Vaccine Research (Nov 2015)
Construction and evaluation of human papillomavirus genotype 18 pseudovirions
Abstract
Introduction: Cervical cancer is the second most common cancer in women worldwide and the role of human papillomavirus (HPV) has been proved in its etiology. The currentley available L1-capsid-protein-based vaccine is highly immunogenic and very high titers of serum antibodies can be obtained by its injection, but unfortunately it is restricted to only a few HPV genotypes and is relatively expensive. Therefore, development of the second-generation HPV vaccines has become the focus of the research and L2 capsid protein that is capable of producing broad spectrum antibodies has become one of the main candidates. Evaluation of the vaccine immunogenicity however, requires develoment of HPV pseudovirions (HPV PvSs) comprised of L1 and L2 virus protein and the present study was an attempt to produce PvSs of HPV18 genotype by an in-house method. Methods: The HPV18 L1/L2 coding plasmid and the reporter plasmid of pEGFP-N1 were amplified in E. coli DH5α and purified using silica oxide method. The plasmids were co-transfected into HEK 293FT cell line and the preliminary analysis of expression was performed using fluorescence microscopy. The PvSs were partially purified using gel filtration chromatography and were used to transduce the 293FT cells to evaluate the infectivity rate of the PvSs. The results were analyzed by fluorescent microscopy, flow cytometry and atomic force microscopy. Results: The results showed that the HPV18 L1/L2 coding plasmid and the pEGFP-N1 reporter plasmid have been successfully co-transfected into HEK 293FT cells and the PvSs were constructed. The 293FT cells were successfully transduced by PvSs that had packaged reporter plasmid. These findings were confirmed by fluorescence microscopy and flow cytometry as well as AFM imaging. Conclusion: In this study, the cotransfection of HPV18 L1/L2 coding plasmid as well as pEGP-N1 reporter plasmid into the HEK 293FT cell led to the assembly of the pseudovirion harboring the reporter gene. The protocols used in this study were easy to perform and relatively inexpensive and did not rely on the commercial kits.
