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Thalia Pacheco-Fernandez,1,* Hannah Markle,1,* Chaitenya Verma,2 Ryan Huston,2,3 Sreenivas Gannavaram,1 Hira L Nakhasi,1 Abhay R Satoskar2,3 1Division of Emerging and Transfusion Transmitted Disease, Center for Biologics Evaluation and Research Food and Drug Administration, Silver Spring, MD, 20993, USA; 2Department of Pathology, Wexner Medical Center, The Ohio State University, Columbus, OH, 43201, USA; 3Department of Microbiology, Wexner Medical Center, The Ohio State University, Columbus, OH, 43201, USA*These authors contributed equally to this workCorrespondence: Abhay R Satoskar, Email abhay.satoskar@osumc.eduAbstract: Leishmaniasis is a neglected tropical disease endemic primarily to low- and middle-income countries, for which there has been inadequate development of affordable, safe, and efficacious therapies. Clinical manifestations of leishmaniasis range from self-healing skin lesions to lethal visceral infection with chances of relapse. Although treatments are available, secondary effects limit their use outside the clinic and negatively impact the quality of life of patients in endemic areas. Other non-medicinal treatments, such as thermotherapies, are limited to use in patients with cutaneous leishmaniasis but not with visceral infection. Recent studies shed light to mechanisms through which Leishmania can persist by hiding in cellular safe havens, even after chemotherapies. This review focuses on exploring the cellular niches that Leishmania parasites may be leveraging to persist within the host. Also, the cellular, metabolic, and molecular implications of Leishmania infection and how those could be targeted for therapeutic purposes are discussed. Other therapies, such as those developed against cancer or for manipulation of the ferroptosis pathway, are proposed as possible treatments against leishmaniasis due to their mechanisms of action. In particular, treatments that target hematopoietic stem cells and monocytes, which have recently been found to be necessary components to sustain the infection and provide a safe niche for the parasites are discussed in this review as potential field-deployable treatments against leishmaniasis.Keywords: leishmaniasis, field-deployable therapies, hematopoietic stem cells, monocytes, ferroptosis, repurposing drugs

Previously, we have demonstrated that deletion of a growth-regulating gene ( LdCen1 ) in the Leishmania donovani parasite ( LdCen1 -/- ) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1 -/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1 -/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1 -/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.

Our developed cell division-specific 'centrin' gene deleted Leishmania donovani (LdCen1 -/- ) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1 -/- immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1 -/- efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to disclose., (Copyright © 2024 Institut Pasteur. All rights reserved.)

Centrin1 gene deleted Leishmania donovani parasite (LdCen1 -/- ) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1 -/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1 -/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1 -/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1 -/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1 -/- , there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1 -/- manufactured under cGMP complaint conditions can be suitable for future clinical trials., (© 2024. The Author(s).)

Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin -deficient Leishmania braziliensis ( LbCen -/- ) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen -/- failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen -/- was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major , L. donovani , and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin -deficient L. donovani ( LdonCen -/- ) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen -/- to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.

Violet-blue light of 405 nm in the visible spectrum at a dose of 270 J/cm 2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani -spiked platelets were evaluated in an immunodeficient Rag2 -/- mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2',7'-Dichlorodihydrofluorescein diacetate (H 2 DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2 -/- mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets.

Cutaneous leishmaniasis (CL) is characterized by extensive skin lesions, which are usually painless despite being associated with extensive inflammation. The molecular mechanisms responsible for this analgesia have not been identified. Through untargeted metabolomics, we found enriched anti-nociceptive metabolic pathways in L. mexicana -infected mice. Purines were elevated in infected macrophages and at the lesion site during chronic infection. These purines have anti-inflammatory and analgesic properties by acting through adenosine receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also found arachidonic acid (AA) metabolism enriched in the ear lesions compared to the non-infected controls. AA is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites., Competing Interests: This article reflects the views of the authors and should not be construed to represent FDA’s views or policies. A.R.S. and The Ohio State University hold a patent #WO2012145734A1 entitled: “Antileishmanial compositions and methods of use”., (© 2023 The Author(s).)

The leishmanin skin test was used for almost a century to detect exposure and immunity to Leishmania, the causative agent of leishmaniasis, a major neglected tropical disease. Due to a lack of antigen used for the intradermal injection, the leishmanin skin test is no longer available. As leishmaniasis control programs are advancing and new vaccines are entering clinical trials, it is essential to re-introduce the leishmanin skin test. Here we establish a Leishmania donovani strain and describe the production, under Good Laboratory Practice conditions, of leishmanin soluble antigen used to induce the leishmanin skin test in animal models of infection and vaccination. Using a mouse model of cutaneous leishmaniasis and a hamster model of visceral leishmaniasis, soluble antigen induces a leishmanin skin test response following infection and vaccination with live attenuated Leishmania major (LmCen -/- ). Both the CD4 + and CD8 + T-cells are necessary for the leishmanin skin test response. This study demonstrates the feasibility of large-scale production of leishmanin antigen addressing a major bottleneck for performing the leishmanin skin test in future surveillance and vaccine clinical trials., (© 2023. The Author(s).)

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether other cell types can be infected. Here, we use unbiased single-cell RNA sequencing (scRNA-seq) to simultaneously analyze host cell and Leishmania donovani transcriptomes to identify and annotate parasitized cells in spleen and bone marrow in chronically infected mice. Our dual-scRNA-seq methodology allows the detection of heterogeneous parasitized populations. In the spleen, monocytes and macrophages are the dominant parasitized cells, while megakaryocytes, basophils, and natural killer (NK) cells are found to be unexpectedly infected. In the bone marrow, the hematopoietic stem cells (HSCs) expressing phagocytic receptors FcγR and CD93 are the main parasitized cells. Additionally, we also detect parasitized cycling basal cells, eosinophils, and macrophages in chronically infected mice. Flow cytometric analysis confirms the presence of parasitized HSCs. Our unbiased dual-scRNA-seq method identifies rare, parasitized cells, potentially implicated in pathogenesis, persistence, and protective immunity, using a non-targeted approach., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Leishmaniasis is a parasitic disease that is prevalent in 90 countries, and yet no licensed human vaccine exists against it. Toward control of leishmaniasis, we have developed Leishmania major centrin gene deletion mutant strains ( LmCen -/- ) as a live attenuated vaccine, which induces a strong IFN-γ-mediated protection to the host. However, the immune mechanisms of such protection remain to be understood. Metabolomic reprogramming of the host cells following Leishmania infection has been shown to play a critical role in pathogenicity and shaping the immune response following infection. Here, we applied untargeted mass spectrometric analysis to study the metabolic changes induced by infection with LmCen -/- and compared those with virulent L. major parasite infection to identify the immune mechanism of protection. Our data show that immunization with LmCen -/- parasites, in contrast to virulent L. major infection promotes a pro-inflammatory response by utilizing tryptophan to produce melatonin and downregulate anti-inflammatory kynurenine-AhR and FICZ-AhR signaling., Competing Interests: The FDA is currently a co-owner of two US patents that claim attenuated Leishmania species with the centrin gene deletion (US7,887,812 and US8,877,213). This article reflects the views of the authors and should not be construed to represent FDA’s views or policies. The remaining authors declare no competing interests.

Leishmaniasis is a tropical disease prevalent in 90 countries. Presently, there is no approved vaccine for human use. We developed a live attenuated L. mexicana Cen -/- (LmexCen -/- ) strain as a vaccine candidate that showed excellent efficacy, characterized by reduced Th2 and enhanced Th1 responses in C57BL/6 and BALB/c mice, respectively, compared to wild-type L. mexicana (Lmex WT ) infection. Toward understanding the immune mechanisms of protection, we applied untargeted mass spectrometric analysis to LmexCen -/- and Lmex WT infections. Data showed enrichment of the pentose phosphate pathway (PPP) in ears immunized with LmexCen -/- versus naive and Lmex WT infection. PPP promotes M1 polarization in macrophages, suggesting a switch to a pro-inflammatory phenotype following LmexCen -/- inoculation. Accordingly, PPP inhibition in macrophages infected with LmexCen -/- reduced the production of nitric oxide and interleukin (IL)-1β, hallmarks of classical activation. Overall, our study revealed the immune regulatory mechanisms that may be critical for the induction of protective immunity., Competing Interests: The FDA is currently a co-owner of two US patents that claim attenuated Leishmania species with the centrin gene deletion (US7,887,812 and US 8,877,213). This article reflects the views of the authors and should not be construed to represent FDA’s views or policies., (© 2023 The Author(s).)

Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen -/- ) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen -/- parasites was mediated by both CD4 + and CD8 + T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4 + and CD8 + T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen -/- parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen -/- MIF -/- immunized group presented higher percentage of CD4 + and CD8 + central memory T cells, increased CD8 + T cell proliferation after challenge compared to LdCen -/- immunization. LdCen -/- MIF -/- immunized group presented elevated production of IFN-γ + and TNF-α + CD4 + T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen -/- group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL., (© 2023. The Author(s).)

No human vaccine is available for visceral leishmaniasis (VL). Live attenuated centrin gene-deleted L. donovani ( LdCen -/- ) parasite vaccine has been shown to induce robust innate immunity and provide protection in animal models. Toll-like receptors (TLRs) are expressed in innate immune cells and are essential for the early stages of Leishmania infection. Among TLRs, TLR-9 signaling has been reported to induce host protection during Leishmania infection. Importantly, TLR-9 ligands have been used as immune enhancers for non-live vaccination strategies against leishmaniasis. However, the function of TLR-9 in the generation of a protective immune response in live attenuated Leishmania vaccines remains unknown. In this study, we investigated the function of TLR-9 during LdCen -/- infection and found that it increased the expression of TLR-9 on DCs and macrophages from ear-draining lymph nodes and spleen. The increase in TLR-9 expression resulted in changes in downstream signaling in DCs mediated through signaling protein myeloid differentiation primary response 88 (MyD88), resulting in activation and nuclear translocation of nuclear factor-κB (NF-κB). This process resulted in an increase in the DC's proinflammatory response, activation, and DC-mediated CD4 + T cell proliferation. Further, LdCen -/- immunization in TLR-9 -/- mice resulted in a significant loss of protective immunity. Thus, LdCen -/- vaccine naturally activates the TLR-9 signaling pathway to elicit protective immunity against virulent L. donovani challenge.

Currently, no licensed vaccine is available for human visceral leishmaniasis (VL), a fatal disease caused by the protozoan parasite Leishmania donovani. Two of our live attenuated L. donovani vaccine candidates, either deleted for Centrin1 (LdCen1 -/- ) or p27 gene (Ldp27 -/- ), that display reduced growth in macrophages were studied to be safe, immunogenic and protective against VL in various animal models. This report involves the identification of differentially expressed proteins, their related pathways and its underlying mechanism in the intracellular stage of these parasites, using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) methods. Out of 50-60 proteins, found to be differentially expressed in these mutant parasites, 36 were found to be common in both the parasites. Such proteins mainly belong to the functional categories viz. metabolic enzymes, chaperones and stress proteins, proteins involved in translation, processing and transport and proteins involved in nucleic acid processing. Proteins known to be host protective, like Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c, calreticulin and those responsible for inducing immune response, namely tubulins, DEAD box RNA helicases, HSP70 and tryparedoxin, have been detected to be modulated in these parasites. Such proteins could be predicted as biomarkers, with further scope of study for their role in growth attenuation. SIGNIFICANCE: This study aims at predicting proteomic biomarkers of Leishmania parasite growth attenuation, that have immunomodulatory role in the disease leishmaniasis. Advanced studies could be helpful in establishing the role of these identified proteins in parasitic virulence and to predict the host interaction at molecular level. Also, these proteins could be exploited as attenuation markers during the development of genetically modified live attenuated parasites as vaccine candidates. These could be cross validated in varied species of Leishmania and other tyrpanosomatids for similar response towards identifying them as universal biomarkers of attenuation., Competing Interests: Declaration of Competing Interest None of the authors has any conflict of interest to disclose., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen -/- ) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen -/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We showed that immunization with LmexCen -/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen -/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen -/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen -/- parasites is safe and efficacious against the Old World visceral leishmaniasis., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Leishmaniasis, caused by an infection of the Leishmania protozoa, is a neglected tropical disease and a major health problem in tropical and subtropical regions of the world, with approximately 350 million people worldwide at risk and 2 million new cases occurring annually. Current treatments for leishmaniasis are not highly efficacious and are associated with high costs, especially in low- and middle-income endemic countries, and high toxicity. Due to a surge in the incidence of leishmaniases worldwide, the development of new strategies such as a prophylactic vaccine has become a high priority. However, the ability of Leishmania to undermine immune recognition has limited our efforts to design safe and efficacious vaccines against leishmaniasis. Numerous antileishmanial vaccine preparations based on DNA, subunit, and heat-killed parasites with or without adjuvants have been tried in several animal models but very few have progressed beyond the experimental stage. However, it is known that people who recover from Leishmania infection can be protected lifelong against future infection, suggesting that a successful vaccine requires a controlled infection to develop immunologic memory and subsequent long-term immunity. Live attenuated Leishmania parasites that are non-pathogenic and provide a complete range of antigens similarly to their wild-type counterparts could evoke such memory and, thus, would be effective vaccine candidates. Our laboratory has developed several live attenuated Leishmania vaccines by targeted centrin gene disruptions either by homologous recombination or, more recently, by using genome editing technologies involving CRISPR-Cas9. In this review, we focused on the sequential history of centrin gene-deleted Leishmania vaccine development, along with the characterization of its safety and efficacy. Further, we discussed other major considerations regarding the transition of dermotropic live attenuated centrin gene-deleted parasites from the laboratory to human clinical trials.

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene-deleted parasite strain ( LmCen -/- ) that induced protection against homologous and heterologous challenges. We demonstrated that the protection is mediated by IFN (Interferon) γ-secreting CD4 + T-effector cells and multifunctional T cells, which is analogous to leishmanization. In addition, in a leishmanization model, skin tissue-resident memory T (TRM) cells were also shown to be crucial for host protection. In this study, we evaluated the generation and function of skin TRM cells following immunization with LmCen -/- parasites and compared those with leishmanization. We show that immunization with LmCen -/- generated skin CD4+ TRM cells and is supported by the induction of cytokines and chemokines essential for their production and survival similar to leishmanization. Following challenge with wild-type L. major , TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice. Furthermore, upon challenge, CD4 + TRM cells induce higher levels of IFNγ and Granzyme B in the immunized and leishmanized mice than in non-immunized mice. Taken together, our studies demonstrate that the genetically modified live attenuated LmCen -/- vaccine generates functional CD4 + skin TRM cells, similar to leishmanization, that may play a crucial role in host protection along with effector T cells as shown in our previous study., Competing Interests: The FDA is currently a co-owner of two US patents that claim attenuated Leishmania species with the Centrin gene deletion (US7,887,812 and US 8,877,213). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ismail, Karmakar, Bhattacharya, Sepahpour, Takeda, Hamano, Matlashewski, Satoskar, Gannavaram, Dey and Nakhasi.)

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., (© 2022. The Author(s).)

Background: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo., Methodology/findings: LdCen-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen-/- parasites by DCs. Neutrophil depletion in LdCen-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4+ T cells in vivo., Conclusions: Collectively, LdCen-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Leishmaniasis is a major public health problem and the second most lethal parasitic disease in the world due to the lack of effective treatments and vaccines. Even when not lethal, leishmaniasis significantly affects individuals and communities through life-long disabilities, psycho-sociological trauma, poverty, and gender disparity in treatment., Areas Covered: This review discusses the most relevant and recent research available on Pubmed and GoogleScholar highlighting leishmaniasis' global impact, pathogenesis, treatment options, and lack of effective control strategies. An effective vaccine is necessary to prevent morbidity and mortality, lower health care costs, and reduce the economic burden of leishmaniasis for endemic low- and middle-income countries. Since there are several forms of leishmaniasis, a pan- Leishmania vaccine without geographical restrictions is needed. This review also focuses on recent advances and common challenges in developing prophylactic strategies against leishmaniasis., Expert Opinion: Despite advances in pre-clinical vaccine research, approval of a human leishmaniasis vaccine still faces major challenges - including manufacturing of candidate vaccines under Good Manufacturing Practices, developing well-designed clinical trials suitable in endemic countries, and defined correlates of protection. In addition, there is a need to explore Challenge Human Infection Model to avoid large trials because of fluctuating incidence and prevalence of leishmanasis.

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania , the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Volpedo, Pacheco-Fernandez, Bhattacharya, Oljuskin, Dey, Gannavaram, Satoskar and Nakhasi.)

MicroRNA-21 (miR-21) inhibits IL-12 expression and impairs the Th1 response necessary for control of Leishmania infection. Recent studies have shown that Leishmania infection induces miR-21 expression in dendritic cells and macrophages, and inhibition of miR-21 restores IL-12 expression. Because miR-21 is known to be expressed due to inflammatory stimuli in a wide range of hematopoietic cells, we investigated the role of miR-21 in regulating immune responses during visceral leishmaniasis (VL) caused by Leishmania donovani infection. We found that miR-21 expression was significantly elevated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection, concomitant with an increased expression of disease exacerbating IL-6 and STAT3. Bone marrow dendritic cells from miR-21 knockout (miR-21KO) mice showed increased IL-12 production and decreased production of IL-10. On L. donovani infection, miR-21KO mice exhibited significantly greater numbers of IFN-γ- and TNF-α-producing CD4 + and CD8 + T cells in their organs that was associated with increased production of Th1-associated IFN-γ, TNF-α, and NO from the splenocytes. Finally, miR-21KO mice displayed significantly more developing and mature hepatic granulomas leading to reduction in organ parasitic loads compared with wild type counterparts. Similar results were noted in L. donovani -infected wild type mice after transient miR-21 depletion. These observations indicate that miR-21 plays a critical role in pathogenesis of VL by suppressing IL-12- and Th1-associated IFN-γ and also inducing disease-promoting induction of the IL-6 and STAT-3 signaling pathway. miR-21 could therefore be used as a potential target for developing host-directed treatment for VL., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen -/- ) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen -/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen -/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen -/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen -/- immunized hamsters. This study demonstrates that the LmCen -/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Leishmaniasis includes a spectrum of diseases ranging from debilitating cutaneous to fatal visceral infections. This disease is caused by the parasitic protozoa of the genus Leishmania that is transmitted by infected sandflies. Over 1 billion people are at risk of leishmaniasis with an annual incidence of over 2 million cases throughout tropical and subtropical regions in close to 100 countries. Leishmaniasis is the only human parasitic disease where vaccination has been successful through a procedure known as leishmanization that has been widely used for decades in the Middle East. Leishmanization involved intradermal inoculation of live Leishmania major parasites resulting in a skin lesion that following natural healing provided protective immunity to re-infection. Leishmanization is however no longer practiced due to safety and ethical concerns that the lesions at the site of inoculation that can last for months in some people. New genome editing technologies involving CRISPR has now made it possible to engineer safer attenuated strains of Leishmania , which induce protective immunity making way for a second generation leishmanization that can enter into human trials. A major consideration will be how the test the efficacy of a vaccine in the midst of the visceral leishmaniasis elimination program. One solution will be to use the leishmanin skin test (LST) that was also used for decades to determine exposure and immunity to Leishmania. The LST involves injection of antigen from Leishmania in the skin dermis resulting in a delayed type hypersensitivity (DTH) immune reaction associated with a Th1 immune response and protection against visceral leishmaniasis. Reintroduction of novel approaches for leishmanization and the leishmanin skin test can play a major role in eliminating leishmaniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pacheco-Fernandez, Volpedo, Gannavaram, Bhattacharya, Dey, Satoskar, Matlashewski and Nakhasi.)

No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene-deleted L. donovani ( LdCen -/- ) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen -/- with neutrophils, we immunized mice intradermally in the ear pinna with LdCen -/- Compared with LdWT infection, LdCen -/- parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6-18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen -/- -immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nβ subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen -/- -immunized mice exhibited much stronger Ag-specific CD4 + T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen -/- parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4 + T cell proliferation in LdCen -/- -immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen -/- -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen -/- parasite vaccine., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163 + CD91 + professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis and is caused by several species of Leishmania parasite. Clinical presentation of CL varies from a self-healing infection to a chronic form of the disease determined by the virulence of infecting Leishmania species and host immune responses to the parasite. Mouse models of CL show contradictory roles of lymphocytes in pathogenesis, while acquired immune responses are responsible for host protection from diseases. To reconcile the inconclusive roles of acquired immune responses in pathogenesis, we infected mice from various genetic backgrounds with two pathogenic strains of Leishmania major, Friedlin or 5ASKH, and assessed the outcome of the infections. Our findings showed that the genetic backgrounds of L. major determine the impact of lymphocytes for pathogenesis. In the absence of lymphocytes, L. major Friedlin induced the lowest inflammatory reaction and pathology at the site of infection, while 5ASKH infection induced a strong inflammatory reaction and severe pathology. Lymphocytes ameliorated 5ASKH mediated pathology, while it exacerbated pathology during Friedlin infection. Excess inflammatory reactions, like the recruitment of macrophages, neutrophils, eosinophils and production of pro-inflammatory cytokines, together with uncontrolled parasite growth in the absence of lymphocytes during 5ASKH infection may induce severe pathology development. Taken together our study provides insight into the impact of differences in the genetic background of Leishmania on CL pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

No vaccine exists against visceral leishmaniasis. Toward developing vaccines against VL, we have reported previously on the immunogenicity of live attenuated LdCen -/- parasites in animal models. Immunization with LdCen -/- parasites has been shown to induce durable protective immunity in pre-clinical animal models. Although the innate immune responses favoring a Th1 type immunity are produced following LdCen -/- immunization, the molecular determinants of such responses remain unknown. To identify early biomarkers of immunogenicity associated with live attenuated parasitic vaccines, we infected macrophages derived from healthy human blood donors with LdCen -/- or LdWT parasites ex vivo and compared the early gene expression profiles. In addition to altered expression of immune related genes, we identified several microRNAs that regulate important cytokine genes, significantly altered in LdCen -/- infection compared to LdWT infection. Importantly, we found that LdCen -/- infection suppresses the expression of microRNA-21 (miR-21) in human macrophages, which negatively regulates IL12, compared to LdWT infection. In murine DC experiments, LdCen -/- infection showed a reduced miR-21 expression with a concomitant induction of IL12. Silencing of miR-21 using specific inhibitors resulted in an augmented induction of IL12 in LdWT infected BMDCs, illustrating the role of miR-21 in LdWT mediated suppression of IL12. Further, exosomes isolated from LdCen -/- infected DCs contained significantly reduced levels of miR-21 compared to LdWT infection, that promoted proliferation of CD4 + T cells in vitro . Similar miR-21 mediated IL12 regulation was also observed in ex vivo human macrophage infection experiments indicating that miR-21 plays a role in early IL12 mediated immunity. Our studies demonstrate that LdCen -/- infection suppresses miR-21 expression, enables IL12 mediated induction of adaptive immunity including proliferation of antigen experienced CD4 + T cells and development of a Th1 immunity, and suggest that miR-21 could be an important biomarker for LdCen -/- vaccine immunity in human clinical trials., One Sentence Summary: Role of miR-21 in vaccine induced immunity., (Copyright © 2019 Gannavaram, Bhattacharya, Siddiqui, Ismail, Madhavan and Nakhasi.)

Background and Objectives: Globally, blood safety interventions have been successful in mitigating risk of the major transfusion-transmitted (TT) viruses. However, strategies that address risk from parasites are comparatively limited. TT parasites are often regional in nature, posing unique challenges; we sought to understand their impact on blood safety., Materials and Methods: An electronic questionnaire was distributed to transfusion medicine leaders in 100 countries. The survey focused on specific questions pertaining to four parasitic diseases: babesiosis, Chagas, leishmaniasis and malaria. Respondents provided data on historical TT cases, local epidemiology, policies to mitigate risk and an assessment of public health perceptions for each aetiologic agent., Results: Twenty-eight (28%) surveys were returned from countries in Europe (n = 13), the Americas (n = 6), Africa (n = 4), Asia (n = 3) and Oceana (n = 2). Historically, no cases of TT leishmaniasis were reported, TT babesiosis was exclusive to Canada and the USA, TT Chagas was limited to the Americas and Spain, while TT malaria was cosmopolitan. Mitigation efforts varied widely; malaria was the most frequently tested parasitic disease. The public's perception of risk for parasitic agents was low, while that of health authorities in endemic countries was higher., Conclusion: The global impact of parasitic infections on blood safety and related mitigation efforts varied widely by parasite epidemiology, test availability, public health priorities and socioeconomic constraints. While parasites continue to pose a risk to blood safety, the successful mitigation of viral risk has elevated the prominence of TT parasites in many locations, thereby requiring consideration of mitigation efforts., (© 2018 International Society of Blood Transfusion.)

Leptin, a pleiotropic protein has long been recognized to play an important role in the regulation of energy homeostasis, metabolism, neuroendocrine function, and other physiological functions through its effects on the central nervous system (CNS) and peripheral tissues. Leptin is secreted by adipose tissue and encoded by the obese ( ob ) gene. Leptin acts as a central mediator which regulates immunity as well as nutrition. Importantly, leptin can modulate both innate and adaptive immune responses. Leptin deficiency/resistance is associated with dysregulation of cytokine production, increased susceptibility toward infectious diseases, autoimmune disorders, malnutrition and inflammatory responses. Malnutrition induces a state of immunodeficiency and an inclination to death from communicable diseases. Infectious diseases are the disease of poor who invariably suffer from malnutrition that could result from reduced serum leptin levels. Thus, leptin has been placed at the center of many interrelated functions in various pathogenic conditions, such as bacterial, viruses and parasitic infections. We review herein, the recent advances on the role of leptin in malnutrition in pathogenesis of infectious diseases with a particular emphasis on parasitic diseases such as Leishmaniasis, Trypanosomiasis, Amoebiasis, and Malaria.

The morphological and biochemical alterations between the two life stages of Leishmania are governed by stage-regulated expression of several genes. Amastigote-specific genes are believed to have a role in the survival and replication of the parasite in the hostile environment of the mammalian host. Previously, we have reported the upregulated expression of A1 gene (LdA1) at the amastigote stage at RNA level. In the present study, we have further characterized LdA1, in order to understand its role in Leishmania. Sequence homology search revealed that LdA1 is unique to the Leishmania genus. Sequence- and structure-level functional annotations predicted the involvement of LdA1 in a range of biological processes critical for survival of the parasites. Western blot confirmed the upregulated expression of LdA1 at the protein level at the amastigote stage. Overexpression of LdA1 in Leishmania did not affect its growth, phenotype, or infectivity. Attempts to generate null mutants of LdA1 by homologous recombination were not successful. Repeated inability to create null mutants of LdA1 was suggestive of gene essentiality. Mutant parasites with a single allele deletion of LdA1 (LdA1 +/- ) showed reduction in motility, size, and growth rate at both the life stages in vitro, which was restored following gene add-back by episomal expression of LdA1 in mutant parasites. Although LdA1 +/- parasites were able to infect macrophages ex vivo, their capacity to survive inside macrophages was reduced significantly (P < 0.01) beyond 72 h of infection. In conclusion, LdA1 is a Leishmania-specific gene having upregulated expression at the amastigote stage and is important for survival of Leishmania parasite.

The protozoan parasite Leishmania has evolved several strategies to undermine host defense mechanisms by inducing Th2-type adaptive immunity and suppressing effector functions of Th1 phenotype. In our earlier studies, using centrin gene-deleted Leishmania (LdCen -/- ) parasites as an immunogen, we have shown induction of an effective Th1-type immunity and robust memory responses that mediate protection against virulent challenge. However, role of inhibitory signals in Leishmania vaccine induced immunity in general, and LdCen -/- in particular has not been studied. Herein, we report that immunization with LdCen -/- parasites produces more functional Th1-type CD4 + T cells via downregulation of CD200-CD200R immune inhibitory axis compared to wild-type infection. We found that expression of CD200 and CD200R was significantly reduced in LdCen -/- infection compared to wild-type infection. Diminished CD200-CD200R signaling in LdCen -/- infection enabled proliferation of CD4 + T cells and resulted in the induction of pro-inflammatory cytokines and suppression of anti-inflammatory response. The effects of diminished CD200-CD200R signaling by LdCen -/- were most evident in the suppression of IL-10-producing CD4 + T cells that helped enhance more Th1 cytokine producing and multi-functional T cells compared to wild-type infection. In vivo blocking of CD200 expression with anti-CD200 treatment in wild-type infected mice limited Th2 response as indicated by reduction of IL-10-producing Tr1 cells and reduced parasite burden. On the other hand, treatment with anti-CD200 improved the LdCen -/- vaccine-induced multifunctional response and reduction in splenic parasite load upon challenge. Taken together, these studies demonstrate the role of CD200-CD200R signals in the protection induced by LdCen -/- parasites.

The visceralizing potential of apparently dermotropic Leishmania donovani in Sri Lanka (L. donovani-SL) was investigated through long-term follow-up of cutaneous leishmaniasis (CL) patients and in vivo and in vitro experimental infection models. CL patients (n = 250) treated effectively with intra-lesional antimony therapy were followed-up six monthly for 4 years. There was no clinical evidence of visceralization of infection (VL) during this period. Infection of BALB/c mice with L. donovani-SL (test) through intra-dermal route led to the development of cutaneous lesions at the site of inoculation with no signs of systemic dissemination, in contrast to the observations made in animals similarly infected with a visceralizing strain of L. donovani-1S (control). Cytokine (IL-10, IFN-γ) release patterns of splenocytes and lymph node cell cultures derived from mice primed with experimental infections (with either test or control parasites) revealed significantly high IFN-γ response associated with test mice with CL, while prominent IL-10 levels were observed in association with control mice with VL. Furthermore, diminished infection efficiency, intracellular growth and survival of L. donovani-SL parasites compared with L. donovani-1S were evident through in vitro macrophage infection experiments. These studies confirm, for the first time, the essential dermotropic nature of L. donovani-SL suggesting natural attenuation of virulence of local parasite strains.

Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1β (IL-1β), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1β in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1β are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease., (Published by Elsevier Inc.)

No vaccine exists against visceral leishmaniasis. To develop effective vaccines, we have previously reported protective role of live attenuated centrin gene-deleted Leishmania donovani ( LdCen -/- ) parasites through induction of Th1 type immune response in mice, hamsters, and dogs. In this study, we specifically explored the role of Th17 cells in LdCen -/- -induced host protection in mice. Our results showed that compared with wild-type L. donovani infection, LdCen -/- parasites induce significantly higher expression of Th17 differentiation cytokines in splenic dendritic cells. There was also induction of IL-17 and its promoting cytokines in total splenocytes and in both CD4 and CD8 T cells following immunization with LdCen -/- Upon challenge with wild-type parasites, IL-17 and its differentiating cytokines were significantly higher in LdCen -/- -immunized mice compared with nonimmunized mice that resulted in parasite control. Alongside IL-17 induction, we observed induction of IFN-γ-producing Th1 cells as reported earlier. However, Th17 cells are generated before Th1 cells. Neutralization of either IL-17 or IFN-γ abrogated LdCen -/- -induced host protection further confirming the essential role of Th17 along with Th1 cytokines in host protection. Treatment with recombinant IL-23, which is required for stabilization and maintenance of IL-17, heightened Th17, and Tc17 responses in immunized mice splenocytes. In contrast, Th17 response was absent in immunized IL-23R -/- mice that failed to induce protection upon virulent Leishmania challenge suggesting that IL-23 plays an essential role in IL-17-mediated protection by LdCen -/- parasites. This study unveiled the role of IL-23-dependent IL-17 induction in LdCen -/- parasite-induced immunity and subsequent protection against visceral leishmaniasis.

Currently, there is no vaccine against visceral leishmaniasis (VL). Toward developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin -/- mutants in naive animal models. In VL endemic areas, asymptomatic carriers outnumber symptomatic cases of VL and are considered to be a reservoir of infection. Vaccination of asymptomatic cases represents a viable strategy to eliminate VL. Immunological correlates of protection thus derived might have limited applicability in conditions where the immunized host has prior exposure to virulent infection. To examine whether LdCen -/- parasites can induce protective immunity in experimental hosts that have low-level parasitemia from a previous exposure mimicking an asymptomatic condition, we infected C57Bl/6 mice with wild-type Leishmania donovani parasites expressing LLO epitope ( LdWT LLO 10 3 , i.v.). After 3 weeks, the mice with low levels of parasitemia were immunized with LdCen -/- parasites expressing 2W epitope ( LdCen -/-2W 3 × 10 6 i.v.) to characterize the immune responses in the same host. Antigen experienced CD4 + T cells from the asymptomatic ( LdWT LLO infected) LdCen -/-2W immunized, and other control groups were enriched using LLO- and 2W-specific tetramers, followed by Flow cytometric analysis. Our analysis showed that comparable CD4 + T cell proliferation and CD4 + memory T cell responses (T CM ) represented by CD62L hi , CCR7 + , and IL-7R + T cell populations were induced with LdCen -/-2W in both asymptomatic and naive animals that received LdCen -/- immunization. Upon restimulation with peptide, T CM cells differentiated into effector T cells and there was no significant difference in the recall response in animals with asymptomatic infection. Following virulent challenge, comparable reduction in splenic parasite burden was observed in both asymptomatic and naive LdCen -/- immunized animals concomitant with the development of multifunctional CD4 + and CD8 + T cells. Further, LdCen -/-2W immunization resulted in complete clearance of the preexisting asymptomatic infection ( LdWT LLO ). Our results demonstrate that LdCen -/-2W immunization could be efficacious for use in asymptomatic VL individuals. Further, immunization with LdCen -/- could help in reducing the parasite burden in the asymptomatic cases and aid in controlling the VL in endemic areas.

No licensed human vaccines are currently available against leishmaniasis. Several anti-leishmanial vaccines are currently undergoing testing, including genetically modified live-attenuated parasite vaccines. Studies with live attenuated Leishmania vaccines such as centrin deleted Leishmania donovani parasites (LdCen -/- ) showed protective immunity in animal models. Such studies typically examined the biomarkers of protective immunity however the biomarkers of attenuation in the parasite preparations have not received adequate attention. As several candidate vaccines enter clinical trials, a more complete product characterization to enable maintenance of product quality will help meet regulatory requirements. Towards this goal, we have determined the complete genome sequence of LdCen -/- and its parent strain Ld1S-2D (LdWT) and characterized the LdCen -/- vaccine strain using bioinformatics tools. Results showed that the LdCen -/- parasites, in addition to loss of the centrin gene, have additional deletions ranging from 350 bp to 6900 bp in non-contiguous loci on several chromosomes, most commonly in untranslated regions. We have experimentally verified a subset of these adventitious deletions that had no impact on the attenuation of the LdCen -/- parasites. Our results identified hitherto unknown features of attenuation of virulence that could be used as markers of product quality in production lots and highlight the importance of product characterization in parasitic vaccines.

The clinical outcome of Leishmania pathogenesis ranges from active skin lesions to fatal visceral dissemination and severely impaired T cell immunity. It is well established that a strong Th1 immune response is protective against cutaneous forms of the disease, however a mixed Th1/Th2 response is most commonly observed against visceral infections as evident from previous studies. Aside from Th1/Th2 cytokines, the pro-inflammatory IL-17 cytokine family plays an important role in the clearance of intracellular pathogens. In Leishmania induced skin lesions, IL-17 produced by Th17 cells is shown to exacerbate the disease, suggesting a role in pathogenesis. However, a protective role for IL-17 is indicated by the expansion of IL-17 producing cells in vaccine-induced immunity. In human visceral leishmaniasis (VL) it has been demonstrated that IL-17 and IL-22 are associated with protection against re-exposure to Leishmania, which further suggests the involvement of IL-17 in vaccine induced protective immunity. Although there is no vaccine against any form of leishmaniasis, the development of genetically modified live attenuated parasites as vaccine candidates prove to be promising, as they successfully induce a robust protective immune response in various animal models. However, the role of IL-17 producing cells and Th17 cells in response to these vaccine candidates remains unexplored. In this article, we review the role of IL-17 in Leishmania pathogenesis and the potential impact on vaccine induced immunity, with a special focus on live attenuated Leishmania parasites., (Published by Elsevier Inc.)

Currently no effective vaccine is available for human visceral leishmaniasis(VL) caused by Leishmania donovani. Previously, we showed that centrin1 and p27gene deleted live attenuated Leishmania parasites (LdCen1(-/-) and Ldp27(-/-)) are safe, immunogenic and protective in animal models. Here, to assess the correlates of protection, we evaluated immune responses induced by LdCen1(-/-) and Ldp27(-/-) in human blood samples obtained from healthy, healed VL (HVL), post kala-azar dermal leishmaniasis(PKDL) and VL subjects. Both parasites infected human macrophages, as effectively as the wild type parasites. Further, LdCen1(-/-) and Ldp27(-/-) strongly stimulated production of pro-inflammatory cytokines including, IL-12, IFN-γ, TNF-α, IL-2, IL-6 and IL-17 in the PBMCs obtained from individuals with a prior exposure to Leishmania (HVL and PKDL). There was no significant stimulation of anti-inflammatory cytokines (IL-4 and IL-10). Induction of Th1 biased immune responses was supported by a remarkable increase in IFN-γ secreting CD4(+) and CD8(+) T cells and IL-17 secreting CD4(+) cells in PBMCs from HVL cases with no increase in IL-10 secreting T cells. Hence, LdCen1(-/-) and Ldp27(-/-) are promising as live vaccine candidates against VL since they elicit strong protective immune response in human PBMCs from HVL, similar to the wild type parasite infection, mimicking a naturally acquired protection following cure.

Background: Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice., Methodology: Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice., Conclusions: Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice., Competing Interests: The authors have declared that no competing interests exist.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani There are no vaccines and available drugs against leishmaniasis are toxic. Immunomodulators that specifically boost the anti-microbial activities of the immune cells could alleviate several of these limitations. Therefore, finding novel immunomodulators for VL therapy is a pressing need. This study is aimed to evaluate the immunomodulatory role of leptin, an adipocyte-derived hormone capable of regulating the immune response, in L. donovani-infected mice. We observed that recombinant leptin treatment reduced splenic parasite burden compared with non-treated infected normal mice. Decrease in parasite burden correlated with an induction of innate immune response in antigen-presenting cells that showed an increase in nitric oxide, enhanced pro-inflammatory cytokine (interferon gamma [IFNγ], interleukin12 [IL]12, and IL1β) response in the splenocytes, indicating host-protecting Th1 response mediated by leptin. Moreover, in infected normal mice, leptin treatment induced IFNγ production from both CD4(+) and CD8(+) T cells, compared with non-treated infected mice. Alternatively, leptin-deficient (Ob/Ob) mice had higher splenic and liver parasite burden compared with the infected normal mice. However, leptin treatment failed to reduce the splenic parasite burden and improve a host-protective cytokine response in these mice. In addition, in contrast to dendritic cells (DCs) from a normal mouse, Ob/Ob mouse-derived DCs showed a defect in the induction of innate immune response on Leishmania infection that could not be reversed by leptin treatment. Therefore, our findings reveal that leptin has a differential immunomodulatory effect in controlling VL in normal and Ob/Ob mice., (© The American Society of Tropical Medicine and Hygiene.)

Transfusion-transmitted infections have been documented for several arboviruses, including West Nile and dengue viruses (1). Zika virus, a flavivirus transmitted primarily by Aedes aegypti mosquitoes that has been identified as a cause of congenital microcephaly and other serious brain defects (2), became recognized as a potential threat to blood safety after reports from a 2013-2014 outbreak in French Polynesia. Blood safety concerns were based on very high infection incidence in the population at large during epidemics, the high percentage of persons with asymptomatic infection, the high proportion of blood donations with evidence of Zika virus nucleic acid upon retrospective testing, and an estimated 7-10-day period of viremia (3). At least one instance of transfusion transmission of Zika virus has been documented in Brazil after the virus emerged there, likely in 2014 (4). Rapid epidemic spread has followed to other areas of the Americas, including Puerto Rico.

Background: The implementation of nucleic acid-based tests for blood donor screening has improved the safety of the blood supply; however, the increasing number of emerging pathogen tests is burdensome. Development of multiplex testing platforms that allow simultaneous screening for different pathogens is a potential solution., Study Design and Methods: The TessArray resequencing microarray is a platform that allows multiplex detection and identification of 97 different blood-borne pathogens in one single test. The objective was to evaluate the lowest concentration detected in blood or plasma, species discrimination, and applicability of the TessArray microarray platform for testing blood donors. Human blood or plasma spiked with selected pathogens (10,000, 1000, or 100 cells or copies/mL), including three viral, four bacterial, and four protozoan pathogens were each tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of pooled specific primers, fragmented, labeled, and hybridized to a microarray. Finally, the detected sequences were identified using an automated genomic database alignment algorithm., Results: The performance of this platform demonstrated detection for spiked bacterial and protozoan pathogens of 100 cells/mL and viral pathogens as low as 100 copies/mL. Coded specimens, including spiked and negative controls, were identified correctly for blood specimens (31/32, 97%) and for plasma specimens (20/22, 91%) demonstrating the effectiveness of the platform., Conclusion: These results indicated that the TessArray microarray platform could be employed for multiplex detection and identification, with a high level of discriminatory power for numerous blood-borne pathogen targets with potential for use in blood safety., (© 2016 AABB.)

No licensed human vaccines are currently available against any parasitic disease including leishmaniasis. Several antileishmanial vaccine formulations have been tested in various animal models, including genetically modified live-attenuated parasite vaccines. Experimental infection studies have shown that Leishmania parasites utilize a broad range of strategies to undermine effector properties of host phagocytic cells, i.e., dendritic cells (DCs) and macrophages (MΦ). Furthermore, Leishmania parasites have evolved strategies to actively inhibit TH1 polarizing functions of DCs and to condition the infected MΦ toward anti-inflammatory/alternative/M2 phenotype. The altered phenotype of phagocytic cells is characterized by decreased production of antimicrobial reactive oxygen, nitrogen molecules, and pro-inflammatory cytokines, such as IFN-γ, IL-12, and TNF-α. These early events limit the activation of TH1-effector cells and set the stage for pathogenesis. Furthermore, this early control of innate immunity by the virulent parasites results in substantial alteration in the adaptive immunity characterized by reduced proliferation of CD4(+) and CD8(+) T cells and TH2-biased immunity that results in production of anti-inflammatory cytokines, such as TGF-β, and IL-10. More recent studies have also documented the induction of coinhibitory ligands, such as CTLA-4, PD-L1, CD200, and Tim-3, that induce exhaustion and/or non-proliferation in antigen-experienced T cells. Most of these studies focus on viral infections in chronic phase, thus limiting the direct application of these results to parasitic infections and much less to parasitic vaccines. However, these studies suggest that vaccine-induced protective immunity can be modulated using strategies that enhance the costimulation that might reduce the threshold necessary for T cell activation and conversely by strategies that reduce or block inhibitory molecules, such as PD-L1 and CD200. In this review, we will focus on the polarization of antigen-presenting cells and subsequent role of costimulatory and coinhibitory molecules in mediating vaccine-induced immunity using live-attenuated Leishmania parasites as specific examples.

Background: Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge., Methods: Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines., Results and Discussion: Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells., Conclusion: The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.

Aims: To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms., Methods and Results: This study presents detailed methods for spiking bacteria or protozoan parasites into whole blood and virus into plasma. Proper methods must start with a documented, reproducible pathogen source followed by steps that include standardized culture, preparation of cryopreserved aliquots, quantification of the aliquots by molecular methods, production of sufficient numbers of individual specimens and testing of the platform with multiple mock specimens. Results are presented following the described procedures that showed acceptable reproducibility comparing in-house real-time PCR assays to a commercially available multiplex molecular assay., Conclusions: A step by step procedure has been described that can be followed by assay developers who are targeting low prevalence pathogens., Significance and Impact of the Study: The development of diagnostic platforms for detection of low prevalence pathogens such as biothreat or emerging agents is challenged by the lack of clinical specimens for performance evaluation. This deficit can be overcome using mock clinical specimens made by spiking cultured pathogens into human matrices. To facilitate evaluation and comparison among platforms, standardized methods must be followed in the preparation and application of spiked specimens., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Background: 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., Methodology/principal Findings: 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., Conclusions: 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.

Live-attenuated parasite vaccines are being explored as potential vaccine candidates since other approaches of vaccination have not produced an effective vaccine so far. In order for live-attenuated parasite vaccines to be tested in preclinical studies and possibly in clinical studies, the safety and immunogenicity of these organisms must be rigorously evaluated. Here we describe methods to test persistence in the immunized host and immunogenicity, and to identify biomarkers of vaccine safety and efficacy with particular reference to genetically attenuated Leishmania parasites.