Both CD4+ and CD8+ T cells respond to antigens fused to anthrax lethal toxin

The lethal toxin produced by Bacillus anthracis is a bipartite toxin in which the first protein, protective antigen (PA), transports the second protein, lethal factor, across the host cell membrane. We have previously shown that CD8(+) T-cell epitopes fused to a nontoxic derivative of lethal factor (LFn) are delivered into the host cell cytosol in a PA-dependent manner. Delivery of these antigens targets them to the intracellular major histocompatibility complex (MHC) class I processing and presentation pathway and leads to the stimulation of antigen-specific CD8(+) T cells in vivo. In this report, we describe the generation and characterization of LFn fusion proteins that include not only a CD8(+) T-cell epitope but also a CD4(+) T-cell epitope. We first show that these fusion proteins induce antigen-specific CD4(+) T-cell responses following incubation with dendritic cells in vitro or injection into mice. Stimulation of CD4(+) T cells by LFn fusion proteins does not require PA but is enhanced by PA in vitro. We also show that a single LFn fusion protein and PA can deliver antigen to both the MHC class II and the MHC class I pathways, resulting in the simultaneous induction of antigen-specific CD4(+) T cells and antigen-specific CD8(+) T cells in the same mouse. These results suggest that this toxin delivery system is capable of stimulating protective immune responses where effective immunization requires stimulation of both classes of T cells.