Abstract
Leishmaniasis is a 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 occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen−/−). Notably, LmCen−/− is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen−/− have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen−/− immunization results in protection and an immune response comparable to leishmanization. LmCen−/− is safe since it is unable to cause disease 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.
Highlights
Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies
The overall strategy of this study is to develop the generation leishmanization that is safer by providing a protective immune response against cutaneous leishmaniasis without causing skin lesions
To delete the centrin gene sequence precisely at the locations determined by the 2 guide RNA sequences flanking the centrin gene without using marker gene replacement, a 50nucleotide oligonucleotide donor DNA sequence was transfected into the promastigotes containing the CRISPR expression vector pLdCNa&b as previously described[33]
Summary
Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. It has been shown in leishmanized mice that rapidly recruited short-lived effector T cells producing IFN-γ confer significant level of protection and could be used as a biomarker of host protection[22,23] These studies collectively show that any effective vaccine should maintain these antigen specific CD4 T cell populations long enough to induce a robust protection against reinfection. The presence of antibiotic resistance genes in any attenuated live vaccine renders the vaccine unacceptable by regulatory agencies for human vaccine trials To overcome these drawbacks, we used CRISPR-Cas genome editing recently established for Leishmania[32,33,34] to generate an attenuated L. major centrin gene deletion mutant (LmCen−/−). Vaccination with LmCen−/− is safe, immunogenic and protective against sand fly transmitted L. major infection, that mimics natural infection in highly relevant cutaneous leishmaniasis animal models meeting efficacy and ethical standards for advancement to human clinical studies
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