Evaluation of a Recombinant Newcastle Disease Virus Expressing Human IL12 against Human Breast Cancer

Abstract

The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 2⁹. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.

Figure 1

Cytotoxic effects of rAF-IL12 and the parental strain AF2240 against: (a) DF-1; (b) MDA-MB231; (c) MCF-7 and; (d) MCF-10A at 24, 48 and 72 h post-inoculation. The values are expressed as the mean values ± standard deviation of triplicate experiments. The mean values as indicated using different superscript letters were significantly different at p < 0.05 when compared between parental AF2240- and rAF-IL12 (Passages 1, 5 & 10)-treated groups.

Figure 2

The expression of human IL-12 in multiple cell lines infected with NDV AF2240 and rAF-IL12 (Passages 1, 5 & 10) determined by ELISA assay at 24, 48 and 72 h. (a) DF-1 cells treated with NDV AF2240 and rAF-IL12 (Passages 1, 5 & 10), (b) MCF-7 cells treated with NDV AF2240 and rAF-IL12 (Passages 1, 5 & 10), (c) MDA-MB231 cells treated with NDV AF2240 and rAF-IL12 (Passages 1, 5 & 10), (d) MCF10A cells treated with NDV AF2240 and rAF-IL12 (Passages 1, 5 & 10). The values are expressed as the mean values ± standard deviation of triplicate experiments. The mean values as indicated using different superscript letters were significantly different at p < 0.05 when compared between parental AF2240- and rAF-IL12 (Passages 1, 5 & 10)-treated groups.

Figure 3

The tumors harvested from untreated, NDV AF2240 and rAF-IL12 passage 10-treated mice (2⁷ HAU/kg) mice. (a) Representative images, (b) average weight and (c) volume of the tumors harvested from untreated, AF2240 and rAF-IL12 passage 10-treated mice. Each value represents the mean values ± S.E.M. There were 9 mice per group. The values are expressed as the mean values ± standard deviation of triplicate experiments. The mean values as indicated using different superscript letters were significantly different at p < 0.05 when compared between parental AF2240- and rAF-IL12 (Passages 1, 5 & 10)-treated groups.

Figure 4

Histological staining (haematoxylin and eosin) of tumors from untreated, NDV AF2240- and rAF-IL12 passage 10-treated mice groups. Condensed round cells stained dark purple represent cells undergoing mitoses. Magnification: X40.

Figure 5

ELISA-based detection of IL-12 levels in the serum of the control (untreated), NDV AF2240- and rAF-IL12 passage 10-treated mice groups. Each value represents the mean values ± S.E.M for n = 9 mice per group. The values are expressed as the mean values ± standard deviation of triplicate experiments. The mean values as indicated using different superscript letters were significantly different at p < 0.05 when compared between parental AF2240- and rAF-IL12 (Passages 1, 5 & 10)-treated groups.

Figure 6

Detection of viral copy number in organs and tumor of treated mice using Taqman quantitative real time PCR. Images represent (a) normal (no tumor) and (b) rAF-IL12 treated mice after 3 days using Taqman quantitative real time PCR, c) detection of viral copy number in organs and tumor in NDV rAF-IL12 passage 10- and AF2240-treated mice after 28 days using Taqman quantitative real time PCR. Each value represents the mean values ± S.E.M for n = 9 mice per group. RBC = Red blood cells. The values are expressed as the mean values ± standard deviation of triplicate experiments. The mean values as indicated using different superscript letters were significantly different at p < 0.05 when compared between parental AF2240- and rAF-IL12 (Passages 1, 5 & 10)-treated groups.