Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

A retrospective serologic survey of hantavirus infections in the county of Cássia dos Coqueiros, State of São Paulo, Brazil

INTRODUCTION: In recent years, hantavirus infections producing severe diseases have obtained an increased attention from public health authorities from the countries of Eurasia to the Americas. Brazil has reported 1,300 cases of hantavirus cardiopulmonary syndrome (HCPS) from 1993 to 2010, with about 80 of them occurring in the northeast of the State of São Paulo, with 48% fatality rate. Araraquara virus was the causative agent of HCPS in the region. Considering that hantaviruses causing human disease in the Americas were unknown until 1993, we have looked for hantavirus infections in the population of Cássia dos Coqueiros county, northeast of the State of São Paulo, Brazil, before this time. This county has about 2,800 inhabitants and an economy based on agriculture, including cultivation of Brachiaria decumbens grass. The grass seeds are an important rodent attraction, facilitating transmission of hantavirus to man. Four HCPS cases were reported so far in the county. METHODS: In this study, 1,876 sera collected from 1987 to 1990 were tested for IgG to hantavirus by IgG-ELISA, using the N recombinant protein of Araraquara virus as antigen. RESULTS: Positive results were observed in 89 (4.7%) samples, which were all collected in 1987. The positivity among urban inhabitants was 5.3%, compared with 4.3% among those living in rural areas. CONCLUSIONS: Our results showed that hantavirus infections occurred in Cássia dos Coqueiros, completely unrecognized, even before hantaviruses were described in the Americas.

Cysticercosis in experimentally and naturally infected pigs: parasitological and immunological diagnosis

Our objective was to evaluate the diagnosis of swine cysticercosis by examining "ante mortem" (inspection of the tongue), "post mortem" (inspection and detailed necropsy) and ELISA for research in serum of antibodies (Ab-ELISA) and antigens (Ag-ELISA). Seven (7) pigs were experimentally infected orally with eggs of Taenia solium and another 10 were naturally infected. In the pigs experimentally infected, inspection of the tongue was negative in all animals, in the routine inspection detailed necropsy and cysticercis were identified in all of them. In pigs with heavy natural infection, inspection of the tongue identified cysticerci in two (20%), while at inspection with necropsy the parasites were identified in large quantities in all animals. In ELISA for antibody search (Ab-ELISA) TS-14 recombinant protein was used, and in search for antigen (Ag-ELISA) a monoclonal antibody against this protein. In animals experimentally infected, blood was collected weekly for 140 days. The Ab-ELISA identified an increase in titers of antibody to cysticerci 21 days after infection, and at the end of the experimental period six animals (86%) were positive to the test. The search for circulating antigens (Ag-ELISA) was positive in two pigs 28 to 91 days after infection. All naturally infected pigs were positive for Ag-ELISA and Ab-ELISA. The search for antibodies and antigens by ELISA in serum from 30 pigs of a local farm and without history of cysticercosis was negative. Thus, the use of TS-14 antigen in ELISA test (Ab-ELISA) can be useful for the diagnosis of cysticercosis in pigs with low infection.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LTG33D), originally produced by some enterotoxigenic Escherichia coil (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LIG33D. Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LTG33D elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LTG33D. Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LTG33D. Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 mu g of rhBMP-2, (3) laser and 7 mu g of rhBMP-2, (4) 7 mu g of rhBMP-2/monoolein gel, (5) laser and 7 mu g rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P < 0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P < 0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone.

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

Human Bone Morphogenetic Protein-2 Use for Maxillary Reconstruction in Cleft Lip and Palate Patients

Background: The conventional methods of maxillary alveolar reconstruction in patient with cleft are the periosteoplasty and autologous bone grafting. As an important alternative of bone substitution, there is the recombinant human bone morphogenetic protein-2 (rhBMP-2). This study compares the rhBMP-2 with periosteoplasty and autologous bone grafting. Methods: Patients with cleft and alveolar defect were divided into 3 groups of 6 patients who underwent to autologous iliac crest bone grafting, resorbable collagen sponge with rhBMP2, and periosteoplasty, respectively. The analysis was performed through computed tomographic scan preoperatively and at months 3, 6, and 12 postoperatively. The variables analyzed were the alveolar defect volume, formed bone volume, bone formation rate, maxillary height repair rate, and the formed bone density mean. Results: The formed bone volume was similar comparing the bone graft and BMP groups at 1-year postoperative analysis (P = 0.58). Both of them had the formed bone volume significantly larger than the periosteoplasty group at 3 and 6 months postoperatively. In this last group, the 1-year follow-up was canceled because the bone formation was insufficient. The bone formation rate, the maxillary height repair rate, and the mean of density of the formed bone were similar in the bone graft and BMP groups at 1-year follow-up with P values of 0.93, 0.90, and 0.81, respectively. Conclusions: The amount of formed bone in the periosteoplasty group was insufficient. There was no difference among the bone graft and rhBMP-2 therapy considering the parameters analyzed.

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODS; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 degrees C for 1 h at pH from 8 to 11.5, while maintaining its biological activity. (C) 2011 Elsevier B.V. All rights reserved.

The protein LJM 111 from Lutzomyia longipalpis Salivary Gland Extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-alpha and IFN-gamma released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry. TNF-alpha and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-alpha and IFN-gamma. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-alpha whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils. (C) 2012 Elsevier B.V. All rights reserved.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway

Background Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Objective Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. Methods We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. Results We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Conclusions and Clinical Relevance Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen-and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm2). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 mu g of pure rhBMP-2; (b) 5 mu g of pure P-1 fraction; (c) 5 mu g of rhBMP-2/monoolein gel; (d) 5 mu g of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 mu g of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 mu g of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. (c) 2011 Wiley Periodicals, Inc.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-beta-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-beta-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 degrees C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan beta-1,3 or beta-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in beta-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 degrees C and 81.3 degrees C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 degrees C. the enzymatic assays demonstrated that XegA is more active in its monomeric state. (c) 2012 Elsevier B.V. All rights reserved.

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.