Highly attenuated modified vaccinia virus Ankara (MVA) as an effective recombinant vector: a murine tumor model

Abstract

Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus (VV) that is unable to replicate in most mammalian cells, was evaluated as an expression vector for a model tumor associated antigen (TAA) and as a potential anti-cancer vaccine. We employed an experimental murine model in which an adenocarcinoma tumor line, CT26.CL25, was stably transfected with a model TAA, beta-galactosidase (beta-gal). Mice injected intramuscularly with a recombinant MVA (rMVA) expressing beta-gal (MVA-LZ), were protected from a lethal intravenous (i.v.) challenge with CT26.CL25. In addition, splenocytes from mice primed with MVA-LZ were therapeutically effective upon adoptive transfer to mice bearing pulmonary metastases of the CT26.CL25 tumor established 3 days earlier. Most importantly, i.v. inoculation with MVA-LZ resulted in significantly prolonged survival of mice bearing three day old pulmonary metastases. This prolonged survival compared favorably to mice treated with a replication competent recombinant VV expressing beta-gal. These findings indicate that rMVA is an efficacious alternative to the more commonly used replication competent VV for the development of new recombinant anti-cancer vaccines.

Figure 1

Intramuscular inoculation of MVA-LZ induces protective immunity to tumor challenge with CT26.CL25. BALB/c mice were inoculated with 10⁸ or 10⁶ (designated *) p.f.u. per mouse of MVA-WT or MVA-LZ. Twenty-one days later, mice received an i.v. challenge with 5×10⁵ tumors cells. Twelve days after tumor inoculation, pulmonary metastases were enumerated in a blinded fashion. A maximum of 500 metastases per lung was recorded. Shown are average numbers of metastases obtained using five mice per group. In a repeat experiment i.m. inoculation with MVA-LZ and VJS6 showed similar results

Figure 2

Successful adoptive immunotherapy of pulmonary metastases established for 3 days using MVA-LZ primed splenocytes. BALB/c mice were inoculated i.v. with 5×10⁵ CT26.WT or CT26.CL25 (β-gal transfected) tumor cells. Three days later, tumorbearing mice were treated with the adoptive transfer of 2×10⁷ splenocytes obtained from mice primed i.m. with 10⁷ p.f.u. of either MVA or MVA-LZ. 10⁷ p.f.u. of VJS6, delivered i.v., was used as a positive control. Twenty-one days after priming, adoptively transferred splenocytes were cultured in the presence of 10 μg ml⁻¹ of the MHC class I-restricted peptide with the sequence TPHPARIGL, corresponding to residues 876–884 of the full-length β-gal protein, for 6 days before adoptive transfer into tumor bearing mice. Mice were killed 12 days after adoptive transfer of splenocytes, and lung metastases were enumerated in a coded blinded fashion. The experiment was repeated and similar results were obtained

Figure 3

Cytokine profile of splenocytes cultured with MHC class I restricted peptides. Splenocytes were prepared from mice that had previously been inoculated i.m. with 10⁷ p.f.u. of the MVA or MVA-LZ viruses. The same dose of VJS6 given i.v. was used as a positive control. Three weeks later, splenocytes were removed and cultured together with 10 μg ml⁻¹ of the class I-restricted β-gal peptide, TPHPARIGL. Two days later, supernatants of the samples were collected and analyzed by ELISA for IL-2, IFN-γ, IL-10, GM-CSF, IL-4, and TNF-α. Results are shown in picograms per ml of supernatant. Note that each flask contained 30 ml of supernatant

Figure 4

Active immunotherapy of pulmonary metastases established for 3 days. BALB/c mice were inoculated with 5×10⁵ CT26.CL25 tumor cells. Three, or 3 and 10 days after tumor inoculation, mice were inoculated with 5×10⁵ p.f.u. i.v. of the designated virus and checked daily for survival. Results shown are from a single experiment, although data is shown in three panels for clarity. Shown are mice receiving two immunizations, 3 and 10 days after tumor inoculation in (A) and (B). Mice receiving a single immunization 3 days after tumor inoculation are shown in C). An independent repeat experiment showed similar results

Figure 5

Down-regulation of β-gal expression of CT26.CL25 pulmonary metastases. In a second survival experiment (data not shown) mice were inoculated with 2×10⁷ tumor cells and treated 3 and 10 days later with 2×10⁷ p.f.u. i.v. with the relevant virus. When mice died their lungs were removed and stained for β-gal expression as described in Materials and Methods. (A) Mouse inoculated with CT26.WT and treated with MVA-LZ, (B) mouse inoculated with CT26.CL25 and treated with FPV, (C) mouse inoculated with CT26.CL25 and treated with MVA-LZ