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Abstract The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL) responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot), further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.