Trypanosoma brucei gambiense excreted/secreted factors impair lipopolysaccharide-induced maturation and activation of human monocyte-derived dendritic cells.

Trypanosoma brucei gambiense, an extracellular eukaryotic flagellate parasite, is the main etiological agent of human African trypanosomiasis (HAT) or sleeping sickness. Dendritic cells (DCs) play a pivotal role at the interface between innate and adaptive immune response and are implicated during HAT. In this study, we investigated the effects of T gambiense and its excreted/secreted factors (ESF) on the phenotype of human monocyte-derived DCs (Mo-DCs). Mo-DCs were cultured with trypanosomes, lipopolysaccharide (LPS), ESF derived from T gambiense bloodstream strain Biyamina (MHOM/SD/82), or both ESF and LPS. Importantly, ESF reduced the expression of the maturation markers HLA-DR and CD83, as well as the secretion of IL-12, TNF-alpha and IL-10, in LPS-stimulated Mo-DCs. During mixed-leucocyte reactions, LPS- plus ESF-exposed DCs induced a non-significant decrease in the IFN-gamma/IL-10 ratio of CD4 + T-cell cytokines. Based on the results presented here, we raise the hypothesis that T gambiense has developed an immune escape strategy through the secretion of paracrine mediators in order to limit maturation and activation of human DCs. The identification of the factor(s) in the T gambiense ESF and of the DCs signalling pathway(s) involved may be important in the development of new therapeutic targets.

Trypanosoma musculi Infection in Mice Critically Relies on Mannose Receptor-Mediated Arginase Induction by a TbKHC1 Kinesin H Chain Homolog.

Arginase activity induction in macrophages is an escape mechanism developed by parasites to cope with the host's immune defense and benefit from increased host-derived growth factor production. We report that arginase expression and activity were induced in macrophages during mouse infection by Trypanosoma musculi, a natural parasite of this host. This induction was reproduced in vitro by excreted/secreted factors of the parasite. A mAb directed to TbKHC1, an orphan kinesin H chain from Trypanosoma brucei, inhibited T. musculi excreted/secreted factor-mediated arginase induction. Anti-TbKHC1 Ab also inhibited T. musculi growth, both in vitro and in vivo. Induction of arginase activity and parasite growth involved C-type lectin receptors, because mannose injection decreased arginase activity induction and parasite load in vitro and in vivo. Accordingly, the parasite load was reduced in mice lacking mannose receptor C-type 1. The T. musculi KHC1 homolog showed high similarity with TbKHC1. Bioinformatics analysis revealed the presence of homologs of this gene in other trypanosomes, including pathogens for humans and animals. Host metabolism dysregulation represents an effective parasite mechanism to hamper the host immune response and modify host molecule production to favor parasite invasion and growth. Thus, this orphan kinesin plays an important role in promoting trypanosome infection, and its neutralization or the lock of its partner host molecules offers promising approaches to increasing resistance to infection and new developments in vaccination against trypanosomiasis.

A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.

BACKGROUND: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. METHODOLOGY/PRINCIPAL FINDINGS: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. CONCLUSION: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

The Trypanosoma brucei gambiense secretome impairs lipopolysaccharide-induced maturation, cytokine production, and allostimulatory capacity of dendritic cells.

Trypanosoma brucei gambiense, a parasitic protozoan belonging to kinetoplastids, is the main etiological agent of human African trypanosomiasis (HAT), or sleeping sickness. One major characteristic of this disease is the dysregulation of the host immune system. The present study demonstrates that the secretome (excreted-secreted proteins) of T. b. gambiense impairs the lipopolysaccharide (LPS)-induced maturation of murine dendritic cells (DCs). The upregulation of major histocompatibility complex class II, CD40, CD80, and CD86 molecules, as well as the secretion of cytokines such as tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-6, which are normally released at high levels by LPS-stimulated DCs, is significantly reduced when these cells are cultured in the presence of the T. b. gambiense secretome. Moreover, the inhibition of DC maturation results in the loss of their allostimulatory capacity, leading to a dramatic decrease in Th1/Th2 cytokine production by cocultured lymphocytes. These results provide new insights into a novel efficient immunosuppressive mechanism directly involving the alteration of DC function which might be used by T. b. gambiense to interfere with the host immune responses in HAT and promote the infectious process.

Serum arginase, a biomarker of treatment efficacy in human African trypanosomiasis.

Arginase serum levels were increased in human African trypanosomiasis patients and returned to control values after treatment. Arginase hydrolyzes l-arginine to l-ornithine, which is essential for parasite growth. Moreover, l-arginine depletion impairs immune functions. Arginase may be considered as a biomarker for treatment efficacy.

Optimization of the arginase activity assay micromethod for macrophages and sera.

OBJECTIVE: We optimized the spectrophotometric micromethod for the determination of arginase activity based on the Corraliza et al. modification of Schimke's method. Arginase activity in sera from patients suffering from human African trypanosomiasis, in macrophage lysates from trypanosome-infected mice, and in purified bovine liver arginase was compared using the conventional and optimized micromethods. RESULTS: The sensitivity of both micromethods was comparable. However, our optimized method has the following advantages: it uses small sample volumes (6 µl per assay vs. 50 µl) and reagent volumes (200 µl vs. 400 µl), it can be carried out in a single microplate well, thereby minimizing handling, and it requires fewer materials and utilizes readily available equipment. Our optimized method proved to be applicable and well suited for small-volume samples and resource-poor laboratories.

Cerebrospinal fluid B lymphocyte identification for diagnosis and follow-up in human African trypanosomiasis in the field.

OBJECTIVES: In human African trypanosomiasis (HAT, sleeping sickness), staging of disease and treatment follow-up relies on white cell count in the cerebrospinal fluid (CSF). As B lymphocytes (CD19 positive cells) are not found in the CSF of healthy individuals but occur in neurological disorders such as multiple sclerosis, B lymphocyte count may be useful for field diagnosis/staging and therapeutic follow-up in HAT. METHODS: Seventy-one HAT patients were diagnosed and 50 were followed-up 6-24 months after treatment. White cell counts were used for conventional staging (stage 1, < or =5 cells/microl CSF, n = 42; stage 2, > or =20 cells/microl, n = 16) and intermediate stage (6-19 cells/microl, n = 13). Slides containing 1 microl of CSF mixed with Dynabeads CD19 pan B were examined microscopically to detect B cell rosettes (bound to at least four beads). RESULTS: Stage 1 patients exhibited zero (n = 37) or one CSF rosette/microl (n = 5), contrary to most stage 2 patients (14/16: > or =2 rosettes/microl). Intermediate stage patients expressed 0 (n = 9), 1 (n = 3) or 2 (n = 1) rosettes/microl of CSF. During follow-up, rosette counts correlated with white cell count staging but were much easier to read. CONCLUSION: B cell rosettes being easily detected in the CSF in field conditions may be proposed to replace white cell count for defining HAT stages 1 and 2 and limit uncertainty in treatment decision in patients with intermediate stage.

Development of a nanoparticulate formulation of diminazene to treat African trypanosomiasis.

There is a real need to develop new therapeutic strategies for African trypanosomiasis infections. In our study, we developed a new drug delivery system of diminazene (DMZ), a trypanocidal drug registered for veterinary use. This drug candidate presents a limited efficacy, a poor affinity for brain tissue and instability. The development of colloidal formulations based on a porous cationic nanoparticle with an oily core ((70)DGNP(+)), has potentially two advantages: stabilization of the drug and potential targeting of the parasite. We analyzed two processes of drug loading: in process (DMZ was added during the preparation of (70)DGNP(+) at 80 °C) and post-loading (DMZ was mixed with a (70)DGNP(+) solution at room temperature). Poor stability of the drug was observed using the in process technique. When using the post-loading technique over 80% drug entrapment efficiency was obtained at a ratio of DMZ:phospholipids (wt:wt) < 5%. Moreover, DMZ loaded into (70)DGNP(+) was found to be protected against oxidation and was stable for at least six months at 4 °C. Finally, in vitro tests on T.b. brucei showed an increased efficacy of DMZ loaded in (70)DGNP(+).

CD23 mediates antimycobacterial activity of human macrophages.

Engagement of surface receptors contributes to the antimicrobial activity of human immune cells. We show here that infection of human monocyte-derived macrophages (MDM) with live Mycobacterium avium induced the expression of CD23 on their membrane. Subsequent cross-linking of surface CD23 by appropriate ligands induced a dose-dependent antibacterial activity of MDM and the elimination of most infected cells. The stimulation of inducible nitric oxide synthase-dependent generation of NO from MDM after CD23 activation played a major role during their anti-M. avium activity. CD23 activation also induced tumor necrosis factor alpha (TNF-alpha) production from MDM. Mycobacteria reduction was partially inhibited by the addition of neutralizing anti-TNF-alpha antibody to cell cultures without affecting NO levels, which suggested the role of this cytokine for optimal antimicrobial activity. Finally, interleukin-10, a Th2 cytokine known to downregulate CD23 pathway, is shown to decrease NO generation and mycobacteria elimination by macrophages. Therefore, (i) infection with M. avium promotes functional surface CD23 expression on human macrophages and (ii) subsequent signaling of this molecule contributes to the antimicrobial activity of these cells through an NO- and TNF-alpha-dependent pathway. This study reveals a new human immune response mechanism to counter mycobacterial infection involving CD23 and its related ligands.

Crosstalk between TAp73 and TGF-ß in fibroblast regulates iNOS expression and Nrf2-dependent gene transcription.

Inducible nitric oxide synthase (iNOS) activity produces anti-tumor and anti-microbial effects but also promotes carcinogenesis through mutagenic, immunosuppressive and pro-angiogenic mechanisms. The tumor suppressor p53 contributes to iNOS downregulation by repressing induction of the NOS2 gene encoding iNOS, thereby limiting NO-mediated DNA damages. This study focuses on the role of the p53 homologue TAp73 in the regulation of iNOS expression. Induction of iNOS by immunological stimuli was upregulated in immortalized MEFs from TAp73-/- mice, compared to TAp73+/+ fibroblasts. This overexpression resulted both from increased levels of NOS2 transcripts, and from an increased stability of the protein. Limitation of iNOS expression by TAp73 in wild-type cells is alleviated by TGF-ß receptor I inhibitors, suggesting a cooperation between TAp73 and TGF-ß in suppression of iNOS expression. Accordingly, downregulation of iNOS expression by exogenous TGF-ß1 was impaired in TAp73-/- fibroblasts. Increased NO production in these cells resulted in a stronger, NO-dependent induction of Nrf2 target genes, indicating that the Nrf2-dependent adaptive response to nitrosative stress in fibroblasts is proportional to iNOS activity. NO-dependent induction of two HIF-1 target genes was also stronger in TAp73-deficient cells. Finally, the antimicrobial action of NO against Trypanosoma musculi parasites was enhanced in TAp73-/- fibroblasts. Our data indicate that tumor suppressive TAp73 isoforms cooperate with TGF-ß to control iNOS expression, NO-dependent adaptive responses to stress, and pathogen proliferation.

Peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis.

Dogs are the main reservoir of zoonotic visceral leishmaniasis. Vaccination is a promising approach to help control leishmaniasis and to interrupt transmission of the Leishmania parasite. The promastigote surface antigen (PSA) is a highly immunogenic component of Leishmania excretory/secretory products. A vaccine based on three peptides derived from the carboxy-terminal part of Leishmania amazonensis PSA and conserved among Leishmania species, formulated with QA-21 as adjuvant, was tested on naive Beagle dogs in a preclinical trial. Four months after the full course of vaccination, dogs were experimentally infected with Leishmania infantum promastigotes. Immunization of dogs with peptide-based vaccine conferred immunity against experimental infection with L. infantum. Evidence for macrophage nitric oxide production and anti-leishmanial activity associated with IFN-γ production by lymphocytes was only found in the vaccinated group. An increase in specific IgG2 antibodies was also measured in vaccinated dogs from 2 months after immunization. Additionally, after challenge with L. infantum, the parasite burden was significantly lower in vaccinated dogs than in the control group. These data strongly suggest that this peptide-based vaccine candidate generated cross-protection against zoonotic leishmaniasis by inducing a Th1-type immune response associated with production of specific IgG2 antibodies. This preclinical trial including a peptide-based vaccine against leishmaniasis clearly demonstrates effective protection in a natural host. This approach deserves further investigation to enhance the immunogenicity of the peptides and to consider the possible engineering of a vaccine targeting several Leishmania species.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns).

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations. HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity. Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal. These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

The separation of trypanosomes from blood by anion exchange chromatography: From Sheila Lanham's discovery 50 years ago to a gold standard for sleeping sickness diagnosis.

Human African trypanosomiasis (HAT), or sleeping sickness, is a neglected tropical disease that is fatal if untreated, caused by Trypanosoma brucei gambiense and T. brucei rhodesiense. In its 2012 roadmap, WHO targeted HAT for elimination as a public health problem in 2020 and for zero transmission in 2030. Diagnosis of HAT is a multistep procedure comprising of clinical suspicion, confirmation, and stage determination. Suspects are identified on clinical signs and/or on screening for specific antibodies. Parasitological confirmation of suspects remains mandatory to avoid unnecessary toxic drug administration. The positive predictive value of the antibody detection tests is low. Simple parasite detection techniques, microscopic examination of lymph node aspirate, or stained thick blood films lack sensitivity, whereas in T. brucei gambiense patients, the number of blood trypanosomes may be very low. Parasite concentration techniques are therefore indispensable. Half a century ago, Sheila Lanham discovered a technique to separate trypanosomes from the blood of infected rodents, based on anion exchange chromatography with diethyl amino ethyl (DEAE) cellulose, a weak anion exchanger. Between pH 6-9, trypanosome surface is less negatively charged than that of blood cells. When blood is poured on top of a DEAE cellulose column, blood cells are retained, whereas parasites pass the column together with the elution buffer. The result is a pure suspension of trypanosomes that retain their morphology and infectivity. Because cell surface charges vary among trypanosome and mammal species, the optimal buffer pH and ionic strength conditions for different combinations of host and trypanosome species were established. Lanham's technique revolutionized the diagnosis of HAT. It is indispensable in the production of the Card Agglutination Test for Trypanosomiasis (CATT), the most used field test for screening in T. brucei gambiense HAT foci and essential to confirm the diagnosis in suspected people. Lumsden and colleagues developed the mini anion exchange centrifugation technique (mAECT). After adaptation for field conditions, its superior diagnostic and analytical sensitivity compared to another concentration technique was demonstrated. It was recommended as the most sensitive test for demonstrating trypanosomes in human blood. At the beginning of the 21st century, the mAECT was redesigned, allowing examination of a larger volume of blood, up to 0.35 ml with whole blood and up to 10 ml with buffy coat. The plastic collector tube in the new kit is also used for detection of trypanosomes in the cerebrospinal fluid. Unfortunately, mAECT also has some disadvantages, including its price, the need to centrifuge the collector tube, and the fact that it is manufactured on a noncommercial basis at only two research institutes. In conclusion, 50 years after Sheila Lanham's discovery, CATT and mAECT have become essential elements in the elimination of HAT.

A role of IgE and CD23/NO immune pathway in age-related resistance of Lewis rats to Plasmodium berghei Anka?

In contrast to young rats, adult rats given i.p. Plasmodium berghei Anka (PbA) control the parasitaemia and repair their anaemia. Here, we investigated whether IgE and CD23/NO immune pathway could be implicated in this age-related resistance of adult rats to PbA. Eight-week-old rats displayed significantly higher levels of plasma total IgE (p=0.01) and soluble CD23 (p=0.003) during the peak of parasitaemia, compared to 4-week-old rats. IgE Fc-binding antibody or aminoguanidine administration to parasitized 8-week-old rats slightly delayed blood parasite clearance or exacerbated anaemia. These data suggest that IgE and CD23/NO could play an important role in the resistance of adult rats experiencing PbA primary intraerythrocytic development.

Virulence and pathogenicity patterns of Trypanosoma brucei gambiense field isolates in experimentally infected mouse: differences in host immune response modulation by secretome and proteomics.

Human African trypanosomiasis is characterised by an important clinical diversity. Although Trypanosoma brucei gambiense field stocks isolated from patients in the same focus did not exhibit apparent genetic variability, they showed marked differences in terms of virulence (capacity to multiply inside a host) and pathogenicity (ability of producing mortality) in experimental murine infections. Two strains exhibiting opposite pathogenic and virulence properties in mouse were further investigated through their host-parasite interactions. In vitro, parasite bloodstream forms or soluble factors (or secretome) from both strains induced macrophage arginase as a function of their virulence. Arginase expression, a hallmark of macrophage alternative activation pathway, favours trypanosome bloodstream forms development. Moreover, a comparative proteomic study of the trypanosome stocks' secretomes evidenced both a differential expression of common molecules and the existence of stock specific molecules. This highlighted the potential involvement of the differential expression of the same genome in the diverse infectious properties of trypanosomes.

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants.

BACKGROUND: A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. METHODS: Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. RESULTS: Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5 degrees C (0.6 degrees C) for P. falciparum infection and 64.6 degrees C (1.8 degrees C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 - 1) or P. falciparum-specific (0.91, 0.8 - 1) primers, or with the microscopy results (0.70, 0.6 - 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. CONCLUSION: This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

Trypanosomatid Infections: How Do Parasites and Their Excreted-Secreted Factors Modulate the Inducible Metabolism of l-Arginine in Macrophages?

Mononuclear phagocytes (monocytes, dendritic cells, and macrophages) are among the first host cells to face intra- and extracellular protozoan parasites such as trypanosomatids, and significant expansion of macrophages has been observed in infected hosts. They play essential roles in the outcome of infections caused by trypanosomatids, as they can not only exert a powerful antimicrobial activity but also promote parasite proliferation. These varied functions, linked to their phenotypic and metabolic plasticity, are exerted via distinct activation states, in which l-arginine metabolism plays a pivotal role. Depending on the environmental factors and immune response elements, l-arginine metabolites contribute to parasite elimination, mainly through nitric oxide (NO) synthesis, or to parasite proliferation, through l-ornithine and polyamine production. To survive and adapt to their hosts, parasites such as trypanosomatids developed mechanisms of interaction to modulate macrophage activation in their favor, by manipulating several cellular metabolic pathways. Recent reports emphasize that some excreted-secreted (ES) molecules from parasites and sugar-binding host receptors play a major role in this dialog, particularly in the modulation of the macrophage's inducible l-arginine metabolism. Preventing l-arginine dysregulation by drugs or by immunization against trypanosomatid ES molecules or by blocking partner host molecules may control early infection and is a promising way to tackle neglected diseases including Chagas disease, leishmaniases, and African trypanosomiases. The present review summarizes recent knowledge on trypanosomatids and their ES factors with regard to their influence on macrophage activation pathways, mainly the NO synthase/arginase balance. The review ends with prospects for the use of biological knowledge to develop new strategies of interference in the infectious processes used by trypanosomatids, in particular for the development of vaccines or immunotherapeutic approaches.

Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids.

The Trypanosomatidae family includes the genera Trypanosoma and Leishmania, protozoan parasites displaying complex digenetic life cycles requiring a vertebrate host and an insect vector. Trypanosoma brucei gambiense, Trypanosoma cruzi, and Leishmania spp. are important human pathogens causing human African trypanosomiasis (HAT or sleeping sickness), Chagas' disease, and various clinical forms of Leishmaniasis, respectively. They are transmitted to humans by tsetse flies, triatomine bugs, or sandflies, and affect millions of people worldwide. In humans, extracellular African trypanosomes (T. brucei) evade the hosts' immune defenses, allowing their transmission to the next host, via the tsetse vector. By contrast, T. cruzi and Leishmania sp. have developed a complex intracellular lifestyle, also preventing several mechanisms to circumvent the host's immune response. This review seeks to set out the immune evasion strategies developed by the different trypanosomatids resulting from parasite-host interactions and will focus on: clinical and epidemiological importance of diseases; life cycles: parasites-hosts-vectors; innate immunity: key steps for trypanosomatids in invading hosts; deregulation of antigen-presenting cells; disruption of efficient specific immunity; and the immune responses used for parasite proliferation.

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs.

Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05), suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053) and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008) were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50) were 8.5, 2.7, 1.6 and 0.12 µM for T. b. brucei 427 90-13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893) and T. b. gambiense Biyamina (MHOM/SD/82), respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.