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We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

The interaction between synthetic peptides and A2.1 class I MHC molecules has been investigated using an inhibition of Ag presentation assay and unbiased peptide sets derived of either viral or eucaryotic origin. For the various sets, strong binding (defined as significant inhibition at the 30 micrograms/ml level) was detected in 7 to 46% of the peptides tested, with an overall frequency of 26%. A set of self-peptides derived from human beta 2 microglobulin was also included in the study. In this case, strong binding was detected in 3 of 15 peptides (20%), thus formally demonstrating a lack of self-/non-self-discrimination at the level of class I molecules. When the whole A2.1-binding database of 105 peptides thus generated was examined by sequence analysis, a significant correlation was found with a recently proposed A2.1-binding motif, whereas no particular positive or negative association was detected between the capacity to bind A2.1 and three different class II alleles (DR1, DR5, and DR7). Finally, using this approach, several peptides capable of binding both A2.1 and multiple DR alleles have been identified, suggesting possible candidates for development of peptide vaccines eliciting both class I and class II restricted responses.