Oncolytic Activity of a Chimeric Influenza A Virus Carrying a Human CTLA4 Antibody in Hepatocellular Carcinoma

Abstract

Oncolytic virotherapy belongs to a kind of active immunotherapy, which could trigger a potent antitumor immune response, showing great potential in clinical application. OVs could induce immune responses through the dual mechanisms of selective tumor killing without destroying normal tissues and induction of systemic antitumor immunity. In this study, we successfully rescued a chimeric oncolytic influenza virus carrying a human CTLA4 antibody in the background of the A/PR/8/34 (PR8) virus. The chimeric virus, called rFlu-huCTLA4, contained the heavy and light chains of the human CTLA4 antibody in the PB1 and PA segments of the PR8 virus, respectively. The first-generation hemagglutination (HA) titers of the rFlu-huCTLA4 virus ranged from 2⁷ to 2⁸, which could be passaged stably in specific pathogen-free (SPF) chicken embryos from P1 to P5. The morphology and size distribution of the chimeric virus were consistent with those of the wt influenza virus. The rFlu-huCTLA4 virus could effectively replicate in various cells in time- and dose-dependent manners. ELISA assay revealed that the secreted huCTLA4 antibody levels in chicken embryos increased gradually over time. Furthermore, MTS and crystal violet analysis showed that the selective cytotoxicity of the virus was higher in hepatocellular carcinoma cells (HepG2 and Huh7) than in normal liver cells (MIHA). In vivo experiments displayed that intratumoral injection with rFlu-huCTLA4 reduced tumor growth and increased the survival of mice compared with the PR8 group. More importantly, in the rFlu-huCTLA4 group, we found that CD4+ and CD8 +T cells were significantly increased in tumor-bearing BALB/c mice. Taken together, these findings demonstrated that the chimeric oncolytic virus rFlu-huCTLA4 could selectively destroy hepatocellular carcinoma cells in vitro and in vivo and may provide a promising clinical strategy for targeted immunotherapy of HCC with the oncolytic flu virus.

Keywords: CTLA4 antibody; HCC; generation hemagglutination; oncolytic virus; rFlu-huCTLA4.

Figure 1

Construction and identification of the recombinant plasmid. (A) Schematic diagram of the experimental model: plasmid construction strategy of recombinant oncolytic influenza viruses pFlu-huCTLA4-PB1 and pFlu-huCTLA4-PA. The recombinant plasmid was co-transfected with the remaining 6 skeleton plasmids into MDCK/COSI cocultured cells, and the recombinant oncolytic virus rFlu-huCTLA4 was saved and the huCTLA4 antibody was produced with the replication of the recombinant virus. (B) Identification of recombinant plasmids pFlu-huCTLA4-PB1 and pFlu-huCTLA4-PA by agarose gel electrophoresis. (C) Identification of 8 transfected plasmids by agarose gel electrophoresis.

Figure 2

Identification of recombinant oncolytic virus rFlu-huCTLA4. (A) The red arrow indicates the virus with regular shape and size, consistent with the morphology and structure of the influenza virus. (B) The size of the rFlu-huCTLA4 virus is mainly between 80 and 120 nm. (C) rFlu-huCTLA4 was continuously amplified in eggs for 5 generations. The titer of hemagglutination was 2⁸~2⁹ and the titer of the virus was 10^8~9 TCID₅₀/ml after five generations on a chicken embryo. It could be passaged stably in the chicken embryo. (D) Growth curve of the recombinant virus. The HA titer of the recombinant virus increased to 2⁷ from 12 to 72 h and decreased to 2⁶ at 96 h. (E) Identification of the rFlu-huCTLA4 gene fragment by RT-PCR. (F) The antibody content of huCTLA4 detected by ELISA increased gradually with time (***P<0.001, note: the dotted line represents the lowest detection line).

Figure 3

Selective cytotoxicity of rFlu-huCTLA4 on different hepatocellular carcinoma cell lines in vitro. (A) MTS showed that rFlu-huCTLA4 had no effect on MIHA but had a selective killing effect on HepG2 and Huh7. The activity of HepG2 cells at 1, 2, and 3 MOI was significantly lower than that at 0.1 MOI 72 h after infection; however, Huh7 cells only 2 and 3 MOI decreased significantly compared with 0.1 MOI cells. (B) The rFlu-huCTLA4 recombinant oncolytic virus detected by crystal violet can significantly inhibit the activity of hepatoma cells but has no effect on normal hepatocytes, which is consistent with the results of MTS. (C) Flow cytometry showed that recombinant oncolytic virus rFlu-huCTLA4 could induce apoptosis of HepG2 cells in a dose-dependent manner. At MOI of 1, the effect of apoptosis of HepG2 cells was more significant than that of MIHA. (**P<0.01,***P<0.001).

Figure 4

rFlu-huCTLA4 caused potential cancer immunotherapy in the H22 cell tumor-bearing model. Flow cytometric analysis of infiltrated CD4+CD69+ T cells (A, B) and CD8+ CD69+ T cells (C, D) in the spleen of the tumor-bearing BALB/c mice with rFlu-huCTLA4 treatment. The percentage of CD4+ CD69+ T cells and CD8+ CD69+ T cells in the spleen of tumor-carrying BALB/c mice treated with rFlu-huCTLA4 was higher than that of the PBS group and PR8 group. (***P<0.001).

Figure 5

The tumor was isolated and the pathological changes observed. (A, B) After 7 days of inoculation, the tumor tissue of mice was isolated, and the tumor tissue of the rFlu-huCTLA4 group was significantly smaller than that of the PBS and PR8 group. (C) After being inoculated with rFlu-huCTLA4, obvious necrosis and dissolution were observed in tumor pathological sections of mice, and no abnormality was observed in liver and lung tissues. Also, there was no significant change in PBS and PR8 groups. (**p<0.01).

Figure 6

Oncolytic effect of recombinant virus rFlu-huCTLA4 in vivo. (A) PBS, PR8, and rFlu-huCTLA4 were injected respectively. The tumor volume was measured every 2 days, and the difference was significant (p < 0.01). rFlu-huCTLA4 could significantly inhibit the growth of the tumor. (B) 40 days later, the PDX mice were killed, tumors were isolated, and tumor volume was measured. rFlu-huCTLA4 significantly inhibited tumor volume growth. (C) Weighing the tumor, rFlu-huCTLA4 significantly inhibited tumor weight growth. (D) The heart, liver, spleen, lung, kidney, brain, and tumor tissue were isolated and viral load measured. (**P<0.01,***P<0.001).