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.

Centrin is a calcium-binding centrosome/basal body–associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.

Leishmaniasis causes significant morbidity and mortality worldwide, and no vaccines against this disease are available. Previously, we had shown that the amastigote-specific protein p27 (Ldp27) is a component of an active cytochrome c oxidase complex in Leishmania donovani and that upon deletion of its gene the parasite had reduced virulence in vivo. In this study, we have shown that Ldp27^−/− parasites do not survive beyond 20 wk in BALB/c mice and hence are safe as an immunogen. Upon virulent challenge, mice 12 wk postimmunization showed significantly lower parasite burden in the liver and spleen. When mice were challenged 20 wk postimmunization, a significant reduction in parasite burden was still noted, suggesting long-term protection by Ldp27^−/− immunization. Immunization with Ldp27^−/− induced both pro- and anti-inflammatory cytokine responses and activated splenocytes for enhanced leishmanicidal activity in association with NO production. Protection in both short- and long-term immunized mice after challenge with the wild-type parasite correlated with the stimulation of multifunctional Th1-type CD4 and CD8 T cells. Adoptive transfer of T cells from long-term immunized mice conferred protection against virulent challenge in naive recipient mice, suggesting involvement of memory T cell response in protection against Leishmania infection. Immunization of mice with Ldp27^−/−also demonstrated cross-protection against Leishmania major and Leishmania braziliensis infection. Our data show that genetically modified live attenuated Ldp27^−/− parasites are safe, induce protective immunity even in the absence of parasites, and can provide protection against homologous and heterologous Leishmania species.

Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modifiedLeishmania donovaniparasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice bothin vitroandin vivo. In vitroinfection of macrophages with live attenuated parasites (compared to that with wild-type [WT]L. donovaniparasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4⁺T cell activationin vitro. Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1β, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WTL. donovani-infected mice. Furthermore, anex vivoantigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4⁺T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen^-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen^-/- parasites as a vaccine against virulent L. donovani. To explore the potential of a combination of LdCen^-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen^-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen^-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Our results demonstrate that an ID vaccination using LdCen^-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization. Leishmaniasis is a disease with a wide spectrum of clinical manifestations caused by different species of protozoa belonging to the Leishmania genus that are transmitted by sand fly vectors. Visceral infections of Leishmania cause significant mortality and morbidity and development of a vaccine to prevent leishmaniasis has become a high priority. We have previously reported that intravenous immunization with a live attenuated parasite vaccine comprised of Leishmania donovani parasites lacking the centrin gene conferred protection in mice, hamsters and dogs. In the current report, we describe the immunological response and associated protection to the ID immunization with attenuated parasites in combination with a sand fly salivary protein (LJM19). We observe that protection against experimental ID challenge with L. donovani resulting from ID immunization with live attenuated parasites in combination with LJM19 is comparable to intracardial immunization and offers improved protective immunity compared to immunization with salivary protein alone and non-immunized hamsters. This study supports the potential use of the genetically attenuated vaccine and a recombinant sand fly salivary protein for control of visceral leishmaniasis.