The role of ISCOMATRIX bilayer composition to induce a cell mediated immunity and protection against leishmaniasis in BALB/c mice

Document Type: Original Article


1 Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Biotechnology Research Center, Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Immunology Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran

5 Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran


Objective(s):Development of new generation of vaccines against leishmaniasis is possible because long-term protection is usually seen after recovery from cutaneous leishmaniasis. ISCOMATRIX is particulate antigen delivery system composed of antigen, cholesterol, phospholipid and saponin. In this study, the role of ISCOMATRIX bilayer composition made by different phase transition temperature (Tc) to induce a type of immune response and protection against leishmaniasis was assessed.
Materials and Methods:ISCOMATRIX formulations with different bilayer compositions consisting of EPC (Tc <0 C), DMPC (Tc 23 C) and DSPC (Tc 54 C) were prepared. Different ISCOMATRIX formulations were mixed with soluble Leishmania antigens (SLA). BALB/c mice were immunized subcutaneously, three times with 2-week intervals.  As criteria for protection, footpads swelling, parasite burden, determination of IgG isotypes and the level of IFN-γ and IL-4 were assessed.
Results: Although the groups of mice immunized with ISCOMATRIX DMPC or ISCOMATRIX DSPC showed the smallest footpad swelling and least parasite burden compared with the buffer group, the difference was not significant. Moreover, the highest level of IFN- γ and IL-4 was observed in the splenocytes of mice immunized with ISCOMATRIX DMPC or ISCOMATRIX DSPC, respectively. After challenge, the mice immunized with ISCOMATRIX DSPC showed the highest elevation of IgG, IgG1 and IgG2a antibodies (P<0.01) compared with control group. However, our results indicated that ISCOMATRIX EPC, DMPC or DSPC generated a mixed Th1/Th2 response that was not protective.
Conclusion: Our results showed that the adjuvanticity of prepared ISCOMATRIX doesn’t influence with different phospholipids at least in our mice model.


1. World Health Organization. Control of the leishmaniases. World Health Organ. Tech Rep Ser 2010; 1- 186.

2. World Health Organization. Control of the Leishmaniases. Report of a WHO expert committee. Technical report series 1990; No. 793.

3. Alvar J, Croft S, Kaye P, Khamesipour A, Sundar S, Reed SG. Case study for a vaccine against leishmaniasis.Vaccine 2013; 31: 244-249.

 4. Thakur CP, Kumar K. Post Kala-azar dermal leishmaniasis: a neglected aspect of kala-azar control programmes. Ann Trop Med Parasitol 1992; 86:355-359.      

5. Bryceson A. A policy for leishmaniasis with respect to the prevention and control of drug resistance. Trop Med Int Health 2001; 6:928–934.

6. Croft SL, Coombs GH. Leishmaniasis current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 2003; 19:502–508.

7. Hadighi R, Mohebali M, Boucher P, Hajjaran H, Khamesipour A, Ouellette M. Unresponsiveness to glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leishmania tropica parasites. Plos Med 2006; 3:659–667.

8. Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2002; 2:845–858.

9. Grencis RK. Th2-mediated host protective immunity to intestinal nematode infections. Philos Trans R Soc Lond B Biol Sci.1997; 352:1377–1384.

10. Modabber F. Vaccines against leishmaniasis. Ann Trop Med Parasitol 1995; 89: 83–88.

11. Khamesipour A, Dowlati Y, Asilian A, Hashemi-Fesharki R, Javadi A, Noazin S, et al.  Leishmanization: use of an old method for evaluation of candidate vaccines against leishmaniasis. Vaccine 2005; 23: 3642–3648.

12. Modabber F, Coler R, Reed SG. Vaccines against Leishmania. In: Modabber, F. (Ed.), New Generation Vaccines. E-Publishing Inc, New York 2009; 273–283.

13. Mutiso JM, Macharia JC, Mutisya RM, Taracha E. Subcutaneous immunization against Leishmania major  infection in mice. Efficacy of formalin-killed promastigotes combined with adjuvants. Rev. Inst. Med. Trop. Sao Paulo 2012; 52: 95–100.

14. Copland MJ, Rades T, Davies NM, Baird MA. Lipid based particulate formulations for the delivery of antigen. Immunol Cell Bio 2005; 83:97–105.

15. Badiee A, Heravi SV, Khamesipour A, Jaafari MR. Micro/nanoparticle adjuvants for anti leishmanial vaccines: present and future trends. Vaccine 2013; 31: 735-749.

16. Hong-Xiang S, Yong X, Yi-Ping Y. ISCOMs and ISCOMATRIX TM .  Vaccine 2009; 27: 4388–4401.

17. Rimmelzwaan GF, Osterhaus ADME. A novel generation of viral vaccines based on ISCOM matrix. In Vaccine Design, the Subunit and Adjuvant Approach (M. F. Powell and M. J. Newman, eds.), New York: Plenum 1995; 543–558.

18. Lövgren K, Morein B, Osterhaus A. ISCOM technology-based Matrix M™ adjuvant: success in future vaccines relies on formulation. Expert Rev Vaccines 2011; 10: 401-403.

19. Sanders MT, Brown LE, Deliyannis G, Pearse MJ. ISCOM-based vaccines: the Second decade. Immunol Cell Biol 2005; 83:119–128.

20. Sjölander A, Drane D, Maraskovsky E, Scheerlinck JP, Suhrbier A, Tennent A, et al. Immune responses to ISCOM formulations in animal and primate models. Vaccine 2001; 19:2661–2665.

21. Barr IG, Sjolander A, Cox JC. ISCOMs and other saponin based adjuvants. Adv Drug Del Rev 1998; 32: 247-271.

22. Sjolander A, Cox JC, Barr IG. ISCOMs: an adjuvant with multiple functions. J leukoc Biol 1998; 64:713-723.

23. Cox JC. Drane D, Suhrbier A. Immunogenic complex and methods relating thereto 2000; PCT/AU00/00110.

24. Behboudi S, Morein B, Vilacres-Eriksson M. In vitro activation of antigen-presenting cells (APC) by defined composition of Quillaja saponaria Molina triterpenoids.  Clin Exp Immunol 1996; 105:26-30.

25. Khamesipour A, Rafati S, Davoudi N, Maboudi F, Modabber F. Leishmaniasis vaccine candidates for development: a global overview. IJMR 2006; 123: 423–438.

26. Scott P, Pearce E,  Natovitz  P, Sher A. Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J Immunol 1987;139: 221-227.

27. Bainor A, Chang L, McQuade TJ, Webb B, Gestwicki JE. Bicinchoninicacid (BCA) assay in low volume. Anal Biochem 2010; 410: 310–402.

28. Liang MT,  Toth I, Davies NM. A novel method for preparing immune stimulating complexes (ISCOMs) by hydration of freeze-dried lipid matrix. Eur J Pharm Biopharm 2008; 68: 840–845.

29. Jaafari MR, Ghafarian A, Farrokh-Gisour A, Samiei A, Kheiri MT, Mahboudi F, et al. Immune response and protection assay of recombinant major surface glycoprotein of leishmania (rgp63) reconstituted with liposomes in BALB/c mice. Vaccine 2006; 24:5708–5717.

30. Titus RG, Marchand M, Boon T, Louis JA. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol 1985; 7: 545–555.

31. Badiee A, Jaafari MR, Khamesipour A. Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes. Exp Parasitol 2007; 115:127–134.

32. Taswell C. Limiting dilution assays for the determination of immuno competent cell frequencies. 1. Data analysis. J Immunol 1981; 126:1614–1619.

33. Jaafari MR, Badiee A, Khamesipour A, Samiei A, Soroush D, Kheiri MT, et al. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome. Vaccine 2007; 25: 6107–6117.

34. Golali E, Jaafari MR, Khamesipour A, Abbasi A, Saberi Z, Badiee A. Comparison of in vivo Adjuvanticity of Liposomal PO CpG ODN with Liposomal PS CpG ODN: Soluble Leishmania Antigens as a Model. Iran J Basic Med Sci 2012; 15:1032-1045.

35. Hejazi H, Tasbihi M, Jaafari M, Badiee A, Pestechian N, Javadi A, et al. The role of liposomal CpG ODN on the course of L. major infection in BALB/C mice. IJP 2010; 5:47-54.

36. Vanloubbeeck Y, Jones DE. The immunology of Leishmania infection and the implications for vaccine development. Ann N Y Acad Sci 2004; 1026: 267-272.    

37. Drane D, Gittleson C, Boyle J, Maraskovsky E. ISCOMATRIX adjuvant for prophylactic and therapeutic vaccines. Expert Rev Vaccines 2007; 6: 761–772.

38. Silva A, Mount A, Krstevska K, Pejoski D, Hardy MP, Owczarek C, et al. The combination of ISCOMATRIX adjuvant and TLR agonists induces regression of established solid tumors in vivo. J Immunol 2015; 194: 2199-2207.

39. Bontempi IA, Vicco MH, Cabrera G, Villar SR, González FB, Roggero EA, et al. Efficacy of a trans-sialidase-ISCOMATRIX subunit vaccine candidate to protect against experimental Chagas disease. Vaccine 2015; 33: 1274-1283.

40. Sjölander A, Baldwin TM,  Curtis JM,  Bengtsson KL, Handman E. Vaccination with recombinant Parasite Surface Antigen 2 from Leishmania major induces a Th1 type of immune response but does not protect against infection. Vaccine 1998; 16:2077-2084.

41. Papadopoulou G, Karagouni E, Dotsika E. ISCOMs vaccine against experimental leishmaniasis. Vaccine 1998; 16: 885-892.

42. Kensil CR. Saponins as vaccine adjuvants. Crit. Rev. Ther. Drug Carrier Syst 1996; 13:1–55.

43. Rönnberg B,  Fekadu M,  Morein B. Adjuvant activity of non-toxic Quillaja saponaria Molina components for use in ISCOM matrix. Vaccine 1995; 13:1375–1382.

44. Drane D, Pearse M. The ISCOMATRIX adjuvant. In: Schijns VE, O’Hagan DT (eds). Immunopotentiators  in Modern Vaccines. Elsevier Academic Press: Amsterdam; Boston 2006; 191–216.