Abstract
Plasmid DNA (pDNA) vaccines are promising means to prevent and treat infectious diseases, such as leishmaniasis, but immunisation protocols require large amounts of supercoiled plasmid DNA (sc-pDNA). Although pDNA can be produced at a reasonable cost in bioreactors; this scale of production may not be the best method at the initial step of a vaccine development when many antigens need to be tested. Then, with the goal of improving the production of VR1012-NH36 and pVAX-NH36 pDNA vaccines against leishmaniasis, the effect of the culture medium and temperature on the pDNA yield was studied in flask cultures. The results indicate that the plasmid volumetric yield increased up to 65 mg/l in flask cultures by using a semi-defined medium, and shifting the culture temperature from 37 to 42°C at the late exponential growth phase. This pDNA production, with at least 80% of sc-pDNA at a laboratory scale seems sufficient to evaluate this and other pDNA vaccine candidates in the initial steps of vaccine development. Key words: Plasmid DNA vaccine, plasmid DNA production, growth medium, flask culture, trace metal optimisation, leishmaniasis.
Highlights
Leishmaniasis is a neglected tropical disease affecting about 12 million poor people in developing countries (Hotez, 2008)
Research devoted to finding alternative methods of preventing and treating this infection has shown that plasmid DNA vaccines containing a DNA fragment encoding a 36 kDa nucleoside hydrolase from Leishmania donovani (VR1012-NH36) induced protective or therapeutic immune responses in a murine model (Aguilar-Be et al, 2005; Gamboa-Leon et al, 2006)
To improve the sc-Plasmid DNA (pDNA) production, E. coli DH5α harbouring pVAX-NH36 was grown in LB, SDCAS, or ZMY505; two semi-defined media previously reported as specific for the production of pDNA
Summary
Leishmaniasis is a neglected tropical disease affecting about 12 million poor people in developing countries (Hotez, 2008). New antigen candidates were identified recently by mining the Leishmania major genome (Herrera-Najera et al, 2009). To assess their potential as pDNA vaccines, pure supercoiled pDNA (sc-pDNA) at mg scale must be obtained for each antigen. The production of pDNA in Escherichia coli depends on many factors, including interactions between the host, plasmid vector and growth conditions, such as media components and the fermentation strategy (O'Kennedy et al, 2000, 2003), as well as downstream processing (Ferreira et al, 2000). The production of pDNA has been a very active study field in the last decade, many attempts to improve the yield and quality of pDNA by engineering E. coli (Borja et al, 2012; Phue et al, 2008), optimising media
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