculated with 108 promastigotes provided consistent results as indicated by an earlier onset and/or larger size of lesions. Three monkeys, which had recovered from skin lesions, were challenge-infected using the same parasite strain/dose; although these animals remained susceptible to homologous infection, lesion size was smaller and healed faster than in the initial infection. The immunologic features during infection were assessed. Levels of IgM and IgG antibodies to promastigote antigens rose during active infection and then declined; immunoblot analyses indicated that numerous leishmanial antigens (predominately >30 kDa) were recognized. Delayed type hypersensitivity (DTH) responses and proliferative responses (PBL) developed during active infection and/or rechallenge. Circulating peripheral T cell subpopulations varied throughout the course of infection. Initially (6–8 weeks p.i.), CD4+ T cells appear to predominate; subsequently (15–21 weeks p.i.), an increase in CD8+ T cells was observed. Pathologic analyses indicated that lesions contained amastigotes with a mononuclear infiltrate of macrophages, lymphocytes, and plasma cells, and formation of tuberculoid-type granulomas. As the progression and resolution of leishmanial infection in rhesus macaques are very similar to those observed in humans, this primate model could be employed for elucidating the mechanisms of protective immunity in cutaneous leishmaniasis. © 1996 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Leishmania amazonensis; Protozoa; Parasitic; Hemoflagellate; Rhesus macaques; Macaca mulatta; Nonhuman primates; Histopathology; Leishmaniasis; CL, cutaneous; DCL, diffuse cutaneous; ML, mucosal; VL, visceral; IFA, Indirect immunofluorescence assay; DTH, delayed-type hypersensitivity; PBL, peripheral blood leukocytes; IL-2, -4, -5, -6, and -10, interleukins 2, 4, 5, 6, and 10, respectively; IFNg, Gamma interferon; p.i., postinoculation; HBBS, Hanks’ balanced salt solution; FBS, fetal bovine serum; PBS, phosphate-buffered saline; SI, stimulation index; PHA, phytohemagglutinin; Con A, concanavalin A.

cerative form of the disease that is accompanied by defective cellular immune responses, has been observed only with parasite species of the L. mexicana complex (reviewed in Grimaldi and Tesh 1993). It is becoming increasingly apparent, however, that the clinical expression and prognosis of leishmanial infection in the human host is dependent on a number of different factors, of which the parasite species is only one. L. amazonensis infection, for instance, can be as-

INTRODUCTION Self-healing CL, accompanied by positive cellular immune responses, has been produced by all the New World Leishmania species. In contrast, DCL, the progressive, anergic, nonul1 Present address: Departamento de Imunobiologia, Universidade Federal Fluminense, Outeiro de São João Batista s/n, Niteroi, RJ 24000-000, Brazil. 2 To whom correspondence should be addressed.

34 0014-4894/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Leishmania amazonensis IN RHESUS MACAQUES sociated with a spectrum of disease manifestations (Barral et al. 1991), depending on the immune status of the host or other external factors. Although a number of antibody-mediated effects against Leishmania have been demonstrated, cell-mediated immunity, rather than humoral immunity, mediates protection against leishmaniasis (Castes et al. 1988; Frankenburg et al. 1993; Holaday et al. 1993). Immune protection to L. major infection in resistant mice is associated with selective activation of the effective CD4+ helper T cells (the Th1 subset) and production of IL-2 and IFNg. In contrast, the progressive leishmanial infection in susceptible mice is correlated with activation of the Th2 CD4+ cell response, which expresses IL-4, IL-5, IL-6, and IL-10 (Locksley and Scott 1991). In addition, contributions by CD8+ T cell to the mediation of resistance has been shown in mice (Hill et al. 1989). Nonhuman primates of several species, although to a lesser extent than inbred strains of mice, have also been used for studying immune response to Leishmania (Lainson and Bray 1966; Lujan et al. 1986, 1987; Dennis et al. 1986; Pung and Kuhn 1987; Olobo et al. 1992; Curry et al. 1994). The availability of these experimental models is desirable, since they will provide valuable data on the efficacy of newly developed vaccines and immunotherapeutic agents against leishmaniasis. In the present study we have examined (i) the susceptibility of Macaca mulatta to L. amazonensis infection and the clinicopathologic changes of the disease and (ii) the immunological profile of the host occurring during primary and challenge infections. MATERIALS

AND

METHODS

Animals. Laboratory-bred and -reared young adult (5 to 7 years old) rhesus macaques (M. mulatta) of both sexes, obtained from the Oswaldo Cruz Foundation Primate Research Center (Rio de Janeiro, Brazil), were used. Experimental animals were housed indoors in individual steel squeeze-back cages in a temperature (18–29°C)- and humidity (60 ± 5%)-controlled environment. Balanced commercial nonhuman primate diet (NUVILAB Primatas, No. 14.915; Ministerio da Agricultura e Reforma Agraria, Brazil) and water were provided ad libitum. In addition, the

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animal diet was supplemented with eggs, fruits, and vegetables. The animal facility is maintained according to the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and the HHS (MD, U.S.A.) guidelines, Guide for the Care and Use of Laboratory Animals. The monkeys were acclimatized to the laboratory conditions for at least 2 to 3 weeks before the experimental procedures were begun. Animals were anesthetized before infection and prior to each sampling or testing procedure. Monkeys were initially restrained in their cages, and subsequently they were given, intramuscularly, Ketamine (Ketalar: Ketamine hydrochloride; Parke Davis; 10– 40 mg/kg body weight) for anesthesia. At the termination of observation, all animals were euthanized using a threefold excess of ketamine anesthesia and tionembutal. Parasites. The strain MHOM/BR/77/LTB0016 of L. amazonensis, which was isolated from a human case of CL, was supplied by Dr. P. Marsden (Federal University of Brasilia, Brazil). The strain has been typed in our laboratories, using both isoenzyme and monoclonal antibody analyses (Barral et al. 1991). The parasite was maintained by serial subcutaneous passage in BALB/c mice. To prepare suspensions of amastigotes for experimental infections, cutaneous nodules were removed aseptically and ground in HBSS in a Ten Broeck tissue grinder. To obtain suspensions of promastigotes, tissue from chronically infected hamsters was cultured initially in NNN blood agar medium with an overlay of HBSS. When promastigotes appeared, the parasites were subinoculated into Schneider’s Drosophila medium (Gibco) (Hendricks et al. 1978), supplemented with 20% heat-inactivated FBS. Parasites were harvested (latelog/stationary phase), washed three times using centrifugation in PBS, and counted in a Neubauer hemocytometer before use for infection. Experimental infections. Primates were infected by intradermal inoculation of variable numbers of promastigotes in 0.1 ml. The various challenge and rechallenge experiments were performed as follows. A study (Experiment 1) was first designed for providing information concerning the minimum infective dose of the parasite. Three monkeys were infected in the left forearm with either 103, 105, and 107 virulent promastigotes (late-log/stationary phase). After recovery from the primary infection, the primates were subsequently challenged (Experiment 2) in the opposite area or upper eyelid with 108 virulent promastigotes [alone or in conjunction with salivary gland lysates from the sand fly Lutzomyia longipalpis (Titus and Ribeiro 1988)] or 106 amastigotes. In Experiment 3, animals (6) were inoculated in the upper eyelid with 108 promastigotes. After recovery from the primary infection, three of the animals (Experiment 4) were challenge-infected in the opposite area with 108 promastigotes. For each experiment, in parallel, either BALB/c mice or hamsters were also inoculated with identical numbers of parasites to determine parasite infectivity. Serologic analyses. Class-specific (IgM and IgG) antileishmanial antibodies were detected using IFA as described

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(Grimaldi et al. 1980). Each individual serum was tested in serial twofold dilutions [1:10 to 1:640] in PBS. Pooled normal monkey serum and pooled human cutaneous leishmaniasis serum were included in each analysis as negative and positive control, respectively. In addition, Western blot analyses were employed for the detection of serum antibodies to leishmanial antigens. Preparation of L. amazonensis promastigote antigens were as previously reported (Leon et al. 1990). Antigens were resolved on SDS–polyacrylamide gel electrophoresis (10%) and transferred electrophoretically to nitrocellulose (Schleicher and Schuell, Keene, NH). The nitrocellulose strips were incubated overnight at 4°C with either immune sera obtained from either primates or (as a positive control) human cutaneous leishmaniasis patients infected with L. amazonensis. Subsequently after washing, strips were incubated with either peroxidaseconjugated goat anti-monkey IgG (U.S. Biochemical Corp., Cleveland, OH) or rabbit anti-human IgG (Sigma). After rinsing, strips were developed in a saturated solution of 3,39-diaminobenzidine in Tris buffer (pH 7.4) containing 0.01% H2O2. Intradermal hypersensitivity test. The evaluation of the DTH response to parasite antigens was done according to the method used for humans (Montenegro skin test). The antigen consisted of heat-killed L. amazonensis promastigotes suspended in PBS with 0.5% phenol. A volume of 0.1 ml containing 5 × 106 parasites was injected into the left forearm. Reactions were measured as skin indurations (caused by erythema and/or edema) at the site of injection at 4 hr and subsequently at 24-hr intervals up to a total period of 96 hr. Leukocyte cultures and proliferation assay. The Rhesus monkey PBL were derived from fresh heparinized venous blood samples (5 ml) and prepared by density gradient centrifugation at 20°C on Ficoll–Hypaque (Histopaque-1077, Sigma). Human PBL (controls) were also separated from healthy volunteers in the same way. The cells were washed twice in cold medium and final suspensions were made in RPMI-1640 (Sigma) supplemented with 20% heatinactivated human AB serum and sodium pyruvate and contained 200 U ml−1 penicillin and 200 mg ml−1 streptomycin. All cultures were performed in triplicate using 96-well flatbottom microtiter plates (Nunc, Demark). Viable cells (as determined by the trypan blue dye exclusion method) were cultured at 2 × 106 cells ml−1 in the presence of optimal culture concentration of mitogens [Con A, Type IV at 16 mg ml−1 (Sigma); PHA-P at 12.5 mg ml−1 (Sigma)] or parasite antigen (10 mg protein/well). The soluble antigens for in vitro blast transformation assays were prepared from L. amazonensis promastigotes, according to the method described by Dennis et al. (1986). Cultures were incubated at 37°C in a humidified atmosphere (5% CO2 and 95% air) for 3 days in the case of mitogens or for 4 days in the case of antigens. Approximately 16 hr before harvesting onto glass fiber filter mats (Titertek, FlowLab, U.S.A.), [3H]thymidine (Amersham, Co., U.K.; 1.0 mCi/well; 5.0 Ci/mM) was added. Radioactive incorporation was measured using a

beta counter (Model 1600 C, Packard, Co., U.S.A.). Results are expressed as the stimulation index (SI, mean cpm stimulated cultures/mean cpm unstimulated cultures). To determine significance, data from the responses of PBL from five uninoculated rhesus macaques (controls) to appropriate mitogens and leishmanial antigens were compared with those determined subsequent to primary and/or challenge infections. The data from the blast transformation studies were analyzed using the unpaired Student t test. A SI $2.5 was considered significant based on the 99th percentile of preinfection values of the monkeys (Lujan et al. 1986). Pathology. The size and appearance of leishmanial lesions, as well as examination for metastases, were evaluated at weekly intervals. Lesion measurements (length and width) were made with the aid of calibrated calipers and the lesion area was calculated using the formula pr1r2 as described by Wilson et al. (1979). An attempt was made to isolate Leishmania organisms from all primates at different intervals postinoculation (p.i.). Biopsy samples from selected cutaneous lesions were inoculated, aseptically, directly into tubes of NNN blood–agar medium overlaid with complete Schneider’s medium. After euthanization, lesions were removed and fixed in 10% buffered formalin. Paraffin sections (5 mM thick) were prepared from central and peripheral zones of the lesion and stained with hematoxylin– eosin. In addition, postmortem histological or culturing examinations of lymphoid tissues (from lymph nodes draining the lesion site, as well as spleen and liver) allowed the assessment of visceralized subclinical infection. Staining and cytofluorographic analysis of PBL. For the analyses of phenotype of circulating T cell subsets of infected animals, the following commercial monoclonal antibodies were employed: CD2 (T11 pan T cell antibody; Coulter Cytometry, Hialeah, FL), CD4 helper/inducer T cell subset (OKT4 antibody; Ortho Diagnostic Systems, Raritan, NJ), and CD8 suppressor/cytotoxic T cell subset (Leu 2a antibody; Becton-Dickinson, San Jose, CA). PBL samples were isolated as indicated above; residual erythrocytes were removed by lysis using 0.15 M NH4Cl. Samples were then washed using Hank’s and incubated in aliquots of 5 × 105 cells for 20 min with a FITC-conjugated monoclonal antibody at 4°C. The cells were then washed and cytofluorographic analyses were performed using a fluorescenceactivated cell sorter (EPIC 751, Coulter). Data are expressed as the percentage of positively staining cells.

RESULTS Establishment of infection and lesion development. In order to establish the infective dose of L. amazonensis required to induce CL in the rhesus monkeys, animals were inoculated with varied numbers of parasites. Animals inoculated (Experiment 1) in the left forearm with either 103, 105, or 107 promastigotes developed a

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Leishmania amazonensis IN RHESUS MACAQUES small erythematous papule by 3 weeks p.i., which resolved on the 4-week p.i. (data not shown). However, hamsters (controls) injected with the identical inocula [103–7] showed progressive lesion development and maintained a chronic, nonhealing infection. In contrast, all primates challenge-infected (Experiment 2) or infected (Experiment 3) in the upper eyelid area with a higher inoculum of the same parasite strain developed more conspicuous skin lesions at the site of inoculation. Some degree of variation in susceptibility between various individual monkeys was observed, but an inoculum of 108 promastigotes of L. amazonensis consistently resulted in establishment of infection (Fig. 1). In the single animal infected together with salivary gland homogenates from Lu. longipalpis, no apparent enhancement of lesion development was found in comparison to the animals receiving parasites alone. However, given the variation in susceptibility to infection and the fact that the inoculating dose (108) employed consistently resulted in infection, it is unclear that sandfly saliva did not provide enhancement of infectivity, as reported in the murine model (Titus and Ribeiro 1988). Further work is required to evaluate this point. Three monkeys from this group (which had recovered from primary skin

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lesions) were challenge-infected (Experiment 4) using the same parasite strain/dose. Although these animals remained susceptible to homologous infection, the lesions were smaller and healed faster than in the initial infection (data not shown). The skin lesions, of an erythematous-papular nature, were first visible at 1–2 weeks p.i. of 108 parasites. Initial growth of the cutaneous lesion progressed rapidly and all lesions ulcerated (after 6 to 8 weeks p.i.). Lesion development progressed (nodules varying between approximately 20 to 120 mm2), peaking at 8 to 15 weeks p.i., and was subsequently followed by regression and healing. Metastases in the extremities were not observed, but some animals developed satellite lesions peripheral to the resolving primary skin lesion. Biopsy samples were positive for parasites (by either culture or direct microscopic evaluation) at the early phase of developing nodular lesions, but negative results were obtained during the healing process. At sacrifice, cultures from the spleen or liver failed to demonstrate visceralization of leishmanial parasites in all animals, but samples from regional lymph nodes draining the lesion site were positive both by culture or tissue sections.

FIG. 1. The course of L. amazonensis infection in naive monkeys (Experiment 3). Animals were infected at the up upper eyelid with 108 L. amazonensis promastigotes. Lesion size development is indicated for animals 6 (j), 7 (+), 8 (× +), 9 (h), 10 (n), and 11 (l).

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Pathology. A firm elastic, yellowish-red nodule developed at the site of the parasite inoculation. After a spontaneous ulceration, the nodular lesion diminished in size; the smaller healing lesions had a whiter more fibrous consistency. All animals (inoculated with either 108 promastigotes or 106 amastigotes of L. amazonensis), when sacrificed had histological evidence of infection. The histopathological findings in the dermis are shown in Fig. 2. Regardless of the lesion size, the nodule was diffusely infiltrated with histiocytes, lymphocytes, and plasma cells. In larger nodular lesions, the chronic inflammatory infiltrate encircled a central area of vacuolated macrophages, with or without intracellular parasites (Figs. 2a and 2d). Neutrophils accompanied the mononuclear infiltrate, apparently associated with lysis of few parasitized macrophages and the liberation of extracellular amastigotes. In early phases of developing ulcerated skin lesions, indistinctly delimited and more or less differentiated macrophage accumulations were found, which evolved to the formation of granulomatous nodules. The granulomas were surrounded by a diffuse mononuclear infiltrate containing giant cells (Fig. 2b). In late stages, fibroblasts proliferated at the periphery of the granulomas and finally invaded them with fibrotic substitution (Fig. 2c). Accompanying this inflammatory process was the reaction of the various elements of the skin to the infection, such as epidermal reactions (e.g., hyperplasia, followed by athrophy and ulceration and reepithelization during lesion regression), neoformation of connective tissue, and vascular reaction. Antibody responses. The antibody responses for animals infected with 108 L. amazonensis promastigotes were monitored with time (Fig. 3). Both IgM and IgG responses varied during infection. IgM titers peaked at 2 weeks postinfection and then declined. During primary infection IgG levels were detectable 2 weeks p.i. and subsequently continued to increase, peaking at 10 weeks postinfection after which levels declined. Following rechallenge, no increases in IgM levels were observed, but the IgG titers increased sharply as compared to primary infec-

tion. Western blots of promastigote homogenates were performed employing immune sera from the monkeys at various times postinfection. The antibodies produced by the animals recognized numerous, distinct leishmanial epitopes associated with components predominantly between 30 and 95 kDa (some weak higher Mr components were also detected). Similar Mr antigens appeared to be recognized by each of the monkeys during the course of infection (Fig. 4), suggesting that these may be immunodominant components. Cell-mediated immune responses. DTH/ Montenegro skin reactions were measured to assess levels of cell-mediated immunity in vivo. While a DTH reaction was elicited in all animals infected with 108 parasites, negative responses (at 9 and 16 weeks p.i.) were observed in monkeys infected with the lower doses (at 103–105 organisms). Specific DTH reactivity was detected as early as 6 weeks p.i. (mean ± SD; 6.40 ± 2.19) and continued up to 21 weeks p.i. (mean ± SD; 6.67 ± 2.89). In addition, a positive reaction (mean ± SD; 7.13 ± 1.84) was detected at 6 weeks after challenge infections, but did not increase significantly in comparison to prechallenge values. Parasite-specific lymphoproliferative response of blood lymphocytes from monkeys was detected during primary and challenge infections. Individual cell-mediated immune responses to leishmanial antigens varied in the course of cutaneous leishmaniasis in L. amazonensis-infected rhesus monkeys (Fig. 5). Proliferative responses were negative (SI < 2.5) in all animals at the initiation of infection, but a positive reaction developed as early as 2 weeks p.i. and, in some cases, persisted beyond apparent self-cure. PBL of animals were comparably responsive to control mitogens (PHA and Con A) prior to and throughout infections (data not shown). Phenotypic analysis of cell surface antigens indicated that the circulating T cell subpopulations from the monkeys vary throughout the course of infection. The baseline ratio of CD4+ to CD8+ T cells in uninfected rhesus monkeys was 0.9. Although distinct from the value de-

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FIG. 2. Histopathology of cutaneous leishmaniasis in rhesus macaques infected with L. amazonensis. Sections from a developing ulcerated skin lesion (animal 4), showing (a) an accumulation of few parasitized and vacuolated macrophages laden with amastigotes (arrows) and (b) a noncompact granulomatous inflammation with giant cells (arrow). Differentiated macrophages, interspersed with more or less numerous lymphocytes and plasma cells, represent the principal feature in this chronic inflammatory process. (c) Section from an older skin lesion (animal 1) in the scarring phase, showing fibrous thickening of the dermal conjunctiva (fibroblastic response) and moderate diffuse infiltration of mononuclear inflammatory cells. Characteristically, parasites are scant or not found in sections. (d) Section from a satellite lesion (animal 3) showing an immature granulomatous inflammatory reaction with few vacuolated macrophages. Hematoxylin and Eosin; bar, 50 mm.

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FIG. 3. Geometric mean of reciprocal (h) IgM and (× +) IgG immunofluorescence titers to promastigote antigen in rhesus macaques infected with 108 L. amazonensis promastigotes. Each point represents the mean value of monkeys tested after primary (6) and challenge (3) infections. No detectable antibody responses to Leishmania were observed in uninfected (controls) monkeys.

fined for humans, this ratio is in agreement with previous studies (Letvin et al. 1983). Initially during infection CD4+ T cells appear to predominate (6–8 weeks p.i.; CD4:CD8 mean ratio, 5.0; range, 2.5–7.1); subsequently, an increase in CD8+ T cells was observed (15–21 weeks, p.i.; CD4:CD8 mean ratio, 1.05; range, 0.37–2.1).

DISCUSSION Rhesus macaques were infected with L. amazonensis in a pilot study to determine the susceptibility and the course of CL in this primate species. The monkeys were evaluated as a model for immunologic studies of leishmaniasis by correlating the progression of cutaneous le-

FIG. 4. Western blot analyses of L. amazonensis promastigote antigens recognized by sera from three infected rhesus monkeys. Methods employed were as described under Materials and Methods. Shown are the reactions of serum antibodies prior to infection [lanes 1, 7, and 13], 13 weeks postinfection [lanes 2, 8, and 14], 15 weeks postinfection [lanes 3, 9, and 15], 17 weeks postinfection [lanes 4, 10, and 16], 19 weeks postinfection [lanes 5, 11, and 17], and 21 weeks postinfection [lanes 6, 12, and 18].

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FIG. 5. Individual lymphoproliferative responses in vitro to soluble leishmanial antigen (disrupted promastigote homogenate at 50 mg/ml) of peripheral blood leukocytes from rhesus macaques prior to and after primary (animals 6 and 10) and challenge (animals 7 and 8) infection with 108 L. amazonensis parasites are indicated. Bars shown indicate the total lesion area occurring during the time course of primary and challenge infection.

sions with both the cellular and the humoral responses during primary and challenge infections. Primate models have been used to study host responses to Leishmania (Lainson and Bray 1966; Dennis et al. 1986; Githure et al. 1986, 1987; Lujan et al. 1987; Pung and Kuhn 1987; Silveira et al. 1990; Olobo et al. 1992; Binhazim et al. 1993). This has been especially important in the case of L. braziliensis complex parasites where the monkey species studied to date are susceptible to infection (Lujan et al. 1987; Silveira et al. 1990; Cuba-Cuba and Marsden 1992) and the murine model is limited. Moreover, studies of L. donovani complex parasites indicate that infected monkeys are highly

susceptible to infection (Dennis et al. 1986; Githure et al. 1986; Binhazim et al. 1993). T cell responses correlate with recovery from and resistance to human leishmaniasis. A lymphocytic response to leishmanial antigens usually develops during both CL and ML but is absent in DCL or VL. Conversely, antiLeishmania antibody titers are generally low in the sera of patients with CL or ML but moderate to high in patients with DCL or VL (reviewed in Grimaldi and Tesh 1993). When tested in vitro, peripheral lymphocytes from patients with CL or ML produced high levels of IFNg in response to parasite antigen (Castes et al. 1988; Frankenburg et al. 1993). There appears to be a mixed

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cytokine profile associated with active CL or ML (Frankenburg et al. 1993; Pirmez et al. 1993) and a dominant T helper (Th) 1-type (Pirmez et al. 1993) and/or CD8+ T cell (DaCruz et al. 1994) responses associated with healing of the disease. The characteristic clinical and pathologic features of the self-healing CL caused by L. amazonensis in humans were observed in rhesus monkeys. DCL that could occur was not observed in the animals during the time course of these experiments. The level of immune responsiveness found, however, suggests that it is unlikely that this anergic form might have subsequently developed. Although self-healing ulcerative cutaneous lesions developed for all animals, variation in the level of susceptibility to leishmanial infection was found. Lower doses of leishmanial parasites allowed for apparently transient infection, whereas an inoculum of 108 late-log promastigotes provided relatively consistent results, including earlier onset and/or larger size of lesions. The characteristic pathologic features of CL in humans were also observed. The cutaneous lesions of infected monkeys contained amastigotes with a mononuclear infiltrate of macrophages, lymphocytes, and plasma cells, with formation of tuberculoidtype granulomas. Similar pathologic features have been observed in infections of another nonhuman primate, Cebus apella with New World Leishmania species (Silveira et al. 1990). Recovery and the development of longlasting resistance to reinfection has been reported to be the case for CL. Some observations suggest, however, that immunity conferred by prior self-resolving leishmanial infection may not always be complete (Saravia et al. 1990). In this study, monkeys which had recovered from skin lesions were challenged using an identical inoculum of the same parasite strain (Experiment 4). Although these primates remained susceptible to homologous reinfection, a partial protective immunity against the parasite seemed to occur. Three rhesus monkeys which had recovered from CL apparently developed acquired resistance, as reflected by a delayed out-

come and smaller size or faster resolution of the lesions than the infected controls. Similar results have been demonstrated in other primates (Dennis et al. 1986; Cuba-Cuba and Marsden 1992). Our results indicate that both humoral and cell-mediated immune responses induced by leishmanial infections in rhesus monkeys correspond to those observed for human CL. Low levels of IgM and IgG antibodies to the parasite were detected in all animals during the course of primary and challenge infections. In addition, immunoblot analyses showed that the monkeys produced antibodies which bound to a number of Leishmania antigens, consistent with human data (Leon et al. 1990). Specific DTH response was detected as early as 6 weeks p.i. and persisted following self-cure and in animals rechallenged; no significant increase in DTH (over that observed in the primary infection) was apparent upon rechallenge. The T cell subpopulations, as determined by FACS analyses of PBL, varied throughout the course of infection. The CD4+ T cell response appeared to correlate with lesion development; with maximal cell levels found at the peak of lesion size. The activation of CD4+ T and/or CD8+ T cells may be essential for the healing that was subsequently observed in these monkeys as has been found in human studies. Further studies are required to elucidate the roles of these cells in a protective response against Leishmania infection. The rhesus monkey model could provide further useful information concerning the mechanisms involved. The findings presented above demonstrate the parallels found between humans and other primates in their responses to leishmanial infection. The parallels found in susceptibility, clinicopathologic changes, and immunologic responses are not surprising, given their close phylogenetic and immunologic relationships (Letvin et al. 1983; Kishino and Hasegawa 1990; Klein et al. 1993). Prospects for human immunoprophylaxis with a new generation of safe and effective subunit vaccines (using either recombinant or synthetic peptides or infectious recombinant vectors) is now within our reach

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Leishmania amazonensis IN RHESUS MACAQUES (reviewed in Grimaldi 1995). As the rhesus monkey is an animal system closer phylogenetically to humans than the murine model and appears to parallel the human responses to infection, this experimental model should provide an indication of the potential success and/or limitations for a human vaccine against leishmaniasis. Success of a vaccine depends not only on the identification of protective antigens but also the adoption of a suitable vaccination protocol (i.e., route of immunization, doses, timing between vaccination and challenge). ACKNOWLEDGMENTS The data presented in this paper have been submitted by Veronica F. Amaral as part of a thesis for a master’s degree in Parasite Biology at the Oswaldo Cruz Institute/ FIOCRUZ, Rio de Janeiro, RJ, Brazil. We greatly acknowledge the help of Ricardo Nogueira, Luiz Eduardo C. Paes, Gerzia Machado, Alvaro Bertho, and Marta Santiago for technical assistance and invaluable support. This work was supported in part by Grants AI-23004 and AI-27811 from the National Institutes of Health and the Fogarty NIH Program (U.S.A.), the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the CNPq/RHAE Program (Brazil).

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Received 11 May 1995; accepted with revision 26 September 1995