ODN 2216 inhibits replication of a retrovirus in vitro
The life cycle of retroviruses is characterized by the reverse transcription of their genomic RNA into DNA and subsequent integration of this viral DNA as provirus into the genome of the host, causing permanent infection most often accompanied by persistent virus production by infected cells. The feline leukemia virus (FeLV), a gammaretrovirus that can be propagated on FEA cells in vitro, affects domestic cats worldwide. As viral replication in chronically infected cats can be lowered by treatment with IFNα[67], the potential of Sup 2216 produced by the PBMCs of five adult cats (c06 and c08 from group 2; c09, c10, c12 from group 3) to inhibit productive infection of FEA cells was analyzed. In initial experiments, this cell line exhibited similar responses as fcwf-4 and CrFK cells to both direct treatment with ODN 2216 and incubation with the different supernatants (Figure3A,4C and D and data not shown). Compared to incubation with medium alone, treatment of FEA cells with Sup 2216 for 24 h prior to inoculation with FeLV followed by repetitive treatments of the cells with this supernatant every 2 days thereafter significantly reduced viral RNA (p < 0.05) and DNA (p < 0.05) measured in the cell culture supernatants and cells respectively as of 4 days post inoculation (Figure6A and B). The antiviral potential of the supernatants from the individual cats was very similar; however the best results were conferred by the Sup 2216 derived from cells of c08 (depicted in Figure6A and B).The kinetic curve of viral RNA loads in cultures of cells treated with Sup 2216 of this cat was similar to those obtained in cells treated with 50U rfeIFNα (data not shown). Also, treatment of the cells with ODN 2216 directly affected neither viral RNA nor viral DNA loads measured in the supernatants and the cells respectively (data not shown). Mx mRNA expression was 80-fold higher in the Sup 2216 treated cells than in all controls 8 days post inoculation, indicating the ability of these supernatants to sustain antiviral mechanisms when applied to the cells repeatedly (Figure6C). Furthermore at this time point, the Sup 2216 treated cells exhibited significantly lower viral DNA loads (p = 0.0313) and produced significantly less virus (p = 0.0313) than cells treated with Sup 2243, Sup Neg or medium alone (Figure6D and E). The extent of Mx transcription conferred by the Sup 2216 of the cats strongly correlated with lower provirus (p = 0.0053) and virus (p = 0.0012) loads measured in the FEA cells and supernatants respectively on day 8 post inoculation (p = 0.0053). Also, the highest Mx mRNA levels in target cells were conferred by Sup 2216 of cat c08 and reflected by the lowest viral and proviral loads measured at this time point in our experiments.
Figure 6 Supernatants derived from ODN 2216-stimulated PBMCs decrease retroviral DNA and RNA loads in target cells. (A) FEA cells were incubated for 24 h with the respective supernatants or medium alone, before inoculation with the feline leukaemia virus (FeLV), as well as every 2 days thereafter. Viral RNA loads were measured at the indicated time points by real time RT-PCR and 45-Ct values are depicted. (B) FeLV DNA loads in the cells were measured at the indicated time points and Ct values were normalized to detection of a housekeeping gene (GAPDH). Mean values from duplicate experiments carried out simultaneously with the supernatants derived from PBMCs of a selected cat (belonging to group 2) and with medium alone are shown as an example. Results for (A) and (B) are indicative of those obtained with supernatants from PBMCs of two additional adult cats (from group 3). Stars represent statistical differences in area under the curve (AUC) measurements between the curves of all three cats obtained in cells incubated with Sup 2216 and each of the other treatments. Mx mRNA expression factors (C), viral loads (45-Ct values depicted) (D) and proviral loads (E) were measured in the FEA cells of five cats (two from group 2 and three from group 3) on day 8 post inoculation. Each dot represents the mean of duplicate measurements for an individual cat. *p < 0.05, **p < 0.01.