Cytotoxicity analysis by LDH assay.

<p>CTL activity of vaccinated BALB/c mice against P815 target cells loaded with synthetic p66 peptide was measured using LDH assay. The splenocytes were stimulated <i>in vitro</i> for 72 hours with p66 peptide, the irrelevant peptide or left un-stimulated and co-cultured with p66-pulsed p815 target cells at different E:T ratios for 6 hours. The effector cells lytic activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049539#s2" target="_blank">Materials and Methods</a>. All data are representative of three independent experiments using pooled spleen cells from five mice and error bars (SD) were calculated based on triplicates. Splenocytes from the mice vaccinated with T7-p66 phage nanoparticles showed a significantly greater cytotoxicity against p66-loaded target cells than the mice receiving p66 emulsified in Freund's adjuvant (p66+FA group). Injection of mice with T7-p66x2 nanoparticles or an emulsion of p66x2 peptide in Freund's adjuvant did not result in a significant cytotoxic activity against target cells compared to negative controls (T7-wt, PBS and CFA/IFA). Similarly, a mixture of T7-wt nanoparticles and p66 peptide only induced a background level of cytotoxic activity in splenocytes similar to negative controls. Data corresponding to T7-wt and PBS have not been shown.