DNA vaccines have demonstrated antitumor efficacy in multiple preclinical 
models, but low immunogenicity has been observed in several human clinical 
trials. This has led to many approaches seeking to improve the immunogenicity of 
DNA vaccines. We previously reported that a DNA vaccine encoding the 
cancer-testis antigen SSX2, modified to encode altered epitopes with increased 
MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional 
CD8(+) T cells in non-tumor-bearing mice. We sought to test whether this 
optimized vaccine resulted in increased antitumor activity in mice bearing an 
HLA-A2-expressing tumor engineered to express SSX2. We found that immunization 
of tumor-bearing mice with the optimized vaccine elicited a surprisingly 
inferior antitumor effect relative to the native vaccine. Both native and 
optimized vaccines led to increased expression of PD-L1 on tumor cells, but 
antigen-specific CD8(+) T cells from mice immunized with the optimized construct 
expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 
expression on tumor cells in vitro. Antitumor activity of the optimized vaccine 
could be increased when combined with antibodies blocking PD-1 or PD-L1, or by 
targeting a tumor line not expressing PD-L1. These findings suggest that 
vaccines aimed at eliciting effector CD8(+) T cells, and DNA vaccines in 
particular, might best be combined with PD-1 pathway inhibitors in clinical 
trials. This strategy may be particularly advantageous for vaccines targeting 
prostate cancer, a disease for which antitumor vaccines have demonstrated 
clinical benefit and yet PD-1 pathway inhibitors alone have shown little 
efficacy to date.