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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.

The protozoan parasite

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

Live attenuated Leishmania donovani parasites such as LdCen(-/-) have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen(-/-) including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4(+) and CD8(+) T cells, IFN-γ production by CD8(+) T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen(-/-) parasites as vaccine candidates, we performed a 24-month follow up of LdCen(-/-) immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen(-/-) (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune(®)). The protection was associated with antibody production and CD4(+) and CD8(+) proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune(®), with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen(-/-) expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune(®) or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.

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.

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 (LdWTLLO 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 (LdWTLLO 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 (LdWTLLO). 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.

Background Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). Methods Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. Results Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. Conclusion This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis. Electronic supplementary material The online version of this article (10.1186/s12879-017-2883-x) contains supplementary material, which is available to authorized users.

Mast Cells (MCs) are one of the first immune cells encountered by invading pathogens. Their presence in large numbers in the superficial dermis, where Leishmania is encountered, suggests that they may play a critical role in immune responses to Leishmania. In this study the interactions of Leishmania donovani, the causative agent of visceral Leishmaniasis, and Leishmania tropica, the causative agent of cutaneous Leishmaniasis with MCs were studied. Co-culture of Leishmania with Peritoneal Mast Cells (PMCs) from BALB/c mice and Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of L. tropica and to a lesser extent of L. donovani. Also, while there was significant uptake of L. tropica by MCs, L. donovani was not phagocytosed. There was significant generation of Reactive Oxygen Species (ROS) by MCs on co-culture with these species of Leishmania which may contribute to their clearance. Interactions of MCs with Leishmania led to generation of MC extracellular traps comprising of DNA, histones and tryptase probably to ensnare these pathogens. These results clearly establish that MCs may contribute to host defences to Leishmania in a differential manner, by actively taking up these pathogens, and also by mounting effector responses for their clearance by extracellular means.

Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis.

Leishmaniasis is a vector-born disease transmitted through sand fly bite with no available vaccine. Vaccination through leishmanization with Leishmania major has been used successfully but is not safe. Recently, we have demonstrated immunization with live attenuated LmCen−/− parasite protects against Leishmaniasis via induction of host cellular immunity. Resident memory T cells (TRMs) are considered the first line of defense against infections invading the host through epithelial barrier. We evaluated the generation and function of skin TRMs post LmCen−/− immunization, compared to that generated by LmWT healed mice. We examined chemokine receptors controlling the generation and survival of skin TRMs, as well as effector and recruitment function of those cells post challenge with WT parasites. We observed that expression of CXCR6 and CXCR3 was significantly higher on T cells from spleens and lymph nodes of immunized mice, compared to those from healed mice. At 15 weeks post immunization, the skin of immunized mice had significantly larger population of TRMs compared to healed mice. In addition, epithelial cytokine production, such as IL-15, IL-33 and TNFa was significantly higher in the skin of immunized mice. Upon virulent challenge, immunofluorescent labeling of skin sections showed rapid recruitment of more T cells in the immunized mice compared to healed mice. IFNγ production by skin TRMs post challenge was significantly higher in immunized mice compared to healed mice. Taken together, these results show that immunization with live attenuated parasites is safer than Leishmanization. In addition, it generates large functional population of skin TRMs which play an important role in protection against the Leishmania infection.

Zoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen(-/-) in a canine infection model and compared it to that of Leishmune(®), a commercially available recombinant vaccine. The immunogenicity of the LdCen(-/-) parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen(-/-) resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen(-/-) vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis.

Leishmania transmission garnered attention due to the virulence of these vector-transmitted parasites. Numerous studies established a remarkable complexity to the infectious inoculum of an infected sand fly bite. Parasites, though they are the etiological agents of leishmaniasis, are but one of several components with potent immunomodulatory properties that shape the immune environment at the bite site. To date, known components include saliva, the parasite secretory gel, and exosomes. Sand fly vector-derived virulence factors are only of consequence in the context of parasite delivery by bite. All of these components participate in creating a milieu that favors parasite establishment. Understanding the immunological consequences of an infected sand fly bite will improve our understanding of disease pathogenesis, and they have implications for novel approaches to leishmaniasis control.

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.

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.

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.

Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β-oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1-mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1(-/-)). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl-CoA, the end-product of the β-oxidation in the Ufm1(-/-) amastigote stage. The Ufm1(-/-) mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re-expression of wild-type Ufm1 with concomitant induction of acetyl-CoA but not by re-expressing the non-conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and β-oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1(-/-) parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amastigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1(-/-) parasites as drug and vaccine targets.

Identifying and characterizingLeishmania donovanigenes and the proteins they encode for their role in pathogenesis can reveal the value of this approach for finding new drug targets. Effective drug targets are likely to be proteins differentially expressed or required in the amastigote life cycle stage found in the patient. Several examples and their potential for chemotherapeutic disruption are presented. A pathway nearly ubiquitous in living cells targeted by anticancer drugs, the ubiquitin system, is examined. New findings in ubiquitin and ubiquitin-like modifiers inLeishmaniashow how disruption of those pathways could point to additional drug targets. The programmed cell death pathway, now recognized among protozoan parasites, is reviewed for some of its components and evidence that suggests they could be targeted for antiparasitic drug therapy. Finally, the endoplasmic reticulum quality control system is involved in secretion of many virulence factors. How disruptions in this pathway reduce virulence as evidence for potential drug targets is presented.

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

Infections of humans with intraerythrocytic parasites of the genus Babesia can be locally prevalent in diverse regions of the United States. Transfusion of blood and blood products collected from donors infected with Babesia may result in a serious illness that can be fatal. In September 2008, the Food and Drug Administration organized a public workshop to discuss the various aspects of transfusion-transmitted babesiosis in the United States including the possible strategies to identify and defer blood donors who may have been infected with Babesia. Discussions were also held on the biology, pathogenesis, and epidemiology of Babesia species. In this article, we summarize the scientific presentations and panel discussions that took place during the workshop.

Molecules and cellular mechanisms that regulate the process of cell division in malaria parasites remain poorly understood. In this study we isolate and characterize the four Plasmodium falciparum centrins (PfCENs) and, by growth complementation studies, provide evidence for their involvement in cell division. Centrins are cytoskeleton proteins with key roles in cell division, including centrosome duplication, and possess four Ca(2+)-binding EF hand domains. By means of phylogenetic analysis, we were able to decipher the evolutionary history of centrins in eukaryotes with particular emphasis on the situation in apicomplexans and other alveolates. Plasmodium possesses orthologs of four distinct centrin paralogs traceable to the ancestral alveolate, including two that are unique to alveolates. By real time PCR and/or immunofluorescence, we determined the expression of PfCEN mRNA or protein in sporozoites, asexual blood forms, gametocytes, and in the oocysts developing inside mosquito mid-gut. Immunoelectron microscopy studies showed that centrin is expressed in close proximity with the nucleus of sporozoites and asexual schizonts. Furthermore, confocal and widefield microscopy using the double staining with alpha-tubulin and centrin antibodies strongly suggested that centrin is associated with the parasite centrosome. Following the episomal expression of the four PfCENs in a centrin knock-out Leishmania donovani parasite line that exhibited a severe growth defect, one of the PfCENs was able to partially restore Leishmania growth rate and overcome the defect in cytokinesis in such mutant cell line. To our knowledge, this study is the first characterization of a Plasmodium molecule that is involved in the process of cell division. These results provide the opportunity to further explore the role of centrins in cell division in malaria parasites and suggest novel targets to construct genetically modified, live attenuated malaria 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.

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

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.

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 coculture 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.

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., Author Summary Leishmaniasis is a disease with a wide spectrum of clinical manifestations caused by different species of protozoa belonging to the Leishmania genus that are transmitted by sand fly vectors. Visceral infections of Leishmania cause significant mortality and morbidity and development of a vaccine to prevent leishmaniasis has become a high priority. We have previously reported that intravenous immunization with a live attenuated parasite vaccine comprised of Leishmania donovani parasites lacking the centrin gene conferred protection in mice, hamsters and dogs. In the current report, we describe the immunological response and associated protection to the ID immunization with attenuated parasites in combination with a sand fly salivary protein (LJM19). We observe that protection against experimental ID challenge with L. donovani resulting from ID immunization with live attenuated parasites in combination with LJM19 is comparable to intracardial immunization and offers improved protective immunity compared to immunization with salivary protein alone and non-immunized hamsters. This study supports the potential use of the genetically attenuated vaccine and a recombinant sand fly salivary protein for control of visceral leishmaniasis.

The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key molecule for plasma cell maintenance and is required in infections where protection depends on antibody response. Here, we report that compared with WT mouse, TACI KO Μϕs expressed lower levels of Toll-like receptors (TLRs), CD14, myeloid differentiation primary response protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLR agonists. Analysis of Μϕ phenotype revealed that, in the absence of TACI, Μϕs adapt the alternatively activated (M2) phenotype. Steady-state expression levels for M2 markers IL-4Rα, CD206, CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, TACI-KO Mϕs were unable to control Leishmania major infection in vitro, and intradermal inoculation of Leishmania resulted in a more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to significantly reduce disease severity. TACI is likely to influence Mϕ phenotype by mediating B cell-activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) signals because both these ligands down-regulated M2 markers in WT but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with BAFF or APRIL enhanced the clearance of Leishmania from cells only when TACI is expressed. These findings may have implications for understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immunodeficiency patients where TACI signaling is ablated.

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.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.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.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.

Five to fifteen percent of visceral leishmaniasis (VL) patients in India develop post kala-azar dermal leishmaniasis (PKDL), usually 1-2 years after apparent clinical cure. There is evidence pointing to a role played by the host immune responses in the disease pathogenesis, however, the contribution of changes in parasite gene expression has not been explored. Highly sensitive gene expression microarray technology was employed to identify genes that are differentially expressed in Leishmania parasites isolated from PKDL patients in comparison with those from VL. Hybridization on Leishmania donovani genomic microarray comprised of unique clones allowed us to identify 46/2268 (2%) clones that showed statistically significant (P

Visceral leishmaniasis (VL) is a neglected vector-borne disease causing fever, fatigue, weight loss, anemia, and hepatosplenomegaly in humans. Untreated, VL is almost 100% fatal within 2 years [1]. VL is transmitted by phlebotomine sand flies and is caused by 2 species of Leishmania. Leishmania infantum (=Leishmania chagasi) is prevalent in the Mediterranean Basin and Latin America and has a zoonotic transmission cycle [1–4]. Leishmania donovani is prevalent in the Indian subcontinent, South Asia, and East Africa [1, 5] and is considered mostly anthroponotic [1, 2]. Most VL studies involve intravenous, intracardiac, or intraperitoneal injection of 106–108 parasites into laboratory animals [6–11]. These routes bypass the skin, an immunologically privileged tissue [12] and the natural delivery site of the parasites. Progressive VL in hamsters was also achieved via intradermal injection of 105 parasites, a route closer to natural transmission by sand fly bites [13, 14]. For cutaneous leishmaniasis, natural transmission by vector bites revealed important distinctions in disease initiation, progression, and response to vaccination compared with needle challenge [15–18]. This likely reflects the complexity of transmission by vector bites. Leishmania are delivered into skin together with molecules that modulate the bite site, including salivary proteins and parasite-derived promastigote secretory gel [19–21]. Additionally, an infected sand fly displays modified feeding behavior with prolonged and persistent probing [22]. All the above initiates a rapid influx of immune cells to the bite site, adding to the complexity of parasite delivery following transmission by bite [16]. The dose delivered by sand flies into skin may also be relevant to disease initiation and evolution [23]. Kimblin et al [24], reported that Phlebotomus duboscqi, a natural vector of L. major, transmits 10 to 105 parasites, whereas Phlebotomus perniciosus and Lutzomyia longipalpis, natural vectors of L. infantum, transmit 8 to 4 × 104 parasites and 4 to 104 parasites, respectively [25, 26]. Despite variability in the dose delivered by individual infected flies, it remains significantly lower compared with the inoculum commonly used in needle-injection models, with the majority of sand flies delivering fewer than 600 parasites [24–26]. Because it mimics clinicopathologic features of active human disease, the hamster needle-injection model of experimental VL has been extensively used for studies on disease pathogenesis and immunosuppression [27–30] and to test efficacy of drugs and vaccines [7–10, 30–32]. To date, there are no models of natural transmission by vector-bite for VL. Studies reporting successful transmission of Leishmania parasites by VL vectors [25, 26] address load delivered by bite without further demonstration of disease progression. We report development of reproducible progressive VL in hamsters following transmission of L. infantum and L. donovani by vector bite and describe disease features unique to sand fly transmission and relevant to naturally acquired VL.

Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within the Indian isolates and to identify differentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could be differentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, differences within the Indian isolates could also be identified. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern blot analysis. Three such fragments were found to represent transcribed sequences that were differentially expressed in the two stages of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in genomic fingerprinting and in identifying differentially transcribed sequences that could potentially contribute to parasite virulence.

In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. However, PCD is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In protozoan parasites and in other unicellular organisms, features of PCD similar to those in multicellular organisms have been reported, suggesting some commonality in the PCD pathway between unicellular and multicellular organisms. However, more extensive studies are needed to fully characterise the PCD pathway and to define the factors that control PCD in the unicellular organisms. The understanding of the PCD pathway in unicellular organisms could delineate the evolutionary origin of this pathway. Further characterisation of the PCD pathway in the unicellular parasites could provide information regarding their pathogenesis, which could be exploited to target new drugs to limit their growth and treat the disease they cause.

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.

Mast cells (MCs) are involved in clearing invading pathogens at the skin surface and play an important role in innate and adaptive defenses. Hence they may have an important role in the control of Leishmaniasis due to the protozoa Leishmania that are inoculated by infected sandfly vector on the skin surface. It is still unclear if their contributions occur directly through active pathogen killing or antigen uptake. Our objectives were to use Leishmania donovoni the causative agent of fatal visceral Leishmaniasis and L. tropica the causative agent of cutaneous leishmaniasis for studying the interactions between the parasite and MCs to clarify if MCs are directly involved in active antigen uptake, and its clearance. We found that co-culture of Leishmania with MCs led to significant killing of L. tropica and to a lesser extent of L. donovoni. Also, while there was significant uptake of L. tropica by MCs, L. donovoni was not phagocytosed by MCs. Uptake increased with time of exposure in case of L. tropica. We also found significant generation of reactive oxygen species by MCs on co-culture with these species of Leishmania, which may contribute to their clearance. However interactions of MCs with Leishmania led to generation of MC extracellular traps comprising of DNA, histones and tryptase probably to ensnare these pathogens. These results clearly establish that MCs may contribute to host defences to Leishmania in a differential manner by actively taking up these pathogens and also by mounting effector responses for their direct clearance by extracellular and non-phagocytic means. This gives us an understanding that MCs can also be used as a target for therapeutic approaches to leishmaniasis.

No vaccine exists against visceral leishmaniasis. Towards developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin−/− mutants in animal models. However, for the use of LdCen1−/− in humans there is a need to develop biomarkers that can predict the immunogenicity of LdCen−/− at an earlier stage of vaccination. Unbiased gene expression profiling of US blood donor’s PBMCs infected with Leishmania parasites (LdWT or LdCen−/−) ex-vivo resulted in the identification of several miRs that regulate important cytokine genes. Importantly, we found a strong induction of miR-21, known to control the expression of IL-12 mRNA, in LdWT infection compared to LdCen−/− infection. IL-12 produced by DCs is critical for priming a host protective Th1 cell response during Leishmania infection. Validation of human PBMC data in a mouse model revealed an enhanced expression of miR-21 in virulent LdWT infected dendritic cells (DCs) compared to LdCen−/−. This correlated with low levels of IL-12 mRNA in WT compared to LdCen−/− infected DCs. Silencing of miR-21 resulted in an augmented induction of IL12 in LdWT infected DCs both in vitro and in vivo, illustrating the role of miR-21 in LdWT infection mediated suppression of IL12. In contrast, LdCen−/− infected DCs showed a strong induction of IL12, and miR-21 silencing resulted in a further increase in IL12 levels. Additionally, blocking of miR-21 enabled strong proliferation of CD4+ T cells in mice infected with LdWT parasites. Thus lower level of miR-21 in a vaccinee may indicate the safety and immunogenicity consistent with a protective response of the vaccine. Our data suggests that miR-21 could be used as a biomarker of protection in anti-leishmanial vaccines.

Leishmaniasis is a neglected tropical disease caused by a group of protozoan parasites of the genus Leishmania. Clinical presentation of leishmaniasis can range from cutaneous, mucocutaneous, or visceral forms depending on the parasite species. Visceral leishmaniasis (VL) caused by L. donovani and L. infantum is the severest and one of the deadliest parasitic diseases of the tropics second only to malaria (1). Nearly, 20,000‐40,000 annual deaths are estimated due to this disease (2). Except for the Indian subcontinent and West Africa, VL is frequent in dogs, which serve as the major reservoir for zoonosis (3). Transmitted by the bite of an infected sandfly, Leishmania endure in the phagolysomal compartment of macrophages by evasion and attenuation of the microbicidal functions of the host (4, 5). Leishmania has evolved as a successful parasite chiefly by its ability to modulate the immunological and cyto-chemical responses of the host following infection. The key strategy for successful pathogenesis is to subvert the nitric oxide burst in the host macrophage. This opportunistic parasite thus establishes a safe niche in the inactivated phagocyte and uncontrolled parasitization in liver, spleen, and bone marrow leads to symptomatic VL characterized by fever, weight loss, hepatosplenomegaly, and anemia (3). Since the pathogenesis of the disease is based on subversion and modulation of both innate and adaptive arms of immunity, the disease is opportunistic to immuno-suppression (6). Hence, commencement of an appropriate immune response is a challenge for the control of VL infection. Indeed, therapeutic drugs like SSG and miltefosine are immuno-modulators that trigger Th1 responses essential for activation of oxidative burst in the macrophages (7, 8). However, major limitations of narrow therapeutic index and increasing incidence of resistance with currently used drugs for VL are encumbrances in effective disease management. Recent approaches like combination therapy, targeted delivery, and use of immune-adjunct are efforts to bring down the effective doses of these toxicity-associated drugs. Most promising are the prospects of various immune-targeted therapeutic approaches for treatment of VL (9). Various leishmanial antigens, cytokines, and antibodies that initiate protective Th1-biased cell-mediated immune responses used singly or as an adjunct to conventional chemotherapy are potent immuno-chemotherapeutic agents for the cure of VL (10, 11). Additionally, medicinal plants and their products have opened new dimensions in search of safer and cheaper anti-leishmanial immuno-modulators. These phytochemicals are not only promising as immuno-chemotherapeutic agents against VL but also have potential as immuno-adjuvants and adjuncts to chemotherapy for a number of other immuno-regulatory diseases (12). Since both cure and resilience to Leishmania infection depend on the immunological status of the host, the antigens that can trigger healing responses can also induce prophylactic immunity. Therefore, identification of immunogens that can induce Th1 responses is the critical aspect of vaccine search against VL (13). Although a number of defined antigens have been reported to impart protective immunity against experimental VL, recent trend of reverse vaccinology is a promising aspect for identification of key immunogens for a successful vaccine (13, 14). This requires rational inputs and algorithm for identification of a promising antigen from the whole proteome data analysis in silico (15). One of the key inputs is to identify the epitopes for activation of both CD4 C Th1 and CD8 C T cells.

Now is a good time to reconsider research priorities as 2015 approaches, the target date originally set for elimination of visceral leishmaniasis. Visceral leishmaniasis is one of the most deadly parasitic diseases and disproportionately affects the poorest and most vulnerable populations. An estimated 200 000–400 000 people contract visceral leishmaniasis every year in developing countries. Spread by sandflies, visceral leishmaniasis can be fought with existing interventions, including treatment and vector control, but, similarly to every other human parasitic disease, no effective vaccine exists.

Previously, we showed that genetically modified live-attenuated Leishmania donovani parasite cell lines (LdCen−/− and Ldp27−/−) induce a strong cellular immunity and provide protection against visceral leishmaniasis in mice. In this study, we explored the mechanism of cross-protection against cutaneous lesion-causing Leishmania mexicana. Upon challenge with wild-type L. mexicana, mice immunized either for short or long periods showed significant protection. Immunohistochemical analysis of ears from immunized/challenged mice exhibited significant influx of macrophages, as well as cells expressing MHC class II and inducible NO synthase, suggesting an induction of potent host-protective proinflammatory responses. In contrast, substantial inhibition of IL-10, IL-4, and IL-13 expression and the absence of degranulated mast cells and less influx of eosinophils within the ears of immunized/challenged mice suggested a controlled anti-inflammatory response. L. mexicana Ag–stimulated lymph node cell culture from the immunized/challenged mice revealed induction of IFN-γ secretion by the CD4 and CD8 T cells compared with non-immunized/challenged mice. We also observed suppression of Th2 cytokines in the culture supernatants of immunized/challenged lymph nodes compared with non-immunized/challenged mice. Adoptively transferred total T cells from immunized mice conferred strong protection in recipient mice against L. mexicana infection, suggesting that attenuated L. donovani can provide protection against heterologous L. mexicana parasites by induction of a strong T cell response. Furthermore, bone marrow–derived dendritic cells infected with LdCen−/− and Ldp27−/− parasites were capable of inducing a strong proinflammatory response leading to the proliferation of Th1 cells. These studies demonstrate the potential of live-attenuated L. donovani parasites as pan–Leishmania species vaccines.

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.

Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp−/−). Ufm1 processing activity was abolished in LdUfsp−/− mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp−/− parasites. Further LdUfsp−/− mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp−/− indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp−/− parasites as drug and vaccine targets., Author Summary Ubiquitin and ubiquitin like proteins (Ubls) and the enzymes that mediate the conjugation/deconjugation reactions have not been well studied in protozoan parasites despite their widely recognized importance in a broad range of cellular functions in eukaryotes. We have previously reported that Ufm1 has distinct protein targets and cellular localization in the human parasite Leishmania donovani and deletion of Ufm1 in L. donovani adversely impacts the pathogenesis suggesting that Ufm1 associated enzymes could be exploited as drug targets. Using sensitive FRET based activity probes we identified the Ufm1 processing peptidase Ufsp in L. donovani. In addition, we show that deletion of Ufsp specifically reduces the survival of amastigotes, the parasite stage that is present in the humans thus altering the pathogenesis. Studies showing inhibition of Ufsp activity by anti-leishmanial drug further suggests that Leishmania Ufsp can serve as a novel target for pharmacological intervention for this parasite that causes deadly disease.

The role of three N-linked glycosylation sites in rubella virus (RV) E1 protein on virion release was analyzed by transfecting Vero 76 cells with infectious RV RNA (Robo302WT) containing isoleucine substitutions at N76, N177, and N209 (individually and in combinations). RV RNAs were detected and found to retain substitutions in the transfected cells, but RV capsid indicative of infection was undetectable, except for in Robo302WT and Robo302-N177I transfected cells. Only culture supernatants of Robo302WT and Robo302-N177I RNA transfected cells were positive for RV, suggestive of the virion release into the culture medium. Further, detection of intracellular RV E1 and newly released virion-associated E1 was possible only from cells previously incubated with Robo302-N177I and Robo302WT culture supernatants, suggesting that N177I substituted virus retained infectivity. These results suggest that while glycosylation at N177 is not critical, N76I and N209I mutations are lethal to RV viability.

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes fromPlasmodium falciparumand evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ × A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae , and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

Very little is known about the cellular factors that are required for the maturation of rubella virus glycoproteins (E2 and E1) in the endoplasmic reticulum of the infected cell. In the present study, we established the interaction of the ER chaperone proteins, calreticulin and calnexin, with the RV E1 and E2 proteins in cells stably expressing the viral proteins. The interaction between E2 and calnexin was significantly higher than with calreticulin. In pulse-chase experiments, the half-life of the E2-calnexin was45 min, whereas the half-life of the calreticulin-E2 interaction was approximately 10 min. Tunicamycin and castanospermine treatments altered the mobilities of intracellular E1 and E2, due to either lack of oligosaccharide ligand addition or trimming of terminal glucose residues, respectively. Further, the drug treatments resulted in a loss of E1 and E2 interaction with calreticulin or calnexin, thereby demonstrating that the interaction is through monoglucosylated forms of RV proteins. These studies suggest that the interaction of RV glycoproteins with the ER chaperone proteins is essential for their maturation in the endoplasmic reticulum.