Introduction 

Most healthy cells express Major Histocompatibility Complex (MHC) class I molecules, which play a vital role in the activation of cytotoxic CD8+ T cells by presenting tumor-associated antigens to the T cell receptors. However, MHC I gene expression is downregulated in 60% of ovarian cancers. The goal of this project is therefore to rescue lost MHC I by forcing expression of NOD-like receptor family CARD domain containing 5 (NLRC5) in ovarian cancer cells. NLRC5 is a co-transcriptional regulator of MHC I and other genes involved in the antigen processing machinery pathway. We hypothesize that, by delivering NLRC5 using a cancer-targeting but not cytotoxic Vaccinia virus, we will recover lost MHC I expression in ovarian cancer cells and tumors. 

Methods 

Three modified strains of Vaccinia virus [modified Vaccinia virus Ankara (MVA), Tian Tan (TT), and the novel Vaccinia backbone SKV)] were tested on human ovarian cancer cell lines to determine the least cytotoxic. Flow cytometry was used to assess the effects of each virus on MHC I expression. In parallel, the coding sequence for NLRC5 was cloned into a J2R plasmid, replacing the viral thymidine kinase with the transgene. 

Results 

TT and SKV have higher infection rates than MVA in OVCAR8 cells, and are more cytotoxic. Furthermore, 95.6% of OVCAR8 cells infected with TT expressed MHC-I compared to 4.73 and 17.6% for MVA and SKV, respectively. Cells treated with IFN-γ for 24h were used as positive control. Since MVA was the least cytotoxic and did not alter MHC-I expression significantly, it was used with the J2R-NLRC5 plasmid to produce the recombinant virus. 

Conclusion 

MVA is the least cytotoxic Vaccinia strain of the three tested viruses on human ovarian cancer cell lines. We anticipate that MVA expressing NLRC5 will effectively transduce ovarian cancer cells, thereby rescuing MHC I expression and restoring the ability for antigen presentation and CD8+ T cell activation.