We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75–84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes). 1. Introduction Combinations of antiretroviral drugs are still the only effective approach to delay the progression to acquired immuno-deficiency syndrome (AIDS) in HIV-1 infected patients. Due to the ability of the virus to introduce and tolerate mutations within the viral proteins, resistance to antiretroviral drug treatment may occur [1–3]. Approximately 10% of the newly diagnosed HIV positive patients, na？ve to drug treatment in Europe, are infected with drug-resistant virus [4, 5]. Effective antiretroviral drug regimens are available; however, to a lesser extent in resource-poor countries than in the industrial parts of the world, whereby a vaccine strategy working in synergy with drug treatment would be beneficial. Previous work has shown that it is possible to induce a cellular immune response against mutated HIV-related epitopes by immunizing with such an epitope [6–8]. The use of short peptides as vaccine candidates is an interesting approach; however, there are immunogenicity problems using only the naked peptide as an immunogen. In order to increase the immunogenicity of short epitopes, novel approaches are needed. We have previously tested the approach of linking peptide to erythrocytes and using these cells as a vessel for transport of peptides to immune cells, as the erythrocytes were treated to be recognized as old by the cells [6]. Despite an increased response, the overall magnitude of the response was weak. We have also previously investigated the use of the B subunit of Cholera toxin (CTB) from the bacterium Vibrio
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