Abstract Infections caused by the protozoan parasite Leishmania include cutaneous (CL), mucosal (ML), and visceral leishmaniasis (VL). The WHO classifies leishmaniasis as a neglected tropical disease with over 12 million current infections globally, and approximately 2 million new cases annually. Patients who recover from leishmaniasis develop protective immunity against reinfection, which altogether indicates that a vaccine is feasible. In the past, leishmanization, a process in which deliberate infections 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 in ZCL-endemic regions. Under the current regulatory environment such practice is not acceptable due to possibility of complications including non-healing lesions. However, these studies suggest that live-attenuated parasites which don not cause a disease could be an effective vaccine for leishmaniasis. Genetically attenuated L. infantum and L. donovani including LdCen-/- have shown promise as a vaccine in animal models. However, using live-attenuated L. donovani as a vaccine in humans could raise safety concerns due to visceralizing potential of this Leishmania species. Attenuated dermatotrophic Leishmania that cross-protects against VL could be a safer vaccine against Leishmania because adverse events (e.g. development of a lesion as vaccination site) will be easy to monitor and can be treated using approved non-pharmacological interventions such as topical thermotherapy. Several clinical and preclinical animal studies have shown that an infection with dermatotrophic Leishmania such as L. major and L. tropica confers cross-protection against VL caused by L. donovani or L. infantum. Using CRISPR-Cas technology, we have generated antibiotic selection marker free centrin gene deficient L. major (LmCen-/-). Whole genome sequencing of LmCen-/- passed through mice multiple times 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 animals. We have also found that immunization with LmCen-/- parasites induces disease protective Th1 response in hamsters and protects them against sand fly transmitted VL caused by L. donovani. Similarly, LmCen-/- immunization also protects against CL caused by L. major. These findings indicate that LmCen-/- is a promising vaccine for leishmaniasis. Our industry partner Gennova Biopharma has already established LmCen-/- production under cGMP conditions at their US-FDA approved facility. In this project, we propose to (Aim 1) perform phenotypic, genetic and biochemical characterization of GMP-LmCen-/-parasites and (Aim 2) determine whether GMP-LmCen-/- induce biomarkers of protection in peripheral blood mononuclear cells (PBMCs) from the patients with active VL, asymptomati...