Dual-scRNA-seq analysis reveals rare and uncommon parasitized cell populations in chronic L. donovani infection.

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.

Leishmania major centrin knock-out parasites reprogram tryptophan metabolism to induce a pro-inflammatory response.

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.

Toll-like Receptor-9 (TLR-9) Signaling Is Crucial for Inducing Protective Immunity following Immunization with Genetically Modified Live Attenuated Leishmania Parasites.

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.

The History of Live Attenuated Centrin Gene-Deleted Leishmania Vaccine Candidates.

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.

Leishmania Major Centrin Gene-Deleted Parasites Generate Skin Resident Memory T-Cell Immune Response Analogous to Leishmanization.

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.

Neutrophil-dendritic cell interaction plays an important role in live attenuated Leishmania vaccine induced immunity.

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.

Determinants of Innate Immunity in Visceral Leishmaniasis and Their Implication in Vaccine Development.

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.

From infection to vaccination: reviewing the global burden, history of vaccine development, and recurring challenges in global leishmaniasis protection.

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.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis.

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.

Revival of Leishmanization and Leishmanin.

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.

Essential Role of Neutrophils in the Protective Immune Response Induced by a Live Attenuated Leishmania Vaccine.

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 Nß 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.

miR-21 Expression Determines the Early Vaccine Immunity Induced by LdCen-/- Immunization.

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.

Leptin Functions in Infectious Diseases.

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.

Gut Microbes Egested during Bites of Infected Sand Flies Augment Severity of Leishmaniasis via Inflammasome-Derived IL-1ß.

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-1ß (IL-1ß), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1ß in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1ß 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.

Live Attenuated Leishmania donovani Centrin Gene-Deleted Parasites Induce IL-23-Dependent IL-17-Protective Immune Response against Visceral Leishmaniasis in a Murine Model.

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.

Immunization with Live Attenuated Leishmania donovani Centrin-/- Parasites Is Efficacious in Asymptomatic Infection.

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 103, i.v.). After 3 weeks, the mice with low levels of parasitemia were immunized with LdCen-/- parasites expressing 2W epitope (LdCen-/-2W 3 × 106 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 (TCM) represented by CD62Lhi, 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, TCM 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.

Live Attenuated Leishmania donovani Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis.

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.

Role of pro-inflammatory cytokine IL-17 in Leishmania pathogenesis and in protective immunity by Leishmania 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.

Modulation of Innate Immune Mechanisms to Enhance Leishmania Vaccine-Induced Immunity: Role of Coinhibitory Molecules.

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-ß, 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.

Methods to Evaluate the Preclinical Safety and Immunogenicity of Genetically Modified Live-Attenuated Leishmania Parasite Vaccines.

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.