Abstract Leishmaniasis is a debilitating and often fatal neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain ( LmCen -/- ). Notably, LmCen -/- is the first genetically engineered gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-target mutations. Mice immunized with LmCen -/- had virtually no visible lesions following challenge with L. major -infected sand flies while non-immunized animals developed large and progressive lesions with a 2-log fold higher parasite burden. LmCen -/- immunization showed protection and an immune response comparable to leishmanization. LmCen -/- is safe since it was unable to cause disease even in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

A Second Generation Leishmanization Vaccine with a Markerless Attenuated Leishmania major Strain using CRISPR gene editing