ODN 2216 induces an antiviral state in the domestic cat in vivo
In a final step, the induction of antiviral responses by ODN 2216 was measured in vivo and characterized using ex vivo assays. The molecule was administered once subcutaneously to two cats (c07 and c08), while two other cats (c05 and c06) received endotoxin-free PBS as a negative control. The injections were well tolerated by all cats: no rise in body temperature, no local reactions at the injection site and no other undesired side effects were noted. Absolute monocyte counts increased 1.5 to 2-fold in the first 12 h in cats that had received ODN 2216 (data not shown). No other treatment-specific alterations were noted in hematological values throughout the experiment. Mx expression was measured in blood at early time points after injection as marker for the induction of antiviral immune processes. mRNA levels of Mx increased by 8.8 and 3.8-fold within 24 h in both treated cats and decreased gradually thereafter until 192 h post treatment (Figure7A). In accordance with observations from in vitro experiments, cat c08 exhibited a stronger response to ODN 2216 than cat c07. No increase in Mx expression was observed in the blood of the control cats (c05 and c06). In a further experiment, plasma was collected from all four cats at regular intervals post injection and was utilized in an in vitro inhibition assay. The plasma obtained from both treated cats 24 and 48 h after administration of the molecule was able to inhibit replication of FCV in vitro (Figure7B). In contrast, plasma from untreated cats seemed to slightly enhance the susceptibility of target cells to FCV inoculation.
Figure 7 Subcutaneous injection of ODN 2216 induces a systemic antiviral state in vivo. (A) The cats received an injection of either 200 μg/kg ODN 2216 (cats 1 and 2) or endotoxin-free PBS (cats 3 and 4). Mx mRNA expression was measured by qPCR in whole blood. Depicted values represent the ratio of mRNA levels for a given cat (c05-c08) at the indicated time point to mRNA levels for the same cat at time point 0 h. (B) fcwf-4 cells were incubated for 24 h in duplicates with plasma collected from the cats 1 to 4 at the indicated time points post injection, and inoculated with the FCV. Indicated are viral inhibition factors calculated as described in the material and methods section. (C) Mx mRNA expression factors measured in the blood of all four cats at time points 8 h-192 h were correlated with the inhibition of FCV induced in vitro by the corresponding plasma samples.