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Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guérrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers
T R A N S A C T I O N S OF T H E ROYAL SOCIETY OF T R O P I C A L M E D I C I N E A N D H Y G I E N E (2003) 97, 365-368
Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Gu6rrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers A. A. K a m i l 1, E. A. G. Khalil 1, A. M. M u s a l, F. M o d a b b e r ~, M. M. M u k h t a r l, M . E. I b r a h i m 1, E. E. Zijlstra 1., D . S a c k s 3, P . G. S m i t h 4, F. Z i c k e r s a n d A. M. E 1 - H a s s a n ~ 1The Leishmaniasis Research Group, Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan; 2Infectious Diseases Research Institute, Seattle, WA, USA; 3National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Parasitic Diseases, Bethesda, MD, USA; 4London School of Hygiene and Tropical Medicine, Keppel Street, London, UK; 5TDR, World Health Organization, Geneva, Switzerland Abstract In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette-Gu6rrin (BCG) vaccine was shown to be safe, but not superior to B C G alone, in protecting against visceral leishmaniasis. From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alumprecipitated A L M + B C G vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM. Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 ~g, 100 tig, 200 ~g, or 400 pg of leishmanial protein in the alum-precipitated A L M + B C G vaccine mixture. Side effects were minimal for all doses and confined to the site of injection. All volunteers in the 10 ~tg, 100 ~g, and 400 ~g groups had a leishmanin skin test (LST) reaction of/> 5 m m by day 42 and this response was maintained when tested after 90 d. Only 1 volunteer out of 5 in the 200 ~g group had a L S T reaction of/> 5mm by day 42 and the reasons for the different L S T responses in this group are unclear. This is the first time that an alum adjuvant with A L M has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers. On the basis of this study we suggest that 100 ~g of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest D T H reaction following vaccination.
Keywords: visceral leishmaniasis, Leishmania major, alum, bacille Calmette-Gu6rrin, vaccine, Sudan Introduction Visceral leishmaniasis (VL) is a major cause of morbidity and mortality in East Africa and the Indian subcontinent; the annual number of cases is about 500000 (E1-Hassan et al., 1993, 1995; El-Hag et al., 1994; Zijlstra et al., 1994). Co-infection with HIV enhances the risk of the disease (Montalban et al., 1990). The only control measure currently available in the Sudan is case detection and treatment with antimonial drugs, which are expensive, not always available and cannot be self-administered. Vector and reservoir control is difficult due to the elusive nature of the vector and the diversity of the animal reservoir. Subclinical infection and development of a T helper cell type 1 (Thl)-type immune response following successful drug treatment suggests that vaccination may provide a possible method for disease control (Badaro et al., 1986; Sacks et al., 1987; Kurtzhals et al., 1992; Ghalib et al., 1993; Zijlstra et al., 1994). Leishmania spp. are good candidates for vaccine development because they undergo minimal, if any, antigenic variation and there is extensive cross-reactivity between Leishmania strains. We observed that individuals who migrated to eastern Sudan from L. major-endemic areas in the west and who were reactive to leishmanin antigen had a lower risk of developing VL (Zijlstra et al., 1994). Dube (et al., 1998) and Misra et al. (2001) showed that vaccination of primates with 2 - 3 injections of autoclaved L. major (ALM) promastigotes + bacille Calmette-Gu6rrin (BCG) or alum-precipitated A L M + BCG, respectively, protects against challenge with L. donovani. This was attributed to extensive antigenic cross-reactivity between Leishmania spp. We recently showed that 1 and 2 doses of A L M + B C G vaccine were safe and immunogenic, but the 2 vaccine doses were not superior to B C G alone in protecting against *Current address: Faculty of Medicine, University of Malawi, Blantyre, Malawi. Address for correspondence: E. A. G. I(halil, Institute of Endemic Diseases, University of Khartoum, Khartoum, P.O. Box 45235, Sudan; phone +249 11 773967, fax +249 11 779712, e-mail [email protected]
VL. These studies also showed that individuals who converted in the leishmanin skin test (LST, induration >~ 5 mm) had a lower risk of VL compared with those with an induration of ~< 5 m m (Khalil et al., 2000a; Satti et al., 2001). It was postulated that reformulation of the vaccine mixture by adding another adjuvant (e.g. alum) would improve the immunogenic potential of ALM. Intramuscularly injected alum is widely used in human vaccines to elicit an early, high, and long-lasting antibody response (Gupta et al., 1993; Gupta & Siber, 1995; Mahon et al., 1996). Studies in monkeys showed that intradermal injections of alum-precipitated A L M + interleukin (IL)-12 or B C G were highly protective against either cutaneous leishmaniasis (CL) (Kenney et al., 1999) or VL (Misra et al., 2001). It is also known that alum promotes the activity of IL-12 as an adjuvant for antibodies as well as T h l responses to HIV gp120 (Jankovic et al., 1997). The present report describes the first study in humans, so far as we are aware, where an alum-precipitated vaccine was injected intradermally, in combination with BCG. These vaccine studies were part of T D R / W H O efforts to develop tools for the control of VL.
Material and Methods Study design This was a dose-escalating, open-labelled, safety and immunogenicity study that was conducted between June 1999 and June 2000. Twenty-four healthy adult volunteers were sequentially allocated to 4 groups: 6 volunteers received 10 pg, 5 received 200 ~g, 5 received 100 ~g, and 8 received 400 pg of leishmanial protein in the alum-precipitated A L M vaccine mixture.
Selection criteria Following informed consent, volunteers aged > 15 years from areas in Sudan non-endemic for CL, mucosal leishmaniasis or VL, with no reactivity to the leishmanin antigen and a reciprocal direct agglutination test (DAT) titre of < 200 were selected. An interview and clinical examination were conducted. The biochemical and haematological profiles of the recruited volunteers were checked.
A.A. KAMILETAL.
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Study prowcol The study protocol was approved by the Ethics Committee of the Institute of Endemic Diseases, University of Khartoum; the Ethics Committee of the Federal Ministry of Health, Sudan; and the Secretariat Committee on Research Involving H u m a n Subjects, W H O , Geneva. Volunteers in the first group were injected intradermally with a small test dose of the vaccine (10 ~xg of leishmanial protein in alum-precipitated A L M + 1/10th of B C G concentration routinely used for tuberculosis vaccination) to assess its tolerability and safety. Volunteers were kept at the leishmaniasis clinic at the Institute of Endemic Diseases, University of Khartoum, for 2 h following vaccination and were followedup daily for the first 8 d and then on days 15, 21, and 60 for local and systemic side effects. The same protocol was repeated for the volunteers in the other 3 groups with increasing doses of 100 lag, 200 ~g, or 400 lag of leishmanial protein in alum-precipitated A L M + 1/10th of B C G concentration. Serum was also collected for antileishmanial antibody estimation.
Vaccine T h e alum-precipitated A L M vaccine was prepared under W H O / T D R auspices by the Razi Institute, Karaj, Iran. It was essentially A L M (Hashemi-Fesharki et al., 1998) precipitated with alum to yield a final concent_ration of 3.6 mg A L M and 11.11 mg a l u m / m L of suspension. From this stock (Lot 11), dilutions were made in pyrogen-free saline to deliver the desired dose. The vaccine and the freshly prepared B C G suspension were mixed just before use and injected intradermally on the deltoid region of the left upper arm.
Leishmanin skin test Th e L S T was scheduled at screening and 21, 42, 60, and 90 d after vaccination, by injecting 0.1 m L of leishmanin antigen (Pasteur Institute, Tehran, Iran, Lot 111) on the volar surface of the left arm using a 1 m L syringe. Volunteers in the 10 pg group were not tested at day 21 and those in the 200/xg group were not tested at day 90. The reactions were measured after 48 h by the ballpoint technique as described by Sokal (1975). A positive result was taken as an induration of /> 5ram.
Direct agglutination test Th e D A T was used to determine the titre of antileishmanial antibodies in the sera of volunteers at screening and 42 and 60 d after injection. The D A T
was performed as described by Harith et aL (1987) and any detectable increase in the titre/> 400 was considered significant.
Results The baseline characteristics of the volunteers in the 4 groups are shown in Table 1. Biochemical (results not shown) and haematological profiles at screening and 42 d after injection were within normal ranges with no significant difference between screening and day 42 values (Table 1).
Tolerability of the vaccine Side effects were minimal (induration, ulceration, etc., usually associated with B C G vaccination) and confined to the site of the injection and did not necessitate medical treatment in any of the 4 groups. All volunteers in the 10 ]xg, 100 lag, and 400 [xg groups developed ulcers with mean diameters of 1.2, 4.3, and 2 mm, respectively, by day 21. Ulcers healed with a mean duration of 9.4 d (range 7 - 2 3 d). There were no systemic reactions. Ulcers developed later in the volunteers in 200 [xg group, at day 42 only 2 had ulcers and 3 still had ulcers by day 60. One volunteer in the 400 pg group refused L S T testing at day 90, because previous reactions were very painful and persisted for > 10 d.
Vaccine immunogenicity (Table 2) 10 Izg group. Four of the 6 volunteers converted in the L S T by day 42 with a mean induration of 15.3 ± 6.9 mm. By day 60, all 6 volunteers converted in the L S T with a mean induration of 12.0 ± 5.4 m m and this continued at day 90 with a mean induration of 10.3 ± 3 mm. No change in antileishmanial antibody titres was detected at days 42 and 60 compared with the baseline values. 100 #g group. All 5 volunteers converted in the L S T by day 21 with a mean induration of 13.8 ± 6.8 mm. The leishmanin positivity persisted when the volunteers were tested at days 42, 60, and 90 with mean indurations of 13.8 ± 2 . 6 , 1 1 . 6 ± 2 . 6 , and 9 . 8 ± 3 m m , respectively. There was no change in the antileishmanial antibody titres at days 42 and 60 compared with the baseline values. 200/zg group. Of the 5 volunteers, 1 converted in the L S T by day 42 with an induration of 7.0 mm. By day 60, three converted in the L S T with a mean induration of 6.3 ± 1.5 mm. Volunteers did not show up for further testing. N o significant increase in the antileishmanial antibody titres was detected in the follow-up samples.
Table 1. B a s e l i n e characteristics and h a e m a t o l o g i c a l proFdes o f S u d a n e s e study v o l u n t e e r s r e c e i v i n g different doses o f l e i s h m a n i a l p r o t e i n in an a l u m - p r e c i p i t a t e d a u t o c l a v e d Leishmania major plus B C G vaccine mixture Autoclaved Leishmania major promastigotes
Mean age (years) Male/female Mean haemoglobin concentration (g/dL) Before injection 42 d post-injection Mean white blood cell count (× 109/L) Before injection 42 d post-injection Erythrocyte sedimentation rate (mm in the first hour) Before injection 42 d post-injection Leishmanin skin test (at screening) (ram) Direct agglutination test titre (at screening) Continuous variables are expressed as mean ~- SD.
10 pg (n = 6)
100 lag (n = 5)
200 lag (n = 5)
400 ~tg (n = 8)
27.6 ± 3.5 5/1
47.2 ± 7.7 4/1
21.3 ± 2 5/0
29.7 ± 5.6 3/5
12.9 ± 1.3 13.5 ± 0.5
14 ± 1.8 13.9 ± 1.1
13.5 ± 0.5 14.0 ± 0.5
13.4 ± 1.9 13.5 ± 1.5
5.6 ± 0.8 5.7 ± 1
5.4 ± 1.8 5.7 4- 1
4.7 ± 1.5 5.4 ± 1.5
5.3 ± 1.2 6.0 4- 1
11.8 4- 3.8 11.1 ± 4.9 0 <200
144-6.5 16.4 + 6.1 0 <200
114-8.5 10 ± 5.0 0 <200
19.34-15.1 15.6 ± 8.2 0 <200
ALUM-PRECIPITATEDAUTOCLAVEDLEISHMANIA MAJOR PLUS BCG VACCINE
367
Table 2. L e i s h m a n i n skin test a n d direct a g g l u t i n a t i o n test results d u r i n g followup o f S u d a n e s e study v o l u n t e e r s after v a c c i n a t i o n w i t h different doses o f l e i s h m a n i a l p r o t e i n in an a l u m - p r e c i p i t a t e d a u t o c l a v e d Leishmania major plus B C G vaccine mixture Leishmanin skin test Volunteer ID 10~g ALV1 ALV2 ALV3 ALV4 ALV6 ALV21 100 btg ALV10 ALV17 ALV19 ALV22 ALV24 200 ~g ALV26 ALV27 ALV29 ALV30 ALV31 400 ~g ALV32 ALV33 ALV34 ALV35 ALV36 ALV38 ALV39 ALV42
Direct agglutination test
Day 21
Day 42
Day 60
Day 90
Day 42
Day 60
ND ND ND ND ND ND
11 10 0 15 0 25
12 10 10 12 6 22
10 9 10 12 6 15
<200 <200 <200 <200 <200 <200
<200 <200 <200 <200 <200 <200
12 10 10 26 11
13 10 17 14 15
11 8 13 11 15
10 8 8 8 15
<200 <200 <200 <200 <200
<200 <200 <200 <200 <200
0 0 0 0 7
0 0 0 0 7
6 5 0 0 8
ND ND ND ND ND
<200 <200 <200 <200 <200
400 <200 <200 400 <200
8 9 8 10 5 10 0 10
10 10 6 7 7 7 8 7
15 8 9 9 7 8 6 12
14 8 5 7 7 Refused a 7 10
<200 <200 <200 <200 <200 <200 <200 <200
<200 <200 <200 800 400 800 1600 800
ND, not done as volunteers were not available for testing. aVolunteer found leishmanin skin test induration unacceptable. 400 l~g group. Of the 8 volunteers, 7 converted in the L S T by day 21. By day 42, all 8 volunteers reacted with a mean induration of 7.7 + 1.4 ram. T h e L S T reaction persisted when the volunteers were tested again at days 60 and 90 with mean indurations of 8.8 -4- 3.3 m m and 8.2 + 2.9 mm, respectively. Five volunteers had an increase in antileishmanial antibody titres (Table 2).
Discussion Visceral leishmaniasis is a major health problem in Sudan; recent epidemics have claimed hundreds of thousands of lives in remote areas with no medical care facilities. Development of a safe and affordable vaccine may provide an immunological control strategy. There is no vaccine currently available for leishmaniasis. Inducing a lesion by live Leishmania at a covered part of the body (known as leishmanization) was practiced in the Middle East and Iran for centuries (Greenblatt, 1988; Modabber, 1989), but, except for Uzbekistan, its use has been discontinued. T h e reasons for choosing an L. major vaccine to protect against VL have been discussed previously (Khalil et al., 2000a). Briefly, it was shown that individuals with previous exposure to L. major, have a lower incidence of visceral disease and cross-reactive antigens amongst Leishmania spp. as well a cross-protection (Dube et al., 1998; Kenney et al., 1999; Misra et al., 2001). In addition, A L M was the only vaccine material which was prepared under the good manufacturing practices required for human use. B C G was included in the vaccine due to its general immunostimulatory activity and ability to enhance cellular responses (Castes et al., 1994) particularly L S T conversion and interferon production (Bahar et al., 1996). B C G alone protects mice against leishmaniasis (Smrkovski & Larson, 1977; Weintraub & Weinbaum,
1977) and, in combination with killed Leishmania promastigotes, protects monkeys against challenge with L. donovani (Misra et al., 2001). Th e combination of killed promastigotes and B C G is used for therapy of leishmaniasis in Venezuela (Convit, 1996). Our recent field study on the efficacy of A L M + B C G vaccine showed that individuals who converted in the L S T (induration /> 5 mm) were better protected than nonconverters (induration ~< 5 mm) (Khalil et al., 2000a). The results of our present study show that using a very small dose of A L M (10 9g) in alum-precipitated A L M + B C G is safe and can induce L S T (delayed type hypersensitivity; D T H ) conversion in 66.6% of volunteers by day 42, and that 100% of volunteers converted by day 60 which persisted at day 90. Using higher doses of A L M in the vaccine mixture was not accompanied by any serious local or systemic side effects. All volunteers in the 100 ~g and 400 ~tg groups converted in L S T by day 42 and this conversion continued at days 60 and 90. This persistence in L S T conversion in the absence of exposure to L. major and/or L. donovani in the volunteers may reduce the need to use booster doses to maintain L S T positivity. We would expect the L S T positivity to persist even longer in endemic areas due to the continuous natural challenge. Th e conversion in the L S T that was seen with 10 9g, 100 gg, and 400 gg of leishmanial protein is superior to L S T conversion rates seen in a phase IflI study that we recently concluded (Khalil et al., 2000b). This is also superior to the 80% conversion rate reported in a Venezuelan study by Castes et al. (1994). In our study the L S T mean induration size for the 100 gg group on days 42 and 60 was greater than that seen in the 400 ~tg group (Table 2). This could have been due to a shift towards antibody production with higher doses of antigen as
368 i n d i c a t e d b y the D A T results. W e c a n n o t explain the low L S T c o n v e r s i o n rates seen in the 200 ~g b u t it m a y have b e e n d u e to the low viability of t h e B C G b a t c h used (transport, storage etc). T h e r e was also a delay in the d e v e l o p m e n t of local reactions in this group w h i c h supports this notion. T h e m e a s u r e m e n t of the antileishm a n i a l antibodies s h o w e d t h a t t h e r e was a n increase in their p r o d u c t i o n with a c h a n g e of reciprocal titre f r o m < 200 to 400 a n d 1600 in 5 of 8 volunteers in the 400 ~g group. As expected, s o m e volunteers d e v e l o p e d antibodies w h e n injected with the h i g h e r c o n c e n t r a t i o n of a n t i g e n (400 l~g). H e n c e , lower doses of t h e vaccine w o u l d b e n e e d e d to i n d u c e a primarily T h l type r e s p o n s e if t h e a n i m a l m o d e l s of e x p e r i m e n t a l leishmaniasis are to b e u s e d as guideline. W e c o n c l u d e t h a t i n t r a d e r m a l l y injected a l u m p r e c i p i t a t e d A L M 4 - B C G at the doses u s e d was safe a n d t h a t the rate of L S T ( D H T ) c o n v e r s i o n is higher t h a n t h a t i n d u c e d b y A L M + B C G , m a k i n g it a p o t e n tially superior c a n d i d a t e vaccine for VL. F u r t h e r s t u d ies are b e i n g c o n d u c t e d to establish the dose for a field efficacy trial o f this vaccine.
Acknowledgement This study received financial support from the U N D P / World Bank/WHO Special Programme for Research and Training in Tropical Diseases. References Badaro, R., Jones, T. C., Carvalho, E. M., Sampaio, D., Reed, S. G., Barral, A., Teixeira, R. & Johnson, W. D. Jr (!986). New perspectives on a subclinical form of visceral leishmaniasis. Journal of Infectious Diseases, 154, 1003-1011. Bahar, K., Dowlati, Y., Shidani, B., Alimohammadian, M. H., Khamesipour, A., Ehsasi, S., Hashemi-Fesharki, R., AleAgha, S. & Modabber, F. (1996). Comparative safety and immunogenicity trial of two killed Leishmania major vaccines with or without BCG in human volunteers. Clinics in Dermatology, 14, 489-495. Castes, M., Blackwell, J., Trujillo, D., Formica, S., Cabrera, M., Zorrilla, G., Rodas, A., Castellanos, P. L. & Convit, J. (1994). Immune responses in healthy volunteers vaccinated with killed leishmanial promatigotes plus BCG. I: Skin-test reactivity, T-cell proliferation and interferon-gamma production. Vaccine; 12, 1041-1051. Convit, J. (1996). Leishmaniasis: immunological and clinical aspects and vaccines in Venezuela. Clinics in Dermatology, 14, 479-487. Dube, A., Sharma, P., Srivastava, J. K., Misra, A., Naik, S. & Katiyar, J. C. (1998). Vaccination of langur monkeys (Presbytis entellus) against Leishmania donovani with autoclaved L. majorplus BCG. Parasitology, 116, 219-221. El-Hag, I. A., Hashim, F. A., E1-Toum, I. A., Homeida, M. M. A., E1-Kalifa, M. Y. & E1-Hassan, A. M. (1994). Liver morphology and fimction in visceral leishmaniasis (kalaazar). Journal of Clinical Pathology, 47, 547 - 551. El-Hassan, A. M., Hashim, F. A., Ali, M. S., Ghalib, H. W. & Zijlstra, E. E. (1993). Kala-azar in western Upper Nile province in the southern Sudan and its spread to a nomadic tribe from the north. Transactions of the Royal Society of Tropical Medicine and Hygiene, 87, 395-398. E1-Hassan, A. M., Zijlstra, E. E., Ismael, A. & Ghalib, H. W. (1995). Recent observations on the epidemiology of kalaazar in the eastern and central states of the Sudan. Tropical and GeographicalMedicine, 47, 151 - 156. Ghalib, H. W., Piuvezam, M. R., Skeiky, Y. A., Siddig, M., Hashim, F. A., E1-Hassan, A. M., Russo, D. M. & Reed, S. G. (1993). Interleukin 10 production correlates with pathology in human Leishmania donovani infections. Journal of Clinical Investigations, 92, 324-329. Greenblatt, C. L. (1988). Cutaneous leishmaniasis: the prospects for a killed vaccine. Parasitology Today, 4, 53-54. Gupta, R. K. & Siber, G. R. (1995). Adjuvants for human vaccines: current status, problems and future prospects. Vaccine, 13, 1263-1276. Gupta, R. K., Relyveld, E. H., Lindblad, E. B., Bizzini, B., Ben-Efraim, S. & Gupta, C. K. (1993). Adjuvants: a balance between toxicity and adjuvanticity. Vaccine, 11, 293-306.
A.A. KAMILETAL. Harith, A. E., Kolk, A. H., Kager, P. A., Leeuwenburg, J., Faber, F. J., Muigai, R., Kiungu, S. & Laarman, J. J. (1987). Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Transactions of the Royal Society of Tropical Medicine, 81, 603-606. Hashemi-Fesharld, R., Ale-Agha, S., Habibi, G. R., Manhouri, H., Esmail-Nia, K., Mohammadi, A. A., Ahourai, P., Vand-Yousefi, G., Bahar, K., Dowlati, Y., Khamesipour, A. & Modabber, F. (1998). Production of inactivated Leishmania major vaccine. Iranian Journal of Medical Sciences, 23, 74-80. Jankovic, D., Caspar, P., Zweig, M., Garcia-Moll, M., Showalter, S. D., Vogel, F. R. & Sher, A. (1997). Adsorption to aluminum hydroxide promotes the activity of IL-12 as an adjuvant for antibody as well as type-1 cytokine responses to HIV-1 gp120. Journal of Immunology, 159, 2409-2417. Kenney, R. T., Sacks, D. L., Sypek, J. P., Vilela, L.~ Gam, A. A. & Evans-Davis, K. (1999). Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis. Journal of Immunology, 163, 4481-4488. Khalil, E. A. G., El-Hassan, A. M., Zijlstra, E. E., Mukhtar, M. M., Ghalib, H. W., Musa, B., Ibrahim, M. E., Kamil, A. A., Elsheikh, M., Babiker, A. & Modabber, F. (2000a). Autoclaved Leishmania major vaccine for the prevention of visceral leishmaniasis: a randomised, double-blind, BCGcontrolled trial in the Sudan. Lancet, 356, 1565-1569. Khalil, E. A. G., E1-Hassan, A. M., Zijlstra, E. E., Mukhtar, M. M. & Ibrahim, M. E. (2000b). Safety and immunogenicity of an autoclaved Leishmania major vaccine. East African Medical Journal, 77, 468-470. Kurtzhals, J. A., Hey, A. S., Theander, T. G., Odera, E., Christensen, C. B., Githure, J. I., Koech, D. K., Schaefer, K. U., Handman, E. & Kharazmi, A. (1992). Cellular and humoral immune responses in a population from the Baringo district of Kenya to Leishmania promastigotes lipophosphoglycan. American Journal of Tropical Medicine and Hygiene, 46, 480-488. Mahon, B. P., Ryan, M. S., Griffin, F. & Mills, K. H. (1996). Interleukin-12 is produced by macrophages in response to live or killed Bordetella pertussis and enhances the efficacy of an acellular pertussis vaccine by promoting induction of T h 1 cells. Infection and Immunity, 64. 5295-5301. Misra, A., Dube, A., Srivastava, B., Sharma, P., Srivastava, J. K., Katiyar, J. C. & Naik, S. (2001). Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG. Vaccine, 19, 3485-3492. Modabber, F. (1989). Experience with vaccines against cutaneous leishmaniasis of man and mice. Parasitology, 98, $49-$60. Montalban, C., Calleja, J. L., Erice, A., Laguna, F., Clotet, B., Podzamczer, D., Coba, J., Mallolas, J., Yebra, M. & Gallego, A. (1990). Visceral leishmaniasis in patients infected with immunodeficiency virus. Co-operative Group for the Study of Leishmaniasis in AIDS. Journal of Infection, 21, 261-270. Sacks, D. L., Lal, S. L., Shrivastava, S. N., Blackwell, J. & Neva, F. A. (1987). An analysis of T-cell responses in Indian kala-azar. Journal of Immunology, 138, 908-913. Satti, I. N., Osman, H. Y., Daifalla, N. S., Younis, S. A., Khalil, E. A. G., Zijlstra, E. E., E1-Hassan, A. M. & Ghalib, H. W. (2001). Immunogenicity and safety of autoclaved Leishmania major plus BCG vaccine in healthy Sudanese volunteers. Vaccine, 19, 2100-2106. Smrkovski, L. L. & Larson C. L. (1977). Antigenic crossreactivity between Mycobacterium bovis (BCG) and Leishmania donovani. Infection and Immunity, 18, 561-562. Weintraub, J. & Weinbaum, F. I. (1977). The effect of BCG on experimental cutaneous leishmaniasis in mice. Journal of Immunology, 118, 2288-2290. Zijlstra, E. E., E1-Hassan, A. M., Ismael, A. & Ghaib, H. W. (1994). Endemic kala-azar in eastern Sudan: a longitudinal study on the incidence of clinical and subclinical infection and post kala-azar dermal leishmaniasis. American Journal of Tropical Medicine and Hygiene, 51, 826-836.
Received 21 March 2002; revised 8 November 2002; accepted for publication 12 November 2002
Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Gu6rrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers A. A. K a m i l 1, E. A. G. Khalil 1, A. M. M u s a l, F. M o d a b b e r ~, M. M. M u k h t a r l, M . E. I b r a h i m 1, E. E. Zijlstra 1., D . S a c k s 3, P . G. S m i t h 4, F. Z i c k e r s a n d A. M. E 1 - H a s s a n ~ 1The Leishmaniasis Research Group, Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan; 2Infectious Diseases Research Institute, Seattle, WA, USA; 3National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Parasitic Diseases, Bethesda, MD, USA; 4London School of Hygiene and Tropical Medicine, Keppel Street, London, UK; 5TDR, World Health Organization, Geneva, Switzerland Abstract In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette-Gu6rrin (BCG) vaccine was shown to be safe, but not superior to B C G alone, in protecting against visceral leishmaniasis. From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alumprecipitated A L M + B C G vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM. Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 ~g, 100 tig, 200 ~g, or 400 pg of leishmanial protein in the alum-precipitated A L M + B C G vaccine mixture. Side effects were minimal for all doses and confined to the site of injection. All volunteers in the 10 ~tg, 100 ~g, and 400 ~g groups had a leishmanin skin test (LST) reaction of/> 5 m m by day 42 and this response was maintained when tested after 90 d. Only 1 volunteer out of 5 in the 200 ~g group had a L S T reaction of/> 5mm by day 42 and the reasons for the different L S T responses in this group are unclear. This is the first time that an alum adjuvant with A L M has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers. On the basis of this study we suggest that 100 ~g of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest D T H reaction following vaccination.
Keywords: visceral leishmaniasis, Leishmania major, alum, bacille Calmette-Gu6rrin, vaccine, Sudan Introduction Visceral leishmaniasis (VL) is a major cause of morbidity and mortality in East Africa and the Indian subcontinent; the annual number of cases is about 500000 (E1-Hassan et al., 1993, 1995; El-Hag et al., 1994; Zijlstra et al., 1994). Co-infection with HIV enhances the risk of the disease (Montalban et al., 1990). The only control measure currently available in the Sudan is case detection and treatment with antimonial drugs, which are expensive, not always available and cannot be self-administered. Vector and reservoir control is difficult due to the elusive nature of the vector and the diversity of the animal reservoir. Subclinical infection and development of a T helper cell type 1 (Thl)-type immune response following successful drug treatment suggests that vaccination may provide a possible method for disease control (Badaro et al., 1986; Sacks et al., 1987; Kurtzhals et al., 1992; Ghalib et al., 1993; Zijlstra et al., 1994). Leishmania spp. are good candidates for vaccine development because they undergo minimal, if any, antigenic variation and there is extensive cross-reactivity between Leishmania strains. We observed that individuals who migrated to eastern Sudan from L. major-endemic areas in the west and who were reactive to leishmanin antigen had a lower risk of developing VL (Zijlstra et al., 1994). Dube (et al., 1998) and Misra et al. (2001) showed that vaccination of primates with 2 - 3 injections of autoclaved L. major (ALM) promastigotes + bacille Calmette-Gu6rrin (BCG) or alum-precipitated A L M + BCG, respectively, protects against challenge with L. donovani. This was attributed to extensive antigenic cross-reactivity between Leishmania spp. We recently showed that 1 and 2 doses of A L M + B C G vaccine were safe and immunogenic, but the 2 vaccine doses were not superior to B C G alone in protecting against *Current address: Faculty of Medicine, University of Malawi, Blantyre, Malawi. Address for correspondence: E. A. G. I(halil, Institute of Endemic Diseases, University of Khartoum, Khartoum, P.O. Box 45235, Sudan; phone +249 11 773967, fax +249 11 779712, e-mail [email protected]
VL. These studies also showed that individuals who converted in the leishmanin skin test (LST, induration >~ 5 mm) had a lower risk of VL compared with those with an induration of ~< 5 m m (Khalil et al., 2000a; Satti et al., 2001). It was postulated that reformulation of the vaccine mixture by adding another adjuvant (e.g. alum) would improve the immunogenic potential of ALM. Intramuscularly injected alum is widely used in human vaccines to elicit an early, high, and long-lasting antibody response (Gupta et al., 1993; Gupta & Siber, 1995; Mahon et al., 1996). Studies in monkeys showed that intradermal injections of alum-precipitated A L M + interleukin (IL)-12 or B C G were highly protective against either cutaneous leishmaniasis (CL) (Kenney et al., 1999) or VL (Misra et al., 2001). It is also known that alum promotes the activity of IL-12 as an adjuvant for antibodies as well as T h l responses to HIV gp120 (Jankovic et al., 1997). The present report describes the first study in humans, so far as we are aware, where an alum-precipitated vaccine was injected intradermally, in combination with BCG. These vaccine studies were part of T D R / W H O efforts to develop tools for the control of VL.
Material and Methods Study design This was a dose-escalating, open-labelled, safety and immunogenicity study that was conducted between June 1999 and June 2000. Twenty-four healthy adult volunteers were sequentially allocated to 4 groups: 6 volunteers received 10 pg, 5 received 200 ~g, 5 received 100 ~g, and 8 received 400 pg of leishmanial protein in the alum-precipitated A L M vaccine mixture.
Selection criteria Following informed consent, volunteers aged > 15 years from areas in Sudan non-endemic for CL, mucosal leishmaniasis or VL, with no reactivity to the leishmanin antigen and a reciprocal direct agglutination test (DAT) titre of < 200 were selected. An interview and clinical examination were conducted. The biochemical and haematological profiles of the recruited volunteers were checked.
A.A. KAMILETAL.
366
Study prowcol The study protocol was approved by the Ethics Committee of the Institute of Endemic Diseases, University of Khartoum; the Ethics Committee of the Federal Ministry of Health, Sudan; and the Secretariat Committee on Research Involving H u m a n Subjects, W H O , Geneva. Volunteers in the first group were injected intradermally with a small test dose of the vaccine (10 ~xg of leishmanial protein in alum-precipitated A L M + 1/10th of B C G concentration routinely used for tuberculosis vaccination) to assess its tolerability and safety. Volunteers were kept at the leishmaniasis clinic at the Institute of Endemic Diseases, University of Khartoum, for 2 h following vaccination and were followedup daily for the first 8 d and then on days 15, 21, and 60 for local and systemic side effects. The same protocol was repeated for the volunteers in the other 3 groups with increasing doses of 100 lag, 200 ~g, or 400 lag of leishmanial protein in alum-precipitated A L M + 1/10th of B C G concentration. Serum was also collected for antileishmanial antibody estimation.
Vaccine T h e alum-precipitated A L M vaccine was prepared under W H O / T D R auspices by the Razi Institute, Karaj, Iran. It was essentially A L M (Hashemi-Fesharki et al., 1998) precipitated with alum to yield a final concent_ration of 3.6 mg A L M and 11.11 mg a l u m / m L of suspension. From this stock (Lot 11), dilutions were made in pyrogen-free saline to deliver the desired dose. The vaccine and the freshly prepared B C G suspension were mixed just before use and injected intradermally on the deltoid region of the left upper arm.
Leishmanin skin test Th e L S T was scheduled at screening and 21, 42, 60, and 90 d after vaccination, by injecting 0.1 m L of leishmanin antigen (Pasteur Institute, Tehran, Iran, Lot 111) on the volar surface of the left arm using a 1 m L syringe. Volunteers in the 10 pg group were not tested at day 21 and those in the 200/xg group were not tested at day 90. The reactions were measured after 48 h by the ballpoint technique as described by Sokal (1975). A positive result was taken as an induration of /> 5ram.
Direct agglutination test Th e D A T was used to determine the titre of antileishmanial antibodies in the sera of volunteers at screening and 42 and 60 d after injection. The D A T
was performed as described by Harith et aL (1987) and any detectable increase in the titre/> 400 was considered significant.
Results The baseline characteristics of the volunteers in the 4 groups are shown in Table 1. Biochemical (results not shown) and haematological profiles at screening and 42 d after injection were within normal ranges with no significant difference between screening and day 42 values (Table 1).
Tolerability of the vaccine Side effects were minimal (induration, ulceration, etc., usually associated with B C G vaccination) and confined to the site of the injection and did not necessitate medical treatment in any of the 4 groups. All volunteers in the 10 ]xg, 100 lag, and 400 [xg groups developed ulcers with mean diameters of 1.2, 4.3, and 2 mm, respectively, by day 21. Ulcers healed with a mean duration of 9.4 d (range 7 - 2 3 d). There were no systemic reactions. Ulcers developed later in the volunteers in 200 [xg group, at day 42 only 2 had ulcers and 3 still had ulcers by day 60. One volunteer in the 400 pg group refused L S T testing at day 90, because previous reactions were very painful and persisted for > 10 d.
Vaccine immunogenicity (Table 2) 10 Izg group. Four of the 6 volunteers converted in the L S T by day 42 with a mean induration of 15.3 ± 6.9 mm. By day 60, all 6 volunteers converted in the L S T with a mean induration of 12.0 ± 5.4 m m and this continued at day 90 with a mean induration of 10.3 ± 3 mm. No change in antileishmanial antibody titres was detected at days 42 and 60 compared with the baseline values. 100 #g group. All 5 volunteers converted in the L S T by day 21 with a mean induration of 13.8 ± 6.8 mm. The leishmanin positivity persisted when the volunteers were tested at days 42, 60, and 90 with mean indurations of 13.8 ± 2 . 6 , 1 1 . 6 ± 2 . 6 , and 9 . 8 ± 3 m m , respectively. There was no change in the antileishmanial antibody titres at days 42 and 60 compared with the baseline values. 200/zg group. Of the 5 volunteers, 1 converted in the L S T by day 42 with an induration of 7.0 mm. By day 60, three converted in the L S T with a mean induration of 6.3 ± 1.5 mm. Volunteers did not show up for further testing. N o significant increase in the antileishmanial antibody titres was detected in the follow-up samples.
Table 1. B a s e l i n e characteristics and h a e m a t o l o g i c a l proFdes o f S u d a n e s e study v o l u n t e e r s r e c e i v i n g different doses o f l e i s h m a n i a l p r o t e i n in an a l u m - p r e c i p i t a t e d a u t o c l a v e d Leishmania major plus B C G vaccine mixture Autoclaved Leishmania major promastigotes
Mean age (years) Male/female Mean haemoglobin concentration (g/dL) Before injection 42 d post-injection Mean white blood cell count (× 109/L) Before injection 42 d post-injection Erythrocyte sedimentation rate (mm in the first hour) Before injection 42 d post-injection Leishmanin skin test (at screening) (ram) Direct agglutination test titre (at screening) Continuous variables are expressed as mean ~- SD.
10 pg (n = 6)
100 lag (n = 5)
200 lag (n = 5)
400 ~tg (n = 8)
27.6 ± 3.5 5/1
47.2 ± 7.7 4/1
21.3 ± 2 5/0
29.7 ± 5.6 3/5
12.9 ± 1.3 13.5 ± 0.5
14 ± 1.8 13.9 ± 1.1
13.5 ± 0.5 14.0 ± 0.5
13.4 ± 1.9 13.5 ± 1.5
5.6 ± 0.8 5.7 ± 1
5.4 ± 1.8 5.7 4- 1
4.7 ± 1.5 5.4 ± 1.5
5.3 ± 1.2 6.0 4- 1
11.8 4- 3.8 11.1 ± 4.9 0 <200
144-6.5 16.4 + 6.1 0 <200
114-8.5 10 ± 5.0 0 <200
19.34-15.1 15.6 ± 8.2 0 <200
ALUM-PRECIPITATEDAUTOCLAVEDLEISHMANIA MAJOR PLUS BCG VACCINE
367
Table 2. L e i s h m a n i n skin test a n d direct a g g l u t i n a t i o n test results d u r i n g followup o f S u d a n e s e study v o l u n t e e r s after v a c c i n a t i o n w i t h different doses o f l e i s h m a n i a l p r o t e i n in an a l u m - p r e c i p i t a t e d a u t o c l a v e d Leishmania major plus B C G vaccine mixture Leishmanin skin test Volunteer ID 10~g ALV1 ALV2 ALV3 ALV4 ALV6 ALV21 100 btg ALV10 ALV17 ALV19 ALV22 ALV24 200 ~g ALV26 ALV27 ALV29 ALV30 ALV31 400 ~g ALV32 ALV33 ALV34 ALV35 ALV36 ALV38 ALV39 ALV42
Direct agglutination test
Day 21
Day 42
Day 60
Day 90
Day 42
Day 60
ND ND ND ND ND ND
11 10 0 15 0 25
12 10 10 12 6 22
10 9 10 12 6 15
<200 <200 <200 <200 <200 <200
<200 <200 <200 <200 <200 <200
12 10 10 26 11
13 10 17 14 15
11 8 13 11 15
10 8 8 8 15
<200 <200 <200 <200 <200
<200 <200 <200 <200 <200
0 0 0 0 7
0 0 0 0 7
6 5 0 0 8
ND ND ND ND ND
<200 <200 <200 <200 <200
400 <200 <200 400 <200
8 9 8 10 5 10 0 10
10 10 6 7 7 7 8 7
15 8 9 9 7 8 6 12
14 8 5 7 7 Refused a 7 10
<200 <200 <200 <200 <200 <200 <200 <200
<200 <200 <200 800 400 800 1600 800
ND, not done as volunteers were not available for testing. aVolunteer found leishmanin skin test induration unacceptable. 400 l~g group. Of the 8 volunteers, 7 converted in the L S T by day 21. By day 42, all 8 volunteers reacted with a mean induration of 7.7 + 1.4 ram. T h e L S T reaction persisted when the volunteers were tested again at days 60 and 90 with mean indurations of 8.8 -4- 3.3 m m and 8.2 + 2.9 mm, respectively. Five volunteers had an increase in antileishmanial antibody titres (Table 2).
Discussion Visceral leishmaniasis is a major health problem in Sudan; recent epidemics have claimed hundreds of thousands of lives in remote areas with no medical care facilities. Development of a safe and affordable vaccine may provide an immunological control strategy. There is no vaccine currently available for leishmaniasis. Inducing a lesion by live Leishmania at a covered part of the body (known as leishmanization) was practiced in the Middle East and Iran for centuries (Greenblatt, 1988; Modabber, 1989), but, except for Uzbekistan, its use has been discontinued. T h e reasons for choosing an L. major vaccine to protect against VL have been discussed previously (Khalil et al., 2000a). Briefly, it was shown that individuals with previous exposure to L. major, have a lower incidence of visceral disease and cross-reactive antigens amongst Leishmania spp. as well a cross-protection (Dube et al., 1998; Kenney et al., 1999; Misra et al., 2001). In addition, A L M was the only vaccine material which was prepared under the good manufacturing practices required for human use. B C G was included in the vaccine due to its general immunostimulatory activity and ability to enhance cellular responses (Castes et al., 1994) particularly L S T conversion and interferon production (Bahar et al., 1996). B C G alone protects mice against leishmaniasis (Smrkovski & Larson, 1977; Weintraub & Weinbaum,
1977) and, in combination with killed Leishmania promastigotes, protects monkeys against challenge with L. donovani (Misra et al., 2001). Th e combination of killed promastigotes and B C G is used for therapy of leishmaniasis in Venezuela (Convit, 1996). Our recent field study on the efficacy of A L M + B C G vaccine showed that individuals who converted in the L S T (induration /> 5 mm) were better protected than nonconverters (induration ~< 5 mm) (Khalil et al., 2000a). The results of our present study show that using a very small dose of A L M (10 9g) in alum-precipitated A L M + B C G is safe and can induce L S T (delayed type hypersensitivity; D T H ) conversion in 66.6% of volunteers by day 42, and that 100% of volunteers converted by day 60 which persisted at day 90. Using higher doses of A L M in the vaccine mixture was not accompanied by any serious local or systemic side effects. All volunteers in the 100 ~g and 400 ~tg groups converted in L S T by day 42 and this conversion continued at days 60 and 90. This persistence in L S T conversion in the absence of exposure to L. major and/or L. donovani in the volunteers may reduce the need to use booster doses to maintain L S T positivity. We would expect the L S T positivity to persist even longer in endemic areas due to the continuous natural challenge. Th e conversion in the L S T that was seen with 10 9g, 100 gg, and 400 gg of leishmanial protein is superior to L S T conversion rates seen in a phase IflI study that we recently concluded (Khalil et al., 2000b). This is also superior to the 80% conversion rate reported in a Venezuelan study by Castes et al. (1994). In our study the L S T mean induration size for the 100 gg group on days 42 and 60 was greater than that seen in the 400 ~tg group (Table 2). This could have been due to a shift towards antibody production with higher doses of antigen as
368 i n d i c a t e d b y the D A T results. W e c a n n o t explain the low L S T c o n v e r s i o n rates seen in the 200 ~g b u t it m a y have b e e n d u e to the low viability of t h e B C G b a t c h used (transport, storage etc). T h e r e was also a delay in the d e v e l o p m e n t of local reactions in this group w h i c h supports this notion. T h e m e a s u r e m e n t of the antileishm a n i a l antibodies s h o w e d t h a t t h e r e was a n increase in their p r o d u c t i o n with a c h a n g e of reciprocal titre f r o m < 200 to 400 a n d 1600 in 5 of 8 volunteers in the 400 ~g group. As expected, s o m e volunteers d e v e l o p e d antibodies w h e n injected with the h i g h e r c o n c e n t r a t i o n of a n t i g e n (400 l~g). H e n c e , lower doses of t h e vaccine w o u l d b e n e e d e d to i n d u c e a primarily T h l type r e s p o n s e if t h e a n i m a l m o d e l s of e x p e r i m e n t a l leishmaniasis are to b e u s e d as guideline. W e c o n c l u d e t h a t i n t r a d e r m a l l y injected a l u m p r e c i p i t a t e d A L M 4 - B C G at the doses u s e d was safe a n d t h a t the rate of L S T ( D H T ) c o n v e r s i o n is higher t h a n t h a t i n d u c e d b y A L M + B C G , m a k i n g it a p o t e n tially superior c a n d i d a t e vaccine for VL. F u r t h e r s t u d ies are b e i n g c o n d u c t e d to establish the dose for a field efficacy trial o f this vaccine.
Acknowledgement This study received financial support from the U N D P / World Bank/WHO Special Programme for Research and Training in Tropical Diseases. References Badaro, R., Jones, T. C., Carvalho, E. M., Sampaio, D., Reed, S. G., Barral, A., Teixeira, R. & Johnson, W. D. Jr (!986). New perspectives on a subclinical form of visceral leishmaniasis. Journal of Infectious Diseases, 154, 1003-1011. Bahar, K., Dowlati, Y., Shidani, B., Alimohammadian, M. H., Khamesipour, A., Ehsasi, S., Hashemi-Fesharki, R., AleAgha, S. & Modabber, F. (1996). Comparative safety and immunogenicity trial of two killed Leishmania major vaccines with or without BCG in human volunteers. Clinics in Dermatology, 14, 489-495. Castes, M., Blackwell, J., Trujillo, D., Formica, S., Cabrera, M., Zorrilla, G., Rodas, A., Castellanos, P. L. & Convit, J. (1994). Immune responses in healthy volunteers vaccinated with killed leishmanial promatigotes plus BCG. I: Skin-test reactivity, T-cell proliferation and interferon-gamma production. Vaccine; 12, 1041-1051. Convit, J. (1996). Leishmaniasis: immunological and clinical aspects and vaccines in Venezuela. Clinics in Dermatology, 14, 479-487. Dube, A., Sharma, P., Srivastava, J. K., Misra, A., Naik, S. & Katiyar, J. C. (1998). Vaccination of langur monkeys (Presbytis entellus) against Leishmania donovani with autoclaved L. majorplus BCG. Parasitology, 116, 219-221. El-Hag, I. A., Hashim, F. A., E1-Toum, I. A., Homeida, M. M. A., E1-Kalifa, M. Y. & E1-Hassan, A. M. (1994). Liver morphology and fimction in visceral leishmaniasis (kalaazar). Journal of Clinical Pathology, 47, 547 - 551. El-Hassan, A. M., Hashim, F. A., Ali, M. S., Ghalib, H. W. & Zijlstra, E. E. (1993). Kala-azar in western Upper Nile province in the southern Sudan and its spread to a nomadic tribe from the north. Transactions of the Royal Society of Tropical Medicine and Hygiene, 87, 395-398. E1-Hassan, A. M., Zijlstra, E. E., Ismael, A. & Ghalib, H. W. (1995). Recent observations on the epidemiology of kalaazar in the eastern and central states of the Sudan. Tropical and GeographicalMedicine, 47, 151 - 156. Ghalib, H. W., Piuvezam, M. R., Skeiky, Y. A., Siddig, M., Hashim, F. A., E1-Hassan, A. M., Russo, D. M. & Reed, S. G. (1993). Interleukin 10 production correlates with pathology in human Leishmania donovani infections. Journal of Clinical Investigations, 92, 324-329. Greenblatt, C. L. (1988). Cutaneous leishmaniasis: the prospects for a killed vaccine. Parasitology Today, 4, 53-54. Gupta, R. K. & Siber, G. R. (1995). Adjuvants for human vaccines: current status, problems and future prospects. Vaccine, 13, 1263-1276. Gupta, R. K., Relyveld, E. H., Lindblad, E. B., Bizzini, B., Ben-Efraim, S. & Gupta, C. K. (1993). Adjuvants: a balance between toxicity and adjuvanticity. Vaccine, 11, 293-306.
A.A. KAMILETAL. Harith, A. E., Kolk, A. H., Kager, P. A., Leeuwenburg, J., Faber, F. J., Muigai, R., Kiungu, S. & Laarman, J. J. (1987). Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Transactions of the Royal Society of Tropical Medicine, 81, 603-606. Hashemi-Fesharld, R., Ale-Agha, S., Habibi, G. R., Manhouri, H., Esmail-Nia, K., Mohammadi, A. A., Ahourai, P., Vand-Yousefi, G., Bahar, K., Dowlati, Y., Khamesipour, A. & Modabber, F. (1998). Production of inactivated Leishmania major vaccine. Iranian Journal of Medical Sciences, 23, 74-80. Jankovic, D., Caspar, P., Zweig, M., Garcia-Moll, M., Showalter, S. D., Vogel, F. R. & Sher, A. (1997). Adsorption to aluminum hydroxide promotes the activity of IL-12 as an adjuvant for antibody as well as type-1 cytokine responses to HIV-1 gp120. Journal of Immunology, 159, 2409-2417. Kenney, R. T., Sacks, D. L., Sypek, J. P., Vilela, L.~ Gam, A. A. & Evans-Davis, K. (1999). Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis. Journal of Immunology, 163, 4481-4488. Khalil, E. A. G., El-Hassan, A. M., Zijlstra, E. E., Mukhtar, M. M., Ghalib, H. W., Musa, B., Ibrahim, M. E., Kamil, A. A., Elsheikh, M., Babiker, A. & Modabber, F. (2000a). Autoclaved Leishmania major vaccine for the prevention of visceral leishmaniasis: a randomised, double-blind, BCGcontrolled trial in the Sudan. Lancet, 356, 1565-1569. Khalil, E. A. G., E1-Hassan, A. M., Zijlstra, E. E., Mukhtar, M. M. & Ibrahim, M. E. (2000b). Safety and immunogenicity of an autoclaved Leishmania major vaccine. East African Medical Journal, 77, 468-470. Kurtzhals, J. A., Hey, A. S., Theander, T. G., Odera, E., Christensen, C. B., Githure, J. I., Koech, D. K., Schaefer, K. U., Handman, E. & Kharazmi, A. (1992). Cellular and humoral immune responses in a population from the Baringo district of Kenya to Leishmania promastigotes lipophosphoglycan. American Journal of Tropical Medicine and Hygiene, 46, 480-488. Mahon, B. P., Ryan, M. S., Griffin, F. & Mills, K. H. (1996). Interleukin-12 is produced by macrophages in response to live or killed Bordetella pertussis and enhances the efficacy of an acellular pertussis vaccine by promoting induction of T h 1 cells. Infection and Immunity, 64. 5295-5301. Misra, A., Dube, A., Srivastava, B., Sharma, P., Srivastava, J. K., Katiyar, J. C. & Naik, S. (2001). Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG. Vaccine, 19, 3485-3492. Modabber, F. (1989). Experience with vaccines against cutaneous leishmaniasis of man and mice. Parasitology, 98, $49-$60. Montalban, C., Calleja, J. L., Erice, A., Laguna, F., Clotet, B., Podzamczer, D., Coba, J., Mallolas, J., Yebra, M. & Gallego, A. (1990). Visceral leishmaniasis in patients infected with immunodeficiency virus. Co-operative Group for the Study of Leishmaniasis in AIDS. Journal of Infection, 21, 261-270. Sacks, D. L., Lal, S. L., Shrivastava, S. N., Blackwell, J. & Neva, F. A. (1987). An analysis of T-cell responses in Indian kala-azar. Journal of Immunology, 138, 908-913. Satti, I. N., Osman, H. Y., Daifalla, N. S., Younis, S. A., Khalil, E. A. G., Zijlstra, E. E., E1-Hassan, A. M. & Ghalib, H. W. (2001). Immunogenicity and safety of autoclaved Leishmania major plus BCG vaccine in healthy Sudanese volunteers. Vaccine, 19, 2100-2106. Smrkovski, L. L. & Larson C. L. (1977). Antigenic crossreactivity between Mycobacterium bovis (BCG) and Leishmania donovani. Infection and Immunity, 18, 561-562. Weintraub, J. & Weinbaum, F. I. (1977). The effect of BCG on experimental cutaneous leishmaniasis in mice. Journal of Immunology, 118, 2288-2290. Zijlstra, E. E., E1-Hassan, A. M., Ismael, A. & Ghaib, H. W. (1994). Endemic kala-azar in eastern Sudan: a longitudinal study on the incidence of clinical and subclinical infection and post kala-azar dermal leishmaniasis. American Journal of Tropical Medicine and Hygiene, 51, 826-836.
Received 21 March 2002; revised 8 November 2002; accepted for publication 12 November 2002