The outcome of Leishmania major experimental infection in BALB/c mice can be modulated by exogenously delivered iron.

We previously established that Leishmania promastigotes express a transferrin receptor and that iron chelators inhibit promastigote growth in vitro. Thus, we were interested in modulating the vertebrate host iron pool and to monitor whether such changes will affect the outcome of L. major infection in BALB / c mice, inoculated in the footpad with 106 stationary phase promastigotes. Treatment of mice with desferrioxamine resulted in a slight delay of the development of cutaneous lesions. In contrast and unexpectedly, systemic iron delivery, at early time points of parasite delivery, significantly limited footpad pathology. Accordingly, parasite loads at the site of parasite delivery, the draining lymph node, liver and spleen were significantly reduced in iron-loaded mice. Importantly, the "protective" effect of iron delivery correlated with the presence, at the site of inoculation, of lower levels of IL-4 and IL-10 transcripts while both IFN-gamma and inducible nitric oxide synthase transcripts were at higher levels. The presence of more type 1 cytokine transcripts was further supported by the increased levels of IgG2a in their sera. These data strongly suggest that susceptibility to L. major as assessed in the footpad model is modifiable by interventions that alter the iron status of the host at early time points of parasite delivery.

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

Use of sequential oligopeptide carriers (SOCn) in the design of potent Leishmania gp63 immunogenic peptides.

The antigenic sequence Ac-IASRYDQL (gp63-SRYD) of the major surface glycoprotein of Leishmania, gp63, was covalently attached to the Lys-N epsilon H2 groups of a new sequential oligopeptide carrier (SOCn), namely, (Lys-Aib-Gly)n (n = 5.6), in order to obtain potent immunogens and site-specific antibodies. It was shown, using 1H-NMR spectroscopy, that the gp63-SRYD octapeptides bound to the SOCn retain their original structural profile outlined by an ionic interaction between R and D side chains and a type 1 beta-turn involving the QNH-->RCO hydrogen bonding. Also, the gp63-SRYD octapeptides linked to the carrier do not experience conformational restrictions, probably because of the favorable conformation of the SOCn. Immunizations of outbred rabbits with the peptide carriers designed resulted in high-titered antibody response to the gp63-SRYD octapeptide and the gp63 cognate protein. Thus, this chemically defined model may be used for incorporating "protective" Leishmania epitopes and ultimately for the design of a multivalent synthetic vaccine against leishmaniosis.