Comparative safety and immunogenicity trial of two killed Leishmania Major vaccines with or without BCG in human volunteers

ELSEVIFR

Comparative Safety and Immunogenicity Trial of Two Killed Leishmania Major Vaccines With or Without BCG in Human Volunteers KAMAL BAHAR, Ph.D.cand., YAHYA DOWLATI, M.D., D. PHARM. BABAK SHIDANI, Ph.D., MOHAMMAD HOSSEIN ALIMOHAMMADIAN, D. PHARM., D. MED. AL1 KHAMESIPOUR, Ph.D., SIEMIN EHSASI, M.D., REZA HASHEMI-FESHARIU, D.V.M., SAEED ALE-AGHA, D.V.M., FARROKH MODABBER, Ph.D. l

LAB.

SCI.,

l

l

T

he variety of epidemiology of different forms of leishmaniases in the world makes it impossible to apply a single control measure universally, except possibly, a cross-protective vaccine. The overwhelming cross-reactive antigens in all pathogenic Leishmanin, and the presence of molecules that cross protect against different species’,2 justify striving for development of a single vaccine. In addition, since other methods of control are costly, not feasible for many epidemiological situations, or require considerable infrastructures (e.g., vector and reservoir control), the UNDP/World Bank/WHO Special Programme (for Research and Training in Tropical Diseases (TDR) has given vaccine development the highest priority for research in leishmaniasis.3 Unlike some other parasites, Leishmania can be grown in cell-free media and large quantities can be obtained with ease. This, plus the use in humans for the past several decades of killed parasites as skin test antigens for diagnosis (leishmanin), has made it possible to try killed organisms with or without adjuvants as vaccines or for immunotherapy in clinical studies (see references 4 and 5 and Convit in this issue). The ultimate vaccine would most likely be a cocktail of several well-defined immunogens rather than a single molecule to overcome genetic restriction of hosts and cover a wider diversity of parasites. Hence, the whole killed parasite mixed with BCG (as an adjuvant) is a first-generation vaccine to be tested while waiting for the second-generation defined vaccines. Many sec-

From the Bakur Medical Laboratory, Tehran, Iran; Center for Research and Training in Skin Diseases and Leprosy, Tehran, Iran; Pasteur Institute of Iran, rchran, lran; Razi State Vaccine 6 Serum Institute, Tehran, Iran; World Health Organization, Special Programme for Research and Training in Tropical Diseases (TDR), Centwa, Switzerland. Address correspondence to K. Bahar, Ph.D. cand.; Bakar Medical Laboratory, No. 249 Kayar Skomali, Tehran 14146, Iran.

0 1996 by K. Bahor et ai. Azw~u~ of the Amwicas,

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York, NY 10010

ond-generation candidate vaccines have been identified, but it will be several years before they can be tested in humans due to requirements for their independent and collective testing towards composing a cocktail vaccine, production under good manufacturing practices (GMP) and toxicity tests. Since 1991, we have taken a systematic and stepwise approach for determining safety, acceptabiiity, and immunogenicity of different doses of killed t. major mixed with different amounts of BCG. These materials could be used directly in human trials as they were in use separately for decades. By necessity we started with a very low dose of killed L. major and a full dose of BCG and gradually decreased the dose of BCG and increased the dose of killed Leishrnania (see Dowlati et al. in this issue). In the present study, we report the final step in the safety and immunogenicity of two vaccines-killed either by autoclave (ALM), or thimerosal plus freezethawing (KLM) in preparation for field efficacy trial. Some preliminary studies on this type of vaccine have been presented earlier.6-8

Materials

and Methods

Vaccines Preparation of a similarly produced killed Leishmania major vaccine (KLM) has been reported previously.’ The KLM used in this study was prepared with a slight modification, as noted below. One large batch was grown for this study and subsequent ongoing efficacy trials. Both ALM and KLM were prepared from this batch. Briefly, promastigotes of L. mnjor (MRHO/IR/ 76/ER, vaccine) from a seed bank at Razi lnstitute were grown in volumes of 50-200 mL in RPM1 (Gibco, Grand Island, NY, USA) with 15% fetal calf serum (Sigma, St. Louis, MO, USA) at 25”C, in roux bottles. Fresh medium was added gradually to reach 200 mL on different days. Parasites were harvested at stationary phase (assessed

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by daily enumeration) on day 16-20 by centrifugation at 3200 rpm for 30 min. Promastigotes were washed five times with pyrogen-free phosphate-buffered saline, pH 7.0-7.2 (PBS) and stored at -70°C. Ten to 12 harvests were pooled to constitute a batch used in these studies. In previous studies (see references 7 and 8, and Dowlati et al. this issue), culture promastigotes were treated with thimerosal (1:5000 v/w) prior to centrifugation in order to kill the parasite and facilitate separation by centrifugation. However, it was noted that protein content of the harvest was drastically reduced in relation to cell count, indicating that thimerosal pretreatment may have caused leakage of proteins during washing. In the present study, live promastigotes were centrifuged at 4°C without thimerosal treatment and harvested immediately. Harvested promastigotes were resuspended in pyrogen-free PBS and stored at -70°C until used. Samples from each harvest and the final lot were assayed for sterility, cell count, and protein concentration. The lot was diluted to desired concentrations (44.44 mg/mL or 11.11 mg/mL) with PBS and divided into two samples. One was treated with thimerosal (1: 10,000) and freeze-thawed five times and kept at -70°C until used and designated KLM. The other sample (ALM) was dispersed into small vials, autoclaved for 15 min at 121”C, (15 PSI) and kept at 4°C. Several methods for protein determination were tried’@” with varying success due to interference by thimerosal or the particulate nature of the vaccines. Finally the method of Lowry et al.‘* was used. Two doses of ALM or KLM were used in this trial, either alone or with a fixed dose of BCG (see below). These were 960 kg protein (low KLM or low ALM) and 3.75 mg protein (high KLM or high ALM).

BCG BCG was kindly donated by the Pasteur Institute, Tehran, Iran. Just prior to use, BCG was reconstituted in its diluent and added to the vaccine to produce a final Table 1. Number of Volunteers

1 2 3 4 5 6 7 8 9 10 Total

BCG* Saline 0.96 mg 3.75 mg 0.96 mg 3.75 mg 0.96 mg 3.75 mg 0.96 mg 3.75 mg

Leishmanin The TDR/WHO reference leishmanin produced by the Pasteur Institute, Tehran, was used throughout.r3 The same isolate of L. rnuio~used for vaccine was employed for its production. Intradermal injections were made with 0.1 mL and the reactions were measured 48-72 h later using the ball point pen method of Sokal.i4 The magnitude of induration was marked by a pen on the skin, which was then transferred to a paper using an adhesive tape.

Purified

Protein

Derivative

(PPD)

The Pasteur Institute of Iran provided the PPD used in these studies. Each volunteer was skin tested with 5 units of PPD on the right forearm and the results were read 24-72 h later as in leishmanin skin test.

Volunteers A seminar was given on the scope of the project and the program of vaccine development in the country by a member of the team (Y.D.) to the staff and workers of an industrial plant and the general audience. Volunteers were sought to participate in a medical and laboratory examination and skin testing with PPD and leishmanin. Following the examination and the results of the tests, those who met the inclusion criteria were then interviewed individually and invited to participate in the program. Volunteers either signed a statement or gave their consent orally. The number of volunteers at each time point and the vaccine composition of each group is shown in Table 1.

and Vaccine Groups (n)

Vaccine

Day 0

Day 42

Day 135

ALM ALM KLM KLM ALM ALM KLM KLM

16 15 16 18 18 15 12 14 11 11

14 13 11 12 13 13 9 11 6 6

14 15 10 8 13 11 5 10 6 7

146

98

69

+ + + +

BCG* BCG* BCG* BCG*

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dilution of 1:lO containing 5 x lo5 colony forming units (CFU)/mL. The BCG given to the control group also contained the same number of organisms. A volume of 0.1 mL was injected intradermally containing 5 x lo4 CFU, equal to a tenth of the normal BCG dose given as a vaccine against tuberculosis.

Volunteers Group

l

*The dose of BCG for all groups was 5 x 104 CFU. This represents 10-l of the dose normally used for vaccination against tuberculosis.

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Protocol The outline of the protocol is shown in Table 2. This was a double-blind randomized placebo controlled trial. The study was repeatedly monitored by TDR consultants throughout the study. Based on clinical examination, laboratory and skin tests, interview and consent, the volunteers were given sequential numbers on the day of vaccination and assigned to different groups (treatments), which were randomized in advance. Clinical evaluation included physical exam for the presence of any regional lymphadenopathy in axillary and cervical areas. Local erythema, induration, edema, ulcer, and necrosis were periodically measured. The extent of reepithelialization and scar formation were recorded as an indication of the healing process at the site of injection. The presence of pain and/or itch was reported by volunteers as a subjective parameter. All parameters were recorded for assessment of safety of the preparation. The site of vaccine injection was photographed on several occasions.

Vaccination Each volunteer received 0.1 mL of injection containing vaccine alone, vaccine mixed with BCG, BCG alone, or PBS intradermally at the base of the left deltoid.

Routine Laboratory Tests for Inclusion Assessment of Safety

and

Blood samples were collected for CBC, ESR, random blood sugar, blood urea, bilirubin, transaminases (ALT and AST), and alkaline phosphates. All CBC were done using Baker-9000 and biochemical tests including blood sugar, urea, creatinine, transaminases, and alkaline phosphatase (AIK I’) was measured by Ciba-Corning Express model 500. Diagnostic kits produced either by Trace or Bahar Medical Laboratories, Tehran, were used for measurements of enzymes. All tests were done usTable 2. Protocol for Vaccine Evaluation Activity

Day

-50 to -10 0 1 3 7 14 28 42-50 45-53 56

Clinical exam and collection of specimens for lab tests*; LSTt and PPDt; evaluation of results, interview and selection of volunteers Vaccination Clinical exam and questionnaire Clinical exam and questionnaire Clinical exam and questionnaire Clinical exam and questionnaire Clinical exam and questionnaire Clinical exam and collection of specimens*; LSTt and questionnaire Reading of LST reactions Clinical exam and questionnaire

* Specimens inch&i blood, urine and stool. f LST = leishmanir skin test

OF TWO

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ing standard medical laboratory techniques under good laboratory practices (GLP). Urine was tested for chemical and cellular elements, especially for hematuria and proteinuria. The acceptable ranges for each test were defined beforehand on the basis of manufacturers’ recommendations Or those determined by Bahar Laboratories, Tehran, Over the years 1991-1994. Exclusion limits were established prior to the initiation of the trial for all tests, as shown in Table 3.

Inclusion

Criteria

1. Induration >5 mm and ~20 mm to IT’D f5 units). 2. Negative reaction to leishmanin; any observable reaction was considered as positive. 3. Without history of major surgery and in good physical condition. 4. Age 16-60 years. 5. Consent to participate.

Exclusion

Criteria

1. Chronic or concomitant diseases. 2. Laboratory tests beyond acceptable normal range. 3. Participation in any other trial during this study. In taking history on chronic and acute infections, particular attention was given to malaria, hypersensitivity, hepatitis, major operations, cardiac problems, and malignancies.

Safety Evaluation The following parameters were recorded: pain, itch, lymphadenopathy, erythema, induration, and duration of healing process of the injection site. Definitions for safety and limits of acceptable or unacceptable reactions were determined in advance of vaccinations. The physicians were not aware of the background of the vaccination during examination and did not have access to the form of previous visit. The following parameters were recorded either by asking the volunteers (degree and duration of itching and pain) or by examination (lymphadenopathy, erythema, induration, and extent of reepithelialization). The extent of side effects were defined as shown in Table 4. Table 3. Limits for exclusion based on laboratory tests Test Total WBC Hct ESR Random blood sugar Urea Total bilirubin ALT and AST Alk Phosphatase Intestinal parasite

Excluded

---I_<2,000 or >1 Z.Ot?(! <33 >50 >180 >60 >1.7 >lOO >7 Entumoebu histolyticu,hookworms

Unit /mm” % mm mg/dL mg/dL

mg/dL U/mL LJ/mL? + or -

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Table 4. Definitions

0 1 2 3

Cytokine

Defined As No discomfort at all Discomfort at the site of injection, not requiring analgesic Discomfort not allowing easy movement of an arm Discomfort preventing normal work

Production

In Vitro

Sterile, heparinized peripheral blood was drawn on days 0,42, and 135 after immunizations from all available volunteers. Lymphocytes were separated by FicollHypaque gradient centrifugation, washed three times with RPM1 1640, adjusted to 2 x 106/mL with the same medium supplemented with 15% fetal calf serum, and cultured for four to five days at 37°C with 5% CO,. Triplicate cultures received either soluble antigen of L. major 20 kg/mL (kindly provided by Dr. D. Sacks, NIH, Bethesda, MD, USA), phytohemagglutinin, @‘HA) 5 Fg/mL (Sigma), or the medium as control. Supernates were collected and assayed for IFN-y and IL-5 by ELISA at Bahar Medical Laboratories, Tehran. The results obtained using kits from Genzyme or monoclonal antibody to IFN-7, kindly provided by DNAX through TDR, were comparable.

Evaluation

of Antibody

EM-INFO (Versions 5.1 initially and 6.02 later) were used for production of all questionnaires, data entry and analyses. Data were double entered and verified.

Results Volunteers The age and sex distribution of the 165 volunteers are shown in Table 5. The mean age was 35 years and 94% of the volunteers were male. There was no significant difference in age and sex distribution of different groups.

Safety Laboratovy Tests The results of initial laboratory tests and those on day 42 were compared. There was no severe reaction in any group at any time. Only a slight increase in transaminase (ALT and AST) enzymes was observed in some volunteers; however, the increase was not statistically significant in any group and they did not exceed the preset limits. There was no change in hematological parameters or urine analysis.

The presence of specific antibody against L&&mania was evaluated by indirect immunofluorescence test (IF) and ELISA. For ELISA, the same soluble antigen used for cell stimulation in lymphocyte culture was employed to coat microtiter plates. Briefly, microtiter plates (Nunc, Denmark) were coated with 0.5 kg/well of Leishmania-soluble antigen in 15 mM PBS pH 7.3. Plates were incubated overnight at 4°C. Free sites were blocked by 100 PL of 3% skim milk in PBS with 0.05% Tween 20. Wells were washed and received 50 PL of 1:lOO dilution of test sera. Plates were incubated at 37°C for 2 h. After three washes, 50 PL biotinylated antihuman Igs (Gibco) in 1:lOOOwere added to each well. Plates were incubated for 1 h at 37”C, washed, and then 50 FL peroxidase-labeled streptavidin (1:lOOO) was added to each well. One hour later plates were washed, chromogen was added, and the reaction stopped by 2 N HCl and read at 450 nm within 30 min (TMB, KPL Inc.). of

Clinical Examination The results of physical examination and measurement of reactions at the site of injection indicated that all side effects were associated with injection of BCG, and addition of killed Leishmaniato BCG did not increase the reaction caused by BCG alone. The extent of pain (and itching) for each day of observation is defined by degree of pain or itching (a numerical designation, see Materials and Methods). In no case were there any severe reactions (degree 3) or one that required treatment. All side effects, except for itching, were associated with

Volunteers

Age (yr)

Male Female Total

15-20

20-30

1 2 3

32 3 35

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Data Analysis

Production

Table 5. Age and Sex Distribution

l

intact promastigotes of Leishmania were used as antigen for IF. Serial twofold dilutions of serum starting with 1:5 were added to each antigen plaque and incubated for 30 min in a humid chamber at 37°C. Slides were washed three times with 15 mM PBS, pH 7.3. Fluorescein-labeled anti-human Igs at optimal dilutions (determined previously) were added to each antigen. Thirty minutes later, slides were taken from humid chamber, washed, and mounted by buffered glycerol pH 7.2. Each slide was evaluated under florescence microscope by two different individuals.

of Degrees of Side Effects

Degree of Reaction

irz Dermatology

3040 79 3 82

40-50

50-59

Total

28 2 30

15 0 15

155 10 165

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BCG. Some minor itching was reported in those receiving the high dose of vaccines, both KLM and ALM. Pain and itching on day 3 are shown as an example in Table 6. All vaccinated volunteers (146) were examined on day 3. Degree 1 is defined as discomfort at the site of injection not requiring analgesic. Degree 2 itching is defined as discomfort interfering with normal use of the arm. The erythematous reactions at the site of injection on day 3 after vaccination are shown in Table 7. Again, much of the reaction is associated with BCG vaccination and not with antigens alone.

Leishmanin Skin Test The results of leishmanin skin test (LST) on days 42 and 135 are shown in Table 8. It is of interest to note that, in general, addition of BCG to ALM or KLM enhances the induction of delayed type hypersensitivity (DTH) to leishmanin. Volunteers who had LST reactions <3 mm in groups 1, 3, 4, 5, and 6 on day 135 were given a booster injection with ALM + BCG. They were skin tested 52 days after the booster injection and the results are also shown in Table 8. A significant increase in LST was seen in groups 3, 4, and 5. Cytokine Production Figure 1 shows the amount of EN-y on days 42 and 135 after injection. For a few volunteers IFN-7 was determined at day 0, prior to vaccination and the results were negligible. No correlation was seen between LST and IFN-1, production, but strong responders were positive for IFN-y. The magnitude of EN-?/ in vaccinated LST individuals was below that of recovered patients whose EN--y response ranged from 780 to 2700 pg (mean 1800).

Discussion The combination of killed L. major (either ALM or KLM) at the highest practicable concentrations (3.9 mg) alone or mixed with BCG produced no significant side effects above those associated with BCG alone. No changes in hematological, biochemical, and renal function tests were noted between day 0 (prior to vaccination) and

C)F TWO

KILLED

LEISHMANlA

MAjOiZ

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Table 6. Pain and Itch Three Days after Vaccinntm Pain degree

Group 1. BCG 2. PBS 3. Low 4. High 5. Low 6. High 7. Low 8. High 9. Low IO. High

in)

Itch degree i ii?

I (4) None I (3) 1 (2) I (4 1 (2) None NOM None I (I)

ALM + BCG ALM + BCG KLM + BCG KLM + BCG ALM ALM KLM KLM

(11)

Yom'

I tli! 111).2(1) I 19) : (7) ! :21 ? ihi

: (1) : i0)

two months after immunization. All parameters checked, e.g., erythema, induration, edema, ulceration, necrosis, pain, itch, lymphadenopathy (cervical and axillary), reepithelialization, and scar formation were indistinguishable between the groups that received the mixed vaccine and the controls receiving BCG alone. The combination of BCG plus ALM or KLM at both doses tested produced a measurable immunological response in about 45-50% of volunteers on days 45 or 135. This is true if IFN-?, and skin test conversion are taken collectively. There is no correlation between the presence or magnitude of these responses, except the high responders for LST were positive for IFN-)I. In general the magnitude of these immune responses produced following vaccination-the indicators of cellular immunity-was lower than those produced by an individual who has recovered from leishmaniasis and presumed protected. Nevertheless, this may be sufficient to prevent the disease, if not the infection. There is no way of knowing whether the response induced by vaccination is protective except by an efficacy trial which is now underway. The responses produced by ALM and KLM were comparable. However, maintenance of ALM is by far simpler and its delivery more convenient as the autodegradation seen with KLM and leishrnanin” has not been observed with ALM. Based on these considerations, ALM is favored as a vaccine and is being tested in field efficacy trials. As the responses of the groups receiving low or high doses of ALM or KLM were not significantly different and considering that low doses of antigen are more

Table 7. Erythema at Site of Injection on Day3 Erythema

___0

I-5 h-10 II-20

21-40 Mean Total -__

BCG 1

Saline 15

ALM

Low + BCG

ALM

High + BCG

KLM

Low + BCG

High + BCG

Low ALM

High ALM

_____.. WI0

Low

High

K 1.M

K1.M

.----_-__-0.00

0.00

0.00

0.00

0.00

3

5 3 9

4 4 8

0.00 7 7

8 I 0.00

LI 5 I1.W

i).iNl

t

1

I 13.0

0.00 2.3

(I.00

il.lMl

O.l)O

I5

2 2

0.00 0.00

5 2

11 0.00 13.3 I6

0.00 0.00 0.00

8 7 15.0

12.9

2 15.6

16

18

I8

15

KLM

12

3.h

l-l

I a,!

1 2 7

LX

II

PLii

II

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Y-IFN (pg/ml ) 1000

800

600

B

S

B+AL

B+AH B+KL B+KH

Figure

1.

IFN--y

likely to produce a Thl-type response,16 we recommended that low dose ALM (about 1.0 mg) be used for efficacy trials. This reduces the cost of the vaccine as compared to the higher doses tested here. The KLM used here was produced similarly to the vaccine used by Mayrink et a1.4,17with New World Leishmtznia.In their studies BCG was not used and three injections of the vaccine produced skin-test conversion in more than 75%. Here, only the first step was tested and clearly the second injection of ALM + BCG caused skin-test conversion in most nonresponders to the first vaccination. Assured by lack of untoward side effects and given the magnitude of leishmaniasis problem in the region, this vaccine is now being tested in a multicenter trial against leishmaniasis as the first step. The results are expected in less than two years. Depending on the results, one or more booster injections may be envisaged in future trials.

production

1. BCG 2. PBS 3. Low 4. High 5. Low 6. High 7. Low 8. High 9. Low 10. High Total

ALM + BCG KLM + BCG KLM + BCG KLM +BCG ALM ALM KLM KLM

ND = not done. *Values in parentheses t Values in parentheses

Day 42* 1.84 (2/13) 0.00 2.0 (5/13) 2.83 (7/13) 1.50 (5/16) 1.81 (3/11) 1.28 0.05 1.50 0.57 22/t%

AH

in vitro.

KL

KH

Acknowledgment We are grateful to Mrs. F. Asadiyoon, Bahar Medical Laboratory, Tehran, Iran; Dr. H. Darabi, Pasteur Institute of Zran, Tehran, Iran, for cell cultures; Miss Sh. Ehsasi,Bahar Medical Laboratory, Tehran, Iran, for local coordination; and Mrs. T. Kuo, World Health Organization, TDR, Geneva, Switzerland, for her redaction of the manuscript in its final f arm. The authors would like to acknowledgethe cooperation of all volunteers and local authorities in Ghani-Abad factory for their kind cooperation and patience during the courseof the study; Dr. D. Sacks,National Institutes of Health, Bethesda, MD, USA, for his assistance,support and guidance; Dr. F. Zicker, World Health Organization/Regional Ofices for the Americas/Pan American Sanitay Bureau, Washington, DC, USA, for his input in the designof the study and its analysis, without whom this trial could not have beendone. This work received financial support from UNDP/World

Table 8. Mean LST on Days 42 and 135 after Primary Iniection of Selected Volunteers Group

AL

I

Vaccination

and on Day 52 after Booster 52 days after boostert

Day 235* 1.21 (2/14) 1.21 (O/15) 3.34 (9/15) 2.26 (5/14) 2.76 (7/14) 2.53 (6/13) 1.50 0.00 1.28 1.70 29/85

are number with LST a3 mm/number tested. indicate only those with LST <3 mm on day 135 received

the booster

1.87 (2/B) ND 3.50 (l/2) 3.90 (5/6) 5.0 (5/7) 2.37 (3/6) ND ND ND ND 16/29

injection.

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Bank/ WHO Special Programme for Research and Training in Tropical Diseases (TDR). References 1.

2.

3.

4.

5.

6.

7.

Rachamim N, Jaffe CL. Pure protein from L. donovani protects mice against both cutaneous and visceral leishmaniasis. J Immunol 1993;150:2322-31. Connell ND, Medina-Acosta E, McMaster RW, et al. Effective immunization against cutaneous leishmaniasis with recombinant bacilli Calmette-Guerin expressing the Leishmaniu surface protease gp63. Proc Nat1 Acad Sci USA 1993;90:11473-7. Tropical Disease Research: Twelfth programme report of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). Geneva: WHO, 1995:13546. Antunes CMF, Mayrink W, Magalhaes PA, et al. Controlled field trials of a vaccine against New World cutaneous leishmaniasis. Int J Epidemiol 1986;15:572-80. Castes M, Trujillo D, Calcagno M, et al. Response Thl-Th2 in human American cutaneous leishmaniasis: Its possible relevance for design of a vaccine. Mem Inst Oswald0 Cruz 1993;88:42-3. Bahar K, Shidani B, Hashemi Fesharki R, et al. Responses and reactions to L. major vaccination. Proceeding of meeting on vaccines against leishmaniasis. Belo Horizonte, University of Minas Gerais and UNDP/World Bank/ WHO Special Program for Research and Training in Tropical Diseases, lo-11 September 1990:20-l. Bahar K, Ehsasi S, Dowlati Y, et al. Overview of human vaccine studies using killed I. major, in session of “Immunology & molecular biology of leishmaniasis”; XIII International Congress for Tropical Medicine & Malaria. Jomtien, Pattaya, Thailand, 29 November4 December 1992.

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8. Bahar K, Ehsasi S, Dowlati Y, et al. Biological activities of Leishmania extracts. XIII International Congress for Tropical Medicine & Malaria. Jomtien, Pattava, Thailand, 29 November4 December 1992. 9. Hashemi-Fesharki R, Ale-Agha S, Ahourai 1’. et al. Vaccine preparation and quality control of killed Leishmnnia major. Arch Inst Razi 1992;42/43:39-50. 10. Bradford M. A rapid and sensitive method for quantisation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:24854. 11. Savory J, Hammond JE. Measurement of proteins in biologic fluids. In: Sonnewirth AC, Jarett I.. editors. Gradwohl’s clinical laboratory method and diagnosis, 8th ed. St. Louis: Mosby, 1990:258-9. 12. Lowry OH, Rosebrough NJ, Farr AL, et (31. Protein measurement with Folin phenol reagent. J Biol Chem 1951; 193:265-75. 13. Alimohammadian MS, Hakimi H, Nikseresht M. The preparation and evaluation of reference ieishmanin from Leishmanin major for use in man for diagnostic and experimental purposes. MJIRI 1993;7:23-8. 14. Sokal JE. Measurement of delayed skin-ti-Lst responses. N Engl J Med 1975;293:501-2. 15. Badaro R, Pedral-Sampaio D, Johnson WD, et al. Evaluation of the stability of a soluble intradermal skin test antigen preparation in American visceral lcishmaniasis. Trans R Sot Trop Med Hyg 1990;84:226--1. of 16. Bretcher PA, Wei G, Menon JN, et al. Establishment stable, cell-mediated immunity that makes ‘susceptible’ mice resistant to Leishmania major. Science i992;257:53942. 17. Mayrink W, Williams I-‘, Da Costa CA, t+ al. An experimental vaccine against American dermal leishmaniasis: Experience in the State of Espirito Santo, Brazil. Ann Trop Med Parasitol 1985;79:259-69.