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.
Highlights
Cell-mediated immunity is considered critical for the prevention and control of many viral infections [1-6]
Sample size may not be of great concern when assessing cytotoxic T lymphocyte (CTL) mediated immune responses against single, small genome viruses such as HIV and HCV, which can be tested in a comprehensive manner using overlapping peptide sets spanning the entire expressed viral genome [9,12]
The present study describes an algorithm by which matrices of optimally defined CTL epitopes derived from five different human viral infections are used in the same enzyme-linked immunospot (ELISpot) assay
Summary
Cell-mediated immunity is considered critical for the prevention and control of many viral infections [1-6]. The approaches developed to detect these responses in vitro have evolved over the years and have provided quantitative and qualitative information on virus-specific T cells for a number of viral infections These assays include, besides others, lymphoproliferative assays using 3H-thymidine incorporation or CFSE staining, limiting dilution precursor-frequency assays for the enumeration of CTL precursor frequencies, intracellular cytokine staining (ICS) and enzyme-linked immunospot (ELISpot) assays [7-10]. Sample size may not be of great concern when assessing CTL mediated immune responses against single, small genome viruses such as HIV and HCV, which can be tested in a comprehensive manner using overlapping peptide sets spanning the entire expressed viral genome [9,12]. For well-studied viruses such as HIV, HCV, EBV and CMV, large sets of such optimally defined CTL epitopes, restricted by common HLA alleles, have been described in the past [14-17], and provide a valuable alternative to measure pathogen-specific CTL responses without the need to synthesize comprehensive peptide sets spanning the entire viral genomes
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