Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50 degrees C. This effect becomes negligible at temperatures higher that ca. 60 degrees C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis. Published by Elsevier B.V.

Sandflies (Diptera, Psychodidae) from forest areas in Botucatu municipality, central western São Paulo State, Brazil

Abstract Background The study of the distribution and ecology of sandfly species is essential for epidemiological surveillance and estimation of the transmission risk of Leishmania spp. infection. Findings In the present study, sandflies were captured in native fragmented forest areas in Rubião Júnior district, Botucatu municipality, São Paulo state, Brazil, between September 2001 and January 2005. A minimum of two automatic light traps were installed per night from 6 pm to 8 am, in different months, resulting in approximately 900 collecting hours. During this period, 216 sandfly specimens of sixteen species were captured. Pintomyia monticola and Brumptomyia guimaraesi were the most abundant with 56 specimens (25.93%) captured per species, followed by Pintomyia fischeri 28 (12.96%) and Psathyromyia pascalei 18 (8.33%). Other captured species were Lutzomyia amarali, Sciopemyia sordellii, Psathyromyia aragaoi, Nyssomyia whitmani, Migonemyia migonei, Pintomyia bianchigalatiae, Pintomyia misionensis, Brumptomyia carvalheiroi, Brumptomyia cardosoi, Brumptomyia cunhai, Brumptomyia nitzulescui, Brumptomyia brumpti and Brumptomyia spp. represented by 58 (26.85%) specimens. Conclusions Although less frequently found, the presence of Pintomyia fischeri, Nyssomyia whitmani and Migonemyia migonei, known vectors of Leishmania braziliensis, indicates risk of American cutaneous leishmaniasis occurrence. Moreover, the absence of Lutzomyia longipalpis-the main vector of Leishmania infantum chagasi, which is the agent of American visceral leishmaniasis-suggests that there is no risk of introduction and establishment of this disease in the studied area.

In vitro efficacy of Coriandrum sativum, Lippia sidoides and Copaifera reticulata against Leishmania chagasi

The increased incidence of visceral leishmaniasis (VL) in Brazil is due to a lack of effective disease control measures. In addition to that, no effective treatment exists for canine VL in response to synthetic drugs. Thus, the objective of this study was to evaluate the effect of the essential oils of Coriandrum sativum and Lippia sidoides, and oleoresin from Copaifera reticulata, on Leishmania chagasi promastigotes and amastigotes. We also examined the toxicity of these treatments on the murine monocyte cell line RAW 264.7. To determine the IC50 a MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on promastigotes, and an in situ ELISA assay was conducted on amastigotes. Here, we demonstrate that oleoresin from C. reticulata was effective against both promastigotes (IC50 of 7.88 µg.mL-1) and amastigotes (IC50 of 0.52 µg.mL-1), and neither of the two treatments differed significantly (p > 0.05) from pentamidine (IC50 of 2.149 µg.mL-1) and amphotericin B (IC50 of 9.754 µg.mL-1). Of the three plant oils tested, only oleoresin showed no toxicity toward monocyte, with 78.45% viability after treatment. Inhibition of promastigote and amastigote growth and the lack of cytotoxicity by C. reticulata demonstrate that oleoresin may be a viable option for analyzing the in vivo therapeutic effects of leishmanicidal plants

Caso alóctone de leishmaniose visceral canina, no município de Campo Mourão, Paraná, Brasil

O presente relato tem por objetivo descrever o primeiro caso alóctone de leishmaniose visceral (LV) no município de Campo Mourão, Paraná, Brasil, em um canino, da raça Boxer, apresentando lesões oculares e cutâneas, linfoadenomegalia e esplenomegalia, atendido no Hospital Veterinário da Faculdade Integrado de Campo Mourão, após ter residido na cidade de Campo Grande, Mato Grosso do Sul. O diagnóstico da enfermidade baseou-se na observação direta de formas amastigotas de Leishmania spp., em linfonodos poplíteos, sugerindo ser um caso de LV, uma vez que o animal era proveniente de área endêmica para a enfermidade. A migração de cães infectados de regiões endêmicas para áreas indenes torna-se um problema para a saúde pública, uma vez que poderá permitir a instalação de novos focos, favorecendo a disseminação da doença em todo o país.