Figure 1.
Vaccines used in this study and characterization of our vaccine candidate.
A) IBDV VP2 is a 452-aa–long protein and is the only component of the T1 symmetry IBDV VLP (VLP-WT). A C-terminal region of HPV-16 E7 (lacking the N-terminal oncogenic domain, OD) encompassing aa 45–98 was cloned in the C-terminal part of VP2, giving rise to a chimeric VLP with the heterologous sequence inside the VLP (VLP-E7). VLP-E7-B was generated by inserting the HPV-16 E7 C-terminal region into the VP2–thus generating a chimeric VLP containing the heterologous sequence inside the VLP–and inserted into the VP2, as indicated in the figure. An illustrative drawing of each construct shows the IBDV VP2 viral capside, according to reported structural data [13] B) Left, the purity of VLP-E7 was analyzed by SDS-PAGE and Coomassie staining. One microgram of the vaccine was loaded onto the protein gel. Right, electron micrograph of a representative purified sample of VLP-E7 showing the virus-like structure. The bar scale (0.2 µm) is shown in the micrograph.
Figure 2.
Immunization with VLP-E7 elicits E7 peptide–directed IFN- γ –secreting splenocytes.
A) IFN- γ –secreting splenocytes from mice (n = 5) immunized twice with either 50 µg of VLP-E7 or VLP-WT or PBS, with or without Sigma Adjuvant System (SAS), were quantified ex vivo by ELISPOT assay. The splenocytes were pooled from each group of mice, plated, and either not stimulated (no peptide) or stimulated with control peptide (11–20) or with two E7 peptides (49–57, H2b epitope and 86–93 HLA-A2 epitope). Bars represent mean ± s.e.m. of six replicates. *p<0.05 compared with VLP-WT. B) IFN- γ –secreting splenocytes from mice (n = 5) immunized twice with either 50 µg of VLP-WT or 50 µg of VLP-E7 alone or with adjuvants (50 µg CpGs or 50 µg Poly-R) were quantified by ELISPOT assay. The splenocytes were pooled from each group of mice, plated, and stimulated as in A. The background values (no peptide and peptide control wells) were subtracted from those stimulated with the 49–57 and 86–93 E7 peptides. Bars represent mean ± s.e.m. of six replicates.
Figure 3.
Therapeutic tumor challenge I.
A) Top, vaccination schedule. Bottom, tumor survival data (n = 10). Black star corresponds to day 0, when tumor cells were inoculated. Black arrows indicate the vaccination days (5 and 12). Symbols are as indicated in the square. B) Evolution of tumor size. Values shown are from individual mice in each group, as indicated in each panel. Animals were sacrificed when tumors reached 1 cm3 (blue line). A representative tumor-bearing control mouse (necropsy) is shown in the photograph.
Figure 4.
Therapeutic tumor challenge II.
A) Top, vaccination schedule. Bottom; tumor survival data (n = 9). Black star corresponds to day 0, when tumor cells were inoculated into the mice. Black arrows indicate the vaccination days (9 and 16). Symbols are as indicated in the square. B) Evolution of tumor size. Values shown are from individual mice in each group, as indicated in each panel. Animals were sacrificed when tumors reached 1 cm3 (blue line). A representative tumor-bearing control mouse (necropsy) is shown in the photograph.
Figure 5.
Therapeutic tumor challenge III.
A) Top, vaccination schedule. Bottom, tumor survival data (n = 7). Black stars correspond to days 0 and +56, when tumor cells (5×105/mouse) were inoculated. Mice vaccinated once (×1, day +9) with VLP-E7 are represented with red (closed) circles. The remaining symbols correspond to the vaccinated mice as indicated in the square in the survival diagram. B) Evolution of tumor size. Values shown are from individual mice in each group, as indicated in each panel. Animals were sacrificed when tumors reached 1 cm3 (blue line). Black stars indicate subcutaneous tumor cell injection days (0 and +56).