Abstract Infections caused by the protozoan parasite Leishmania include cutaneous (CL), mucosal (ML), and visceral leishmaniasis (VL). Over 12 million people currently suffer from leishmaniasis, and approximately 2 million new cases occur annually. Currently no vaccine is available for this disease for humans. However, patients who recover from leishmaniasis develop immunity against reinfection indicating that a vaccine is feasible. In the past, leishmanization, a process in which deliberate infection with a low dose of Leishmania major, etiologic agent of zoonotic cutaneous leishmaniasis (ZCL) causes a controlled skin lesion and provides > 90% protection against reinfection, was a common practice. Although such practice may not be acceptable under the current regulatory environment due to possibility of complications, these observations suggest that live- attenuated parasites that provide a complete array of antigens without causing disease could be an effective vaccine for leishmaniasis. Genetically attenuated L. infantum and L. donovani have shown promise as a vaccine in animal models. However, using these parasites in humans could raise safety concerns due to their visceralizing potential. Attenuated dermatotrophic Leishmania that cross-protects against VL could be a safer vaccine because potential adverse events (e.g. development of a lesion at vaccination site) can be easily monitored and effectively treated using approved topical interventions. Several clinical as well as animal studies have shown that an infection with dermatotrophic Leishmania such as L. major or immunization with antigens from these parasites confers significant cross-protection against VL. However, it is not known whether immunization with attenuated cutaneous disease causing species such centrin gene deficient L. major will protect against VL. Using CRISPR-Cas technology, we have generated antibiotic selection marker free centrin gene deficient L. major (LmCen-/-). In this project, we propose to use a novel canine model of VL to test the safety and efficacy of GLP-grade LmCen-/-. Whole genome sequencing of LmCen-/- has confirmed stable deletion of centrin gene without other mutations in the parasite genome. Our preliminary findings show that LmCen-/- are highly attenuated and fail to cause disease in immunocompromised mice. We have also found that immunization with LmCen-/- parasites induces a disease protective Th1 response in hamster as well as mice and completely protects against homologous challenge with virulent L. major. Our industry partner Gennova Biopharma has already established LmCen-/- production under GLP conditions at their US-FDA approved facility. In this project, we propose to (Aim 1) optimize GLP-LmCen-/- immunogenicity and immunization protocol and determine its safety in dogs and (Aim 2) evaluate efficacy of GLP-LmCen-/- as a vaccine using a novel model of canine VL in which dogs are naturally exposed to bites of L. infantum infected wild Phlebotomus pernicious in VL- hyperendemic regions of Tunisia. The scientific promise of this project, if successful, could provide the foundation for advancing LmCen-/- parasites as a vaccine against leishmaniasis in humans.