Plasmid DNA is currently being considered for immunization as an alternative approach to protein-based vaccines. The prevailing opinion is that the linear and open-circular forms of plasmid DNA are less effective than the supercoiled isoform in inducing immune responses. However, it can be argued that the plasmid is likely to be nicked and relaxed during its transport into the nucleus, regardless of the level of supercoiling present. This article investigates the validity of the recommendations regarding supercoiling requirements for optimal efficiency of DNA vaccines.
Some studies appear to be consistent with the notion that cell transfection with plasmids is better with the supercoiled than the relaxed open-circular isoform. Other studies have shown that there is a minimum limit of supercoiled plasmid that needs to be present for potent in vivo responses to DNA vaccines, with the limit being reported to be >70% in one study. However, other data indicate that gene transfer is not significantly affected by the level of supercoiling of the gene, and cationic lipids and microparticles can be used to deliver both isoforms without significant loss of efficiency in vivo.
To further address this issue, we evaluated the in vitro and in vivo potency of DNA plasmids encoding the HIV proteins, gp140 env and p55 gag. The in vitro results showed no relationship between the degree of supercoiling and cellular transfection efficiency. Similarly, the antibody titers measured in mice immunized with fully relaxed open-circle plasmids were not significantly different from responses elicited with supercoiled plasmid. However, linearized DNA was ineffective.
Based on the findings of ourselves and others that the relaxed open-circular form of a plasmid DNA is no less efficient in transfecting cells or in eliciting antibody responses than its supercoiled form, we propose that it is possible to lower the requirement for a high percentage of supercoiled plasmid in a DNA vaccine formulation.