Fig. 5. Detoxified, lyophilized iVAX reactions produce conjugate vaccines.
(A) iVAX lysates were detoxified via deletion of lpxM and expression of F. tularensis LpxE in the source strain used for lysate production. (B) Growth rates of CLM24 WT and CLM24 ∆lpxM strains. No growth defects were observed across two clones of the knockout strain. Values represent means and error bars represent SDs of n = 4 replicates. ns, not significant. (C) The resulting lysates exhibited significantly reduced endotoxin activity, as measured by activation of human TLR4 in HEK-Blue hTLR4 reporter cells. **P = 0.003, as determined by two-tailed t test. Values represent means and error bars represent SDs of n = 3 replicates. (D) FtO-PS conjugate vaccines produced and purified from detoxified iVAX reactions contained 0.21 ± 0.3 EU/10-μg dose, as measured by human TLR4 activation. Dashed line represents endotoxin levels reported in commercial conjugate vaccines (<12 EU/dose). Value represents mean and error bars represent SD of n = 6 replicates. (E) iVAX reactions producing sfGFP217-DQNAT were run immediately or following lyophilization and rehydration. (F) Glycosylation activity was preserved following lyophilization, demonstrating the potential of iVAX reactions for portable biosynthesis of conjugate vaccines. Top panel shows signal from probing with αHis to detect the carrier protein, middle panel shows signal from probing with αFtO-PS, and bottom panel shows αHis and αFtO-PS signals merged. Images are representative of at least three biological replicates. Molecular weight ladder is shown at the left of each image.