Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4+ T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-γ production. Furthermore, a potent IFN-γ-inducing, Db-restricted CD8+ epitope was identified using MHC class I mutant B6.C-H-2bm13 mice and intracellular IFN-γ and whole blood CD8+ T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this Db-restricted PstS-3 epitope. IFN-γ ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4+ and CD8+ T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2b, p, and f haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2d, k, r, s, and q haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2bm12 mice could be overcome by DNA vaccination. IFN-γ-producing CD8+ T cells could also be detected against the Db-restricted epitope in H-2p haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4+ and particularly CD8+ T cell responses against mycobacterial Ags.

Human papillomavirus type 16 is commonly implicated in cervical cancers. The viral genome encodes potential targets like the oncoprotein E7, expressed in transformed cells but thought to represent a poorly immunogenic antigen. We describe in this work a DNA-based vaccination protocol aimed at inducing an efficient anti-E7 immune response in vivo. Plasmids allowing the expression of the E7 protein in distinct cellular compartments were generated and assayed in an in vivo model of tumor growth. Our data demonstrate that mice vaccinated with a plasmid encoding for an E7 protein fused to a domain of the MHC class II-associated invariant chain (IiE7) were protected against tumor challenge. Mice immunized against an ubiquitinated form of E7 (Ub(Ala)E7) failed to control tumor growth. Protection induced by IiE7 was correlated with the development of CD8 + CTL and required the presence of CD4 + cells. In vitro studies confirmed that the IiE7 fusion protein was expressed at high levels in the endosomal compartment of transfected cells, while the natural and the ubiquitin-modified form of E7 were mainly nuclear. The present study suggests that an efficient anti-tumor response can be induced in vivo by DNA constructs encoding for E7 protein forms localizing at the endosomal compartment.