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. 2003 Nov;134(2):303–308. doi: 10.1046/j.1365-2249.2003.02299.x

Immune response measured in human volunteers vaccinated with autoclaved Leishmania major vaccine mixed with low dose of BCG

M MAHMOODI *,†, A KHAMESIPOUR *, Y DOWLATI *, S RAFATI ‡, A Z MOMENI §, M EMAMJOMEH §, H HEJAZI §, F MODABBER
PMCID: PMC1808866  PMID: 14616791

Abstract

The immune responses induced against Leishmania antigens in volunteers who were vaccinated in a double-blind, randomized field efficacy trial of a preparation of autoclaved Leishmania major (ALM) mixed with a low dose of Bacille Calmette–Guerin vaccine (BCG) who developed either a cutaneous leishmaniasis (CL) lesion due to exposure to infected sandfly bite(s) or did not develop a lesion during the course of the trial were studied and compared with those of non-vaccinated controls. Blood samples were also assayed from different groups including volunteers with history of CL and volunteers with previous positive or negative leishmanin skin test (LST) without a history of CL. The vaccinated volunteers had received a single dose of either ALM mixed with a low dose of BCG or the same dose of BCG alone. The LST and in vitro proliferative response (stimulation index, SI), interferon gamma (IFN-γ) production and, in a few cases, interleukin (IL)-4 production of peripheral blood mononuclear cells to soluble Leishmania antigens were measured. The results indicated that volunteers who developed CL in the vaccine arm showed a slightly higher SI than cases who received BCG alone. Volunteers with history of CL and volunteers with positive LST demonstrated the strongest proliferation indices and IFN-γ production. The data suggest that a single dose of ALM + BCG induces a weak Th1 response in vaccinated volunteers that is far lower than that in volunteers with prior subclinical infection or volunteers with history of CL, who are presumed to be immune.

Keywords: interferon gamma, Leishmania vaccine, proliferation response

INTRODUCTION

Leishmaniases are vector-borne zoonotic or anthroponotic parasitic diseases with many distinctive manifestations, ranging from a self-healing cutaneous leishmaniasis to a lethal visceral form. Vector and reservoir control are not always possible or practical in the case of zoonotic diseases, or require infrastructure beyond the means of the affected population. Even if successful, these measures are not maintained because of cost [1–3]. The first-line drug (antimonials) is toxic, costly and requires a prolonged series of daily injections, and resistance is developing rapidly in many countries [4]. Leishmania infection usually induces a life-long immunological memory and protection against further infections [5]. In experimental murine models of leishmaniasis, control of infection requires generation of Th1 response represented by production of interferon (IFN)-γ in the absence of Th2 response, which is associated with interleukin (IL)-4 or IL-10 [6,7]. Most Leishmania are easily cultured, hence, unlike many other parasites, production of vaccine using the parasite or its components is feasible. For all these reasons, first-generation (killed parasite) vaccine development for leishmaniasis has received considerable attention [8]. Clinical trials have been conducted in Brazil, Colombia, Ecuador, Iran and Sudan [9–11]. Randomized double-blind trials were carried out in Iran to evaluate the safety, immunogenicity and efficacy of autoclaved Leishmania major vaccine (produced by the Razi Institute, Iran) mixed with Bacille Calmette–Guerin vaccine (BCG) [9–11]. The efficacy trial of a single dose of the combined autoclaved Leishmania major (ALM) + BCG vaccine compared with BCG alone against zoonotic cutaneous leishmaniasis (ZCL) in Iran was conducted in a high-risk population coming to live in a hyperendemic focus [11]. A total of 2453 volunteers participated in the trial. Because evaluation of the immune responses of all participants in this type of clinical trial is beyond the funds and infrastructure available, it was decided to sample consenting adults with parasitologically confirmed cutaneous leishmaniasis (CL) as soon as they were diagnosed during the follow-up period of active case detection. In addition to these patients, consenting adults who had not developed a lesion and new as well as recovered cases that were not part of the trial but who agreed to participate were also tested. By comparing these groups, we hoped to find out if vaccination had induced sensitization to Leishmania antigens, so that upon infection a more rapid Th1 response would be triggered.

MATERIALS AND METHODS

Study design

The present study was part of a large randomized double-blind and BCG-controlled field efficacy trial [11], performed north-east of Isfahan where the incidence rate of ZCL had been consistently high (4–6% per year in 5 previous years). The population is new to the endemic area because this is the residential area of a military base; the residents stay there for only a few years. Briefly, a tota1 of 2310 healthy volunteers, who had no leishmanin skin test (LST) reaction, met the inclusion/exclusion criteria, were vaccinated randomly, either with ALM + BCG or with a 10th of the normal dose of BCG alone. The protocol had received clearance from the Ethical Board of the Institute, the Ethical Committee of the Iranian Ministry of Health, the Secretariat Committee on Research Involving Human Subjects, WHO, Geneva.

Vaccine

The ALM vaccine was produced under the guidelines of good manufacturing practices at Razi Serum Vaccine Institute, Hessarak, Iran [12]. Based on previous safety and dose-finding studies [10], each injection of 0·1 ml given intradermally at the left deltoid consisted of either 1 mg of whole-cell autoclave-killed Leishmania major (ALM) protein mixed with roughly 5 × 104 colony forming units (CFU) of BCG (1/10 of normal dose of BCG used for vaccination against tuberculosis) or the same dose of BCG alone [9,10]. BCG was freshly resuspended and mixed with ALM just prior to injection. The TDR/WHO reference leishmanin, produced by the Pasteur Institute, Tehran [13], was used in this trial to perform LST. All the assays reported here were completed and data locked before the codes for the vaccine trail became available for the present study.

Selection of participants

A case was defined only if smear or culture were positive for Leishmania parasites. Blood samples were taken from the volunteers, at least 17 years of age, who were willing to participate, sign the informed consent and donate blood. A total of 117 blood samples were collected during the 2-year follow-up. As the immune response evaluation of all participants in this type of vaccine efficacy trial is impossible, volunteers in different groups were not selected randomly. During the follow-up of the vaccine efficacy trial [11], whenever a case was detected we asked for his/her consent and if given then a blood sample was collected and processed. Usually those accompanying the case also agreed and donated blood; the selection may introduce unintentional bias in the results, but there are no apparent differences between these and all other patients to indicate a bias.

The following groups of volunteers were selected: (1) n = 21 volunteers who were injected with ALM + BCG and developed CL later due to natural exposure (transmitted by infected sandflies); (2) n = 19 volunteers who were injected with BCG alone and developed CL later due to natural exposure; (3) n = 18 volunteers who received ALM + BCG and did not develop any Leishmania lesion; (4) n = 14 volunteers who were injected with BCG alone and did not develop CL; (5) n = 7 volunteers from the non-injected group who developed CL due to natural exposure; (6) n = 7 volunteers with active CL from a group of volunteers who were excluded from the trial due to a positive LST; (7) n = 17 volunteers with chronic CL described as non-healing patients: these volunteers were resistant to multiple courses of antimonial therapy (lesion(s) lasting from 1 to 20 years); and (8) n = 9 volunteers with a history of CL who were cured from the disease and described as volunteers with history of CL.

Isolation of peripheral blood mononuclear cells

Heparinized peripheral blood samples (12–20 ml) were obtained from each volunteer at an early stage of diagnosis (2–9 weeks after development of lesion(s) as indicated by the volunteers), peripheral blood mononuclear cells (PBMCs) were purified by centrifugation over Ficoll-Hypaque gradients (Histopaque 1077, Sigma Chemical Corp., St Louis, MO, USA), similar to the method of Boyum [14]. PBMCs were stored in freezing media (mixture of 80% fetal calf serum (FCS), 12% RPMI-1640 and 8% dimethyl sulphoxide) and kept frozen in liquid nitrogen.

In vitro lymphocyte proliferation assay

The PBMCs were recovered from liquid nitrogen, thawed rapidly and washed with RPMI-1640 (Sigma). The viability of mononuclear cells was examined by trypan blue exclusion test. The cells were resuspended in complete culture medium (RPMI-1640 supplemented with 5% heat-inactivated, type AB positive, human serum, 1%l-glutamine, 100 IU/ml of penicillin and 100 µg/ml streptomycin). The cells were cultured in U-bottomed 96-well plates (Costar, Cambridge, MA, USA), using 2 × 105 cells in 200 µl volume per well in triplicate. The cells were either cultured alone or stimulated with phytohaemagglutinin (PHA; Sigma), 10 µg/well or 25 µg/well of soluble L. major antigen (SLA, gift of Dr David L. Sacks, National Institutes of Health), and was prepared by freeze-thawed, whole-cell lysate of promastigotes in stationary phase. The cells were incubated in a humidified atmosphere at 37°C and 5% CO2 for 5 days and pulsed with 1·0 µCi [3H]-thymidine (Amersham, Aylesbury, UK) per well during the last 18 h of culture. Cells were harvested onto glass fibre filters (Whatman, Reeve Angel, Clifton, NJ, USA). [3H]-thymidine incorporation was determined by liquid scintillation counting. Proliferation response is expressed as the stimulation index (SI), which represents the ratio of mean counts per minute in stimulated cells in triplicate wells to the mean counts per minute in unstimulated cells in control wells.

Cytokine assay

Culture supernatant fluids were collected at 4 days post-stimulation for both antigen-treated and PHA-treated wells and controls. Supernatants were assayed for IFN-γ using a double-sandwich ELISA technique according to the procedure of Curry et al. [15]. The IFN-γ concentration was determined by comparison to a human recombinant IFN-γ standard (Sigma); the detection range of this procedure was 20 pg/ml to 2 ng/ml. IL-4 was measured by a competitive enzyme immunoassay (EIA) method (Chemoicone Inc., CA, USA), following the manufacturer's instructions. The assay was calibrated with IL-4 standard to detect this cytokine within the range of 100–2000 pg/ml.

Statistical analysis

Statistical analysis was performed using SPSS version 9. The magnitude of the responses in different subject groups was compared by the Mann–Whitney U-test and Wilcoxon's rank test. Pearson's correlation coefficient (r) was used to determine whether significant correlations existed between the different response parameters. P < 0·05 was considered significant.

RESULTS

Lymphocyte proliferation response and cytokine production by stimulated PBMCs of injected (ALM + BCG/BCG) cases

In vivo LST response and in vitro lymphocyte proliferation response and IFN-γ production of SLA-stimulated PBMCs of active CL cases who had received ALM + BCG (Group 1) and the control groups (Groups 2 and 5) are summarized in Table 1. The mean proliferative responses to SLA were significantly higher (P < 0·05) in Group 1, who received ALM + BCG (mean ± s.d., 3·77 ± 1·96) compared to Group 2 (BCG-injected cases) or non-injected cases (Group 5), or those vaccinated who did not develop any lesion (Group 3) (2·39 ± 3·58). IFN-γ production from the cases who received ALM + BCG (Group 1) was not significantly higher compared to the control groups; BCG-injected cases (Group 2) or non-injected cases (Group 5). The results of LST on days 80 and 360 showed a small increase in diameter of indurations in ALM + BCG injected in Group 1 compared to BCG-injected CL cases in Group 2 (2·38 ± 2·46 versus 1·23 ± 1·98 at day 80 and 6·73 ± 4·98 versus 5·92 ± 5·14 at day 360) and this was not statistically significant (Table 1). No IL-4 was detectable in any of the culture supernatants except from cells of non-healing patients (Group 7). No correlation was found between proliferation response of ALM + BCG-injected cases (Group 1) with the magnitude of LST conversion at day 80 or 1 year post-injection. In vitro stimulation of PBMCs by SLA indicated that there was no significant difference (P = 0·08) between the proliferation response of volunteers who were injected with ALM + BCG but did not develop any lesion (Group 3; 2·39 ± 3·58) and BCG-injected healthy volunteers (Group 4; 2·13 ± 1·56).

Table 1.

Stimulation indices (SI) of in vitro lymphocyte proliferation response and IFN-γ production of stimulated peripheral blood mononuclear cells with soluble Leishmania antigen (SLA) or mitogen (PHA) from injected (ALM + BCG/BCG) cases, non-injected cases and volunteers with history of cutaneous leishmaniasis (CL) and the magnitude of leishmanin skin test (LST) reaction (mm) at day 80 and 1 year post-injection

Groups Types of vaccine injected Age ranges (years) LST induration mean (mm) at day 80 post-injection LST induration mean (mm) at 1 year post-injection SIPHA SISLA IFN-γ (pg/ml)
1 ALM + BCG(21)* 18–45 2·38 ± 2·46 6·73 ± 4·98  85·22 ± 115·18 3·77 ± 1·96   76·6 ± 9·20
2 BCG(19) 18–44 1·23 ± 1·98 5·92 ± 5·14  76·46 ± 82·8 2·72 ± 1·81  30·66 ± 53·11
3 ALM + BCG(18) 18–47 2·97 ± 2·73 5·03 ± 4·64  46·25 ± 11·87 2·39 ± 3·58  113·8 ± 155·9
4 BCG(14) 18–47 2·04 ± 3·79 4·85 ± 3·33  73·08 ± 89·12 2·13 ± 1·56    6·5 ± 18·3
5 (7) 18–46 n.d. 4·37 ± 2·68  62·02 ± 70·55 2·15 ± 2·14     20 ± 34·6
6 (7) 18–48 n.d. n.d.  73·04 ± 64·62 9·17 ± 2·79 350·50 ± 288·51
7 (17) 33–60 n.d. n.d.  42·06 ± 26·17 1·98 ± 1·30   19·3 ± 51·3
8 (9) 18–55 n.d. n.d. 114·96 ± 172·66 9·26 ± 3·51 573·33 ± 323·89
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Group 1: injected with ALM + BCG and later on developed Leishmania lesion(s) due to natural infection. Group 2: injected with BCG and developed Leishmania lesion(s) later due to natural infection. Group 3: injected with ALM + BCG and did not develop any Leishmania lesion. Group 4: injected with BCG and did not develop any Leishmania lesion. Group 5: non-injected volunteers who developed Leishmania lesion(s). Group 6: volunteers with active Leishmania lesion(s) and with previous positive LST reaction. Group 7: volunteers with chronic Leishmania lesion(s). Group 8: volunteers with history of cutaneous leishmaniasis.

*

Number of volunteers; n.d.: not determined.

Proliferative responses and cytokine production (IFN-γ and IL-4) of PBMCs stimulated with SLA from leishmanin-positive cases, non-healing patients of CL and volunteers with history of the disease

The individuals who were excluded from the vaccination trial due to reaction to leishmanin (mean ± s.d., 4·36 ± 2·32 mm) on screening day (Group 6) were also followed in the trial. This group, who developed CL lesion(s), showed significantly higher stimulation indices (mean ± s.d., 9·17 ± 2·79) compared to any other group except the volunteers with history of CL (Group 8), P < 0·005 compared to non-injected control cases (Group 5) or P < 0·002 compared to vaccine-injected cases (Group 1). IFN-γ production in these LST-positive patients (mean ± s.d., 350·50 ± 288·51 pg/ml) was higher than that observed in the ALM + BCG-injected CL cases (Group 1; P < 0·05). The proliferative responses of 17 patients with non-healing lesions (Group 7) revealed a low stimulation index (SI) (1·98 ± 1·30). PBMCs from volunteers with history of CL (Group 8) exhibited a strong lymphocyte proliferation response (9·26 ± 3·51) (Fig. 1) when stimulated with SLA, and this was significantly higher compared to all other groups (P < 0·005) except LST-positive non-injected cases (Group 6). Absence or low IFN-γ production (ranging from less than 8 to 150 pg/ml) was observed in supernatants of PBMC cultures in response to SLA from non-healing patients (Group 7, Fig. 2), whereas the highest level of IFN-γ was produced by cells from volunteers with history of CL (Group 8), P < 0·01 compared with group 1, which correlated well with the lymphocyte proliferation response (Table 1 and Fig. 2). The measurement of IL-4 in the cultured supernatants of PBMCs from non-healing patients (Group 7) stimulated with SLA showed a relatively high level of this cytokine (mean ± s.d., 1064·3 ± 817·6 pg/ml). No antigen-specific IL-4 was detected in PBMC cultures of volunteers with a history of CL.

Fig. 1.

Fig. 1

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Lymphocyte proliferation response and interferon-gamma production of SLA-stimulated PBMCs from vaccine-injected (Group 1), BCG-injected (Group 2) and volunteers with history of cutaneous lieshmaniasis (Group 8).

Fig. 2.

Fig. 2

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Interferon-gamma and interleukin-4 production of soluble Leishmania antigen-stimulated PBMCs from non-healing patients of cutaneous lieshmaniasis (Group 7) and volunteers with history of the disease (Group 8).

DISCUSSION

The results of this study, although not highly significant, indicate that the CL cases who were injected with ALM + BCG had slightly higher SI and IFN-γ to soluble Leishmania antigen compared to BCG-injected or CL cases from non-injected control groups (Table 1). This is compatible with the low severity of the disease in the vaccinated group reported earlier [11].

ALM + BCG-injected volunteers who did not develop any lesion had lower SIs compared with the ALM + BCG-injected CL cases (2·39 ± 3·58 versus 3·77 ± 1·96, P < 0·01). In an approach to produce an effective vaccine against New World CL, similar results were obtained with a vaccine consisting of killed promastigotes of five American Leishmania strains Leish vaccine 5. The PBMC proliferative responses showed a significant enhancement in vaccinees [16]. In another study, conducted in Brazil [17], killed promastigotes of five or six Leishmania strains (Leish vaccine 5 or Leish vaccine 6), alone or mixed with Corynebacterium parvum as an adjuvant, was used and the results showed that SIs are significantly higher in the vaccinated group compared to the placebo control group. Although the composed Leish vaccine was used with a different adjuvant (Corynebacterium parvum) the results are consistent with the results of the current study, in which BCG was used as an adjuvant. In the current study the level of IFN-γ production was assessed in vitro in PBMC culture as an indication of induction of Th1 response. There was no positive correlation between PBMC proliferation responses of ALM + BCG-injected volunteers (healthy volunteers) with the magnitude of LST conversion at day 80 or 1 year post-injection.

The results indicated that the cases who received ALM + BCG did not produce significantly higher levels of IFN-γ compared to the cells from BCG-injected cases or cells from the non-injected control group. Considering stimulation indices and IFN-γ as markers of immunity, no significant difference was observed between ALM + BCG, BCG alone and non-injected controls, suggesting that the vaccine did not prime Th1 cells, or at the time of exposure to the Leishmania-infected sandfly the prime cells had disappeared.

The results of this efficacy field trial indicated that the injection of a single dose of ALM + BCG does not offer a significant protection against natural infection compared with BCG as control. BCG was used in the control arm of the trial to assure the blindness of the study but it may not have been an ideal control, as evidence exists that BCG alone gives some protection against experimental leishmaniasis in murine models [18,19], improves recovery in patients with American tegumentary leishmaniasis [20] and contains antigens that are cross-reactive with Leishmania [19]. However, injection of this vaccine did induce significantly higher rates of LST conversion (36·2%versus 7·9% for the vaccine and BCG groups, respectively, on day 80, and on day 360 the rates were 33%versus 19%), which was associated with a lower (P < 0·03) incidence of the disease in the vaccine group (35% lower incidence than non-responders) [11].

No IL-4 was detected in the supernatants of SLA-stimulated PBMC culture of ALM + BCG injected cases. IL-4 was measured in order to investigate the possibility of induction of a Th2 response (undesired immune response) by this vaccine, because in experimental models and certain conditions mice given Leishmania antigens produced exacerbating disease when challenged with live L. major [21].

In the original efficacy trial, the candidates who showed any reaction to LST at the time of recruitment were excluded. The LST-positive individuals were excluded because at the time it was believed that positive LST correlates with protection against the disease and moreover LST-positive individuals were excluded due to safety reasons. Follow-up of these individuals showed that cases of CL developed among LST-positive individuals even with the highest magnitude of reaction to leishmanin. The relatively high level of IFN-γ production and strong proliferation response (9·17 ± 2·79) during the active stage of the lesion in this group would probably indicate a prior exposure to the parasite resulting in subclinical infection.

The strongest responses were observed among the volunteers with a history of CL (SI: 9·26 ± 3·51, P < 0·005) (Fig. 1) who experienced a long active infection with L. major. These individuals may still harbour viable organisms, despite clinical cure, and is an indication of the ability of most of these subjects in controlling the infection. These results are consistent with the data obtained by Carvalho and his colleagues [22].

Non-healing cases of CL showed the least proliferation responses against SLA and the lowest IFN-γ production. A relatively high level of IL-4 was detected in SLA-stimulated PBMC culture of these cases compared to that in non-exposed individuals (P = 0·001). In human visceral leishmaniasis, it has been reported that the absence of lymphocyte proliferation and IFN-γ generation are associated with progression of the disease [23]. In the present study, the absence of Th1-like response to Leishmania antigen in these patients would probably explain their inability to control infection.

Overall, the results indicate that the combination of ALM plus BCG produced a measurable immunological response, but the magnitude of these immune responses following vaccination was lower than those produced by volunteers with a history of CL (presumably protected) or the cases that had a previous subclinical CL. This finding points to the fact that a sufficiently powerful Th1 response to confer protection against ZCL was not induced by ALM + BCG vaccine. The administration of multiple vaccine doses and use of alternative adjuvants may induce a Th1 immune response powerful enough to protect against natural infection. Additionally, leishmanization (live challenge) seems to act as a very useful tool for testing Leishmania vaccine.

Acknowledgments

The authors are grateful to Dr David L. Sacks, NIH for his valuable guidance and constant assistant throughout this project; and to Dr A. R. Zohoor (Kerman University of Medical Sciences, Kerman, Iran) for his statistical analysis of this study. This project was supported by UNDP/Word Bank/WHO Special programme for Research and Training in Tropical Diseases (TDR).

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