Fig. 7. iVAX-derived conjugates protect mice from lethal F. tularensis challenge.

(A) Groups of five BALB/c mice were immunized intraperitoneally (IP) with PBS or 10 μg of purified, cell-free synthesized unmodified or FtO-PS–conjugated carrier proteins. FtO-PS–conjugated carriers prepared in living E. coli cells using PCGT were used as positive controls. Mice were boosted on days 21 and 42 with identical doses of antigen. IN, intranasally. (B) On day 56, FtLPS-specific IgG titers were measured by ELISA in serum of individual mice immunized with PBS or anti–F. tularensis conjugate vaccines (FtO-PS-CV) (black dots) with F. tularensis LPS immobilized as antigen. Mean titers of each group are also shown (red lines). Only iVAX-derived conjugates elicited significantly higher levels of FtLPS-specific IgG compared to PBS immunized controls across all carrier proteins tested (**P < 0.01, Tukey-Kramer post hoc test; CV, conjugate vaccine). On day 66, mice were challenged intranasally with 6000 CFU (60 times the intranasal LD₅₀) F. tularensis subsp. holarctica LVS Rocky Mountain Laboratories and monitored for survival for an additional 25 days. Kaplan-Meier curves for immunizations with (C) MBP^4xDQNAT, (D) PD^4xDQNAT, and (E) EPA^DNNNS-DQNRT as the carrier protein are shown. iVAX-derived vaccines protected mice from lethal pathogen challenge as effectively as vaccines synthesized using the state-of-the-art PGCT approach. *P < 0.05; **P < 0.01, Fisher’s exact test.