Figure 6 – Immunogenicity and potency evaluation of PWCV. A-C) Comparison of different fermentation strategies by the administration of two subcutaneous doses of PWCV in two weeks interval. D-F) Comparison of different downstream processes and storage temperatures by the administration of one subcutaneous dose of PWCV. One PWCV dose is composed of 100 µg protein + 200 µg Alum. Mouse blood was collected twelve days after the last immunization. A and D) IgG antibody inductionin vivo ; B and E) IL-17A production in vitro ; C and F) Survival after challenge. Numbers represent different PWCV lots used in immunization assay: 1- adjuvant Alum alone (negative control ); 2- Engineering lot 007/09 produced at 60 L bioreactor (positive control , prepared as described by Gonçalves et al., 2014); 3- Batch fermentation () with cells harvested at the highest OD (10); 4- Batch fermentation () with cells harvested at OD 6.5; 5- Fed-batch fermentation () with cells harvested at the highest OD (14); 6- Perfusion-batch fermentation () with cells harvested at the highest OD (30); 7- Fed-batch fermentation () with cells harvested at the highest OD (14) using hollow fiber system and preserved at -80°C; 8- Same fed-batch lot () preserved at 4°C; 9- Fed-batch fermentation () with cells harvested at the highest OD (14) by centrifugation; 10- Batch fermentation () with cells harvested at OD 6.5 by hollow fiber system and preserved at -80°C; 11- Batch fermentation () with cells harvested at OD 6.5 by centrifugation and preserved at -80°C. Bars represent median. Significant difference is presented between adjuvant group and PWCV or between vaccines obtained from different fermentation strategies when indicated. Statistics was calculated by the Mann-Whitney U test and shown by asterisks: * P< 0.05, ** P < 0.01, and *** P< 0.001. Survival was analyzed by Kaplan-Meier method and the log-Rank test to compare the curves.