Development and Characterization of an Avirulent Leishmania major Strain

Leishmania major causes cutaneous leishmaniasis. An antileishmanial vaccine for humans is unavailable. In this study, we report development of two attenuated L. major strains—5ASKH-HP and LV39-HP—by continuous culture (high passage) of the corresponding virulent strains (low passage). Both avirulent strains showed similar changes in proteome profiles when analyzed by surface-enhanced laser desorption ionization mass spectrometry. Liquid chromatography–mass spectrometry and microarray characterization of 5ASKH strains revealed substantially altered gene and protein expression profiles, respectively. Both virulent and avirulent L. major strains grew comparably in culture, but the avirulent strain survived significantly less in BALB/c-derived peritoneal macrophages. Both attenuated strains failed to infect BALB/c mice and elicited IFN-g, but not IL-4 and IL-10, responses. 5ASKH-HP parasites failed to induce significant infection even in severely immunocompromised- SCID or inducible NO synthase–, CD40-, or IL-12–deficient mice, indicating attenuation. The avirulent strain induced less IL-10, but higher IL-12, in macrophages. The avirulent strain failed to reduce CD40 relocation to the detergent-resistant membrane domain and to inhibit CD40-induced phosphorylation of the kinases Lyn and protein kinase C-b and MAPKs MKK-3/6 and p38MAPK or to upregulate MEK-1/2 and ERK-1/2 in BALB/c-derived peritoneal macrophages. The virulent and the avirulent strains reciprocally modulated CD40-induced Ras-mediated signaling through PI-3K and Raf-1. Avirulent 5ASKH-primed BALB/c mice were protected against virulent L. major challenge infection. The loss of virulence accompanied by substantially altered proteome profiles and the elicitation of host-protective immune responses indicate plausibly irreversible attenuation of the L. major strain and its potential use as a vaccine strain.