Fig. 5. Increased safety and translational potential of a cPANHPVAX variant lacking the E7 pRb-binding sites.

a Schematic representation of the cPANHPVAX antigen variants b Numbers of IFN-γ spots per 10⁶ splenocytes, measured upon stimulation with the E7_(49-57) peptide, were compared in 7 groups of mice immunized with antigen cPANHPVAX, wo/pRb-cPANHPVAX, mo/pRb-cPANHPVAX, wo/pRb-cPANHPVAX-7R, mo/pRb-cPANHPVAX-7R, wo/pRb-cPANHPVAX-9R or mo/pRb-cPANHPVAX-9R, respectively. Shown are the mean and SD of triplicate values on each mouse. Statistical significance was assessed using the nonparametric Mann–Whitney test. P-values ≤ 0.05 are considered as significant and are labeled as follows: *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001; ****P-value < 0.0001. c The BRK assay was performed to evaluate the oncogenic transforming potential of different antigen candidates. All the genes encoding antigen candidates were cloned into pcDNA 3.1 (+) under the control of the cytomegalovirus (CMV) immediate early promoter. The BRK assay was performed by using the 7 different pcDNA 3.1 (+) based constructs (3 independent experiments), co-transfected with EJ-ras. The plasmid encoding HPV16 E7 is used as a positive control (P.C.). The empty vector pcDNA 3.1 (+) is used as a negative control (N.C.). d Relative quantification of the results of the BRK assay. The number of colonies is expressed as the percentage of colonies obtained in cells transfected with plasmids HPV16 E7 and EJ-ras, which is taken as 100%.