Altered antigen-presentation and antiviral T cell responses by co-expression of a viral antigen and cell death modulating proteins.

LORENA ITATÍ IBAÑEZ; MICHAEL SCHOTSAERT; PETER VANDENABEELE; XAVIER SAELENS

Institute Pasteur, Paris France

Congreso; ECDO Euroconference on Apoptosis; 2009

Gene vaccination is a simple method to induce adaptive immune responses against microbial or tumor antigens. However, clinical success of DNA vaccination is still limited mainly as a result of poor immune responses. In this study we have used the conserved nucleoprotein (NP) of influenza A virus as a model T-cell antigen to analysis the effect of co-expression of two cell death modulating genes: FADD and RIP1. This rational was based on the observation that antigenic material associated with or released from dying cells has a strong immunostimulatory capacity and hence might improve the magnitude and quality of the NP-specific immune response. Dual promoter plasmids were constructed that allow expression of NP with or without FADD, FADD-DED, FADD-DD, RIP-1, RIP1K45R, RIP1deltaDD or RIP1K45RdeltaDD. Functional analysis of the constructs was assessed in vitro, using transient transfection of HEK-293 cells to document expression, and NFkB reporter gene induction. LPS-free plasmid DNA was prepared and used to immunize Balb/c mice via the intramuscular route. The DNA vaccine was administered in three doses of 100 ìg per mouse, following different schemes of vaccination. Splenic NP-specific CD8+ and CD4+ were quantified by ELISPOT and intracellular cytokine staining, focusing on IFN-gamma production in the presence of a dominant H2-Kd-restricted NP-CTL epitope peptide. The protective efficacy of the different gene vaccine constructs was compared by challenging the mice with a 4 LD50 PR8 virus. Following challenge, morbidity and mortality were monitored on a daily basis for two weeks. ELISPOT experiments showed that splenocytes from mice injected with constructs that have a death domain, i.e. FADD-DD, RIP-1 and RIP1K45R produced less IFNg than splenocytes from mice injected with plasmids expressing only NP. Conversely, gene vaccination with constructs lacking a death domain produced more IFNg. Mice vaccinated with plasmids expressing RIP1K45 and RIP1K45RdeltaD together with NP were protected as well as mice receiving plasmid expressing only NP, after challenge with PR8 virus. We are currently focusing on the extent of memory T cell responses that are induced by the NP-RIP1 vaccine constructs.