Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH(-) (107 dogs), (2) IFAT((-))/DTH(+) (18 dogs), and (3) IFAT((+))/DTH(+) (13 dogs), providing an overall prevalence of infection of 43 % (138/320). Thus, the specific prevalence of IFAT ((+)) /DTH ((-)) 33.4 % (107/320) was higher than those of IFAT ((-)) /DTH ((+)) 5.6 % (18/320) and IFAT ((+)) /DTH ((+)) 4.0 % (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT ((+)) /DTH ((-)) rate of 77.5 % (107/138) was also higher than those of 13.0 % (18/138) of IFAT ((-)) /DTH ((+)) and 9.5 % (13/138) of IFAT ((+)) /DTH ((+)) rates. The frequency of asymptomatic dogs (76 %-105) was higher than those of symptomatic (16.6 %-23) and oligosymptomatic ones (7.4 %-10). A total of 16 (11.6 %) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT ((+)) /DTH ((-)) profile: 41 % (9/22) from symptomatic, 33.3 % (3/9) from oligosymptomatic, and 5.2 % (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76 %-105), the majority (72.4 %-76) was characterized by the IFAT ((+)) /DTH ((-)) profile with a doubtful immunogenetic resistance against infection.

Recuperação e purificação do antígeno 503 de Leishmania i. chagasi expresso em E. coli e remoção de endotoxina utilizando adsorção em leito expandido

The growing interest and applications of biotechnology products have increased the development of new processes for recovery and purification of proteins. The expanded bed adsorption (EBA) has emerged as a promising technique for this purpose. It combines into one operation the steps of clarification, concentration and purification of the target molecule. Hence, the method reduces the time and the cost of operation. In this context, this thesis aim was to evaluate the recovery and purification of 503 antigen of Leishmania i. chagasi expressed in E. coli M15 and endotoxin removal by EBA. In the first step of this study, batch experiments were carried out using two experimental designs to define the optimal adsorption and elution conditions of 503 antigen onto Streamline chelating resin. For adsorption assays, using expanded bed, it was used a column of 2.6 cm in diameter by 30.0 cm in height coupled to a peristaltic pump. In the second step of study, the removal of endotoxin during antigen recovery process was evaluated employing the non-ionic surfactant Triton X-114 in the washing step ALE. In the third step, we sought developing a mathematical model able to predict the 503 antigen breakthrough curves in expanded mode. The experimental design results to adsorption showed the pH 8.0 and the NaCl concentration of 2.4 M as the optimum adsorption condition. In the second design, the only significant factor for elution was the concentration of imidazole, which was taken at 600 mM. The adsorption isotherm of the 503 antigen showed a good fit to the Langmuir model (R = 0.98) and values for qmax (maximum adsorption capacity) and Kd (equilibrium constant) estimated were 1.95 mg/g and 0.34 mg/mL, respectively. Purification tests directly from unclarified feedstock showed a recovery of 59.2% of the target protein and a purification factor of 6.0. The addition of the non-ionic surfactant Triton X-114 to the washing step of EBA led to high levels (> 99%) of LPS removal initially present in the samples for all conditions tested. The mathematical model obtained to describe the 503 antigen breakthrough curves in Streamline Chelanting resin in expanded mode showed a good fit for both parameter estimation and validation steps. The validated model was used to optimize the efficiencies, achieving maximum values of the process and of the column efficiencies of 89.2% and 75.9%, respectively. Therefore, EBA is an efficient alternative for the recovery of the target protein and removal of endotoxin from an E. coli unclarified feedstock in just one step.

The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil

To identify natural infections by Leishmania spp. in insect vectors of cutaneous and visceral leishmaniasis, we performed field studies in natural and anthropic environments in the Guaicurus Settlement (Bodoquena Range) of the Bonito municipality, Mato Grosso do Sul state, Brazil. From October 2002 to October 2003, a total of 1395 sandfly females were captured with Shannon and light traps and dissected in search of flagellates. The sample is composed of a total of 13 species, with Lutzomyia almerioi (59.9%) and Lutzomyia longipalpis (31.4%) predominant. Infections by flagellates were directly observed in three of the dissected of Lu. almerioi females (0.36%). To increase the sensitivity of detection, DNA extracted from pools of the 1220 dissected females (Lu. almerioi 808, Lu. longipalpis 399 and Nyssomyia whitmani 13) was subjected to small subunit rRNA-based polymerase chain reactions (SSU-PCR). DNA from Leishmania (L.) infantum chagasi was detected in at least 0.37% of Lu. almerioi females and in 0.25% of Lu. longipalpis females. The DNA of the Leishmania (Viannia) sp. was detected in 0.12% of Lu. almerioi and in 0.70% of Lu. longipalpis. Leishmania (L.) amazonensis was found in 1.25% of Lu. longipalpis. Mixed infections of L. (Leishmania) sp. and L. (Viannia) sp. were found in 0.50% of Lu. longipalpis. When considering that each positive pool contained at least a single infected specimen, we found a 1.23% rate of Leishmania spp. infection among the total population of dissected female sand flies as determined by PCR. This is the first report of natural infection by L. (L.) infantum chagasi and L. (Viannia) sp. in Lu. almerioi. It is also the first report of infection by L. (Viannia) sp. in Lu. longipalpis. The observation that Lu. longipalpis and Lu. almerioi are naturally infected by agents of both cutaneous and visceral leishmaniases suggests that these two species play a role in the transmission (continua) (continuação) of these diseases within the study area. Furthermore, the finding that Lu. longipalpis has been naturally infected by L. (L.) amazonensis and L. (Viannia) sp., and Lu. almerioi by L. (L.) infantum chagasi and L. (Viannia), suggests their participation as permissive vectors

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MU) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p > 0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p > 0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p > 0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells. (C) 2011 Elsevier B.V. All rights reserved.

Leishmania sp identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.

Phlebotomine sandfly fauna and natural Leishmania infection rates in a rural area of Cerrado (tropical savannah) in Nova Mutum, State of Mato Grosso in Brazil

Introduction American cutaneous leishmaniasis (ACL) has been reported in every municipality of the State of Mato Grosso, Brazil, but the transmission epidemiology remains poorly understood. Our study was developed in a rural area of the Nova Mutum municipality where four autochthonous cases of ACL were reported in 2009. Our aims were to describe the local phlebotomine sandfly fauna and to investigate the infection rates and infecting Leishmania species in the captured sandflies. Methods Entomological captures were performed bimonthly at 10 fixed sites close to the edge of a forested area between June 2011 and April 2012. Results A total of 3,743 phlebotomine sandflies belonging to 31 distinct species were captured. Approximately 75% of the specimens were females. The most abundant species (45.4%) was Lutzomyia antunesi, which was consistently captured at every site. Species that are epidemiologically important for ACL, such as L. flaviscutellata, L. whitmani and L. umbratilis, were also captured. L. antunesi and L. ubiquitalis were naturally infected by Leishmania braziliensis or Le. guyanensis, with minimum infection rates of 0.88% and 6.67%, respectively. Surprisingly, L. antunesi was infected by Le. infantum (synonym chagasi). Conclusions The natural infection of L. antunesi and L. ubiquitalis by Leishmania sp. suggests that these species might play a role in the zoonotic cycle of ACL in Nova Mutum. The presence of Le. infantum in L. antunesi suggests that there may be a risk of an outbreak of visceral leishmaniasis (VL) in Nova Mutum.

Susceptibility of laboratory-reared female Lutzomyia longipalpis (Lutz & Neiva, 1912) to infection by different species and strains of Leishmania Ross, 1903

A study was undertaken to compare the susceptibility of laboratory-reared female Lutzomyia longipalpis to infection by different species or strains of New World Leishmania. The sand flies proved to be highly susceptible to infection by a strain of Le. guyanensis, with flagellates developing in all (18/18) of the specimens examined. A lower infection rate of 37 per cents (10/27) was recorded in flies exposed to infection by a strain of Le. amazonensis. Flagellates developed in 13 per cents (6/46) of the sand flies that glood fed on dogs in the earlly stage of experimental infection with an old laboratory strain of Le. chagasi. In contrast, promastigotes did not develop in sand flies that blood fed on dogs with naturally acquired Le. chagasi. The naturally infected dogas were in an advanced stage of disease. Flagellates developed in 9// (3/32) of the sand flies that blood fed on lesions of hamsters infected with a strain of Le. braziliensis and in 9 per cents (3/34) of those that fed on hamsters with lesions due to a parasite fo the mexicana complex (strain MHOM/BR/73/BH121). Sand flies did not develop flagellate infections after blood feeding on hamsters bearing lesions induced by strain MHOM/BR/71/BR49. Factors influencing the susceptibility of Lu. longipalpis to infection by New World species of Leishmania are discussed.

Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC. The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae)

Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Activity of Brazilian and Bulgarian propolis against different species of Leishmania

Extracts of propolis samples collected in Brazil and Bulgaria were assayed against four Leishmania species - Leishmania amazonensis, L. braziliensis, L. chagasi from the New World, and L. major from the Old World - associated to different clinical forms of leishmaniasis. The composition of the extracts has been previously characterized by high temperature high resolution gas chromatography coupled to mass spectrometry. Considering the chemical differences among the extracts and the behavior of the parasites, it was observed significant differences in the leishmanicidal activities with IC50/1 day values in the range of 2.8 to 229.3 µg/ml . An overall analysis showed that for all the species evaluated, Bulgarian extracts were more active than the ethanol Brazilian extract. As the assayed propolis extracts have their chemical composition determined it merits further investigation the effect of individual components or their combinations on each Leishmania species.

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37% when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2% formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil

The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L) chagasi. This study proved the occurrence of infection with Leishmania (L) chagasi in felines from Andradina municipality, São Paulo State. (C) 2010 Elsevier B.V. All rights reserved.