To determine whether, and if so how, adjuvants effected immunogenicity of these peptides, HLA-A*1101 transgenic mice were immunized with pools of peptides that included all of the three peptides (KS9, AM9, RS9) alone or with varying adjuvants. HLA-A*1101 transgenic mice were immunized with: (1) CD8 + epitope peptide pool, (2) peptide pool plus PADRE, (3) peptide pool Figure 1 Schematic representation of the synthetic lipopeptide immunogens used in this study. The C-terminal end of a promiscuous CD4 + T cell peptide epitope (PADRE) was joined in sequence with the N-terminal end of one of three different T. gondii CD8 T cell epitopes: SAG1 224-232 (A), GRA6 164-172 (B); GRA7 134-142 (C) or three epitopes linked together (D) with a three alanine linker. The N-terminal end of each resulting CD4-CD8 peptide was extended by a lysine covalently linked to one molecule of palmitic acid. This results in a four lipopeptides construct. The abbreviation Lp is used throughout the manuscript whenever the lipopeptide (Lp) has been studied. When there is a mixture of components or undivided components they are named individually. Sequence of PADRE is AKFVAAWTLKAAA. Structure of Pam 2 Cys is PAM 2 KSS. a = abbreviation for name of protein from which peptide is derived. Figure 1 . The mice were immunized twice at intervals of three weeks. Ten to fourteen days after the last immunization, spleen cells were separated from immunized mice and stimulated by appropriate peptides in an ex vivo IFN-γ ELISpot assay. Data presented are averages of three independent replicate experiments. *, P < 0.05; **, P < 0.01. (Figure 3b ) were used as immunogens. For the lipopeptide immunizations, the mice were vaccinated twice at intervals of three weeks. For the immunizations containing individual components including the same peptides, the mice were vaccinated three times at intervals of two weeks. Ten to fourteen days after the last immunization, spleen cells were separated from immunized mice and stimulated by the appropriate peptide in an ex vivo IFN-γ ELISpot assay. Data presented are a representative example from three independent experiments. *, P < 0.05; **, P < 0.01. plus PADRE emulsified with GLA-SE, and (4) peptide pool plus PADRE and Pam 2 Cys emulsified with GLA-SE. Mice were inoculated three times at intervals of two weeks. Eleven to fourteen days post immunization, spleen cells were isolated and exposed to each individual peptide. A peptide was considered immunogenic if it induced IFN-γ spot formation that was significantly higher in the immunization group compared with the group inoculated with PBS. After the immunizations, only KS9 and AM9 were found to be immunogenic in HLA-A*1101 transgenic mice, and only when the iniversal CD4 + helper T cell peptide epitope PADRE was included. Robust responses were observed when GLA-SE was added in the vaccine. Greater responses were elicited when Pam 2 Cys was used as an adjuvant for some peptides but did not enhance responses to all of them, and in fact reduced the effect of some peptides. Figure 4 shows the representative data of IFN-γ spot formation from the four immunization groups which were stimulated by individual peptides.