Resistance Against Leishmania donovani Induced with an Aluminum Hydroxide Vaccine

ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 18, No. 2 Copyright 1988, Institute for Clinical Science, Inc. Resistance Against Leishmania donovani Induced with an Aluminum Hydroxide Vaccine J. C. JARECKI-BLACK,* K. L. HALLMAN,* E. R. JAM ES,! Ph.D., and A. B. GLASSMAN, M.D.*$ *Department of Pathology and Laboratory Medicine, fdepartment of Ophthalmology, Medical University of South Carolina, Charleston, SC 29425 ABSTRACT Mice immunized with a subcutaneous protocol combining killed parasites and aluminum hydroxide gel exhibited significant resistance against subsequent challenge with Leishmania donovani promastigotes. Protection was greatest using 25 mg of aluminum hydroxide per injection. Resistance elicited by this killed parasite and aluminum hydroxide protocol was as effective on day 14 as that provided by immunization with a glucan and killed parasite preparation, and more effective in hepatic amastigote reduction at day 28. The effectiveness of aluminum hydroxide as an adjuvant appears to result, at least in part, from its ability to activate macrophages, thus aiding in the elimination of this intracellular parasite. Introduction Leishmania donovani is an obligate intracellular parasite which multiplies in culture or the sandfly vector as a prom astigote or flagellated form. W hen introduced into a vertebrate host, the parasite is engulfed by phagocytic cells, transforms to the amastigote stage, and disseminates throughout the reticuloendothelial system. The disease in man is often fatal, although spontaneous recovery may occur.12 Current treatment consists of chemotherapeutic intervention in the form of pentavalent antimonial compounds, but such drugs are relatively toxic and treatm ent failure som etim es occurs.17 The screening of potential new drugs21 and advances in the methods of drug delivery16 are being investigated, but vaccin e-in d u ced resistan ce w ould be a major step in the eventual control and prevention of human visceral leishmaniasis. R ecently in our laboratory, it was demonstrated that such immunoprophylaxis is feasible using formalin-killed parasites administered with glucan, a (31,3- polyglucose derivative of yeast cell walls, as an adjuvant. Protection was elicited not only by intravenous administration of the glucan-killed parasite combination4 but also with a subcutaneous immunization protocol.3 72 0091-7370/88/0100-0072 $00.90 Institute for Clinical Science, Inc. t Present address: Senior Vice-President for Medical Affairs, M ontefiore Medical Center, 111 East 210th Street, Bronx, NY 10467

LEISHMANIA DONOVANI RESISTANCE BY ALUMINUM HYDROXIDE 73 Only lim ited trials with glucan have been conducted, and it is not currently approved for human use. This study was undertaken, therefore, to examine the efficacy of aluminum hydroxide gel, an adjuvant already available in commercial vaccines, in p o te n tia tin g resistance against v isceral leish m an iasis. O ur results demonstrate that such an immunization protocol can successfully elicit significant resistance against L. donovani, thus indicating that immunoprophylaxis may eventually be possible. Materials and Methods E x p e r im e n t a l M o d e l a n d P a r a s it e L eish m a n ia d o n o vani, stra in 2S (WR371), is maintained in our laboratory in axenic c u ltu re.19 G row th m edium consists of Media 199 supplemented 10 percent with lysed, defibrinated rabbit blood and 14.6 mg L-glutamine per litre. C u ltu res are in itiate d from m inced mouse spleen and passaged at four to five day intervals. Prom astigotes for immunization or challenge are harvested within 10 serial passage of culture initiation. F em ale C 57B L/6 m ice* w ere housed in air-conditioned quarters with food and water provided ad libitum. The mice were eight to 10 weeks old at the beginning of each experiment. A d ju v a n t P r e p a r a t io n Glucan (G)t and gelatinous aluminum hydroxide (AL)$ were purchased as standard formulations. The glucan consisted of a particulate suspension in dextrose at a concentration of 5 mg per ml, and alum inum hydroxide gel was suspended in Earle s Balanced Salt Solution (EBSS) at a concentration of 250 mg per ml. * Jackson Laboratories. t Accurate Chemical and Scientific Corporation. $ Fisher Scientific Company. I m m u n iz a t io n P r o t o c o l Promastigotes were harvested during stationary phase growth by centrifugation (900 g, 15 min) as a previous study has shown that stationary-phase parasites are more im m unogenic.7 The parasites were washed three times in EBSS, and killed in 0.1 percent formalin for 30 min at room tem perature. Form alin-killed parasites (P) were kept at 4 C overnight, washed three times, counted in a hemocytometer and resuspended in EBSS at a concentration of 108 per ml. Animals were immunized with adjuvant (25 mg AL or 0.5 mg G) combined with 107 parasites, adjuvant alone (AL or G), parasites alone (P), or were untreated (NT). The potentiation of resistance to L. donovani infection as a function of dosage response to alum inum hydroxide was also examined by immunizing additional groups of mice with either 25 mg, 12.5 mg, or 6.25 mg Al combined with 107 killed parasites. All animals received three isovolumetric injections subcutaneously at four day intervals (weekly for the dosage response experiment), and were challenged via the tail vein 21 days post-immunization with 107 prom astigotes per mouse. M o n it o r in g o f I n f e c t io n C o u r s e G roups of m ice w ere sacrificed in chloroform vapor 14 and 28 days postc h a llen g e. Body, sp le en an d liv e r weights were recorded and impression slides were made from liver and spleen. Slides w ere fixed in methanol, stained with Giemsa, and parasite burdens in liv e r and sp le e n w ere d e te rm in e d according to Stauber s m ethod.20 Results were analyzed by the Mann-W hitney u- test10 and were considered significant at a p < 0.05.

74 JARECKI-BLACK, HALLMAN, JAMES, AND GLASSMAN Results Mice immunized subeutaneously with aluminum hydroxide and killed parasites (ALP) dem onstrated significant resistance (p < 0.001) to leishmanial infection as compared to controls receiving adjuvant alone (AL), parasite alone (P), or untreated mice (table I). The administratio n of a lu m in u m h y d ro x id e alone re s u lte d in a n o n sp e c ific im m une response against L. donovani infection as evidenced by reduced parasite burdens in AL mice as com pared to untreated animals (p < 0.05), but this non-specific protection was no longer significant at day 28. The ALP group, however, still exhibited resistance against infection at day 28 post-challenge, and this protection was significant compared to all control groups (p < 0.001). Results of mice im munized with the ALP protocol, compared to mice receiving glucan and killed parasites (GP), are shown in table II. Controls were immunized subeutaneo usly w ith adjuvant alone (G or AL), killed parasites alone (P), or were untreated. At day 14 postchallenge, both the ALP and GP preparations resulted in a significant reduction T A B L E I Resistance Against LeM>kma.yu.a. donovani Elicited by an Aluminum Hydroxide and Killed Parasite Vaccine Hepatic Amastigote Burdens (X 10^) Group Day 14 Day 28 ALP *5.95 ± 0.6 *3.91 ± 0.9 AL *17.61 + 2.1 12.37 ± 1.9 P 22.62 ± 2.9 10.05 ± 2.4 NT 22.66 ± 2.3 13.78 ± 1.6 Mice received three subcutaneous injections at four day intervals followed by intravenous challenge (107 viable promastigotes per mouse) 21 days later. ALP: aluminum hydroxide gel (25 mg) with killed parasites (10^); AL: aluminum hydroxide alone; P: killed parasites alone; NT: untreated. Results reported as mean ± standard error; N = 7 mice per group. *p < 0.05 compared to untreated controls. TABLE I I Comparison of Vaccines Combining Killed Parasites with Aluminum Hydroxide or with Glucan Hepatic Amastigote Burdens (X 107) Group Day 14 Day 28 I Adjuvant Groups GP *4.80 + 0.75 3.10 + 0.81 ALP *4.88 + 0.52 *0.73 + 0.30 II Control Groups AL 7. 33 + 0.63 *1.73 + 0.29 G 6.10 + 0.53 4.93 + 2.1 P 5.11 + 0.63 2.64 + 0.58 NT 6. 76 + 0.57 3.48 + 0.61 Mice received three subcutaneous injections of adjuvant alone (G = 0.6 mg; AL = 25 mg), killed promastigotes alone (10^), killed promastigotes plus adjuvant, or were untreated. Injections were given at four day intervals prior to challenge with 107 viable promastigotes 21 days after the last immunization. AL: aluminum hydroxide gel, G: glucan, P: killed parasites, NT: untreated. Results reported as mean ± standard error; N = 7 mice per group. *p < 0.05 as compared to untreated controls. in hepatic am astigote proliferation in recipients as compared to all controls (p < 0.05). The level of resistance induced in the recipients of the two different immunization protocols was similar. By day 28 post-challenge, however, the GP group no longer dem onstrated a difference in amastigote infection as compared to untreated mice or other controls. Both the ALP (p < 0.001) and AL (p < 0.01) treatments still provided significant protection against infection at day 28, again demonstrating that the administration of alum inum hydroxide alone may elicit non-specific resistance against L. donovani. Mice immunized with 25 mg Al combined with killed parasites exhibit optimal resistance to L. donovani challenge as compared to groups receiving lesser dosages (12.5 mg or 6.25 mg) of the adjuvant (p < 0.01) as shown in table III. Mice immunized with 12.5 mg of aluminum hydroxide and killed parasites also d em o nstrated increased resistance

Group LEISHMANIA DONOVANI RESISTANCE BY ALUMINUM HYDROXIDE 75 T A BLE III Potentiation of Resistance Against LesiihmarUa donova.ru. as a Function of Dose Response to Aluminum Hydroxide Hepatic Parasite. Burden (X 10&) Day 30 a l 25p *18.6 ± 5.5 ALi2P *30.5 ± 7.7 AL6P 30.0 ± 9.9 P 50.7 ± 9.5 NT 121.7 ± 59.1 Mice received three subcutaneous injections at weekly intervals combining killed parasites with aluminum hydroxide (25 mg, 12.5 mg or 6.25 mg respectively). Intravenous challenge (107 promastigotes per mouse) was 30 days post immunization. Results are reported as mean ± standard error; N = 7 mice per group. *p < 0.05 as compared to untreated controls. against infection; however, such resistance was not as significant as that evidenced in mice receiving the higher dosage of adjuvant (p < 0.05). H epatic amastigote burdens of mice immunized with 6.25 mg Al and killed parasites did not differ from those of untreated controls. Discussion This report demonstrates that immunization against Leishmania donovani using killed parasites and alum inum hydroxide in a subcutaneous immunization protocol may be a practical method for the prevention of visceral leishmaniasis. The feasibility of immunoprophylaxis as a method of control for leishmaniasis is not new. Im munization against Leishmania tropica with live cultures has been evaluated previously, and successful field trials of frozen stored vaccine have been undertaken in Israel.10 Cutaneous leishm aniasis still rem ains the only protozoan disease for which vaccination is practical; however, other experim e n ta l im m u n iz a tio n tria ls h av e recently met with some success. Howard et al utilized irradiated (150 Krad) L. tropica promastigotes in an intravenous protocol to induce protection against challenge in Balb/c mice, assessing protection both by healing of the lesion and survival of the mouse.5 Our laboratory has previously demonstrated protection against L. donovani challenge using a subcutaneous immunization protocol, combining the adjuvant glucan with killed promastigotes. In this and a previous study employing an intravenous protocol,4 no protection resulted from the administration of dead promastigotes alone. The present study confirms that the inclusion of an adjuvant is essential for protection to be conferred against L. donovani in our model. The experiments described herein assess the resistance resulting from the administration of a protocol combining aluminum hydroxide and killed parasites and compare the results of that subcutaneous immunization protocol to one utilizing glucan as the adjuvant. The glucan and killed parasite protocol provided significant protection to recipients at day 14 post-challenge; how ever, resistan ce against infection was no longer evident at day 28. Protection provided by the alum inum hydroxide and killed parasite protocol was significant throughout the entire time course of our study. The mechanisms responsible for the success of alum inum hydroxide as an adjuvant still remain to be identified. It is known that cellular responses to the in jectio n of antig en in alu m in a gel include granulom a form ation and the influx of effector cells such as macrophages and plasma cells.14 Antibody production probably plays a limited role in eliciting protection against visceral leishmaniasis, as it appears that cell mediated im m une m echanism s are of prim ary im portance in controlling infection,15 and in promoting vaccine-induced resistance.6 The im m une response w hich occurs when an adjuvant combined with parasite antigen is processed by macrophages ultimately augments the lympho

76 JARECKI-BLACK, HALLMAN, JAMES, AND GLASSMAN cytic immune response, resulting in the proliferation and differentiation of these cells. Lym phocytes activated in this m anner produce soluble factors, and the production of such factors also results in the activation of m acrophages.9 Such activated phagocytes are able to kill Leishmania amastigotes which otherwise can survive and replicate in resident macrophages.13 The present study confirms that the adm inistration of aluminum hydroxide with a killed parasite vaccine results in decreased infection in vivo. Additionally, the presentation of leishmanial antigens in the context of a killed parasite vaccine should prom ote the macrophage processing essential for enhanced immune responsiveness. A further advantage of the adjuvanticity of aluminum hydroxide is its availability and proven safety. Although limited human trials utilizing glucan have been conducted,11 it is presently not approved for other than experim ental use. A lum inum hydroxide is already available as a commercial adjuvant, and has proven to be safe when used with antigens such as tetanus and diptheria toxoids.18 Antibody responses elicited by an aluminum hydroxide-absorbed cholera vaccine have also been shown to be higher and longer lasting than those following administration of the plain vaccin e.8 In d eed, the effectiveness and safety of aluminum hydroxide as an adjuvant has not exceeded.2 It has been shown by the present authors that an im m unization protocol combining killed parasites with the adjuvant aluminum hydroxide can successfully induce resistance against L. donovani infection. For such immunoprophylaxis to be acceptable, however, immunization must be as innocuous as possible. Protection elicited by a subcutaneous immunization protocol appears to have considerable practical advantages for attainment of such a goal, and th e d em o n strated ad ju v an ticity and safety of aluminum hydroxide suggest that further evaluation of this vaccine is deserved. Acknowledgments Thanks are extended to Mrs. Emily Davidson for editorial services and to Dr. Janies Liberatos for helpful discussion. This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. References 1. G r e e n, M. D., K a r k, J. D., W it z t u m, E., G r e en b l a t t, C. L., and Sp ir a, D. T.: Frozen stored Leishmania tropica vaccine: The effect of dose, route of administration and storage on the clinical lesion. Trans. Royal. Soc. Trop. Med. Hyg. 77:152-159, 1983. 2. H lllem AN, M. R.: Newer directions in vaccine d e v elo p m en t and u tiliz a tio n. J. Inf. D is. 252:407-20, 1985. 3. H olbrook, T. W. and C oo k, J. A.: Immunization of mice against Leishmania donovani by subcutaneous injections of dead promastigotes. Amer. J. Trop. Med. H yg. 32:51-53, 1983. 4. H o l b r o o k, T. W., C o o k, J. A., a n d Pa r k e r, B. W.: Im m unization against Leishmania donovani: glucan as an adjuvant w ith killed p ro m astigotes. Am er. J. Trop. M ed. H yg. 30:762-768, 1981. 5. H o w a r d, J. G., L i e w, F. Y., H a l e, C. and N ic k l in, S.: Prophylactic immunization against experimental leishmaniasis. II. Further characterization of the protective im m unity against fatal Leishmania tropica infection induced by irradiated promastigotes. J. Immun. 732:450-455, 1984. 6. J a r e c k i-b la c k, J. C., G l a ssm a n, A. B., and J a m e s, E. R.: Adoptive transfer of vaccineinduced resistance to Leishm ania donovani. Amer. J. Trop. Med. Hyg. 34:1095-1097, 1985. 7. Ja r e c k i-b l a c k, J. C., Ja m e s, E. R., K ir s h t e in, J. W., K ir s h t e in, J. D., and G l a ssm a n, A. B.: Leishmania donovani: Im m unization against infection as a function of parasite growth phase. Amer. J. Trop. Med. Hyg. 35:1117-1120, 1986. 8. Joo, I.: Antigenicity testing of plain and aluminum hydroxide-absorbed whole-cell cholera vaccines. Acta Tropica 36:151-156, 1979. 9. K e n d e, M.: Role of macrophages in the expression of immune responses. J. Amer. Vet. Med. Assoc. 182:1037-1042, 1982. 10. M a n n, H. B. and W h it n ey, D. R.: On a test of whether one of two random variables is stochastically larger than the other. Ann. Math. Stat. 28:52-54, 1947. 11. M a n s e l l, P. W. A., Ro w d e n, G., and H a m m er, G.: Clinical experience with the use of glu-

LEISHMANIA DONOVANI RESISTANCE BY ALUMINUM HYDROXIDE can. Immune Modulation and Control of Neoplasia by Adjuvant Therapy. Chiragos, M.A., ed. New York, Raven Press, 1978, pp. 225-280. 12. M a n s o n -B a h r, P. E. C.: Immunity in kala-azar. Trans. Roy. Soc. Trop. M ed. Hyg. 55:550-555, 1961. 13. N acy, C. A.: M acrophage activation to kill Leishmania donovani: Characterization of a T cell-derived factor that suppresses lymphokineinduced intracellular destruction of amastigotes. J. Immunol. 233:448-453, 1984. 14. OSEBOLD, J. W.: M echanism s of action b y im m unologic adjuvants. J. Amer. Vet. Med. Assoc. 181:9 8 3-9 8 7, 1982. 15. Poulter, L. W.: M echanisms of im m unity to leishmaniasis: Evidence for a changing basis of protection in self-limiting disease. Clin. Exp. Immunol. 39:14-26, 1980. 16. R e e d, S. G., Barral-N e t t o, M., and I n v e r so, J. A.: Treatment of experimental visceral leishmaniasis with lymphokine encapsulated in liposomes. J. Immunol. 232:3116 3119, 1984. 17. Re e s, P. H., Ka g e r, P. A., W e l l d e, B. T., and H ockm EYER, W. T.: The response of Kenyan kala-azar to treatm ent with sodium stibogluconate. Amer. J. Trop. Med. Hyd. 33:357-361, 1984. 18. Sarosa, J. A., Ba h r a w i, W., W it ja k s o n o, H., B u d ia r so, R. L. P., B r o to w a sisto B e n c ic, Z., D e W it t, W. E., and G o m e z, C. Z.: A controlled field trial of plain and alum inum hyodroxide-adsorbed cholera vaccines in Suraboya, Indonesia during 1973 75. B ull. W HO 56:619 627, 1978. 19. St ä u b e r, L. A.: Characterization of strains of Leishmania donovani. Exp. Parasit. 28:1-11, 1966. 20. Stä u b er, L. A.: Leishmaniasis in the hamster. Some Physiological Aspects and Consequences of Parasitism. Cole, W. H., ed. New Brunswick, NJ, Rutgers University Press, 1955, pp. 76-90. 21. St e c k, E. A. andklnnamon, K. E.: Leishmania donovani, Plasmodium berghei, Trypanosoma rhodesiense: Anti-protozoal effects of some amidine types. Exp. Parasit. 52:404-413, 1981.