Leishmaniasis is a debilitating and often fatal neglected tropical disease caused by 29 Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with 30live Leishmania major has been used successfully but is no longer practiced because it resulted in 31 unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR 32 genome edited L. major strain (LmCen -/-). Notably, LmCen -/is the first genetically engineered 33 gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-34 target mutations. Mice immunized with LmCen -/had virtually no visible lesions following 35 challenge with L. major-infected sand flies while non-immunized animals developed large and 36 progressive lesions with a 2-log fold higher parasite burden. LmCen -/immunization showed 37 protection and an immune response comparable to leishmanization. LmCen -/is safe since it was 38 unable to cause disease even in immunocompromised mice, induces robust host protection against 39 vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials. 40 41 42 Leishmaniasis is a neglected disease caused by infection with protozoans of the genus Leishmania 43 that is transmitted by infected sand flies 1 . Worldwide, an estimated 1 billion people are at risk of 44 infection in tropical and subtropical countries where up to 1.7 million new cases in 98 countries 45 occur each year 2,3 . The disease pathology ranges from localized skin ulcers (cutaneous 46 leishmaniasis, CL) to fatal systemic disease (visceral leishmaniasis, VL), depending on the species 47 of the infecting Leishmania parasite 1,4 . Treatment options for both VL and CL are limited and there 48 is poor surveillance in the most highly endemic countries 1,5 . A prophylactic vaccine would be an 49 effective intervention for protection against this disease, reducing transmission and supporting the 50 elimination of leishmaniasis globally. Currently there are no available vaccines against any form 51 of human leishmaniasis.
52Unlike most parasitic infections, patients who recover from leishmaniasis naturally or following 53 drug treatment develop immunity against reinfection indicating that the development of an 54 effective vaccine should be feasible 6-8 . Furthermore, leishmanization, a process in which 55 deliberate infections with a low dose of virulent Leishmania major provides greater than 90% 56 protection against reinfection and has been used in several countries of the Middle East and the 57 former Soviet Union 9-11 . Leishmanization is however no longer practiced because it is ethically 58 unacceptable due to the resulting skin lesions that last for months at the site of inoculation. The 59 overall strategy of this study is to develop the next generation leishmanization that is safer by 60 providing a protective immune response against cutaneous leishmaniasis without causing skin 61 lesions.
4In case of leishmaniasis cell mediated immunity is critical, and...
