This letter presents a method which employs surface acoustic wave induced acoustic streaming to differentially peel treated red blood cells (RBCs) off a substrate based on their adhesive properties and separate populations of pathological cells from normal ones. We demonstrate the principle of operation by comparing the applied power and time required to overcome the adhesion displayed by healthy, glutaraldehyde-treated or malaria-infected human RBCs. Our experiments indicate that the method can be used to differentiate between various cell populations contained in a 9 μl droplet within 30 s, suggesting potential for rapid diagnostics.

BACKGROUND: A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of a malaria DNA vaccine is presented. A 40 kHz ultrasonic atomization device was used to create the microparticles from a feedstock containing 5 volumes of 0.5% w/v PLGA in acetone and 1 volume of condensed DNA which was fed at a flow rate of 18 ml h −1 . The plasmid DNA vectors encoding a malaria protein were condensed with a cationic polymer before atomization.

The absence of a validated surrogate marker for the immune state has complicated the design of a subunit vaccine against asexual stages of Plasmodium falciparum. In particular, it is not known whether the capacity to induce antibodies that inhibit parasite growth in vitro is an important criterion for selection of P. falciparum proteins to be assessed in human vaccine trials. We examined this issue in the Plasmodium yoelii rodent malaria model using the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1 19 ). To examine the relationship between inhibitory antibodies in immunized mice and the immune state, as indicated by resistance to a blood-stage challenge, we used an allelic replacement strategy to generate a transgenic P. falciparum line that expresses MSP1 19 from P. yoelii. We show that MSP1 19 is functionally conserved across these two divergent Plasmodium species, and replacing PfMSP1 19 with PyMSP1 19 has no detectable effect on parasite growth in vitro. By comparing growth rates of this transgenic line with a matched transgenic line that expresses the endogenous PfMSP1 19 , we developed an assay to measure the specific growth-inhibitory activity directed exclusively to the PyMSP1 19 protein in the sera from vaccinated animals. To validate this assay, sera from rabbits immunized with recombinant PyMSP1 19 were tested and showed specific inhibitory activity in a concentration-dependent manner. In mice that were immunized with recombinant PyMSP1 19 , the levels of PyMSP1 19 -specific inhibitory activity did not correlate with the total antibody levels measured by enzymelinked immunosorbent assay. Furthermore, they did not correlate with resistance to subsequent blood-stage infection, and some mice with complete protection showed no detectable inhibitory activity in their prechallenge sera. These data indicated that growth-inhibitory activity measured in vitro was not a reliable predictor of immune status in vivo, and the reliance on this criterion to select vaccine candidates for human clinical trials may be misplaced. The transgenic lines further offer useful tools for comparing the efficacy of MSP1 19 -based vaccines that utilize different immunization regimens and antigen formulations.

Development of a safe, effective and affordable malaria vaccine is central to global disease control efforts. One of the most highly regarded proteins for inclusion in an asexual blood stage subunit vaccine is the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1(19)). As production of vaccine antigens in plants can potentially overcome cost and delivery hurdles, we set out to produce MSP1(19) in plants, characterise the protein and test its immunogenicity using a mouse model. Plasmodium yoelii MSP1(19) (PyMSP1(19)) was produced in Nicotiana benthamiana using the MagnICON® deconstructed TMV-based viral vector. PyMSP1(19) yield of at least 23% total soluble protein (TSP;3-4 mg/g Fwt) were achieved using a codon-optimised construct that was targeted to the apoplast. Freeze-dried leaf powder contained at least 20 mg PyMSP1(19) per gram dry weight and the protein retained immunogenicity in this form for more than 2 years. Characterisation studies, including SDS-PAGE, mass spectrometry and circular dichroism, indicated that the plant-expressed PyMSP1(19) was similar to its Escherichia coli- and Saccharomyces cerevisiae-expressed counterparts. Purified plant-made PyMSP1(19) induced strong immune responses following intraperitoneal immunisation, although titres were lower than those induced by an equivalent dose of purified E. coli-expressed PyMSP1(19). The reason for this is uncertain but may be due to differences in the oligomerisation profile of the vaccines. The plant-made PyMSP1(19) vaccine was also found to be orally immunogenic when delivered alone or following immunisation with a PyMSP1(19) DNA vaccine. This study adds to an increasing body of research supporting the feasibility of plants as both a factory for the production of malaria antigens, and as a safe and affordable platform for oral delivery of a temperature-stable malaria vaccine.

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP1(19)) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1(19) is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h(-1). High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 microm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

The efficiency of delivery of DNA vaccines is often relatively low compared to protein vaccines. The use of superparamagnetic iron oxide nanoparticles (SPIONs) to deliver genes via magnetofection shows promise in improving the efficiency of gene delivery both in vitro and in vivo. In particular, the duration for gene transfection especially for in vitro application can be significantly reduced by magnetofection compared to the time required to achieve high gene transfection with standard protocols. SPIONs that have been rendered stable in physiological conditions can be used as both therapeutic and diagnostic agents due to their unique magnetic characteristics. Valuable features of iron oxide nanoparticles in bioapplications include a tight control over their size distribution, magnetic properties of these particles, and the ability to carry particular biomolecules to specific targets. The internalization and half-life of the particles within the body depend upon the method of synthesis. Numerous synthesis methods have been used to produce magnetic nanoparticles for bioapplications with different sizes and surface charges. The most common method for synthesizing nanometer-sized magnetite Fe3O4 particles in solution is by chemical coprecipitation of iron salts. The coprecipitation method is an effective technique for preparing a stable aqueous dispersions of iron oxide nanoparticles. We describe the production of Fe3O4-based SPIONs with high magnetization values (70 emu/g) under 15 kOe of the applied magnetic field at room temperature, with 0.01 emu/g remanence via a coprecipitation method in the presence of trisodium citrate as a stabilizer. Naked SPIONs often lack sufficient stability, hydrophilicity, and the capacity to be functionalized. In order to overcome these limitations, polycationic polymer was anchored on the surface of freshly prepared SPIONs by a direct electrostatic attraction between the negatively charged SPIONs (due to the presence of carboxylic groups) and the positively charged polymer. Polyethylenimine was chosen to modify the surface of SPIONs to assist the delivery of plasmid DNA into mammalian cells due to the polymer's extensive buffering capacity through the "proton sponge" effect.

No abstract

Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.