Ranadhir Dey

Intracellular Replication-Deficient Leishmania donovani Induces Long Lasting Protective Immunity against Visceral Leishmaniasis

No vaccine is currently available for visceral leishmaniasis (VL) caused by Leishmania donovani. This study addresses whether a live attenuated centrin gene-deleted L. donovani (LdCen1−/−) parasite can persist and be both safe and protective in animals. LdCen1−/− has a defect in amastigote replication both in vitro and ex vivo in human macrophages. Safety was shown by the lack of parasites in spleen and liver in susceptible BALB/c mice, immune compromised SCID mice, and human VL model hamsters 10 wk after infection. Mice immunized with LdCen1−/− showed early clearance of virulent parasite challenge not seen in mice immunized with heat killed parasites. Upon virulent challenge, the immunized mice displayed in the CD4+ T cell population a significant increase of single and multiple cytokine (IFN-γ, IL-2, and TNF) producing cells and IFN-γ/IL10 ratio. Immunized mice also showed increased IgG2a immunoglobulins and NO production in macrophages. These features indicated a protective Th1-type immune response. The Th1 response correlated with a significantly reduced parasite burden in the spleen and no parasites in the liver compared with naive mice 10 wk post challenge. Protection was observed, when challenged even after 16 wk post immunization, signifying a sustained immunity. Protection by immunization with attenuated parasites was also seen in hamsters. Immunization with LdCen1−/− also cross-protected mice against infection with L. braziliensis that causes mucocutaneous leishmaniasis. Results indicate that LdCen1−/− can be a safe and effective vaccine candidate against VL as well as mucocutaneous leishmaniasis causing parasites.

Cholesterol depletion associated with Leishmania major infection alters macrophage CD40 signalosome composition and effector function

CD40, a costimulatory molecule expressed on macrophages, induces expression of interleukin 12 (IL-12) in uninfected macrophages and IL-10 in macrophages infected with Leishmania major. IL-12 suppresses, whereas IL-10 enhances, L. major infection. The mechanisms that regulate this difference in CD40-induced cytokine production remain unclear, but it is known that L. major depletes cholesterol. Here we show that cholesterol influenced the assembly of distinct CD40 signalosomes. Depletion of membrane cholesterol inhibited the assembly of an IL-12-inducing CD40 signalosome containing the adaptors TRAF2, TRAF3 and TRAF5 and the kinase Lyn and promoted the assembly of an IL-10-inducing CD40 signalosome containing the adaptor TRAF6 and the kinase Syk. Thus, cholesterol depletion might represent an immune-evasion strategy used by L. major.

Immunity to Visceral Leishmaniasis Using Genetically Defined Live-Attenuated Parasites

Leishmaniasis is a protozoan parasitic disease endemic to the tropical and subtropical regions of the world, with three major clinical forms, self-healing cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). Drug treatments are expensive and often result in the development of drug resistance. No vaccine is available against leishmaniasis. Subunit Leishmania vaccine immunization in animal models has shown some efficacy but little or none in humans. However, individuals who recover from natural infection are protected from reinfection and develop life-long protection, suggesting that infection may be a prerequisite for immunological memory. Thus, genetically altered live-attenuated parasites with controlled infectivity could achieve such memory. In this paper, we discuss development and characteristics of genetically altered, live-attenuated Leishmania donovani parasites and their possible use as vaccine candidates against VL. In addition, we discuss the challenges and other considerations in the use of live-attenuated parasites.

Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence

Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein‐encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co‐immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene‐deleted parasites (Ldp27−/−) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27−/− parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.

Live Attenuated Leishmania donovani p27 Gene Knockout Parasites Are Nonpathogenic and Elicit Long-Term Protective Immunity in BALB/c Mice

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.

Induction of Host Protective Th1 Immune Response by Chemokines in Leishmania donovani-infected BALB⁄c Mice

The resolution from leishmanial infection is dependent on the coordinated interactions between the components of the cell mediated immune system and the activation of T‐cell population into appropriate cytokine production and the activation of macrophages. Earlier reports established that C‐C chemokines particularly macrophage inflammatory protein (MIP)‐1α and macrophage chemoattractant protein (MCP)‐1 restrict the parasitic burden via the regulation of impaired protein kinase C (PKC) signalling and induction of free‐radical generation in murine leishmaniasis. This study explored the role of MIP‐1α and MCP‐1 in the induction of T helper 1 (Th1) immune response and suppression of T helper 2 (Th2) response in Leishmania donovani‐infected BALB/c mice. These chemokines induced the known pro‐inflammatory cytokine interleukin (IL)‐12 secretion and inhibited the secretion of anti‐inflammatory cytokines IL‐10 and transforming growth factor‐β in infected macrophages. Impaired antigen presentation capability of infected macrophages was also restored by the chemokine treatment. C‐C chemokine treatment resulted in reduced levels of mRNA expression of IL‐10, but increased levels of mRNA expression of IL‐12p40, interferon (IFN)‐γ, tumour necrosis factor‐α and inducible nitric oxide synthase in both liver mononuclear cells as well as in splenocytes, reflecting a switch of CD4+ differentiation from Th2 to Th1. Flow cytometric analysis of infected spleen cells suggested that C‐C chemokine treatment enhances the CD4+ T cells to produce increased levels of IFN‐γ. These studies hypothesize a promising immuno‐prophylactic effect of chemokines against leishmaniasis by induction of Th1 cytokine release imparting a long‐term resistance.

A New Model of Progressive Visceral Leishmaniasis in Hamsters by Natural Transmission via Bites of Vector Sand Flies

BACKGROUND Visceral leishmaniasis (VL) is transmitted by sand flies. Protection of needle-challenged vaccinated mice was abrogated in vector-initiated cutaneous leishmaniasis, highlighting the importance of developing natural transmission models for VL. METHODS We used Lutzomyia longipalpis to transmit Leishmania infantum or Leishmania donovani to hamsters. Vector-initiated infections were monitored and compared with intracardiac infections. Body weights were recorded weekly. Organ parasite loads and parasite pick-up by flies were assessed in sick hamsters. RESULTS Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared with uninfected organs and had geometric mean parasite loads (GMPL) comparable to intracardiac inoculation of 10(7)-10(8) parasites, although vector-initiated disease progression was slower and weight loss was greater. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, 45.6%, 50.0%, and 33.3% of sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%-82%) rather than total GMPL (2.0 × 10(4)-8.0 × 10(4)) per midgut. CONCLUSIONS This model provides an improved platform to study initial immune events at the bite site, parasite tropism, and pathogenesis and to test drugs and vaccines against naturally acquired VL.

Biomarkers of Safety and Immune Protection for Genetically Modified Live Attenuated Leishmania Vaccines Against Visceral Leishmaniasis – Discovery and Implications

Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood-borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, subunit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in Leishmania donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters, and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines, e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen−/− in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated in normal individuals. In addition, comparative analysis of biomarkers in PBMCs from asymptomatic or healed visceral leishmaniasis individuals in response to vaccine candidates including live attenuated parasites may provide clues about determinants of protective immunity and be helpful in shaping the final Leishmania vaccine formulation in the clinical trials.

Leishmania donovani-Induced Ceramide as the Key Mediator of Akt Dephosphorylation in Murine Macrophages: Role of Protein Kinase Cζ and Phosphatase

ABSTRACT Leishmania donovani is an intracellular protozoan parasite that impairs the host macrophage immune response to render it suitable for its survival and establishment. L. donovani-induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a pleiotropic second messenger playing an important role in regulation of several kinases, including mitogen-activated protein kinase and phosphatases. We observed that the endogenous ceramide generated during leishmanial infection led to the dephosphorylation of protein kinase B (PKB) (Akt) in infected cells. The study of ceramide-mediated Akt phosphorylation revealed that Akt was dephosphorylated at both Thr308 and Ser473 sites in infected cells. Further investigation demonstrated that ceramide was also responsible for the induction of PKCζ, an atypical Ca-independent stress kinase, as well as the ceramide-activated protein phosphatases (e.g., protein phosphatase 2A [PP2A]). We found that Akt dephosphorylation was mediated by ceramide-induced PKCζ-Akt association and PP2A activation. In addition, treatment of L. donovani-infected macrophages with PKCζ-specific inhibitor peptide could restore the translocation of phosphorylated Akt to the cell membrane. This study also revealed that ceramide is involved in the inhibition of proinflammatory cytokine tumor necrosis factor alpha release by infected macrophages. These observations strongly suggest the importance of ceramide in the alteration of normal cellular functions, impairment of the kinase/phosphatase balance, and thereby establishment of leishmaniasis in the hostile macrophage environment.

Regulation of impaired protein kinase C signaling by chemokines in murine macrophages during visceral leishmaniasis.

ABSTRACT The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 h. In this study, we investigated the role of MIP-1α and MCP-1 in the regulation of impaired PKC activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.