Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis


1 Department of Parasitology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz; Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Center for Research and Training in Skin Disease and Leprosy, Tehran University of Medical Sciences, Iran

4 Department of Parasitology and Mycology, School of Medicine; Skin Disease and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM) and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM). In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Materials and Methods: L. major (MRHO/IR/75/ER) from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN) medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS) 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins.
Results: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major.
Conclusion: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.


Alvar J, Vélez I, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis Worldwide and Global estimates of its incidence. PLoS ONE 2012;7:e35671.  Back to cited text no. 1
Alvar J, Croft S, Kaye P, Khamesipour A, Sundar S, Reed S. Case study for vaccine against leishmaniasis. Vaccine 2013;31:B244-9.  Back to cited text no. 2
Firooz A, Khamesipour A, Ghoorchi M, Nassiri-Kashani M, Eskandari S, Khatami A, et al. Imiquimod in combination with meglumine antimoniate for cutaneous Leishmaniasis: A randomized assessor-blind controlled trial. Arch Dermatol 2006;142:1575-9.  Back to cited text no. 3
Hadighi R, Boucher P, Khamesipour A, Meamar A, Roy G, Ouellette M, et al. Glucantime-resistant Leishmania tropica isolated from Iranian patients with cutaneous Leishmaniasis are sensitive to alternative anti Leishmania drugs. Parasitol Res 2007;101:1319-22.  Back to cited text no. 4
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:e162.  Back to cited text no. 5
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;23:3642-8.  Back to cited text no. 6
Khamesipour A, Rafati S, Davoudi N, Mahboudi F, Modabber F. Leishmaniasis Vaccine Candidates for Development: Global Overview. Indian J Med Res 2006;123:423-38.  Back to cited text no. 7
[PUBMED]  Medknow Journal  
Mahmoodi M, Khamesipour A, Dowlati Y, Rafati S, Momeni A, Emamjomehi M, et al. Immune response measured in human volunteers vaccinated with autoclaved Leishmania major vaccine mixed with low dose of BCG. Clin Exp Immunol 2003;134:303-8.  Back to cited text no. 8
Clarisa B, Palatnik-de-Sousa. Vaccines for leishmaniasis in the fore coming 25 years. Vaccine 2008;26:1709-24.  Back to cited text no. 9
Shama U, Singh S. Immunobiology of Leishmaniasis. Indian J Exp Bio 2009;47:412-23.  Back to cited text no. 10
Fesharki RH, Agha SA, Ahourai P, Djavadian E, Taghavi A, Golabi A, et al. Vaccine preparation and quality control of killed Leishmania major. Arch Inst Razi 1992;43:39-50.  Back to cited text no. 11
Chakavarti B, Chakavarti D. Electrophoretic separation of proteins. J Vis Exp 2008;16:758.  Back to cited text no. 12
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;27:4747-53.  Back to cited text no. 13
Noazin S, Modabber F, Khamesipour A, Smith P, Moulton LH, Nasseri K, et al. First generation Leishmaniasis vaccines: A review of field efficacy trials. Vaccine 2008;26:6759-67.  Back to cited text no. 14
Bahar K, Dowlati Y, Shidani B, Alimohammadian M, Khamesipour A, Ehsasi S, et al. Comparative safety and immunogenicity trial of two killed Leishmania major vaccines with or without BCG in human volunteers. Clin Dermatol 1996;14:489-95.  Back to cited text no. 15
Rhee EG, Mendez S, Shah JA, Wu C-y, Kirman JR, Turon TN, et al. Vaccination with heat-killed Leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J Exp Med 2002;195:1565-73.  Back to cited text no. 16
Okwor I, Kuriakose S, Uzonna J. Repeated inoculation of killed Leishmania major induces durable immune response that protects mice against virulent challenge. Vaccine 2010;28:5451-7.  Back to cited text no. 17
Ogunkolade B, Monjour L, Vouldoukis I, Rhodes-Feuillette A, Frommel D. Inoculation of BALB/c mice against Leishmania major infection with Leishmania-derived antigens isolated by gel filtration. J Chromatogr 1998;440:459-65.  Back to cited text no. 18
Hejazi S, Soleymanifard S, Yousefi H, Ghavaminejad A. Investigation and comparison of L.major and L.gerbilli protein content by sodium dodecyl sulfate polyacrylamide gel electrophoresis. J Isfahan Med Sch 2000;17:14-7.  Back to cited text no. 19
Male D, Brostoff J, Roth D, Roitt I. Immunology. (seventh ed.)Mosby, Philadelphia 2006.  Back to cited text no. 20