Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment

Abstract

Human papillomavirus (HPV) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.