Therapy for Mucosal Leishmaniasis
• JID 2006:194 (15 October) • 1151
M A J O R A R T I C L E
Immunotherapy for Drug-Refractory
Roberto Badaro,1,3Iza Lobo,1Alvaro Muno ˜s,1Eduardo M. Netto,1Farrokh Modabber,2Antonio Campos-Neto,4
Rhea N. Coler,5and Steven G. Reed5
1Federal University of Bahia, Salvador, Brazil;
San Diego, San Diego;
2Drugs for Neglected Diseases Initiative, Geneva, Switzerland;
4The Forsyth Institute, Boston, Massachusetts;5Infectious Disease Research Institute, Seattle, Washington
3University of California,
toxic, and relapses are common. Immunotherapy using a mixture of killed parasites, with or without bacille
Calmette-Gue ´rin, is an alternative but is used sporadically because of inconsistent results.
We developed a define immunotherapeutic antigen preparation for use in an observational, open-
label trial to treat 6 patients with ML with a history of Sbvtherapy failure. All patients were treated with the
antigens thiol-specifi antioxidant, Leishmania major stress inducible protein 1, Leishmania elongation initiation
factor, and Leishmania heat shock protein 83, plus granulocyte-macrophage colony-stimulating factor. Patients
underwent clinical and pathological evaluations before the initiation of immunotherapy and at 3, 6, 9, 12, 18, 24,
and 60 months after.
One month after the third injection, 1 patient showed complete clinical remission (CC) and remained
disease free for the duration of the study. At the 9-month follow-up examination, 5 patients showed CC, and all
patients were asymptomatic at a subsequent 5-year follow-up examination.
These data support the concept that vaccine therapy with a define antigen combination, used
with standard chemotherapy, is a safe and effective approach to treat drug-refractory ML.
Pentavalent antimony (Sbv) is the mainstay therapy for mucosal leishmaniasis (ML), but it is
Mucosal leishmaniasis (ML) is a chronic, disfigurin
disease, often difficul to treat . Once granulomas
develop in the mucosa, patients may become refractory
to conventional chemotherapy (pentavalent antimony
[Sbv]) [2, 3] or to alternative drugs (amphotericin B,
pentamidine, and miltefosine) [4–7]. Immunotherapy
with crude Leishmania antigen preparations, alone or
in combination with bacille Calmette-Gue ´rin, has been
partially successful, producing dramatic healing in
some patients [8–11], and has been recommended as
an alternative approach to treat ML . However,
because these vaccines are undefine mixtures, impos-
sible to standardize, and can cause severe local or sys-
Received 8 March 2006; accepted 9 June 2006; electronically published 15
Presented in part: Third World Congress on Leishmaniasis, plenary lecture, 14
April 2005, Palermo-Terrasini, Sicily, Italy (session 2, abstract 1).
Potential conflicts of interest: none reported.
Financial support: National Institutes of Health (grants AI25038 and AI36810;
and Health Biotechnology Fellowship Grant GM08347 to R.N.C.).
Reprints or correspondence: Dr. Steven G. Reed, Infectious Disease Research
Institute, 1124 Columbia St., Suite 400, Seattle, WA 98104 (email@example.com).
The Journal of Infectious Diseases
? 2006 by the Infectious Diseases Society of America. All rights reserved.
temic adverse side effects , it is unlikely that they
will become widely used.
In the present study, we treated patients with drug-
refractory ML with a combination of leishmanial re-
combinant antigens—thiol-specifi antioxidant (TSA)
, Leishmania major stress inducible protein 1
(LmSTI1) [15, 16], Leishmania elongation initiation
factor (LeIF) [17–19], and Leishmania heat shock pro-
tein 83 (Lbhsp83) —chosen on the basis of studies
demonstrating their recognition by patients’ serum
samples and/or cells in vitro and of animalstudiesdem-
onstrating either protection or therapeutic efficac in
tor (GM-CSF) was included to enhance antigen uptake
and processing [21–23]. We report results of a 5-year
PATIENTS AND METHODS
trial conducted during 1995–2000 at the outpatient
clinic of the University Hospital, Federal University of
Bahia, Salvador, Brazil. Ethics approval was obtained
from the University Institutional Review Board. In-
This was an observational, open-label
1152 • JID 2006:194 (15 October) • Badaro et al.
formed consent was obtained beforeenrollmentofeachpatient.
Six patients with active ML acquired
in endemic areas of southern Bahia wereenrolled.Clinicalchar-
acteristics of the patients are presented in table 1. Most patients
had granulomatous lesions in the nasal mucosa, 1 had septal
destruction, and 1 had oropharyngeal and laryngeal involve-
ment. The median duration of ML was 4 years (range, 1–5
years). All patients reported previous cutaneous lesions, at an
average of 11 years before the appearance of ML. Previous Sbv
treatment ranged from 1–15 courses of 20 mg/kg/day for 20–
30 days. All patients reacted strongly to the Montenegro skin
test (table 1). In 4 patients, Leishmania parasites wereidentifie
by biopsy before immunotherapy. Specifi IgG antileishmanial
antibodies were detected in all patients, with titersrangingfrom
1.7 to 22.1 times above the cutoff of the ELISA, performed as
described elsewhere .
Patients were enrolled using the following
entry criteria: (1) active ML characterized by granulomatous
or ulcerative lesions involving the nose, pharynx, palate,larynx,
or lips; (2) active lesions for 16 months; (3) positive Monte-
negro skin test (15 mm induration, as described elsewhere [24,
25]); (4) histopathological finding compatible with ML or
demonstration of Leishmania parasites in affected tissues; (5)
positive titers of antibody to Leishmania antigens; (6) HIV
negative; and (7) no history of malignancy or immunosup-
TSA, LmSTI1, Lbhsp83, and LeIF (an inducer of interleukin
[14–20] and 50 mg of the cytokine GM-CSF (Leukine; Amgen)
were prepared in 0.5 mL and administered subcutaneously(sc)
in the anterior surface of the forearm.
Evaluation of clinical response and follow-up.
munotherapy, patients were evaluated by endoscopy to docu-
ment lesions at baseline. The criteria used to evaluate clinical
response were as follows: (1) complete clinical remission (CC),
define as scarring of the affected tissue as documented by
subsequent endoscopies; (2) clinicalimprovement(CI),define
as clear amelioration of the lesions, compared with the baseline
evaluation, including scar tissue; (3) failure (F), define as no
improvement and/or new areas of mucosal granulomatous re-
action; and (4) relapse (R), define as expansion of original
lesions or appearance of new lesions after previous documen-
tation of complete or partial response.
Sbv(Glucantime) was administered intravenously (iv)
(20 mg/kg/day for 20 days) to patients who experienced im-
Serum samples of patients were tested for the pres-
ence of antileishmanial (soluble leishmania antigen [SLA]) an-
tibody by ELISA, as described elsewhere .
Measurement of delayed-type hypersensitivity
(DTH) was performed using crude antigen, as described else-
where , or the individual recombinant antigens. Reactions
were measured 48–72 h after intradermal injection of 0.1 mL
of antigen. A minimum of 1 week elapsed between the skin
test and the firs dose of immunotherapy.
was performed in all patients by use of a 3-mm punch. Sections
were fixe in 10% formalin, embedded in paraffin and stained
Proliferation and cytokine assays.
by gradient centrifugation and used in proliferation assays.
Briefl , PBMCs (cells/well) were incubated in 96-well
plates (Costar) in medium (RPMI 1640 with 10% pooled hu-
man serum and gentamycin [50 mg/mL]), with or without an-
tigens (10 mg/mL), for 5 days at 37?C in 5% CO2, and pulsed
with 1 mCi of [3H]-thymidine (Amersham) for an additional
18 h. Levels of supernatant interferon (IFN)–g were analyzed
by sandwich ELISA (PharMingen) . For this assay, culture
supernatants were obtained 72 h after the initiation of the
Student’s t test (2-tailed) was used to
compare IFN-g and IL-5 cytokine levels elicited from patient
PBMCs before and after immunotherapy.
ered to be significant
Biopsy of mucosal lesions
PBMCs were isolated
was consid-P ! .05
Overall Clinical Response and Follow-Up
All 6 patients were treated with 3 consecutive doses (1 month
apart) of immunotherapy and underwent periodic clinical fol-
low-up examinations, including endoscopy. Patients who did
not respond well received further series of immunotherapy in
combination with a 20-day course of Sbv. Table 1 presents
clinical and laboratory profile of the 6 patients before im-
munotherapy. The overall outcome and treatment intervention
followed the firs course of immunotherapy for all 6 patients
and is presented in table 2.
of CL in the left leg 4 years before the onset of symptoms in
the nose. He received treatment for the nasal discharge and
oral cephalosporin at a local health post. One year later, he had
difficult swallowing because of inflammatio of oral mucosae.
He came to our clinic and had a presumptive diagnosis of ML.
He received a full course of 30 days of Glucantime(3ampoules/
intramuscularly/iv/daily). Sixty days later, there was significan
improvement in his nose, and the oral lesions disappeared. Six
months later, the lesions reappeared and spread in the oral and
nasal mucosae, resulting in destruction of his nasal septum.
Before the initiation of immunotherapy, a biopsy found amas-
tigotes, and diagnosis of ML was made. Laboratoryevaluations,
Fifty-one-year-old man with a previous history
Therapy for Mucosal Leishmaniasis • JID 2006:194 (15 October) • 1153
Table 1. Clinical characteristics of the study patients.
age in years)
courses of Sbv
LST, mm of
in lesionsHistopathological analysis Before Rx After Rx
01 (M, 51
02 (M, 51)
03 (M, 25)
04 (M, 54)
05 (F , 45)
06 (F , 60)
destruction; OM, oral mucosal; OPLM, oropharyngeal-laryngeal mucosal; PC, penile cutaneous; Rx, immunotherapy; Sbv, pentavalent antimony.
aELISA optical density (OD) reading of patient’s serum at a 1:25 dilution divided by the OD of a pool of serum from healthy individuals living in the same
geographical area as the patients. Antibody to soluble leishmania antigen was measured.
F , female; LST, leishmanin skin-test reaction with Leishmania crude antigen; M, male; NC, nasal cutaneous; NM, nasal mucosal; NSD, nasal septal
leishmanin skin test, and serological analysis were performed
before the firs dose of the immunotherapy. Three consecutive
doses of immunotherapy were administered 1 month apart.
During the second month, there was significan improvement.
One month after the third injection, complete cicatrization of
the lesions in nasal and oral mucosal areas was observed. He
remained asymptomatic at the 5-year follow-up examination.
Fifty-one-year-old man with a history of CL
in the penis 2 years before mucosal involvement. He recalled
being bitten by a mosquito on his penis and a month later
noted a small ulcerative lesion. He was empirically treated with
penicillin for a sexually transmitted infection. Noimprovement
was noted. He then received a presumptive diagnosis of CL in
the local health post. He received Glucantime daily for 21 days.
He noted improvement after this treatment but was not com-
pletely cured. Three more courses of Sbvwere administered 1
month apart. One year later, he received another 30-day course
of Glucantime, and modest improvement was noted. Two more
courses of Glucantime were administered,andnoimprovement
was noted. He declined additional Sbvtherapy because of severe
and bleeding. Three months after this, the penis lesion ulcer-
ated, and an ulcer appeared in his nasalseptum.Hewasreferred
to our clinic. ML was confi med by leishmanin skin test (15-
mm induration). He received 3 consecutive courses of Glu-
cantime (20/kg/day for 21 days/course);again,noimprovement
was noted. At the 3-month follow-up examination, a nasal
biopsy found ML with documented amastigotes. Because he
had been exposed to several courses of Sbv, we offered him the
experimental immunotherapy. After the firs course of 3 doses
of immunotherapy alone, no improvement was noted. Thiswas
followed by a combination of Sbvand immunotherapy. After
3 months, dramatic improvement was noted. The lesion in the
penis was completely healed, and 80% of the nasal ulceration
scarred. The following month, new areas of ulceration were
noted in the nose. Another course of this combination treat-
ment was administered. Again, significan improvement was
noted. At 9 months (1 month after the last treatment), no
lesions were noted in the nose. From then through the 5-year
follow-up examination, he remained completelyasymptomatic.
Twenty-five-yea -old man with a history of a
self-healing ulcer in his knee 5 years before oral ulcers. After
4 months of ulcerative lesions in the palate and buccal mucosa,
he received a presumptive diagnosis of unspecifi recurrent
stomatitis. Topical corticosteroidwasindicatedbyapharmacist,
without improvement. He became hoarse and visited our hos-
pital. On physical examination, an infiltrativ lesion in the pal-
ate extending through the pharynx and larynx was found. The
biopsy found an exudative granulomatous lesion with amas-
tigotes. The patient was treated with 3 courses 1 month apart
of 20 mg/kg/day of Glucantime, without significan improve-
ment. He was hospitalized and administered a 28-day course
of Glucantime. Because there was no improvement,weinitiated
the immunotherapy treatment; significan improvement was
noted. When he returned for evaluation 1 month later, new
ulcerative areas were noted in the larynx and palate. He was
treated with 3 more doses of immunotherapy. At the 9-month
follow-up examination, no active lesions were found. At the 5-
year follow-up examination, he remained asymptomatic.
Fifty-four-year-old man with a history of nasal
obstruction 2 years before visiting a doctor. He had a positive
leishmanin test and documented ML with anterior septal de-
struction. At that time, he received a 40-day course of 28 mg/
kg/day of Glucantime. He reported no bleeding episodes for 1
year. He was referred to our clinic for retreatment after nasal
bleeding, and the obstruction reappeared. A biopsy found a
granulomatous exudative infiltration No amastigotes were ob-
served; both serological analysis and leishmanin skin test were
positive. Because the patient refused to receive Sbv, we offered
treatment with immunotherapy. After the third dose of im-
munotherapy, he had a complete scarringoflesionsintheseptal
Clinical responses and follow-up examinations of the study patients.
duration in years/
no. of courses
1 month after completion of immunotherapy
1 course Sbv+ 3 doses immunotherapy
1 course Sbv+ 3 doses immunotherapy
1 course Sbv+ 3 doses immunotherapy
1 course Sbv+ 3 doses immunotherapy
1 course Sbv+ 3 doses immunotherapy
1 course Sbv
One course of pentavalent antimony (Sbv) p 20 mg/kg/day of Sbvfor 20 days. CC, complete clinical remission; CI, clinical improvement; F , treatment failure; R, relapse.
Therapy for Mucosal Leishmaniasis • JID 2006:194 (15 October) • 1155
Patient with mucosal leishmaniasis, before (A) and after (B)
sensitivity (DTH) reactions to recombinant
antigens before and after immunotherapy.
Elicitation of delayed-type hyper-
Induration reaction to antigen
LmSTI1 TSA Lbhsp83LeIF
of DTH skin test performed with individual recombi-
nant antigens before/after the third dose of the im-
munotherapeutic preparation. Lbhsp83, Leishmania
heat shock protein 83; LeIF , Leishmania elongation
initiation factor; LmSTI1, Leishmania major stress in-
ducible protein 1; ND, not done; TSA, thiol-specific
Data are millimeters of indurationreaction
mucosae. He remained completely asymptomatic during the 5-
year follow-up examination.
Forty-five-yea -old woman with a history of
cutaneous nasal and septal inflammatio and redness of the
nose for 1 year. Because she had a family member with ahistory
of leishmaniasis, she asked to be treated at the health post.
After 1 course of chemotherapy, her nose improved but ul-
cerated again 3 months later. She self-administered three 15-
day courses of Glucantime, again with intermittent improve-
ment. Because scarring was still incomplete, she visited our
clinic. We administered Sbvand immunotherapy because she
had several prior suboptimal courses of Sbv. She received a 28-
day course of Sbvand 3 doses of the immunotherapy. At the
end of the treatment, she had significan improvement with a
few areasof infiltration Atthe6-monthfollow-upexamination,
new active areas in the septum were noted. She refused further
combination therapy, so another full 28-day course of Sbvwas
follow-up examination. She remained symptom free at the 5-
year follow-up examination.
Sixty-year-old woman with no history of CL.
Five years before the initiation of immunotherapy, she devel-
oped an ulceration in the nose. She self-treated with local herbs
for 6 months, with scarring of the lesion. However, the nasal
obstruction remained. One year before enrollmentinthisstudy,
she had relapses of the nose lesion with complete ulceration of
the nasal septum. She received a presumptive diagnosis of ML
from a local doctor on the basis of the clinical aspect of the
lesion and a strongly positive leishmanin skin test. She received
2 courses of Sbv. She reported improvement of the cutaneous
lesion in the nose but not with the nasal obstruction and had
relapses that ended with a septal perforation. At the hospital,
her prior electrocardiogram evaluation showed prolongationof
PR intervals (i.e., the time between the beginning of the P wave
and the beginning oftheQRScomplexinanelectrocardiogram)
and left bundle block, so we administeredimmunotherapyonly.
Three doses of immunotherapy were given, resulting in com-
plete healing of the nasal lesion and some crusty lesions in the
nasal septum. At the 9-month follow-up examination, these
lesions were completely healed, with no further signs of ML.
The patients’ clinical outcome can be summarizedasfollows:
1 month after the third dose of the immunotherapeutic prep-
aration, 1 patient (01) was considered to have achieved CC, 2
patients (04 and 06) had visible CI, and 3 patients (02, 03, and
05) did not respond (F) to the immunotherapy. No further
intervention was administered to the patients with CC or CI.
The 3 patients who did not respond received the combination
of 3 injections of the immunotherapeutic preparation and a
20-day course of Glucantime. At the 6-month follow-up ex-
amination, patient 01 (CC) remained asymptomatic, and pa-
tient 04, initially classifie as CI, became CC. Patient 06 still
showed evidence of inflammatio around the scarred area in
the nose; no additional treatment was provided because she
indicated that the symptoms were improving. Patients 02, 03,
and 05 demonstrated dramatic improvement of the lesions at
the end of the firs retreatment. However, at the 6-month fol-
low-up examination, all 3 patients had evidence of new active
lesions adjacent to thescarred areas.Patients02and03accepted
3 additional consecutiveinjectionsoftheimmunotherapyprep-
aration in combination with another 20-day course of Sbv.
Patient 05 could not remain in the hospital and preferred re-
ceiving Sbvtherapy in her hometown. At the 9-month follow-
up examination, only patient 05 remained incompletely cured;
however, dramatic CI was noted, and no additional treatment
was provided. At the 12-month follow-up examination, all 6
patients showed CC and remained asymptomatic until the last
follow-up examination 5 years later.
chemotherapy with Sbv, produced sustained long-term CC of
ML. Figure 1A and 1B shows the active lesion of patient 05
before and after treatment, respectively. Complete healing
of the septum can be clearly seen after the therapy.
1156 • JID 2006:194 (15 October) • Badaro et al.
antigens. One month after the third administration of the immunotherapeutic preparation, cytokine production was determined by stimulating PBMCs
with recombinant leishmanial proteins (10 mg/mL Leishmania major stress inducible protein 1 [LmSTI1], thiol-specific antioxidant [TSA], Leishmania
elongation initiation factor [LeIF], Leishmania heat shock protein 83 [Lbhsp83], or soluble leishmania antigen [SLA]). Interferon (IFN)–g (A) andinterleukin
(IL)–5 (B) production was determined by ELISA from supernatants taken after 72 h. Cultures were performed in triplicate. Significant differences
() between cells isolated from patients at baseline (before immunotherapy) and after immunotherapy are indicated by the asterisks.P ! .5
Cytokine production by peripheral-blood mononuclear cells (PBMCs) from patients with mucosal leishmaniasis in response to leishmanial
Skin Test with Recombinant Antigens
Four patients underwent skin testing before and after immu-
notherapy. The other 2 patients were tested only after the third
dose of immunotherapy. Table 3 shows the results of skin re-
actions (in millimeters of induration) at 48 h after antigen
administration. Three patients responded to LmSTI1 and to
TSA, all 4 to Lbhsp83, and only 1 to LeIF after 3 doses of
immunotherapy. The skin-test reaction to Lbhsp83 was signif-
icantly stronger than the reactions to the other 3 antigens
( ).P ! .001
Figure 2 shows the cytokine production of the patients before
and after immunotherapy. In the 4 patients tested, an increase
in antigen-specifi proliferation of PBMCs in response to
all patients tested responded with increased INF-g production
after in vitro stimulation with each of the immunizingantigens.
PBMCs collected before immunization produced significan
amounts of IL-5 but not IFN-g when stimulated with SLA. Of
particular interest, this SLA-elicited IL-5 production decreased
Therapy for Mucosal Leishmaniasis • JID 2006:194 (15 October) • 1157
to background levels after CC. There was no IFN-g or IL-5
antigen-specifi cytokine production in PBMCs collected from
uninfected, unimmunized individuals from the United States
(data not shown). Levels of antibody to whole leishmania de-
creased in all patients after immunotherapy (table 1).
After the firs dose of the immunotherapeutic preparation, all
6 patients had mild systemic adverse reactions such as fever,
headache, malaise, or somnolence. All 6 patientspresentedlocal
site-of-injection reactions characterized by redness and edema
resembling leishmanin skin-test induration. The difference in
leishmanin skin tests was the lack of demarked granulomatous
reactions. The same pattern of reactivity occurred after the
second and third doses of immunotherapy. Such reactionshave
been described elsewhere after sc administration of GM-CSF
. There was no correlation between the intensity of the
local or systemic reactions and the number of doses given to
the patients. However, 1 patient presented a necrotic skin re-
action at the site of injection after the third dose,whichresolved
promptly (within 2 weeks) with topical steroid therapy.
Immunotherapy for ML was introduced almost a century ago
. The earliest report of this intervention dates from 1912,
which coincides with the time of introduction of antileish-
manial Sb chemotherapy by Gaspar Vianna . After its in-
troduction, immunotherapy was inconsistently practiced for
the next 50 years because Sb was efficaciou in the majority of
patients. However, because of problems related to drug toxicity
and drug resistance, Convit et al.  and Mayrink et al. 
revisited immunotherapy and reported on the treatment of CL
and ML by use of vaccines consisting of whole promastigote
preparations. Cure rates from 76% to 94% were reported.
The rationale of inducing antigen-specifi immuneresponses
in patients who already have responses to the parasite [29, 30]
may seem paradoxical. However, beneficia effects from mod-
ulating the immune system by whole Leishmania antigens have
been documented [8, 9]. Although questions remain regard-
ing which immune regulatory mechanisms promoted by im-
munotherapy result in clinical responses, several interesting
points were observed in the present study. The most signif-
icant finding in patient PBMC responses were (1) an increase
in the production of IFN-g to the antigens contained in the
immunotherapeutic preparation after immunization, without
an increase in the production of IL-5 to these antigens, and
(2) a significan decrease in anti–whole parasite IL-5 pro-
duction after immunization. Because the immunized patients
had been previously treated with Sbv, it is likely that this
down-regulation of IL-5 production was associated with the
The proteins contained in the immunotherapeutic prepa-
ration used in this study have been shown to elicit responses
from patient PBMCs (Lbhsp83) , to protect mice against
leishmaniasis (LmSTI1 and TSA) [14, 15, 32–36], and to stim-
ulate IL-12 from mouse and human cells in vitro and to be
effective in the treatment of leishmaniasis in mice or humans
in vivo [35, 37]. At the time this study was initiated, there were
no adjuvants available for clinical use with the ability to selec-
tively promote Th1 responses of the type thought to be essential
for resistance to leishmaniasis. We selected GM-CSFforreasons
of convenience and because of reported adjuvant and anti-
leishmanial activity [38–41]. Although there is evidence for
complementary therapeutic effects of GM-CSF and Sb in leish-
maniasis [42–44], it is unlikely that GM-CSF alone was re-
sponsible for the clinical resolutions seen. Our previous ex-
perience using GM-CSF, with or without Sb, to treat patients
with refractory ML was not successful (R. Badaro, unpublished
In conclusion, these data support the concept that vaccine
therapy is a safe and effective approach to treat drug-refractory
ML. For the firs time, a define antigen combination has been
shown to be effective in the treatmentof ML,eveninthepatient
population that is most difficul to treat. This opens the pos-
sibility of developing a safe, affordable, and standardized im-
munotherapeutic for broad application. Among the most sig-
nifican finding was the observation of decreased Th2 profil
in response to parasite lysate after the completion of the firs
series of immunotherapy. This supports the concept that ML
is characterized by a mixed Th1/Th2 cytokine profil  and
indicates that clinical cure is more strongly associated with an
antigen-specifi decrease in Th2 responses rather than an in-
crease in Th1 responses to the parasite. Of significanc is that
after immunization, although none of the antigens induced IL-
5. It is interesting to note that the recombinant antigens were
not recognized strongly by patients with ML before immu-
notherapy, perhaps indicating immunodominance by nonpro-
Since the initiation of this study, improvements have been
made in the vaccine candidate. This has resulted in the pro-
duction of a polyprotein, Leish-111f, formulated in mono-
phosphoryl lipid A with squalene oil emulsion (MPL-SE) [34–
36]. This vaccine candidate has been manufactured undergood
manufacturing practice conditions and is currently undergoing
testing under US investigational new drug regulationsinseveral
countries. Among the indications for which the vaccine is being
evaluated are CL and ML in South America. It is our intention
to extend these studies as appropriate and modify the vaccine
as needed to optimize the development of an immunothera-
1158 • JID 2006:194 (15 October) • Badaro et al.
peutic for leishmaniasis to be used to enhance chemotherapy.
This could be achieved by demonstrating efficac in patients
with drug-refractory ML, the approach used in the present
study, or by decreasing the amount of chemotherapy needed
We thank the study participants, who were outpatients at the clinic of
the Division of Infectious and Parasitic Diseases, University Hospital, Fed-
eral University of Bahia, Salvador, Bahia,Brazil,andwhounderwentrounds
of clinical assessments and treatments.
1. Marsden PD. Mucosal leishmaniasis (“espundia”Escomel,1911).Trans
R Soc Trop Med Hyg 1986;80:859–76.
2. Rocha RA, Sampaio RN, Guerra M, et al. Apparent Glucantime failure
in fiv patients with mucocutaneous leishmaniasis. J Trop Med Hyg
3. Sampaio RN, Sampaio JH, Marsden PD. Pentavalent antimonial treat-
ment in mucosal leishmaniasis. Lancet 1985;1:1097.
4. Berman JD. Treatment of New World cutaneous and mucosal leish-
maniases. Clin Dermatol 1996;14:519–22.
5. Di Giorgio C, Faraut-Gambarelli F, Imbert A, Minodier P, Gasquet M,
Dumon H. Flow cytometric assessmentofamphotericinBsusceptibility
in Leishmania infantum isolates from patients with visceral leishman-
iasis. J Antimicrob Chemother1999;44:71–6.
6. Netto EM, Costa JM, Vieira JB, Marsden PD. An attempt to treat skin
ulcer caused by Leishmania braziliensis with ketoconazole. Rev SocBras
Med Trop 1989;22:105–6.
7. Soto J, Arana BA, Toledo J, et al. Miltefosine for new world cutaneous
leishmaniasis. Clin Infect Dis 2004;38:1266–72.
8. Convit J, Castellanos PL, Rondon A, et al. Immunotherapy versus che-
motherapy in localised cutaneous leishmaniasis. Lancet 1987;1:401–5.
9. Convit J, Castellanos PL, Ulrich M, et al. Immunotherapy of localized,
intermediate, and diffuse forms of American cutaneous leishmaniasis.
J Infect Dis 1989;160:104–15.
10. Genaro O, de Toledo VP, da Costa CA, Hermeto MV, Afonso LC,
Mayrink W. Vaccine for prophylaxis and immunotherapy, Brazil. Clin
11. Mayrink W, Magalhaes PA, Michalick MS, et al. Immunotherapy as a
treatment of American cutaneous leishmaniasis: preliminary studiesin
Brazil. Parassitologia 1992;34:159–65.
12. Convit J, Ulrich M, Zerpa O, et al. Immunotherapy of American cu-
taneous leishmaniasis in Venezuela during the period 1990–99. Trans
R Soc Trop Med Hyg 2003;97:469–72.
13. Machado-Pinto J, Pinto J, da Costa CA, et al. Immunochemotherapy
for cutaneous leishmaniasis: a controlled trial using killed Leishmania
(Leishmania) amazonensis vaccine plus antimonial. Int J Dermatol
14. Webb JR, Campos-Neto A, Ovendale PJ, et al. Human and murine
immune responses to a novel Leishmania major recombinant protein
encoded by members of a multicopy gene family. Infect Immun 1998;
15. Webb JR, Kaufmann D, Campos-Neto A, Reed SG. Molecular cloning
of a novel protein antigen of Leishmania major that elicits a potent
immune response in experimental murine leishmaniasis. J Immunol
16. Webb JR, Campos-Neto A, Skeiky YA, Reed SG. Molecular character-
ization of the heat-inducible LmSTI1 protein of Leishmania major.Mol
Biochem Parasitol 1997;89:179–93.
17. Probst P, Skeiky YA, Steeves M, Gervassi A, Grabstein KH, Reed SG.
A Leishmania protein that modulates interleukin (IL)-12, IL-10 and
tumor necrosis factor-alpha production and expression of B7-1 in hu-
man monocyte-derived antigen-presenting cells. Eur J Immunol 1997;
18. Skeiky YA, Guderian JA, Benson DR, et al. A recombinant Leishmania
antigen that stimulates human peripheral blood mononuclear cells to
express a Th1-type cytokine profil and to produce interleukin 12. J
Exp Med 1995;181:1527–37.
19. Skeiky YA, Kennedy M, Kaufman D, et al. LeIF: a recombinant Leish-
mania protein that induces an IL-12-mediated Th1 cytokine profile J
20. Skeiky YA, Benson DR, Costa JL, Badaro R, Reed SG. Association of
Leishmania heat shock protein 83 antigen and immunoglobulin G4
antibody titers in Brazilian patients with diffuse cutaneous leishman-
iasis. Infect Immun 1997;65:5368–70.
21. Follador I, Araujo C, Orge G, et al. Immune responses to an inactive
vaccine against American cutaneous leishmaniasis together with gran-
ulocyte-macrophage colony-stimulating factor. Vaccine 2002;20:1365–8.
22. Tarr PE, Lin R, Mueller EA, Kovarik JM, Guillaume M, Jones TC. Eval-
uation of tolerability and antibody response after recombinant human
granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and a
single dose of recombinant hepatitis B vaccine. Vaccine 1996;14:
23. Jones T, Stern A, Lin R. Potential role of granulocyte-macrophage
colony-stimulating factor as vaccine adjuvant. Eur J Clin Microbiol
Infect Dis 1994;13(Suppl 2):S47–53.
24. Kadaro AY, Ghalib HW, Ali MS, et al. Prevalence of cutaneous leish-
maniasis along the Nile River north of Khartoum (Sudan) in the af-
termath of an epidemic in 1985. Am J Trop Med Hyg 1993;48:44–9.
25. Zijlstra EE, Daifalla NS, Kager PA, et al. rK39 enzyme-linked immu-
nosorbent assay for diagnosis of Leishmania donovani infection. Clin
Diagn Lab Immunol 1998;5:717–20.
26. Badaro R, Reed SG, Barral A, Orge G, Jones TC. Evaluation of the
micro enzyme-linked immunosorbent assay (ELISA) for antibodies in
American visceral leishmaniasis: antigen selection for detection of in-
fection-specifi responses. Am J Trop Med Hyg 1986;35:72–8.
27. Row R. Curative value of Leishmania culture vaccine in Oriental sore.
Br Med J 1912;9:540–1.
28. Caronia G. L’implego di movi preparati de antimonio per via intra-
musculare nella cura della leishmaniosi infantile. La Pediatria 1916;
29. Carvalho EM, Johnson WD, Barreto E, et al. Cell mediated immunity
in American cutaneous and mucosal leishmaniasis. J Immunol 1985;
30. Reed SG, Scott P. T-cell and cytokine responses in leishmaniasis. Curr
Opin Immunol 1993;5:524–31.
31. Skeiky YA, Benson DR, Guderian JA, et al. Immune responses of leish-
maniasis patients to heat shock proteins of Leishmania species and
humans. Infect Immun 1995;63:4105–14.
32. Campos-Neto A, Porrozzi R, Greeson K, et al. Protection against cu-
taneous leishmaniasis induced by recombinant antigens in murine and
nonhuman primate models of the human disease. Infect Immun 2001;
33. Campos-Neto A, Webb JR, Greeson K, Coler RN, Skeiky YA, Reed SG.
Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanialfu-
sion proteins confers protection against Leishmania major infection in
susceptible BALB/c mice. Infect Immun 2002;70:2828–36.
34. Coler RN, Skeiky YA, Bernards K, et al. Immunization with a poly-
protein vaccine consisting of the T-cell antigens thiol-specifi antiox-
idant, Leishmania major stress-inducible protein 1, and Leishmania
elongation initiation factor protects against leishmaniasis. Infect Im-
35. Coler RN, Reed SG. Second-generation vaccines against leishmaniasis.
Trends Parasitol 2005;21:244–9.
36. Skeiky YA, Coler RN, Brannon M, et al. Protective efficac of a tan-
demly linked, multi-subunit recombinant leishmanial vaccine (Leish-
111f) formulated in MPL adjuvant. Vaccine 2002;20:3292–303.
Therapy for Mucosal Leishmaniasis • JID 2006:194 (15 October) • 1159
37. Badaro R, Lobo I, Nakatani M, et al.Successful useofadefine antigen/
GM-CSF adjuvant vaccine to treat mucosal leishmaniasis refractory to
antimony: a case report. Braz J Infect Dis 2001;5:223–32.
38. Almeida RP, Brito J, Machado PL, et al. Successful treatment of re-
fractory cutaneous leishmaniasis with GM-CSF and antimonials. Am
J Trop Med Hyg 2005;73:79–81.
39. Boente P, Sampaio C, Brandao MA, Moreira ED, Badaro R, Jones TC.
Local peri-lesional therapy with rhGM-CSF for Kaposi’s sarcoma.Lan-
40. Handman E, Burgess AW. Stimulation by granulocyte-macrophagecol-
ony-stimulating factor of Leishmania tropica killing by macrophages.
J Immunol 1979;122:1134–7.
41. Robson M, Kucukcelebi A, Carp SS, et al. Effects of granulocyte-mac-
rophage colony-stimulating factor on wound contraction. Eur J Clin
Microbiol Infect Dis 1994;13(Suppl 2):S41–6.
42. Almeida R, D’Oliveira A Jr., Machado P, et al. Randomized, double-
blind study of stibogluconate plus human granulocyte macrophage
colony-stimulating factor versus stibogluconate alone in the treatment
of cutaneous leishmaniasis. J Infect Dis 1999;180:1735–7.
43. Badaro R, Nascimento C, Carvalho JS, et al. Recombinant human
granulocyte-macrophage colony-stimulating factor reverses neutrope-
nia and reduces secondary infections in visceral leishmaniasis. J Infect
44. Badaro R, Nascimento C, Carvalho JS, et al. Granulocyte-macrophage
colony-stimulating factor in combination with pentavalent antimony
for the treatment of visceral leishmaniasis. Eur J Clin Microbiol Infect
Dis 1994;13(Suppl 2):S23–8.
45. Pirmez C, Yamamura M, Uyemura K, Paes-Oliveira M, Conceicao-
Silva F, Modlin RL. Cytokine patterns in the pathogenesis of human
leishmaniasis. J Clin Invest 1993;91:1390–5.