Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial

TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(2000)94,698-703

Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial

Abstract

The safety and immunogenicity of an intramuscular (IM) and intradermal (ID) formulation of autoclaved Leishmania (Leishmania) amazonensis vaccine was evaluated in 296 volunteers in a randomized, placebocontrolled, double-blind trial in Colombia. There were 4 vaccination groups: IM vaccine, IM placebo, ID vaccine, and ID placebo. The ID formulations were mixed with BCG as adjuvant at the time of injection. For each group, 3 vaccinations were given with a 20-day interval between injections, and adverseevents were monitored at 20 min, and at 2,7 and 21 days after each injection. BCG-induced adverse reactions resulted in cancellation of the third vaccine administration in the ID groups. Antibody titres did not differ significantly between the groups. Montenegro skin-test conversion was achieved by 86.4% and 90% of the IM vaccine group and by 25% and 5% of the IM placebo group 80 days and 1 year after vaccination, respectively. A significant increase in mean Leishmania-antigen lymphocyte proliferation indexes was observed after IM vaccine immunization, but not after IM placebo immunization, 80 days and 1 year after vaccination. Significant levels of IFNy but not IL10 were observed 1 year after vaccination in the IM vaccine group compared to the IM placebo group. The good safety profile and evidence of Thl immune reactions due to IM vaccination in this phase-I/II study suggest that a population-based phase-III efficacy trial of the IM vaccine should be initiated. cutaneous leishmaniasis, vaccine, Lkshmania amazonensis,clinical trial, BCG adjuvant, immune responses, safety, Colombia

Keywords:

Introduction

New World cutaneous leishmaniasis is endemic and an important public health problem throughout most of South America. The diseasepresents with single, diffuse or mucocutaneous lesions. Recommended treatment based on pentavalent antimonials is expensive, of long duration, requires several intramuscular injections and may induce -severe side-effects (BERMAN; 1988; 0~ LIARO&BRYCESON, 1993;V~~Ez et al.. 1997).Cureis often associated with production of scars and sequelae. Unresponsiveness of parasites to antimonials and drug resistance are growing problems (OLLIARO & BRYCESON, 1993), and second-line drugs are highly toxic. The development of a leishmaniasis vaccine would be a desirable control measure to prevent human leishmaniasis. The development of a ‘first-generation vaccine’, based on killed whole-cell promastigote forms with or without adjuvant, was initiated at least 2 decadesago (MAYRINK et al., 1979). In Brazil. an initial vaccine formulation containing 5 Leishmanid specieswas tested in a field trial. There was a significant reduction in leishmaniasis incidence in those vaccinees who converted their Leishmanin skin-test responses compared to the placebo group (ANTUNES et al., 1986). In Venezuela, heat-killed Leishmania promastigotes plus BCG have been used as immunotherapy of cutaneous leishmaniasis (CONWT, 1996). Immunization with autoclaved leishmanial promastigotes plus BCG as adjuvant was found to induce significantly (P < 0.001) raised levels of gamma-interferon (IFNy) production, lymphocyte proliferation and skin-test conversion (CASTES et al., 1994). Other trials that looked at immune response and efficacy compared multi-species versus single-species formulations (MARZOCHI et al., 1995; GENAROet aE., 1996; -1~0s et al., 1998) and merthiolate-killed versus autoclaved preparations (DE LUCA et al., 1999). Although there is substantial evidence that vaccination may be effective for inducing a protective immune response against Leishmania infection, the optimal vaccine formulation, immunization scheme and use of BCG *Author for correspondence; fax +57 45716675, e-mail [email protected]

are not yet clear. The present study was designed to evaluate the safety and immunogenicity in healthy adult volunteers of an intramuscular (IM) and intradermal (ID) formulation of an autoclaved preparation of L. (L.) amazonensis, with and without BCG as adjuvant. Materials and Methods Study design

This was a randomized, placebo-controlled, doubleblind trial to assessadverseevents and immune responses of a Leishmania candidate vaccine formulated for IM and ID use. Volunteers were randomly assigned to 4 groups: 2 experimental groups (IM and ID) and 2 corresponding placebo control groups. The trial was approved by the ethical committee of the University of Antioquia and conducted following Good Clinical Practices (GCP) standards. Partkipants

The volunteers consisted of 204 soldiers of the Colombian Army and 92 undergraduate students from the University of Antioquia, Medellin, Colombia. Inclusion criteria

The inclusion criteria were: Montenegro skin test (MST) c 3 mm; no lesion that suggested active cutaneous or mucosal leishmaniasis; no history of cutaneous or mucosal leishmaniasis; no severe concomitant medical condition; age > 18 years; agreement to participate in the study with written informed consent; agreement to be present at scheduled examination dates. Vaccine/placebo The Lekhmania vaccine, based on whole-cell autoclaved L. (L.) amazonensis strain MHOM/BR/67/PHS,

was prepared under Good Manufacturing Practice (GMP) by BIOBRAS @, Brazil (MODABBER, 1995). The IM vaccine (batch 7031028-1V) was administered in a volume of 1.0 mL (total nitrogen 0.38 mg/mL, Kjedhal). The ID vaccine (batch 7031027-1V) was dispensed in 2.7-n& vials (total nitrogen 2.9 mg/mL, Kjedhal) and mixed with 0.3 mL of BCG vaccine (l/10 of the normal BCG vaccine dose) at the moment of injection and administered in a volume of 0.1 mL. The National Institute of Health of Colombia produced BCG

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vaccine (batch 288B from live Calmette Guerin Mycobacterium tuberculosis var. bovis (Pasteur 1173 P2 bacilli strain). Phosnhate buffer saline (PBS) solution, pH 7.4 (batch 20429) was used as placebo and was supplied by BIOBRAS in vials identical to those in which vaccine was supplied. Schemes of vaccination Volunteers were randomly assigned to 4 groups: IM vaccine; IM placebo; ID vaccine; ID placebo. Each volunteer received 3 doses of his/her respective formulation with a 20-day interval between each injection. The ID groups received IM injections (vaccine or placebo) for the third injection. Clinical follow-up The participants were examined by independent blinded clinicians for local and systemic adverse events at 20 min, and at 2, 7 and 21 days after each injection. The presence of erythema, induration, itching, pain, ulcer formation, and systemic reactions (fever, malaise, adenopathy) was graded from 0 to 3. Grade 0 corresponded to absence of symptoms; 1 = mild, not requiring medical attention; 2 = moderate, requiring medical attention; and 3 = severe, limiting normal activities. Participants who presented any grade-3 reaction were excluded from further vaccination. Samples were collected for haematological and biochemical tests (haemoglobin, white blood cell count, platelets, glucose, creatinine, AST, ALT) before vaccination and on day 80 after the first injection. Immune responses were evaluated bv the Montenegro and indirect fluorescent antibody (IEAT) tests on alivolunteers, and by lymphoproliferation and cytokine tests on 25 IM vaccine and 25 IM placebo volunteers. Immune response Montenegro skin test. The skin-test antigen (batch 6560002) was derived from the same strain used for the vaccine preparation (total nitrogen 0.040 mg/mL, Kjedhal) and 0.1 mL was injected ID. Induration was measured 48 h after application by the ball-pen technique and the cut-off point was 2 5 mm (WHO, 1990). Lymphocyte proliferation assay. This procedure was carried out as described by SARAVIA et al. (1989). In brief, peripheral blood mononuclear cells (PBMC) were obtained in Ficoll-Hypaque gradients (Histopaque 1077, Sigma Chemical Co., St Louis, USA), washed with PBS (GIBCO Labs, Grand Island, NY, USA), counted and resuspended in RPMI 1640 medium (GIBCO Labs) supplemented with 20% heat-inactivated human serum from healthy male donors. L. (L.) amazonensispromastigotes were harvested at stationary phase (day 5 of culture). After 2 washes with PBS, promastigotes were counted and resuspended in RPM1 medium, without serum or plasma, at 1 X lo6 parasites/ml. PBMC (1 X 105) were added to flat-bottomed 96-well microtitre plates, containing 1 X lo5 freeze-thawed promastigotes/well or phytohaemagglutinin (PI-IA; Sigma Chemical Co.) 5 pg/mL in RPM1 1640 medium. Each experimental situation (Leishmaniu antigen or PHA) was evaluated in triplicate. Cells and parasites were incubated at 37”C, in 5% CO,, for 6 days. Cultures with PI-IA were incubated under identical conditions for 4 days. Tritiated thymidine (1 uCi, Amersham, UK) diluted in RPM1 1640 medium was added to each microtitre well culture 18 h before harvesting with a multiple automated sample harvester. Glass fibre filter strips (Whatman, Reeve Angel, Clifton, NJ, USA) were air dried and the discs containing radiolabelled PBMC were counted utilizing a toluene-Omnifluor cocktail (New England Nuclear) and beta scintillation counter (Beckman). Results were expressed as the proliferation index (PI), defined as the ratio between mean counts per minute (cpm) of triplicate experimental and mean cpm

of non-stimulated cells. A PI > 2 was considered positive. Cytokine assay: IFNy sandwich-ELISA. IFNy was determined as described by ROBLEDO (1998), 80 days and 1 year after vaccination. The PBMC from the 50 vaccinated volunteers were cultivated in 24-well flatbottomed plates at 1 X lo6 cells/well and stimulated with L. (L.) amazonensis soluble antigen at a 2: 1 parasite-cell ratio. Cells and antigen were incubated at 37°C in 5% CO,, for 3 days at which time culture supematants were collected. For the IFNy determination, wells of flatbottomed 96-well microtitre plates were coated overnight at 4°C with anti-human IFNy monoclonal antibody (Genzyme, Cambridge, MA, USA). After blocking and washing, samples or recombinant human IFNy (Boehringer Mannheim) were incubated for 1 h at room temperature. The wells were subsequently incubated with rabbit anti-human IFNy polyclonal anti-serum (Genzyme) for 1 h at room temperature, and, after washing, wells were treated with a peroxidase conjugate goat anti-rabbit IgG antibody (Jackson Immunoresearch, West Grove, PA, USA) for 30 min at room temperature. ABTS (2,2’-azinobis(3-ethylbenzthiazoline-sulphonic acid; KPL, Gaithersburg, MD, USA) was used as substrate and plates were incubated at room temperature for 30 mm. Absorbance was read at 410 nm within 30 mm using an ELISA reader (BioRad). The lower limit of the assay was 0.8 U IFNyImL. Cytokine assay: interleukin-10 (IL-1 0). The assay was conducted as described for IFNy. Antihuman IL-10 monoclonal antibody, clone Jes3-9D7 (Pharmingen, San Diego, CA, USA) was used as capture antibody and samples were subsequently incubated with biotinylated anti-human IL- 10 monoclonal antibody, clone Jes3-12G8. Indirectfluorescent antibody test. The test was conducted according to procedures and reagents routinely employed in our laboratory (VELEZ & AGUDELO, 1996). Antigen for the IFAT was prepared from a 5-day-old culture of strain UA140 of L. (V.) panamensis in NNN modified medium. Data analysis A database was built using ACCESS Microsoft’@ and data processing was carried out with SPSS (version 9.0, Chicago, IL, USA). We explored whether or not the data were normally distributed-with the Kolmogorove-Smirnov Z test. We used the Mann-Whitnev non-narametric test for cytokine analyses. Other variables (used for skintest reactions, T cell-proliferation indexes and antibody determination) were log-transformed and Student’s ttest for differences between means was applied. A P value < 0.05 was considered as statistically significant. Results A total of 296 volunteers with normal haematological values, liver function tests, and urea and creatinine tests were enrolled, randomly distributed into the 4 groups, and given the first dose of vaccine: 239 (80.7%) volunteers-received the second dose and 199‘(67*2%) completed the full 3-dose vaccination schedule. Vaccination of both ID BCG groups was terminated after the second dose owing to local reactions. After the first ID injection, adverse events prevented subsequent vaccination in 14 (23.0%) of 61 volunteers in the placebo-BCG ID group and in 25 (41.7%) of 60 in the vaccine-BCG ID group. After the second dose, 2 additional participants were withdrawn from the placebo-BCG ID group and 5 from the vaccine-BCG ID group because of adverse events. The remaining members of the ID injection groups received their third injection according to IM procedures, and no adverse events were observed after the third injection. In addition, no participant in the vaccine IM or placebo IM groups interrupted the vaccination schedule owing to adverse reactions. Compliance with the full 3-dose vaccination was over 80% for the IM

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groups and only 557% for the placebo-BCG ID group and 35% for vaccine-BCG ID group (Table 1). Adverse events No haematological or biochemical abnormality was observed in any participant after immunization. The frequency and intensity of adverse events in the vaccineBCG ID group and placebo-BCG ID group are summarized in Table 2. Reactions at the site of injection were more frequent and intense in the vaccine-BCG ID group, starting from the second day after the first dose of vaccination, and were characterized by induration, pain, itching, and then ulceration/abscess formation. No systemic reactions were seen. Volunteers withdrawn from the study were followed-up periodically and were seen until they healed. The mean time to healing of the abscess was 2 months. Immune response Montmegro skin test. All volunteers were MST negative before vaccination. Mean skin test conversion at day 80 after the first vaccine dose was statistically higher for the vaccine IM group than for placebo IM group (P< 0.0001). High conversion rates were also observed for individuals in the vaccine-BCG ID and placebo-BCG ID groups. One year after the vaccination the MST was evaluated in the same sample population previously selected for proliferation and cytokine quantification. Skin-test conversion continued to be significantly ditferent between groups (P < O*OOO1) (Table 3). Lymphocyte proliferation assay. The results for PHA testing were within normal ranges, indicating a normal immune response in all participants. Before vaccination the geometric means of the proliferation index with soluble Leishmania antigen were negative in both intramuscularly injected groups, with borderline statistical difference (P = 0.048) between the groups. After vaccination, the geometric mean of the proliferation index was positive in the vaccine IM group and negative in the placebo IM group. Eighty days after the first vaccine dose geometric means of the proliferation index were 1a96 & 0.87 and 0.34 f 0.44 for the vaccine IM and placebo IM group, respectively (P < 0.0001). One year later, cellular immunity persisted in the volunteers who received vaccine IM and the difference between vaccine and placebo groups was statistically significant (P= 0.021) (Table 4, Figure). Cytokine assay: ZFNy production. Eighty days after vaccination all samples were below the minimum detection level of the technique. One year after vaccination, small levels of IFNy were found in the IM vaccine group (a mean of 88.9 pg/mL) whereas the value for the placebo IM group was 26.5 pg/mL. The difference between the groups was statistically significant with a mean rank of 20.6 for vaccine IM group and 13.33 for placebo IM group [Mann-Whitney U test = 80.00, P (2 tailed) = 0.0481. Cytokine assay: IL-l Oproduction. IL1 0 production was measured 1 year after vaccination. The mean rank for the vaccine IM group was 20.86 and for the placebo group was 18.32 [Mann-Whitney U test = 158, P (2 tailed) = 0.4951. The lack of statistical difference indicates that IM vaccination did not induce a Th2 response. Indirect fluorescent antibody test. The IFAT was performed in 265 paired serum samples obtained before and on day 80 after the first dose (Table 5). No statistically significant differences were observed between vaccine and placebo groups either before or after vaccination. These data were confirmed by dot-ELISA. The geometric means of antibody titres 80 days after vaccination for vaccine IM and placebo IM were 1: 1.9 and 1:2.29, respectively (P = 0.502). Discussion This study was the first phase-II Leishmania vaccine clinical trial carried out in Colombia. Overall, this trial

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Table 2. Number of adverse events and intensity grade after each dose of intradermal with BCG, or L. amazonensis vaccine with BCG

placebo

Second dose

First dose Day 2

preparations:

Day 21

Day 7

Day 1

Day 7

Day 2 12

123123123123123

Day 21 3

123

Adverse event Erythema Placebo Vaccine Pain Placebo Vaccine Induration Placebo Vaccine Itching Placebo Vaccine Fever Placebo Vaccine Ulceration Placebo Vaccine

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11 22

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1

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3

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11 22

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16 9

1 2

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3 4

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11 22

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4-51-

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Values given are numbers of individuals with a specified adverse event at intensity grade of 1 (mild), 2 (moderate) or 3 (severe), at various times after preparation injection. No BCG was used in the third dose. After the third dose no adverse events were observed.

Table 3. Montenegro skin-test (MST) conversion after vaccination with and without BCG (positives defined as 2 5 mm induration)

with L. amazonen&

80 days after vaccination Total tested

Group Vaccine IM Placebo IM Vaccine-BCG Placebo-BCG

ID ID

59 64 28 16

IM, intramuscular; ID, intradermal; “t=7.616,d.f. 113~2,P
Positive MST n (%) 51 16 19 13

(86.4) (25.0) (67.9) (81.3)

One year after vaccination

Geometric mean f SD 2.24 1.13 2.28 1.76

vaccine or placebo,

f * f &

0.66” 0.94” 0*48b 0.59b

Total tested

Positive MST n P/A)

Geometric mean f SD

20 20

18 (90.0)

2.03 310.52 0.67 zt 0.84’ ND ND

l$go ND

ND, not done.

‘t = 3.167, d.f. 37, P= 0.003. ‘t=

6.139,d.f.

31.6, P< 0.0001.

demonstrated that the IM L (L.) amazonensis vaccine formulation prepared by BIOBRAS has a good safety and immunogenicity profile. In this study, no adverse reaction was seen with the use of the soluble IM vaccine. Although vaccines containing killed Leishmavliu organisms have been tested by the ID route, in association with BCG as adjuvant (CASTES et al., 1994; SHARPLJLSet al., 1994; ARMIJOS et al., 1998), in this trial the frequency and intensity of adverse events with the ID formulation were unacceptable. Nodules and ulcers at the injection site were more frequent in the vaccine-BCG ID group than in the placebo-BCG ID group and were more intense than expected with BCG alone. This enhanced reactogenesis of vaccine-BCG ID injections may be related to previous natural sensitization of this population to Mycobucterium sp. or to previous BCG vaccination. Unfortunately, no PPD testing was performed on enrolment to allow a correlation between PPD response and adverse reaction, but 99% of the volunteers had a BCG vaccine scar. The in-vivo immunogenicity measured by skin-test conversion to Leishmaniu antigen in the vaccine IM

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0.1 Vaccine Placebo Figure. Proliferation index (PI) of peripheral blood mononuclear cells with Leishmania antigen before (squares), 80 days after (triangles), and 1 year after (circles) first dose in the intramuscular L. amazonensis vaccine and intramuscular placebo groups. Data points represent the stimulation index, defined as the ratio between the mean counts of stimulated and nonstimulated cultures. The horizontal line indicates the sample mean.

ID ID

46 52 12 23

Total tested

Values given are numbers (%) of individuals IM, intramuscular; ID, intradermal.

Vaccine IM Placebo IM Vaccine-BCG Placebo-BCG

Group

Table 5. Antibody titres against wltb or without BCG

7 (28) 1 (4)

(87.0) (84.6) (91.7) (87.0)

23 (96) 3 (14)

: t=; 0 1 (4.3)

. 1 (8.3) 2 (8.7)

1:16

0 0

1 (1.9)

O

1:32

i

:

1:64

::

z:

Total tested

8 (40)

16 (80)

5 (45.5) 13 (56.5)

Negative 4 8 3 4

(8.9) (15.7) (27.3) (17.4)

1:8

4 (8.9) 8 (15.7) 1 (9.1) 4 (17.4)

1:16

80 days after vaccination

: 1 (4.3)

1 (4.3)

0

1:64

2 (18.2)

1 (2.0)

3 (6.7)

1:32

or placebo,

0.67 f l-00”

1.40 f O-91”

Geometric mean f SD

vaccine

Positive PI II (%)

of the first dose of L. amazonensis

20 20

or placebo

One year after vaccination

vaccine

Total tested

L. amazonensis

1.96 f 0087~ 0.34 f 0.44b

before and 80 days after the application

t g;;

1:8

with the antibody titres indicated.

40 44 11 20

Negative

obtained

Before vaccination

antigens

2;

0.14 f 0.56” -0.19 f 0.58”

with

Geometric mean f SD

Positive PI n (%)

Total tested

Geometric mean f SD

Positive PI n (%)

L. panamensis

25 25

Vaccine Placebo

Pt = 2.030, d.f. 48, P = 0.048. b t = 8.073, d.f. 35, P < 0.0001. ‘t=2.415,d.f.38, P=O.O21.

Total tested

Group

vaccination

80 days after vaccination

80 days after, and one year after intramuscular

Before vaccination

(PI) before,

index

Table 4. Proliferation

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group (86.4% and 95% 80 days and 1 year after vaccination, respectively) was higher than in the placebo IM group (25% and 5%, respectively). Both vaccine and placebo ID groups receiving BCG presented high skintest conversion. BCG is known to have antigens crossreacting with Leishmaniu and to improve recovery in cutaneous leishmaniasis patients, so some degree of Leishmaniu-directed immune response was expected (DOWLATI et al., 1996). Similar results have been observed in other countries. In Ecuador, a cocktail vaccine with 3 phenol-killed Leishmuniu species given in 2 ID injections plus BCG induced 85% skin-test conversion. BCG alone induced skin-test conversion in 20% (ARMIJOS et al., 1998). In Venezuela, the combination of BCG and Leishmuniu antigen was more immunogenic than autoclaved Leishmaniu antigen alone or BCG alone (CASTES et al., 1994). In Iran, BCG enhanced the induction of MST conversion by autoclaved and thimerosal-killed L. muior antigen (BAHAR et al.. 1996; MOMEN1 et&., 1999j. ” . Before vaccination, we found a low index of lymphocyte proliferation in response to Leishmuniu antigens with borderline siznificance between vaccine IM and placebo IM groups. After vaccination, there was a satisfactory immune response in vitro as determined by lymphocyte proliferation and cytokine (IFNy) for the IM vaccine group, whereas no significant change was recorded for the IM placebo group. In contrast, antibody production subsequent to immunization in the IM vaccine group was poor. Therefore, a Thl response and not a Th2 response seems to be elicited by the IM vaccine, as expected since Leishmaniu infection is primarily associated with cellular immune responses. Autoclaved L. (L.) umuzonensis vaccine induced a measurable cellular immune response in this study. However, some consideration should be given as to whether further studies should be conducted with autoclaved or nonautoclaved vaccine preparations. In 1 study, a nonautoclaved preparation induced a higher rate of skin-test conversion and of IFNy production than did an autoclaved preparation that lost stability within a short period of time (DE LUCA et al., 1999). The BIOBBAS L. (L.) amuzonensis vaccine production process has been standardized with reproducible growth of cultures. In this phase-I/II trial, the BIOBRAS vaccine demonstrated promising safety and immunogenicity data. These encouraging manufacturing develouments and earlv clinical results suggest that a nhase-III trial should be undertaken to evaiiate the protective efficacy of the vaccine with respect to the incidence and severity of American cutaneous leishmaniasis in Colombia. Acknowledgements This investieation was SuDDOITed bv UNDPWorld Bank/ WHO Special Programme for kesearcdand Training in Tropical Diseases PDR) grant number ID 970173 and Universidad de Antioquia. The authors express their gratitude to the Fuerzas Armadas de Colombia for their kind cooperation and to the volunteers included in the study for their collaboration. References Antunes, C. M. F., Mayrink, W. & Magalhaes, P. A. (1986). Controlled field trial of a vaccine against New World cutaneous leishmaniasis. InternationalJournal of Epidemiology, 15, 572-580. Armijos, R., Weigel, M., Aviles, H., Maldonado, R. & Racines, J. (1998). Field trial of a vaccine against New World cutaneous leishmaniasis in an at-risk child population: safety, immunogenicity and efficacy during the first 12 months of follow up. Journal of Infectious Diseases, 177, 1352-1357. Bahar, K., Dowlati, Y., Shidani, B., Alimphammadian, M. H., Khamesipour, L. I., Ehsasi, S., Hashemi-fesharki, R., Ale-

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Received 28 July 1999; revised 24 May 2000; accepted for publication 24 May 2000