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.

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain ofLeishmania major. Two fresh strains ofL. majorfrom Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen^−/−) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen^−/− parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen^−/− amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen^−/− in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen^−/− parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen^−/− is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen^−/− parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

The leishmanin skin test (LST) has been used for decades to detect exposure and immunity to the parasiteLeishmania, the causative agent of the neglected tropical disease leishmaniasis. In the LST,Leishmaniaantigen (leishmanin) is intradermally injected into the forearm. In an individual who has been previously infected, a delayed-type hypersensitivity (DTH) reaction results in a measurable induration at the site of the injection, indicating that previous exposure toLeishmaniahas resulted in the development of cell-mediated immunity. LST positivity is associated with long-lasting protective immunity against reinfection, most notably as reported for visceral leishmaniasis (VL). Despite efforts over the past few decades, leishmanin antigen is no longer produced under good manufacturing practice (GMP) conditions anywhere in the world. Consequently, the use of the LST in epidemiological studies has declined in favor of serological and molecular tests. In this review, we provide a historical overview of the LST and justification for the reintroduction of leishmanin. A GMP-grade leishmanin can be used to detect immunity in vivo by the LST and can be investigated for use in an interferon-γ release assay (IGRA), which may serve as an in vitro version of the LST. The LST will be a valuable tool for surveillance and epidemiological studies in support of the VL elimination programs and as a surrogate marker of immunity in vaccine clinical trials. Methods: A review of the literature was conducted using PubMed as the primary database, with MeSH terms “leishmanin skin test” OR “Montenegro test” OR “Montenegro skin test.” Articles written in English that describe the history or standardization of leishmanin, the use of leishmanin in an IGRA, or the use of the LST in epidemiological studies or vaccine trials were prioritized in our appraisal of the literature.

Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His ₆ -tagged exbB-exbD and S-tagged tonB , we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli . Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB ₄ -ExbD ₂ complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB ₄ -ExbD ₁ -TonB ₁ . Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB ₄ -ExbD ₁ -TonB ₁ . Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization. IMPORTANCE Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB ₄ -ExbD ₁ -TonB ₁ complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

Leishmaniasis is a neglected tropical disease endemic in over 90 countries. The disease has two main pathologies; cutaneous leishmaniasis (CL) that generally self-heals, and visceral leishmaniasis (VL) that is fatal if untreated. The majority of VL cases, concentrated on the Indian subcontinent (ISC) and East Africa, are caused by Leishmania donovani. However, recent foci of CL on the ISC have been attributed as an atypical phenotype of L. donovani including a recent outbreak in Himachal Pradesh, India. Whole genome sequencing and phylogenetic analysis was undertaken to investigate the origins and genetic factors leading to this pathology atypical of L. donovani. Here we demonstrate the isolate from Himachal Pradesh is derived from a genetic hybridization between two independent L. donovani parents from the ‘Yeti’ ISC1 divergent clade of parasites, identified in the Nepalese highlands. This reveals that intraspecies L. donovani hybrids can give rise to a novel strain associated with CL.