To produce cytotoxic T lymphocyte (CTL) expansion in vivo, a two-step antibody delivery system for the delivery of pre-formed HLA/peptide complexes to the abundant CD20 mols. on the surface of B cells, co-opting the role of B cells as antigen-presenting cells was used.However, to avoid the need for sequential injections and to simplify the process to a one-step procedure, the anti-CD20 B9E9 scFvSA fusion protein could be prebound to biotinylated recombinant HLA-A2 class I monomers containing the HLA-A*201 restricted Gag peptide ex vivo, and then administered as a preformed complex.The ability of this modified protein to expand HIV-1-specific CTL ex vivo in a one-step procedure was tested.In consecutive HLA-A2-pos. HIV-1-pos. individuals, the single-step ex-vivo immunization procedure resulted in clear expansion of tetramer staining CD8-pos. T cells after a single stimulation cycle, a situation not observed previously.The tetramer-pos. CD8-pos. cells were CD27/28-/69+/45Ra+, and the percentage of gated tetramer-specific cells that fell into this category approached 100%, significantly greater than previously.HIV-neg. HLA-A2-neg. individuals failed to show such expansion.These data strongly suggest that a one-step procedure may be optimal both in terms of expansion and patient preference.