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Journal of Virology, February 2001, p. 1339-1347, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1339-1347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Rapid Definition of Five Novel HLA-A*3002-Restricted Human Immunodeficiency Virus-Specific Cytotoxic T-Lymphocyte Epitopes by Elispot and Intracellular Cytokine Staining Assays

Philip J. R. Goulder,1,2,* , Marylyn M. Addo,1 Marcus A. Altfeld,1 Eric S. Rosenberg,1 Yanhua Tang,1 Ugene Govender,3 Nolwandle Mngqundaniso,3 Ken Annamalai,3 Thorsten U. Vogel,4 Mike Hammond,5 Michael Bunce,6 Hoosen M. Coovadia,3 and Bruce D. Walker1

Partners AIDS Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 021291; Division of Infectious Diseases, The Children's Hospital, Boston, Massachusetts 021152; Department of Paediatrics, University of Natal, Durban,3 and Natal Blood Transfusion Service, Pinetown,5 South Africa; Wisconsin Regional Primate Center, Madison, Wisconsin 537154; and Oxford Transplant Centre, Churchill Hospital, Oxford OX3 7LJ, United Kingdom6

Received 31 July 2000/Accepted 15 November 2000

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) play a major role in control of viral replication. To understand the contribution of this antiviral response, an initial step is to fully define the specific epitopes targeted by CTL. These studies focused on CTL responses restricted by HLA-A*3002, one of the HLA-A molecules most prominent in African populations. To avoid the time-consuming effort and expense involved in culturing CTL prior to defining epitopes and restricting alleles, we developed a method combining Elispot assays with intracellular gamma interferon staining of peripheral blood mononuclear cells to first map the optimal epitopes targeted and then define the HLA restriction of novel epitopes. In two A*3002-positive subjects whose CTL responses were characterized in detail, the strongest response in both cases was to an epitope in p17 Gag, RSLYNTVATLY (residues 76 to 86). Using this method, CTL epitopes for which there were no motif predictions were optimized and the HLA restriction was established within 48 to 72 h of receipt of blood. This simple and convenient approach should prove useful especially in the characterization of CTL responses specific to HIV and other viruses, particularly in localities where performing cytotoxicity assays would be problematic.

* Corresponding author. Present address: Department of Paediatrics, Nuffield Department of Medicine, Level 7, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. Phone: 44-1865-221335. Fax: 44-1865-220993. E-mail: philip.goulder{at}ndm.ox.ac.uk.

Journal of Virology, February 2001, p. 1339-1347, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1339-1347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.