Fig. 4
Panel a: The levels of E7-specific IgG and SIgA antibodies in serum (left) and vaginal discharges (right), respectively, on day 41; the sera and vaginal discharge samples were tested by indirect ELISA using recombinant human papillomavirus type 16 protein E7 as the coating antigen. Goat-anti-mouse IgG-H&L (HRP) antibody and Goat Anti-Mouse IgA alpha chain (HRP) were used as a secondary antibody. The absorbance of each microwell was read on a spectrophotometer at 450 nm. Error bars represent the mean ± standard error value of each group (P < 0.001). Panels b and c: Detection of E7-specific T-lymphocytes using an IL-2 (panel b) and IFN-γ (panel c) ELISPOT assay in the splenocytes (left) and intestinal mucosal lymphocytes (right); female C57BL/6 mice were immunized orally with 109 CFU of Lactococcus lactis containing rE7 on days 1, 2, 3, 14, 15, 16, 29, 30, and 31. In order to measure cytokine production from the cells, on day 41 (ten days after the last immunization), intestinal mucosal lymphocytes and splenocytes were collected and stimulated with MHC class I - restricted peptides and MHC class II - restricted peptides (HPV-16 E749–57 and HPV-16 E730–67, respectively). Then, the level of E7-specific IFN-γ-producing CD8+ and CD4+ T cells and E7-specific IL-2-producing CD8+ and CD4+ T cells was calculated via mouse IFN-γ and IL-2 ELISPOT kit, respectively. The diagram demonstrates the number of spots per 104 splenocytes and intestinal mucosal lymphocytes after stimulation. The results are presented as mean ± SD (n 10). PBS (Control group); LLEV: Induced L. lactis harboring pNZ8123; LLE7BF: Induced L. lactis harboring pNZ8123-HPV16-E7 produced through batch fermentation; LLE7FBF: Induced L. lactis harboring pNZ8123-HPV16-E7 produced through fed-batch fermentation; LLOE7BF: Induced L. lactis harboring pNZ8123-HPV16-optiE7 produced through batch fermentation; and LLOE7FBF: Induced L. lactis harboring pNZ8123-HPV16-optiE7 produced through fed-batch fermentation