Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response

Abstract

We developed recombinant variants of oncolytic vaccinia virus LIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other in vitro and in vivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. In vitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. In vivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic T cells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice. The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer.

Keywords: IL-15/IL-15Rα complex; LIVP; breast adenocarcinoma; colon carcinoma; interleukin-15; interleukin-15 receptor alpha; oncolytic virus; vaccinia virus.

Graphical abstract

Figure 1

Construction of recombinant LIVP strains expressing IL-15 and IL-15Rα by homologous recombination (A) Scheme of genetic modification of TK gene. Microphotographs of BHK21 cells (B) and 4T1 cells (C) infected with LIVP-RFP recombinant strain (the same picture was obtained for LIVP-IL15-RFP and LIVP-IL15Ra-RFP; data are not shown). Red immunofluorescence merged with bright field microscopy. (D) Western blotting analysis in cell lysate/supernatant of infected cells by LIVP-IL15-RFP (left panel) normalized the protein concentration to β-actin (right panel). Description: Sprntnt Ctrl, Lysate Ctrl, a supernatant and a lysate of the control (non-infected) cells; IL-15 VV Inf. Suprntnt, IL-15 VV Inf. Lysate, a supernatant and a lysate of IL-15 VV or IL-15Ra infected cells, respectively.

Figure 2

Cytotoxicity and replication characteristics of the developed strains (A) Viability of BHK-21, 4T1, and CT26 cells through 120 h after infection (h.p.i) by MOI 1 of LIVP-RFP, LIVP-IL15-RFP, LIVP-IL15Rα-RFP, and combination of LIVP-IL15-RFP with LIVP-IL15Rα-RFP (LIVP-IL15-RFP + LIVP-IL15Rα-RFP). (B) Kinetics of recombinant viral strains in different cell lines (BHK-21,4T1, CT26). ANOVA was performed for statistical analysis, ∗p < 0.05 and ∗∗p < 0.01 indicate significance.

Figure 3

Virotherapy by the recombinant strains in vivo (A) Subcutaneous CT26 colon carcinoma and 4T1 breast tumor progression in control treated with PBS and experimental groups treated with LIVP-RFP, LIVP-IL15-RFP, LIVP-IL15Rα-RFP, and a combination of LIVP-IL15-RFP+ LIVP-IL15Rα. ✝ represents the sacrifice of the animal. (B) The overall survival rate of BALB/c mice bearing CT26 colon carcinoma and 4T1 breast tumors treated by the recombinant viruses and the control group based on the Kaplan-Meier method. For statistical analysis, ANOVA and survival analysis were performed; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 indicate significance.

Figure 4

Recombinant strains alter immune system response Analysis of tumor-infiltrated lymphocytes 7 days after complete treatment of 4T1 breast carcinoma-bearing mice (n = 3). The levels of F4/80⁺, CD45⁺, CD4 T⁺, and CD8⁺ T cells were measured. ANOVA was performed for statistical analysis; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 indicate significance.

Figure 5

Cytokine analysis of the serum samples from the 4T1 tumor-bearing mice treated by LIVP-RFP, LIVP-IL15-RFP, LIVP-IL15Rα-RFP, and combination of LIVP-IL15-RFP+ LIVP-IL15Rα in comparison with the untreated mice (control group) Statistical analysis was performed using a t test, with ∗p < 0.05 indicating significance. Designation: −1, 1 day after the last intratumoral VV injection; −7, 7 days after the last intratumoral VV injection.

Figure 6

Cytokine analysis of the tumor samples from the 4T1 tumor-bearing mice treated by LIVP-RFP, LIVP-IL15-RFP, LIVP-IL15Rα-RFP, and combination of LIVP-IL15-RFP+ LIVP-IL15Rα in comparison with the untreated mice (control group) Statistical analysis was performed using a t test, with ∗p < 0.05 indicating significance. Designation is the same as in Figure 5.

Figure 7

ELISA of IL-15/IL-15Rα complex produced by viruses in tumor-infiltrated fluids of 4T1 breast cancer syngeneic mice in vivo Designation: (−1), samples collected 24 h after complete treatment; (−7), samples collected 7 days after complete treatment. Statistical analysis was performed using a t test, with ∗p < 0.05 indicating significance.

Figure 8

Histological analysis of tumor and organs of the treated mice Histology analysis of the liver, spleen, and the tumor treated by recombinant viruses expressing IL-15 or IL-15Rα and their combination compared with the parental virus (LIVP-RFP) and the control group.