Myxoma virus expressing human interleukin-12 does not induce myxomatosis in European rabbits

Abstract

Myxoma virus (MV) is a candidate for oncolytic virotherapy due to its ability to selectively infect and kill tumor cells, yet MV is a species-specific pathogen that causes disease only in European rabbits. To assess the ability of MV to deliver cytokines to tumors, we created an MV (vMyxIL-12) that expresses human interleukin-12 (IL-12). vMyxIL-12 replicates similarly to wild-type MV, and virus-infected cells secrete bioactive IL-12. Yet, vMyxIL-12 does not cause myxomatosis, despite expressing the complete repertoire of MV proteins. Thus, vMyxIL-12 exhibits promise as an oncolytic candidate and is safe in all known vertebrate hosts, including lagomorphs.

FIG. 1.

Construction of pBS 135-136 hIL-12 EGFP. The cloning plasmid pBS 135-136 EGFP contains a unique PstI site between M135R and a poxviral synthetic early/late promoter into which a PacI linker was inserted. PacI was also added to the primers used to amplify hIL-12. The IL-12 cassette with both subunits was inserted into pCR2.1 TOPO. In addition, the minimal sequence for the poxviral P11 promoter was inserted before the p40 subunit of hIL-12. Both vectors were digested with PacI, gel purified, and ligated to form the pBS 135-136 hIL-12 EGFP plasmid that was used to create vMyxIL-12 in MV-infected BGMK cells by homologous recombination. CMV, cytomegalovirus; IRES, internal ribosomal entry site.

FIG. 2.

Viral growth, replication, and secreted hIL-12 production. Single-step growth analysis of vMyxIL-12 (dashed line and circles) and vMyxgfp (solid line and triangles) was evaluated on BGMK (A), 8484 (B), and RK-13 (C) cells. Cells were infected with virus at an MOI of 3. Cells were harvested at the times indicated, and the titers of infectious virus present at each time point were determined by subsequent infection on BGMK cells. All growth analyses were performed in triplicate. The production of secreted hIL-12 from infected BGMK cells was measured by enzyme-linked immunosorbent assay (D). Cells were infected at the indicated MOI, and the cellular supernatant was collected 48 hpi. RK-13 cells (E) were infected at an MOI of 3, and cell supernatants were collected at the indicated time points.

FIG. 3.

vMyxIL-12 pathogenesis in rabbits. Three rabbits were infected with vMyxIL-12, two rabbits with vMyx, and two rabbits with vMyxgfp. The rabbits were each infected with 2,000 FFU, with 1,000 FFU injected into each hind flank. Rabbits were monitored daily for the clinical symptoms associated with myxomatosis. Scores increase as the disease progresses. Animals are normally sacrificed within 24 to 48 h following a score of 15. Certain symptoms, such as severe orthopnea, mouth breathing, cyanosis, and lack of food and/or water intake for >24 h, required that the rabbits be euthanized for ethical reasons.