Sphingomyelin liposome bearing whole Leishmania lysate antigens induce strong Th2 immune response in BALB/c mice

Document Type : Original Article


1 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Immunology, 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 Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Whole Leishmania lysate antigens (WLL) has been shown to be effective to tackle leishmaniasis in murine models. Although liposomes can be considered as promising vaccines, the activity of phospholipase-A (PLA) in WLL, breeds difficulties to preparing stable liposomal WLL. One strategy to overcome this shortcoming is to use lipids such as sphingomyelin (SM) which is resistant against PLA. This study aim is formulating stable SM liposomes containing WLL and comparing their adjuvant effects with another first generation vaccine , i.e. solube Leishmania Antigen (SLA) liposomes in BALB/c mice.
Materials and Methods: BALB/c mice were immunized subcutaneously, three times with 2-week intervals, with Empty-liposome (E-lipo), Particulate WLL, Liposome-WLL, Liposome-SLA and control Buffer, three times every 2-week. Protection was assessed through measuring the swollen footpads and the load of parasites in the spleen. Other factors were used to assess the response of immune system by means of IgG subclasses, IL-4 and IFN-γ levels and intracellular cytokine assay in cultured splenocytes.
Results: Although liposomal WLL were stable in terms of physicochemical properties, mice received Liposome-WLL did not reduce footpad swelling. The load of parasites in spleen and levels of IL-4- were also higher compared to other immunized groups. In terms of IgG isotypes, no considerable difference observed in mice received Liposome-WLL or other formulations.  
Conclusion: Liposome-WLL could be a suitable vaccine delivery system when a Th2 response is desired. Also, further studies are warranted to fully understand the role of sphingomyelin in inducing an immune response.


1. Croft SL, Sundar S, Fairlamb AH. Drug resistance in leishmaniasis. Clin Microbiol Rev 2006; 19:111-126.
2. Firooz A, Khamesipour A, Ghoorchi MH, Nassiri-Kashani M, Eskandari SE, Khatami A, et al. Imiquimod in combination with meglumine antimoniate for cutaneous leishmaniasis: a randomized assessor-blind controlled trial. Arch Dermatol 2006; 142:1575-1579.
3. Firooz A, Khatami A, Dowlati Y. Itraconazole in the treatment of cutaneous leishmaniasis. Int J Dermatol 2006; 45:1446-1447.
4. Khalil EA, El Hassan AM, Zijlstra EE, Mukhtar MM, Ghalib HW, Musa B, et al. Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan. Lancet 2000; 356:1565-1569.
5. Momeni AZ, Jalayer T, Emamjomeh M, Khamesipour A, Zicker F, Ghassemi RL, et al. A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran. Vaccine 1999; 17:466-472.
6. Sharifi I, FeKri AR, Aflatonian MR, Khamesipour A, Nadim A, Mousavi MR, et al. Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet 1998; 351:1540-1543.
7. Noazin S, Khamesipour A, Moulton LH, Tanner M, Nasseri K, Modabber F, et al. Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis: a meta-analysis. Vaccine 2009; 27:4747-4753.
8. Ejazi SA, Ghosh S, Bhattacharyya A, Kamran M, Das S, Bhowmick S, et al. Investigation of the antigenicity and protective efficacy of Leishmania promastigote membrane antigens in search of potential diagnostic and vaccine candidates against visceral Leishmaniasis. Parasit Vectors 2020; 13:272.
9. Hojatizade M, Badiee A, Khamesipour A, Jaafari MR. Evaluation of immune response against Leishmaniasis in BALB/c mice immunized with cationic DOTAP/DOPE/CHOL Liposomes containing soluble Leishmania major Antigens. Iran J Parasitol 2019; 14:68-77.
10. Ikeogu NM, Akaluka GN, Edechi CA, Salako ES, Onyilagha C, Barazandeh AF, et al. Leishmania immunity: advancing immunotherapy and vaccine development. Microorganisms 2020; 8:1201.
11. Afrin F, Rajesh R, Anam K, Gopinath M, Pal S, Ali N. Characterization of Leishmania donovani antigens encapsulated in liposomes that induce protective immunity in BALB/c mice. Infect Immun 2002; 70:6697-6706.
12. Rivier D, Bovay P, Shah R, Didisheim S, Mauël J. Vaccination against Leishmania major in a CBA mouse model of infection: role of adjuvants and mechanism of protection. Parasite Immunol 1999; 21:461-473.
13. Bhowmick S, Ali N. Identification of novel Leishmania donovani antigens that help define correlates of vaccine-mediated protection in visceral leishmaniasis. PLoS One 2009; 4:e5820.
14. Askarizadeh A, Badiee A, Khamesipour A. Development of nano-carriers for Leishmania vaccine delivery. Expert Opin Drug Deliv 2020; 17:167-187.
15. Zahednezhad F, Saadat M, Valizadeh H, Zakeri-Milani P, Baradaran B. Liposome and immune system interplay: challenges and potentials. J Control Release 2019; 305:194-209.
16. Ravindran R, Maji M, Ali N. Vaccination with liposomal leishmanial antigens adjuvanted with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) confers long-term protection against visceral leishmaniasis through a human administrable route. Mol Pharm 2012; 9:59-70.
17. Barenholz Y. Sphingomyelin and cholesterol: from membrane biophysics and rafts to potential medical applications. Subcell Biochem 2004; 37:167-215.
18. Semple SC, Leone R, Wang J, Leng EC, Klimuk SK, Eisenhardt ML, et al. Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity. J Pharm Sci 2005; 94:1024-1038.
19. Claassen E, Westerhof Y, Versluis B, Kors N, Schellekens M, van Rooijen N. Effects of chronic injection of sphingomyelin-containing liposomes on lymphoid and non-lymphoid cells in the spleen. Transient suppression of marginal zone macrophages. Br J Exp Pathol 1988; 69:865-875.
20. Melendez AJ. Sphingosine kinase signaling in immune cells: potential as novel therapeutic targets. Biochim Biophys Acta 2008; 1784:66-75.
21. Chavoshian O, Biari N, Badiee A, Khamesipour A, Abbasi A, Saberi Z, et al. Sphingomyelin Liposomes containing soluble Leishmania major antigens induced strong Th2 immune response in BALB/c Mice. Iran J Basic Med Sci 2013; 16:965-972.
22. Pawlowic MC, Zhang K. Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence. Mol Biochem Parasitol 2012; 186:11-20.
23. Rezvan H, Moafi M. An overview on Leishmania vaccines: a narrative review article. Vet Res Forum 2015; 6:1-7.
24. Gillespie PM, Beaumier CM, Strych U, Hayward T, Hotez PJ, Bottazzi ME. Status of vaccine research and development of vaccines for leishmaniasis. Vaccine 2016; 34:2992-2995.
25. Handman E. Leishmaniasis: current status of vaccine development. Clin Microbiol Rev 2001; 14:229-243.
26. Giunchetti RC, Reis AB, da Silveira-Lemos D, Martins-Filho OA, Corrêa-Oliveira R, Bethony J, et al. Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis. Res Vet Sci 2008; 85:106-112.
27. Firouzmand H, Badiee A, Khamesipour A, Heravi Shargh V, Alavizadeh SH, Abbasi A, et al. Induction of protection against leishmaniasis in susceptible BALB/c mice using simple DOTAP cationic nanoliposomes containing soluble Leishmania antigen (SLA). Acta Trop 2013; 128:528-535.
28. Jafari I, Heravi Shargh V, Shahryari M, Abbasi A, Jaafari MR, Khamesipour A, et al. Cationic liposomes formulated with a novel whole Leishmania lysate (WLL) as a vaccine for leishmaniasis in murine model. Immunobiology 2018; 223:493-500.
29. Working Group on Research Priorities for Development of Leishmaniasis Vaccines, Costa CH, Peters NC, Maruyama SR, de Brito EC Jr, Santos IK. Vaccines for the leishmaniases: proposals for a research agenda. PLoS Negl Trop Dis 2011; 5:e943.
30. Giunchetti RC, Corrêa-Oliveira R, Martins-Filho OA, Teixeira-Carvalho A, Roatt BM, de Oliveira Aguiar-Soares RD, et al. Immunogenicity of a killed Leishmania vaccine with saponin adjuvant in dogs. Vaccine 2007; 25:7674-7686.
31. Lasri S, Sahibi H, Sadak A, Jaffe CL, Rhalem A. Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes. Vet Res 1999; 30:441-449.
32. Mayrink W, Genaro O, Silva JC, da Costa RT, Tafuri WL, Toledo VP, et al. Phase I and II open clinical trials of a vaccine against Leishmania chagasi infections in dogs. Mem Inst Oswaldo Cruz 1996; 91:695-697.
33. Soto M, Ramírez L, Pineda MA, González VM, Entringer PF, de Oliveira CI, et al. Searching genes encoding Leishmania antigens for diagnosis and protection. Scholarly Res Exch 2009; 2009:173039.
34. Moafi M, Rezvan H, Sherkat R, Taleban R. Leishmania vaccines entered in clinical trials: a review of literature. Int J Prev Med 2019; 10:95.
35. Saleem K, Khursheed Z, Hano C, Anjum I, Anjum S. Applications of nanomaterials in Leishmaniasis: a focus on recent advances and challenges. Nanomaterials 2019; 9:1749.
36. Silverman JA, Deitcher SR. Marqibo®(vincristine sulfate liposome injection) improves the pharmacokinetics and pharmacodynamics of vincristine. Cancer Chemother Pharmacol 2013; 71:555-564.
37. Bhat HB, Kishimoto T, Abe M, Makino A, Inaba T, Murate M, et al. Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains. J Lipid Res 2013; 54:2933-43.
38. Hafez IM, Maurer N, Cullis PR. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther 2001; 8:1188-1196.
39. Brgles M, Habjanec L, Halassy B, Tomasić J. Liposome fusogenicity and entrapment efficiency of antigen determine the Th1/Th2 bias of antigen-specific immune response. Vaccine 2009; 27:5435-5442.
40. Copland MJ, Rades T, Davies NM, Baird MA. Lipid based particulate formulations for the delivery of antigen. Immunol Cell Biol 2005; 83:97-105.
41. Ignatius R, Mahnke K, Rivera M, Hong K, Isdell F, Steinman RM, et al. Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo. Blood 2000; 96:3505-3513.
42. London E. How principles of domain formation in model membranes may explain ambiguities concerning lipid raft formation in cells. Biochim Biophys Acta 2005; 1746:203-20.
43. Pralle A, Keller P, Florin EL, Simons K, Hörber JK. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol 2000; 148:997-1008.
44. Lonez C, Vandenbranden M, Ruysschaert JM. Cationic liposomal lipids: from gene carriers to cell signaling. Prog Lipid Res 2008; 47:340-7.
45. Ballou LR, Laulederkind SJ, Rosloniec EF, Raghow R. Ceramide signalling and the immune response. Biochim Biophys Acta 1996; 1301:273-287.
46. Maceyka M, Spiegel S. Sphingolipid metabolites in inflammatory disease. Nature 2014; 510:58-67.
47. El Alwani M, Wu BX, Obeid LM, Hannun YA. Bioactive sphingolipids in the modulation of the inflammatory response. Pharmacol Ther 2006; 112:171-183.
48. Chalfant CE, Spiegel S. Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling. J Cell Sci 2005; 118:4605-4612.
49. Ohanian J, Ohanian V. Sphingolipids in mammalian cell signalling. Cell Mol Life Sci 2001; 58:2053-2068.
50. Martinova EA. Influence of sphingolipids on T lymphocyte activation. Biochemistry (Mosc) 1998; 63:102-110.
51. Pettus BJ, Chalfant CE, Hannun YA. Sphingolipids in inflammation: roles and implications. Curr Mol Med 2004; 4:405-418.
52. Li SY, Chen C, Zhang HQ, Guo HY, Wang H, Wang L, et al. Identification of natural compounds with antiviral activities against SARS-associated coronavirus. Antiviral Res 2005; 67:18-23.
53. Xu Y, Casey G, Mills GB. Effect of lysophospholipids on signaling in the human Jurkat T cell line. J Cell Physiol 1995; 163:441-450.
54. Hauser JM, Buehrer BM, Bell RM. Role of ceramide in mitogenesis induced by exogenous sphingoid bases. J Biol Chem 1994; 269:6803-6809.
55. Feng Z, Lai Y, Ye H, Huang J, Xi XG, Wu Z. Poly (γ, L-glutamic acid)-cisplatin bioconjugate exhibits potent antitumor activity with low toxicity: a comparative study with clinically used platinum derivatives. Cancer Sci 2010; 101:2476-2482.
56. Choi CM, Lerner EA. Leishmaniasis as an emerging infection. J Investig Dermatol Symp Proc 2001; 6:175-182.
57. Nixon GF. Sphingolipids in inflammation: pathological implications and potential therapeutic targets. Br J Pharmacol 2009; 158:982-993.
58. Bartlett G.R. Phosphorous assay in column chromatography. J Biol Chem 1959; 234: 466-468.