Currently no vaccine is available against hepatitis C virus (HCV) and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin (PRF). In this study we looked to determine the mechanism of cell death induced by PRF in antigen-expressing cells, as well as examine its adjuvant potential in a large pre-clinical model, the pig.
We used a bicistronic DNA vaccine that encoded the HCV NS3 protein under the control of the CMV promoter and PRF controlled by the weaker SV40 promoter, as our previous work has shown that the effect of PRF was dependent on the level of antigen expression and the timing of cell death after expression of the immunogen. Here, we show that PRF is co-localised with NS3 in DNA-transfected cells and that the expression of PRF resulted in non-apoptotic cell death, most likely by necrosis, mediated through the RIP1 kinase pathway. Our data and other studies support the notion that over-expression of intracellular NS3 triggers apoptotic cell death, but over-expression of PRF within the same cell alters the NS3/PRF balance towards necrosis, resulting in enhanced immunogenicity of NS3
Vaccination studies showed that, compared with the canonical DNA vaccine and a bicistronic vaccine encoding NS3 and the proapoptotic gene NSP4 from rotavirus, the PRF-containing vaccine elicited enhanced cell mediated immune responses against the NS3 protein in vaccinated mice and pigs. Co-expression of PRF significantly enhanced T cell-mediated immune responses, as determined by increased responses to all NS3 peptides in ELISpot and increased numbers of effector memory CD8 T cells that secreted IFNγ, IL-2 and TNF-a in response to stimulation by NS3 peptides. The addition of PRF also broadened the immune response by enhancing T-cell responses to non-dominant epitopes. Conversely, pro-apoptotic NSP4 failed to enhance immune responses, but instead reduced the strength of the immune response to some NS3 epitopes. A mouse challenge model suggested that the PRF immunity and clearance of the NS3 antigen from hepatocytes, was CD8+ T cell dependent.
Importantly, the adjuvant activity of PRF was also evident in the enhanced NS3 responses observed in the pre-clinical, large animal model. As shown in the pig vaccination studies, our strategy increased the magnitude of the initial effector cell response, and as this dictates the magnitude of the population of central memory T cells, measures employed to optimize this response will increase overall vaccine efficacy. These studies and other preclinical studies ongoing in our laboratory suggest that a DNA vaccine encoding a suitable immunogen and PRF is a candidate for human clinical trials.