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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 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.)

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.)

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 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.

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 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.

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