Fig 1.
Design and purification of the vaccine antigen.
(A) Scheme of the designed E6E7 antigen. Ala-Ala-Tyr (AAY) spacers were inserted between the epitopes. (B) SDS-PAGE of purified antigen stained with Coomassie Brilliant Blue. MW: molecular mass marker.
Table 1.
E6 and E7 epitopes used to assemble in the E6E7 protein.
Fig 2.
Anti-tumor responses elicited in mice immunized with E6E7 multi-epitope protein.
Mice were immunized with three doses of PBS/2M urea (PBS) or E6E7 in intervals of 14 days. Mice were challenged with TC-1 tumor cells two weeks after the last dose. (A) Groups of five C57BL/6 mice were immunized as described. (B) TLR4 KO mice (C57BL10/ScCr) were vaccinated with PBS/2M urea (5 mice, PBS) or E6E7 (6 mice). (C) Groups of eight CD4 KO mice were vaccinated with PBS/2M urea (PBS) or E6E7. (D) Groups of five CD8 KO mice were vaccinated with PBS/2M urea (PBS) or E6E7. In parallel, C57BL/6 wild-type mice (5 mice) were used as a control of tumor growth competence. Asterisk indicates statistically significant percentage of tumor-free animals when compared to control group (** p < 0.005).
Fig 3.
Therapeutic anti-tumor responses elicited in mice immunized with E6E7.
(A) Groups of five C57BL/6 mice were treated with three doses of PBS/2M urea (PBS) or E6E7 in intervals of seven days. The first dose was administered three days after the challenge with TC-1 tumor cells. (B) Groups of TLR4 knockout mice were treated with three doses of PBS/2M urea (4 mice, PBS) or E6E7 (4 mice) in intervals of seven days. In parallel, C57BL/6 wild-type mice (5 mice) were used as a control of tumor growth competence. The tumor size in each animal was monitored daily and is given in mm2 (length x width).
Fig 4.
Induction of antigen-specific IFN-γ secretion responses in mice immunized with E6E7.
(A) C57BL/6 mice were immunized with three doses of PBS/2M urea (PBS) or E6E7 protein vaccine and challenged with TC-1 tumor cells two w3eeks after the last dose. The ELISPOT assay was performed with PBMC twenty days after the challenge. (B) TLR4 knockout mice were immunized with three doses of PBS/2M urea (PBS) or E6E7 and challenged with TC-1 tumor cells two weeks after the last dose. The ELISPOT assay was performed with PBMC twenty days from the challenge. The cells were stimulated with E6 (VYDFAFRDL49-57; DKKQRFHNI127-135) and E7 (RAHYNIVTF49-57; LCVQSTHVD67-75) peptides. (C) Comparative IFN-γ production between PBMC stimulated with E649-57 and/or E749-57 specific peptides (Stimul.); Ø –Unstimulated cells.