Immunity toward exogenous and expressed hFIX.

Mice were injected in utero or as adults with AdhFIX then repeatedly rechallenged with hFIX protein and AdhFIX as described in Figure 2 while hFIX antibody concentrations were measured. The y-axis shows blood hFIX antibody concentrations (ratio versus control hyperimmune serum, 1/10 000 dilution) after in utero or adult injection of AdhFIX (phase 1), repeated injection of hFIX protein to the adult mice (phase 2, shaded), and repeated injection of AdhFIX to the adult mice (phase 3). The x-axis shows the experimental time course in days after first injection. Arrowheads indicate injection points. The D15 and D17 fetal AdhFIX groups are mice initially injected in utero with AdhFIX at days 15 and 17 after conception, respectively. The adult AdhFIX group contains mice initially injected as adults with AdhFIX. The protein challenge control group did not receive prior injection of AdhFIX. The AdhFIX challenge group received neither prior injections of AdhFIX nor hFIX protein. Diamonds show the analysis of individual mice at a given time point. The number of samples with no detectable antibodies at each time point is depicted as a diamond and number, both lying beneath the lower dotted line. The number of samples exceeding an absorbance ratio of 1.5 at each time point is depicted as a diamond and number, both lying above the upper dotted line. The inset shows values of ratio versus control hyperimmune serum using 3 dilutions (1/20, 1/100, and 1/10 000) for individual mice after the seventh injection of hFIX protein.