Leishmania chagasiT-Cell Antigens Identified through a Double Library Screen

Abstract

Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1␥, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L.

Identification of Differentially Expressed Proteins from Leishmania amazonensis Associated with the Loss of Virulence of the Parasites

“…a ) Spots match ID number obtained from ImageMaster Platinum;b ) Name of the identified protein;c ) Uniprot identification code;d ) Experimentally predicted and expected isoelectric point ( pI );e ) Experimentally predicted and expected molecular weight ( Mr , in kDa);f ) Number of identified peptides by MS;g ) Percentage of the protein sequence covered by identified peptides;h ) Normalized data from R0 represented by mean values of each condition divided by R30 value;i ) Fold represents the maximum spot intensity mean value of the conditions divided by the smallest value;j ) One-way ANOVA ( P <0.01) obtained from spot analysis;k ) Biological functions according to NCBI, UniProt, and Gene Ontology databases;l ) Biological activity and/or immunological application described in other studies: [22] Tull et al, 2010; [23] Oliveira et al, 2006; [24] Daifalla et al, 2011; [25] Iyer et al, 2008; [26] Hunger-Glaser et al, 1999; [27] Werbovetz et al, 1999; [28] Feng et al, 2011; [29] Niemirowicz et al, 2007; [30] Hunger-Glaser et al, 1997; [31] Alcolea et al, 2009; [32] Bhaskar et al, 2012; [33] Khanra et al, 2012; [34] Berberich et al, 2003; [35] Moore et al, 1996; [36] Swenerton et al, 2011; [37] Hummadi et al, 2006; [38] Martins et al, 2006; [39] Burns et al, 1993; [40] Kushawaha et al, 2011; [41] Misra et al, 2005; [42] Bringaud et al, 1995; [43] Eggleson et al, 1999; [44] Mureev et al, 2007; [45] Steiner et al, 2007; [46] Martínez-Rodríguez et al, 2012; [47] Drummelsmith et al, 2004; [48] Achour et al, 2002; [49] Buda et al, 2013; [50] Alcolea et al, 2011; [51] Martín et al, 2009; [52] Silva et al, 2012. The proteins were identified through the data included in ...…”

Recovery and purification of recombinant 503 antigen of Leishmania infantum chagasi using expanded bed adsorption chromatography

“…E. coli cultivation for 503 antigen production E. coli M15 strain expressing the His-tagged 503 antigen was kindly provided by Dr. Mary Wilson from the University of Iowa (Iowa, USA) [8]. The clone was cultured in 2xTY medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl, pH 7.0) supplemented with ampicillin (0.1 g/L) and kanamycin (0.025 g/L) at 37 • C and 400 rpm on a bioreactor (Biostat B., B. Braun Biotech International) with a working volume of 1.5 L. The expression of the recombinant protein was induced by the addition of lactose to the cultivation medium at a final concentration of 10 g/L when the optical density at 600 nm reached approximately 0.5 [25].…”

“…Consequently, distinct host immune responses occur to antigens expressed during the intracellular amastigote stage, which are difficult to produce in bulk [7]. The 503 antigen, a protein with 100% identity to the elongation factor 1␥ (EF-1␥) of L. infantum, was identified through screening a cDNA library from the intracellular amastigote form of Leishmania i. chagasi [8]. This antigen constitutes a potential target for the immune response during mammalian infection, suggesting that the 503 antigen can be utilized in the development of diagnostic agents for serological tests or of vaccines against VL [8].…”

Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives

“…Importantly, the levels of anti-A2 antibodies are higher in asymptomatic animals as compared with the symptomatic ones [70,71], thus indicating a potential role in protective immunity elicited in infection with L. infantum. The A2 antigen also contains B and T cell epitopes recognized by human cells, which is an important requirement for induction of protection against leishmaniasis [64,70,72].…”