Type I interferon gene therapy protects against cytomegalovirus-induced myocarditis

Abstract

Type I interferons (IFNs) are produced early in response to viral infection and modulate adaptive immunity. Previously we demonstrated localized protection against murine cytomegalovirus (MCMV) infection in IFN DNA-inoculated mice. Here we examine the effect of seven IFN subtypes (IFNA1, A2, A4, A5, A6, A9 and B), administered by DNA inoculation, on systemic MCMV infection and myocarditis. IFN transgene expression altered the pathogenesis of MCMV infection with regard to virus titre and myocarditis. IFNA6 treatment reduced MCMV replication whilst IFNA5 and A2 enhanced virus replication. IFNA6, A9, and B treatment inhibited acute myocarditis. A T helper type 1-like, antibody and cytokine, response correlated with decreased virus titre and myocarditis. In addition, IFNA6 was able to reduce chronic cardiac inflammation. This research into the effectiveness of seven type I IFNs, using DNA gene therapy, highlights the need for correct subtype usage in the treatment of disease. We demonstrate effective subtypes for treatment in both the acute and chronic phases of MCMV infection and the resultant development of myocarditis.

Figure 1

IFN transgenes expressed in the mammalian expression vector pkCMVint. MuIFNA1, A2, A4, A5, A6, A9 and B carrying the full-length MuIFN subtype genes (black) including the signal sequence (shaded grey) located upstream of the mature protein. Flanking primer sequences with incorporated restriction enzyme sites used for subcloning of each transgene cassette are shown.

Figure 2

Effect of IFN DNA treatment on MCMV replication in vivo. MCMV titres in the spleen (day 3 p.i.), liver (day 3 p.i.), and salivary glands (SG) (day 7 p.i.). Mice were inoculated with 10⁴ PFU MCMV i.p. at 2 weeks post-DNA vaccination. Virus titres are expressed as percentage control titre (average PFU/g tissue, five mice per time-point ±SE) found for mice vaccinated with the vehicle. *Statistically significant differences between treatment groups and vehicle (P<0·05) are shown and are representative of two independent experiments.

Figure 3

DNA treatment with IFNA6, IFNA9 and IFNB reduces MCMV-induced myocarditis. BALB/c mice were inoculated with 10⁴ PFU of MCMV i.p. at 2 weeks post-DNA vaccination. (a) The average number of inflammatory foci/heart section from groups of five mice per time-point ±SE are shown at day 7. *Statistically significant differences between treatment groups and vehicle (P<0·05) are shown and are representative of two independent experiments. (b) Histopathology of murine cardiac tissue (H & E stained, × 160). Heart sections for normal or vehicle, IFNA1-, IFNA2-, IFNA4-, IFNA5-, IFNA6-, IFNA9- and IFNB-treated, MCMV-infected mice (day 7 p.i.) are as indicated.

Figure 4

Cytokine expression in sera of IFN DNA-treated and MCMV-infected mice. Mice were inoculated with 10⁴ PFU MCMV i.p. at 2 weeks post-DNA vaccination. The cytokines IFN-γ, IL-4, IL-6 and IL-10 were determined in the sera at day 7 p.i. by ELISA and are expressed as the average serum titre (pg/ml) ± SE (five mice per group). *Statistically significant differences between treatment groups and vehicle (P<0·05) are shown.

Figure 5

Effect of IFN DNA treatment in chronic phase of MCMV infection. BALB/c mice were inoculated with 10⁴ PFU of MCMV i.p. at 2 weeks post-DNA vaccination. (a) MCMV titre in the salivary glands (SG) of mice was determined day 30 p.i. Virus titres are expressed as percentage control titre (average PFU/g tissue, five mice per time point ±SE) found for mice vaccinated with the vehicle. (b) The average number of inflammatory foci/heart section from groups of five mice per time-point ±SE are shown at day 30 and day 56. *Statistically significant differences between treatment groups and vehicle (P<0·05) are shown and are representative of two independent experiments.