Expression cloning for the discovery of viral antigens and epitopes recognized by T cells.
Knowledge of the antigens that are recognized by virus-specific T cells can identify candidate subunit vaccine compounds. Viral antigens are frequently used in cross-sectional and longitudinal studies of the immune response in pathogenesis research. Peptide epitopes are required for the construction of fluorescent major histocompatibility complex-peptide tetramers, which are used to track specific T-cell responses. Expression cloning is a family of methods of antigen discovery that are particularly suitable for DNA viruses with large genomes. Libraries of viral nucleic acid are expressed in formats suitable for presentation to either CD4 or CD8 T cells. Pools of antigens representing fragments of viral open reading frames are loaded into cells expressing the necessary antigen processing and presentation machinery. Highly sensitive T-cell readouts such as lymphokine secretion, proliferation, and gene activation are used to detect active pools, which are then broken down in a reiterative process until a single active clone can be isolated and sequenced. These methods are most applicable if T-cell clones reactive with the whole virus can be obtained by in vitro restimulation or sampling of infected tissues. Alternative methods of antigen discovery are better suited for nonculturable viruses. Expression cloning methods are somewhat generic and are adaptable between infectious diseases, autoimmunity, and tumor immunology research projects.
Koelle
David M
DM
Department of Medicine, University of Washington, Seattle, WA 98104, USA. viralimm@u.washington.edu
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000235:genetics* / Q000276:immunology; D015967:Gene Expression Regulation, Viral / Q000276:immunology*; D006801:Humans; D015533:Transcriptional Activation; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology
D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D014765:Viral Vaccines