Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.     

Drug Targeting to Macrophages With Solid Lipid Nanoparticles Harboring Paromomycin: an <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation Against <Emphasis Type="Italic">L. major</Emphasis> and <Emphasis Type="Italic">L. tropica</Emphasis>

Leishmaniasis is a worldwide disease that leads to high mortality and morbidity in human populations. Today, leishmaniasis is managed via drug therapy. The drugs that are already in clinical use are limited to a number of toxic chemical compounds and their parasite drug resistance is increasing. It is therefore essential, in order to circumvent the current difficulties, to design a new anti-leishmanial drug treatment strategy. Besides producing new, active anti-leishmanial entities, another promising strategy could be developing novel delivery systems and formulations of the existing pharmaceutical ingredients to improve drug efficacy. In the present study, paromomycin sulfate (PM), as one of the promising anti-leishmanial drugs, was formulated in solid lipid nanoparticles (SLN), and its in vitro efficacy was investigated against different strains of Leishmania using a MTT test, Parasite-Rescue-Transformation-Assay, SYTO Green staining, and fluorescent microscope imaging. The results show that PM-loaded SLN is significantly more effective than PM in inhibiting parasite propagation (P?<?0.05) and that cytotoxicity of PM-SLN formulations is size dependent. According to our results, delivery of the drugs to the macrophages via nanoparticle utilization seems to be an accessible and practical approach.     

Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.     

Critical role of aldehydes in cigarette smoke-induced acute airway inflammation

Background

Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation.

Methods

BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs.

Results

Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein.

Conclusion

Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.     

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Background

We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects.

Methodology/Principal Findings

Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b^-CTE, pcDNA-cpa/b^-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b^-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies.

Conclusions/Significance

This report demonstrates cSLN''s ability to boost immune response magnitude of cpa/cpb^-CTE cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines.     

Paromomycin loaded solid lipid nanoparticles: Characterization of production parameters

Paromomycin has been shown to have anti-leishmaniasis activity; however, its clinical use is restricted to some content owing to its poor skin penetration. To identify innovative methods of dermal administration of paromomycin and controlling the release delivery system, paromomycin was loaded into the solid lipid media as nanoparticles. Type of the method; microemulsion or solvent diffusion, the type of lipid; cetyl palmitate or stearic acid, were comparatively investigated on the average diameter, size distribution and entrapment efficiency of the lipid nanoparticles to maximize entrapment efficiency, reduce the particle size and its distribution. Three quantitative factors, paromomycin content, weight fraction of Tween 80 and drug to lipid ratio, were also investigated at two levels for Solid Lipid Nanoparticles (SLNs) formulation in a fractional factorial design. The results indicated that microemulsion was the most efficient method and stearic acid was the preferred lipid for SLNs formulation. The average size of the particles was reduced to 299.08 nm and the entrapment efficiency was enhanced from immediate release to 24 h.