Development of plasmid dna formulations for application as microspheric dna vaccines

The advent of DNA vaccines has heralded a new technology allowing the design and elicitation of immune responses more adequate for a wider range of pathogens. The formulation of these vaccines into the desired dosage forms extends their capability in terms of stability, routes of administration and efficacy. This thesis describes an investigation into the fabrication of plasmid DNA, the active principle of DNA vaccines, into microspheres, based on the tenet of an increased cellular uptake of microparticulate matter by phagocytic cells. The formulation of plasmid DNA into microspheres using two methods, is presented. Formulation of microspheric plasmid DNA using the double emulsion solvent evaporation method and a spray-drying method was explored. The former approach involves formation of a double emulsion, by homogenisation. This method produced microspheres of uniform size and smooth morphology, but had a detrimental effect on the formulated DNA. The spray-drying method resulted in microspheres with an improved preservation of DNA stability. The use of polyethylenimine (PEI) and stearylamine (SA) as agents in the microspheric formulation of plasmid DNA is a novel approach to DNA vaccine design. Using these molecules as model positively-charged agents, their influence on the characteristics of the microspheric formulations was investigated. PEI improved the entrapment efficiency of the plasmid DNA in microspheres, and has minimal effect on either the surface charge, morphology or size distribution of the formulations. Stearylamine effected an increase in the entrapment efficiency and stability of the plasmid DNA and its effect on the micropshere morphology was dependent on the method of preparation. The differences in the effects of the two molecules on microsphere formulations may be attributable to their dissimilar physico-chemical properties. PEI is water-soluble and highly-branched, while SA is hydrophobic and amphipathic. The positive charge of both molecules is imparted by amine functional groups. Preliminary data on the in vivo application of formulated DNA vaccine, using hepatitis B plasmid, showed superior humoral responses to the formulated antigen, compared with free (unformulated) antigen.

The effects of the environment on the stability and expression of genetically engineered plasmids

The lac promoter is widely used in plasmid expression systems, even though it is prone to catabolite repression. As a consequence glycerol is often used as an alternative carbon source. Three plasmids containing various sizes of the staphylococcal protein A (SPA) gene, which are under the control of the lac promoter were investigated in continuous culture, to evaluate the effects of nutrient limitations on their stability and expression. The fears of catabolite repression were dispelled as a low expression plasmid (pPA16) produced a greater amount of truncated SPA under glucose limiting conditions (11 ug mg-1 cell protein) when compared to that using glycerol (8 ug mg-1 cell protein). Segregational instability was also observed under glycerol limiting conditions at all the dilution rates investigated. Whereas pPA16 was relatively stable under glucose limiting conditions, with SPA production being continuous. Experiments using excess glycerol with limited ammonium increased the stability of pPA16, (when compared to limited glycerol) with expression of SPA being continuous but reduced (6 ug mg-1 cell protein). With excess glucose and limited ammonium the copy numbers remained high but expression of SPA paralled that produced under glucose limiting conditions. This might indicate that the higher levels of glucose are reducing expression (catabolite repression) or that the low level of ammonium is affecting protein production. A high expression plasmid (pPA31) produced nearly 100 ug full length SPA mg-1 cell protein, while another high expression plasmid (pPA34) producing truncated SPA proved to be very unstable. An ELISA was developed to detect the SPA produced by these experiments, which could be adapted for western blotting or immunogold probing using electron microscopy. SPA was localised in electron lucent areas present in the periplasmic space of the E. coli host harbouring pPA16. While in the same host containing pPA31, SPA was localised not only in electron lucent areas but also around the whole of the outer-membrane.