Optimization of Naked DNA Delivery for Interferon Subtype Immunotherapy in Cytomegalovirus Infection

Abstract

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200mg IFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMV gB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases.

Fig. 1

The effect of DNA dosage in IFN transgene treatment of MCMV infection and myocarditis. Bupivacaine-treated BALB/c mice were inoculated with doses of either 50μg or 100μg of Blank, IFNA6 or IFNB DNA at 14 days prior to viral challenge with 1x104 pfu of MCMV i.p. Viral titres were determined by plaque assay in (A) spleen and (B) liver at day 3 p.i. and (C) salivary glands at day 7 p.i. Results are expressed as mean pfu/g ± SEM (n=5). (D) Myocarditis was evaluated at day 7 p.i. and is expressed as the number of foci per heart section ± SEM (n=5). *, Statistical significance (p≤0.05) compared with Blank treatment groups.

Fig. 2

Co-treatment with 400μg IFN subtype DNA does not affect acute-phase MCMV infection or myocarditis. Bupivacaine treated BALB/c mice were inoculated with Blank vector or combinations of IFNA6/Blank, IFNA9/Blank, IFNB/Blank, IFNA6/A9, IFNA6/B, and IFNA9/B DNA at a dosage of 200μg per transgene to a total of 400μg DNA, bilaterally via the TA muscle. At 14 days post-inoculation, mice were challenged with 1x104 pfu MCMV i.p. Viral titres were determined in (A) spleen and (B) liver at day 3 p.i. and (C) salivary glands at day 7 p.i. and are expressed as pfu/g tissue ± SEM (n=5). (D) Myocarditis was evaluated at day 7 p.i. and is expressed as the number of foci per heart section ± SEM (n=5). *, Statistical significance (p≤0.05) compared with Blank treatment groups. Bl. denotes Blank expression vector.

Fig. 3

IFN DNA inoculation without bupivacaine treatment is ineffective against MCMV infection and myocarditis. BALB/c mice were either not treated (No Bp) or treated (Bp) with bupivacaine 5 days prior to DNA inoculation with 200μg of either Blank or IFNA6 vector DNA and subsequently challenged with 1x104 pfu of MCMV i.p 14 days later. Viral titres were determined by plaque assay in (A) spleen and (B) liver at day 3 p.i., and (C) salivary glands at day 7 p.i. Results expressed as % Blank treatment group ± SEM (n=5). (D) Myocarditis was evaluated at day 7 p.i. and is expressed as the number of foci per heart section ± SEM (n=5). *, Statistical significance (p≤0.05) compared with Blank treatment groups.

Fig. 4

Combinational IFN DNA co-administration with gB improves vaccine efficacy in vivo. BALB/c mice were vaccinated with either Blank, gB, or gB/IFNA6/IFNB DNA (200μg/mouse) 14 days prior to i.p. inoculation with 1x104 pfu of MCMV. Viral titres were determined by plaque assay in (A) spleen at day 3 p.i. and (B) liver at day 7 p.i. Myocarditis was evaluated at (C) day 10 p.i. and (D) day 56 p.i. Myocarditis is expressed as the number of foci per heart section ± SEM (n=5). *, Statistical significance (p≤0.05) compared with gB DNA vaccinated group.