Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis, and antiviral therapy

Abstract

In 2001, Jackson et al. reported that murine IL-4 expression by a recombinant ectromelia virus caused enhanced morbidity and lethality in resistant C57BL/6 mice as well as overcame protective immune memory responses. To achieve a more thorough understanding of this phenomenon and to assess a variety of countermeasures, we constructed a series of ECTV recombinants encoding murine IL-4 under the control of promoters of different strengths and temporal regulation. We showed that the ECTV-IL-4 recombinant expressing the highest level of IL-4 was uniformly lethal for C57BL/6 mice even when previously immunized. The lethality of the ECTV-IL-4 recombinants resulted from virus-expressed IL-4 signaling through the IL-4 receptor but was not due to IL-4 toxicity. A number of treatment approaches were evaluated against the most virulent IL-4 encoding virus. The most efficacious therapy was a combination of two antiviral drugs (CMX001(®) and ST-246(®)) that have different mechanisms of action.

Figure 1

IL-4 expression by ECTV recombinant viruses as a function of promoter strength. (A) A schematic of the pMBEV1.1 plasmid indicating the location of the fragmented CHO gene insertion site flanked by EV014 and EV015 genes. Numbers represent the position in bp of the respective sites. Hind III and Bgl I insertion sites are also indicated (Chen, Danila, Feng, Buller, Wang, Han, Lefkowitz, & Upton, 2003). (B and C) 3 × 10⁴ CV-1 cells were infected with each ECTV recombinant at 10 PFU/cell. The supernatants were collected at 24 hr p.i. Comparable volumes of supernatants were quantified for IL-4 by Western blot (B) and ELISA and bioassay with CT.4R cells (C). BV-IL-4 indicates baculovirus produced IL-4. (D) Groups of 6 C57BL/6 mice were infected by the FP route with 3,000 PFU of the indicated viruses. At 6 days p.i., the mice were bled from the orbital sinus and sera were assayed for IL-4 by ELISA.

Figure 2

Histopathology of liver from ECTV-7.5E/L-IL-4 infected C57BL/6 mice at 7 days p.i. ECTV-7.5E/L-IL-4 infection. Panel A. Confluent area of necrosis. Mag. 400×; Panel B. A hepatic vessel filled with macrophages. The arrows delineate the boundary of the vessel. Mag. 200×; ECTV-WT infection. Panel C. Discrete focus of necrosis (irregular line) containing inflammatory/immune cells and surrounded by intact hepatocytes (arrows), Mag. 400×; Panel D. The irregular line encompasses a focus of lymphocytes. Arrows show macrophages containing phagocytized erythrocytes. Mag 600×.

Figure 3

Transmissibility of ECTV-11KL-IL-4 in C57BL/6 mice. Four C57BL/6 mice were infected with 5 × 10³ PFU of ECTV-WT or ECTV-11KL-IL-4 in the left rear FP. The infected mice were transferred every 2 days into a new cage containing 6 naïve, disease susceptible A/NCR mice. Infection was monitored by death or seroconversion.

Figure 4

Efficacy of ST-246^® and CMX001^® combination treatment against ECTV-11KL-IL-4 in A/NCR mice. Female A/NCR (n=5) mice were inoculated intranasally with 200 PFU of ECTV-11KL-IL-4. Commencing at 6 hrs p.i., mice were treated by gavage daily for 14 days with placebo, ST-246^® (100 mg/kg) and/or CMX001^® (4 mg/kg). Mice were weighed individually 3 times a week. For clarity, error bars have not been shown; however, they do indicate similar kinetics of weight change.