Merozoite-Protein Loaded Liposomes Protect against Challenge in Two Murine Models of Plasmodium Infection.

Liposomes loaded with GPI-anchored proteins induce a strong and effective response against protozoan pathogens when immunized in mice. On the basis of results using Plasmodium falciparum merozoite proteins loaded on liposomes, we tested whether a similar approach would be protective in the P. yoelii XL and P. berghei NK65 models of lethal murine malaria infections. We also analyzed the enhancing capacity of additional adjuvants. As expected, merozoite protein loaded liposomes provided strong humoral responses (IgG titers of 105 against MSP119) in BALB/c and C57BL/6 host backgrounds and showed total protection in lethal challenges (100% survival) with P. yoelii, while protection against lethal challenge with P. berghei NK was at least partial (11%-20% in C57BL/6 mice and no protection in BALB/c). Thus, immunization with proteoliposomes is not only highly immunogenic but also provides a protective response in widely used murine models of malaria.

Liposomes loaded with P. falciparum merozoite-derived proteins are highly immunogenic and produce invasion-inhibiting and anti-toxin antibodies.

The formulation of an effective vaccine against malaria is still a significant challenge and the induction of high anti-parasite antibody titers plus a sustained T cell response is mandatory for the success of such a vaccine. We have developed a nanoliposome-based structure which contains plasma membrane-associated proteins (PfMNP) of Plasmodium falciparum merozoites on its surface. Incorporation of parasite-derived proteins led to a significant increase in the size and dispersity of particles. Immunization of particles in BalbC and C57BL/6 mice led to high anti-MSP119 IgG titers (10(4)) after the first dose and reached a plateau (>10(6)) after the third dose. While very high titers were observed against the C-terminal domain of the vaccine candidate MSP1, only modest titers (≤10(3)) were detected against MSP2. The induced antibodies showed also a strong growth-inhibiting effect in reinvasion assays. In addition, PfMNP immunization generated antibodies which partially blocked the inflammatory response, probably by blocking TLR-induced activation of macrophages by malarial toxins such as GPI anchors. The results underline the potential of nanoliposome-based formulations as anti-malarial vaccines.

Nanobiotechnologic approach to a promising vaccine prototype for immunisation against leishmaniasis: a fast and effective method to incorporate GPI-anchored proteins of Leishmania amazonensis into liposomes.

Liposomes are known to be a potent adjuvant for a wide range of antigens, as well as appropriate antigen carriers for antibody generation response in vivo. In addition, liposomes are effective vehicles for peptides and proteins, thus enhancing their immunogenicity. Considering these properties of liposomes and the antigenicity of the Leishmania membrane proteins, we evaluated if liposomes carrying glycosylphosphatidylinositol (GPI)-anchored proteins of Leishmania amazonensis promastigotes could induce protective immunity in BALB/c mice. To assay protective immunity, BALB/c mice were intraperitoneally injected with liposomes, GPI-protein extract (EPSGPI) as well as with the proteoliposomes carrying GPI-proteins. Mice inoculated with EPSGPI and total protein present in constitutive proteoliposomes displayed a post-infection protection of about 70% and 90%, respectively. The liposomes are able to work as adjuvant in the EPSGPI protection. These systems seem to be a promising vaccine prototype for immunisation against leishmaniasis.

Nanopharmaceutical approach of epiisopiloturine alkaloid carried in liposome system: preparation and in vitro schistosomicidal activity.

Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma. This disease control has been widely made by praziquantel-reference drug, but resistance to this drug has already been found. There has been the finding of an imidazole alkaloid in jaborandi leaves-epiisopiloturine, which has known activity against adult, young and egg forms of Schistosoma mansoni. This alkaloid is an apolar molecule with difficult solubility; therefore, the liposomal structure of epiisopiloturine was proposed. Liposomes are carrying structures of drugs that may enhance solubility of compounds such as epiisopiloturine. In this work, we report in vitro epiisopiloturine-loaded liposomes effect formed by different concentrations of lipids 9:1 (weight ratio) dipalmitoylphosphatidylcholine:cholesterol and 8:2 (weight ratio) dipalmitoylphosphatidylcholine:cholesterol. Results have showed that epiisopiloturine extraction and isolation have been successful through high-performance liquid chromatography-HPLC and its purity confirmed through mass spectrometry has showed 287 Da molecular mass. Formulations from 9:1 DPPC:cholesterol and 8:2 DPPC:cholesterol with loaded EPI (300 microg/ml) have killed parasites at 100% after incubation 96 h and 120 h, respectively. Confocal microscopy employed to observe morphological alterations in the tegument of adult form of Schistosoma mansoni. Details from interaction, between epiisopiloturine and liposome, have been achieved by semi-empirical AM1 calculations, which have showed that epiisopiloturine inside is more stable than the outside form, at least 10 kcal. This is first time that schistosomicidal activity has been reported for epiisopiloturine-loaded into liposome.

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid-rich extract and membrane models.

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA- KAAGQAALGAL-NH(2) , DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis.

Using multidimensional projection techniques for reaching a high distinguishing ability in biosensing.

Recent advances in the control of molecular engineering architectures have allowed unprecedented ability of molecular recognition in biosensing, with a promising impact for clinical diagnosis and environment control. The availability of large amounts of data from electrical, optical, or electrochemical measurements requires, however, sophisticated data treatment in order to optimize sensing performance. In this study, we show how an information visualization system based on projections, referred to as Projection Explorer (PEx), can be used to achieve high performance for biosensors made with nanostructured films containing immobilized antigens. As a proof of concept, various visualizations were obtained with impedance spectroscopy data from an array of sensors whose electrical response could be specific toward a given antibody (analyte) owing to molecular recognition processes. In addition to discussing the distinct methods for projection and normalization of the data, we demonstrate that an excellent distinction can be made between real samples tested positive for Chagas disease and Leishmaniasis, which could not be achieved with conventional statistical methods. Such high performance probably arose from the possibility of treating the data in the whole frequency range. Through a systematic analysis, it was inferred that Sammon's mapping with standardization to normalize the data gives the best results, where distinction could be made of blood serum samples containing 10(-7) mg/mL of the antibody. The method inherent in PEx and the procedures for analyzing the impedance data are entirely generic and can be extended to optimize any type of sensor or biosensor.

Biosensors for efficient diagnosis of leishmaniasis: innovations in bioanalytics for a neglected disease.

The need for reliable, fast diagnostics is closely linked to the need for safe, effective treatment of the so-called "neglected" diseases. The list of diseases with no field-adapted diagnostic tools includes leishmaniasis, shigella, typhoid, and bacterial meningitis. Leishmaniasis, in particular, is a parasitic disease caused by Leishmania spp. transmitted by infected phlebotomine sandfly, which remains a public health concern in developing countries with ca. 12 million people infected and 350 million at risk of infection. Despite several attempts, methods for diagnosis are still noneffective, especially with regard to specificity due to false positives with Chagas' disease caused by Trypanosoma cruzi . Accepted golden standards for detecting leishmaniasis involve isolation of parasites either microscopically, or by culture, and in both methods specimens are obtained by invasive means. Here, we show that efficient distinction between cutaneous leishmaniasis and Chagas' disease can be obtained with a low-cost biosensor system made with nanostructured films containing specific Leishmania amazonensis and T. cruzi antigens and employing impedance spectroscopy as the detection method. This unprecedented selectivity was afforded by antigen-antibody molecular recognition processes inherent in the detection with the immobilized antigens, and by statistically correlating the electrical impedance data, which allowed distinction between real samples that tested positive for Chagas' disease and leishmaniasis. Distinction could be made of blood serum samples containing 10(-5) mg/mL of the antibody solution in a few minutes. The methods used here are generic and can be extended to any type of biosensor, which is important for an effective diagnosis of many other diseases.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application.

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine's formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 nm, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis.

Incorporation of antigenic GPI-proteins from Leishmania amazonensis to membrane mimetic systems: influence of DPPC/cholesterol ratio.

The reconstitution of membrane proteins into liposomes is a useful tool to prepare antigenic components that induce immunity. We have investigated the influence of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol molar ratio on the incorporation of a GPI-protein from Leishmania amazonensis on liposomes and Langmuir monolayers. The latter system is a well behaved and practical model, for understanding the effect of variables such as surface composition and lipid packing on protein incorporation. We have found that the DPPC/cholesterol molar ratio significantly alters the incorporation of the GPI-protein. In the absence of cholesterol, reconstitution is more difficult and proteoliposomes cannot be prepared, which we correlated with disruption of the DPPC layer. Our results provide important information that could be employed in the development of a vaccine system for this disease or be used to produce other GPI-systems for biotechnological application.

Hypoxia modulates expression of the 70-kD heat shock protein and reduces Leishmania infection in macrophages.

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O2, 5% CO2, and balanced N2) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected with Leishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity.

Effect of hypoxia on macrophage infection by Leishmania amazonensis.

In the present study, we compared the effect of 5% oxygen tension (hypoxia) with a normal tension of 21% oxygen (normoxia) on macrophage infection by the protozoan parasite Leishmania amazonensis. Macrophages from different sources (human cell line U937, murine cell line J774, and murine peritoneal macrophages) exposed to hypoxia showed a reduction of the percentage of infected cells and the number of intracellular parasites per cell. Observations on the kinetics of infection indicated that hypoxia did not depress L. amazonensis phagocytosis but induced macrophages to reduce intracellular parasitism. Furthermore, hypoxia did not act synergistically with gamma-interferon and bacterial lipopolysaccharides in macrophages to induce killing of parasites. Experiments also indicated no correlation between nitric oxide production and control of infection in macrophages under hypoxic condition. Thus, we have provided the first evidence that hypoxia, which occurs in various pathological conditions, can alter macrophage susceptibility to a parasitic infection.